CN109520803A - A kind of method and application reducing microballoon non-specific adsorption - Google Patents
A kind of method and application reducing microballoon non-specific adsorption Download PDFInfo
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- CN109520803A CN109520803A CN201811648131.5A CN201811648131A CN109520803A CN 109520803 A CN109520803 A CN 109520803A CN 201811648131 A CN201811648131 A CN 201811648131A CN 109520803 A CN109520803 A CN 109520803A
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention belongs to detection technique fields, more particularly, to a kind of method and application for reducing microballoon non-specific adsorption.First, the modification of the high molecular polymer containing carboxyl or ester group is carried out to the surface for the amino microballoon being prepared, then the modification of the small organic molecule containing carboxyl or acid anhydrides is carried out to the microballoon after modification again, carboxyl microballoon after modifying twice significantly reduces microballoon to the non-specific adsorption of DNA fragmentation and/or albumen during biologic applications.Method provided by the present invention is easy to operate, is easy to control, and preparation-obtained carboxyl microballoon can widely be incorporated in the fields such as biomarker, bio-separation and in-vitro diagnosis.
Description
Technical field
The invention belongs to detection technique fields, more particularly, to a kind of method for reducing microballoon non-specific adsorption and answer
With.
Background technique
In recent years, detection of nucleic acids is widely used in the fields such as medical diagnosis on disease, science of heredity, criminology, food safety, with microballoon
It is even more to become research hotspot for the DNA detection of carrier (such as: magnetic microsphere, fluorescent microsphere etc.).
Microballoon will cause detection sensitivity reduction to the non-specific adsorption of DNA fragmentation and albumen, more even will cause inspection
Survey result mistake.In actually detected, especially multiple determination, non-specific adsorption not can avoid again, containing more in sample
Kind DNA fragmentation or multiple protein, it is easy to cause non-specific adsorption.
The main reason for causing non-specific adsorption is hydrophobic effect and electrostatic interaction；Mainly change in terms of two at present
On the one hand non-specific adsorption is to be closed using sealer to microsphere surface non-specific sites, hinders non-specificity
Absorption；It on the other hand is to be modified to microsphere surface, this method inherently changes non-specific adsorption.
The surface modification for reducing non-specific adsorption is mainly the following mode: being carried out using organic matter to magnetic microsphere
Surface modification, such as the microballoon of PEG modification, have effectively resisted non-specific adsorption；Using natural macromolecule to microballoon into
Row surface modification, such as Yang-Chuang Chang etc. are prepared for microballoon using chitosan；Surface is carried out using inorganic molecule to repair
Decorations, utilize SiO2Microballoon is wrapped up, microballoon is can protect and is not corroded by external environment, in SiO2Surface carries out functionalization,
It not only improves the application range of microballoon but also non-specific adsorption is effectively reduced；Surface is carried out to microballoon using biomolecule to repair
Decorations reduce non-specific adsorption using the biomolecule of special nature.
The above method is complicated for operation, and application range is narrow, and therefore, urgent need provides a kind of reduction microballoon non-specificity suction
Attached method and application.
Summary of the invention
The first purpose of this invention is, for the deficiencies in the prior art, provides a kind of non-spy of reduction microballoon
The method of opposite sex absorption.
For this purpose, above-mentioned purpose of the invention is achieved through the following technical solutions:
A method of microballoon non-specific adsorption being reduced, the method for reducing microballoon non-specific adsorption includes: in amino
The surface of microballoon first modifies one layer of high molecular polymer containing carboxyl perhaps ester group and then modifies having containing carboxyl or acid anhydrides again
The amino of microsphere surface is simultaneously completely converted into carboxyl to reduce microballoon to the non-specific of DNA fragmentation and/or albumen by machine small molecule
Property absorption.
While by adopting the above technical scheme, the present invention can also be used or be combined using technology further below
Scheme:
Preferably, the carboxylic high molecular polymer include: in polyacrylic acid, polymethylacrylic acid and poly extremely
Few one kind；The high molecular polymer containing ester group includes: polymethyl methacrylate.
Preferably, the small organic molecule include: in acetic anhydride, succinic anhydride, maleic anhydride, acetic acid and propionic acid at least
It is a kind of.
Second object of the present invention is, for the deficiencies in the prior art, provides a kind of microballoon.
For this purpose, above-mentioned purpose of the invention is achieved through the following technical solutions:
A kind of microballoon, the microballoon are prepared by the previously described method for reducing microballoon non-specific adsorption.
A further object of the invention is, for the deficiencies in the prior art, provides a kind of microballoon to DNA piece
Application in terms of section and/or the non-specific adsorption of albumen.
For this purpose, above-mentioned purpose of the invention is achieved through the following technical solutions:
A kind of application of microballoon as previously described in terms of the non-specific adsorption to DNA fragmentation and/or albumen.
The present invention provides a kind of method, preparation-obtained microballoon and application for reducing microballoon non-specific adsorption, firstly,
The modification that the high molecular polymer containing carboxyl or ester group is carried out to the surface for the amino microballoon being prepared, then again to modification
Microballoon afterwards carries out the modification of the small organic molecule containing carboxyl or acid anhydrides, and the carboxyl microballoon after modifying twice is in biologic applications
In the process, microballoon is significantly reduced to the non-specific adsorption of DNA fragmentation and/or albumen.Method behaviour provided by the present invention
Make simple, be easy to control, preparation-obtained carboxyl microballoon can widely be incorporated in biomarker, bio-separation and external
The fields such as diagnosis.
Detailed description of the invention
Fig. 1 is potential diagram of the microballoon before modification, after PAA modification and after SAA modification in the embodiment of the present invention 1.
Fig. 2 is the microballoon in the embodiment of the present invention 2 in the non-specific adsorption after PAA and SAA modification and before modification
Result figure.
Fig. 3 is that PAA and the microspheres quality ratio before modification are respectively 1:10,1:6.67,1:3.33 in the embodiment of the present invention 3
And the mass ratio of the microballoon before 1:2, SAA and modification be 1:1.4 ratio under modify after microballoon non-specific adsorption
Result figure.
Specific embodiment
The present invention is described in further detail referring to the drawings and specific embodiments.
Embodiment 1
(1) 3 mg polyacrylic acid solutions (PAA) are weighed to be dissolved in 1 mL MEST buffer solution (10 mM, pH 5.0), are added
10 mg microballoons (the Subjective and Objective structure formed by carboxyl bulbec and amino mother bulb), after mixing, react on vertical mixed instrument
30 min.After reaction, 10 mg EDC are weighed and are dissolved in 1 mL MEST buffer solution.EDC/MEST solution is added to above-mentioned
In mixed solution, after mixing, 2 h are reacted on vertical mixed instrument.After reaction, washing three times, it is super to be dispersed in 1 mL
In pure water.
(2) it weighs 3.57 mg succinic anhydrides (SAA) to be dissolved in 2 mL dehydrated alcohols, microballoon prepared by step (1)
Magneto separate removes supernatant, and 2 mL SAA/ ethanol solutions are added, react 24 h on vertical mixed instrument.After reaction, three are washed with ethyl alcohol
It is secondary, then wash in the ultrapure water for being dispersed in 1 mL three times.As shown in FIG. 1, FIG. 1 is the microballoons in the embodiment of the present invention 1 to repair
Adorn forward and backward potential diagram；Before modification, the current potential of microballoon is -29.5 mV, and after PAA is modified, the current potential of microballoon is -31.2 mV,
The current potential of microballoon is -40.3 mV after SAA is modified, that is to say, bright introduce carboxylic group in microsphere surface.
Embodiment 2
(1) take 10 mg microballoons in 3 centrifuge tubes respectively, Magneto separate removes supernatant, weighs 9 mg PAA and is dissolved in 3 mL, 10 mM
In MEST, every pipe is added 1 mL PAA/MEST solution and reacts 30 min on vertical mixed instrument after mixing.Reaction terminates
Afterwards, 30 mg EDC are weighed to be dissolved in 3 mL, 10 mM MEST.Every pipe is added into 1 mL EDC/MEST solution, is uniformly mixed
Afterwards, 2 h are reacted on vertical mixed instrument.After reaction, washing three times, is dispersed in the ultrapure water of 1 mL.
(2) 3.57 mg SAA are weighed to be dissolved in 2 mL dehydrated alcohols.Microballoon Magneto separate prepared by step (1) is gone
Clearly, 2 mL SAA/ ethanol solutions are added in every pipe, react 24 h on vertical mixed instrument.After reaction, it is washed three times with ethyl alcohol, then
Washing three times, is dispersed in the ultrapure water of 1 mL.
(3) 2 μ g of microballoon after taking step (2) to modify and the 2 μ g of microballoon that step (1) and (2) are modified is not carried out, use respectively
Hybridization solution (0.2 M Tris pH 7.4) washs 1 time.It is separately added into 10 μ L non-specific amplification products again, 88 μ L hybridization solutions,
At 50 DEG C, hybridize 30 minutes.It washed once, be dispersed in 50 μ L ultrapure waters with hybridization solution, carry out flow cytometer detection, streaming result
As shown in Fig. 2, showing: the microballoon after experience PAA and SAA modification, non-specific adsorption value significantly reduce.It is connected on amplified production
Fluorescent material can be detected by the channel streaming APC, and the fluorescence intensity detected is higher, illustrates that non-specific adsorption is got over
By force.
Embodiment 3
(1) it weighs the mg polyacrylic acid solution of 2 mg, 3 mg, 6 mg, 10 (PAA) to be dissolved separately in 1 mL MEST, then distinguishes
10 mg microballoons are added, after mixing, 30 min are reacted on vertical mixed instrument.After reaction, it is molten that 40 mg EDC are weighed
In 4mL MEST buffer solution.1 mL EDC/MEST solution is separately added into above-mentioned mixed solution, after mixing,
2 h are reacted on vertical mixed instrument.After reaction, washing three times, is dispersed in the ultrapure water of 1 mL.
(2) 14.28 mg SAA are weighed to be dissolved in 8 mL dehydrated alcohols.Microballoon Magneto separate prepared by step (1) is gone
Supernatant, every pipe are added 2 mL SAA/ ethanol solutions, react 24 h on vertical mixed instrument.After reaction, it is washed three times with ethyl alcohol,
It is washed in the ultrapure water for being dispersed in 1 mL three times again.
(2) microballoon for taking 2 μ g to be prepared by step (2) respectively washs 1 with hybridization solution (0.2 M Tris pH 7.4)
It is secondary.10 μ L non-specific amplification products are added, 88 μ L hybridization solutions are added, at 50 DEG C, hybridize 30 minutes.One is washed with hybridization solution
It is secondary, it is dispersed in 50 μ L ultrapure waters, carries out flow cytometer detection, streaming result is as shown in figure 3, show: the mass ratio of microballoon and PAA
When for 10:1.5, non-specific adsorption value is minimum.It is connected with fluorescent material on amplified production, can be carried out by the channel streaming APC
Detection, the fluorescence intensity detected is higher, illustrates that non-specific adsorption is stronger.
Above-mentioned specific embodiment is used to illustrate the present invention, is merely a preferred embodiment of the present invention, rather than to this
Invention is limited, and in the spirit and scope of protection of the claims of invention, any modification for making to invention is equally replaced
It changes, improve, both fall within protection scope of the present invention.
Claims (5)
1. a kind of method for reducing microballoon non-specific adsorption, which is characterized in that the side for reducing microballoon non-specific adsorption
Method includes: first to modify one layer of high molecular polymer containing carboxyl or ester group on the surface of amino microballoon, is then modified again containing carboxylic
The small organic molecule of base or acid anhydrides and the amino of microsphere surface is completely converted into carboxyl with reduce microballoon to DNA fragmentation and/
Or the non-specific adsorption of albumen.
2. the method according to claim 1 for reducing microballoon non-specific adsorption, which is characterized in that the carboxylic height
Molecularly Imprinted Polymer includes: at least one of polyacrylic acid, polymethylacrylic acid and poly；The macromolecule containing ester group
Polymer includes: polymethyl methacrylate.
3. the method according to claim 1 for reducing microballoon non-specific adsorption, which is characterized in that the small organic molecule
It include: at least one of acetic anhydride, succinic anhydride, maleic anhydride, acetic acid and propionic acid.
4. a kind of microballoon, which is characterized in that the microballoon is by the method described in claim 1 for reducing microballoon non-specific adsorption
It is prepared.
5. a kind of application of microballoon as claimed in claim 4 in terms of the non-specific adsorption to DNA fragmentation and/or albumen.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811648131.5A CN109520803B (en) | 2018-12-29 | 2018-12-29 | Method for reducing nonspecific adsorption of microspheres and application |
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| CN201811648131.5A CN109520803B (en) | 2018-12-29 | 2018-12-29 | Method for reducing nonspecific adsorption of microspheres and application |
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| CN109520803A true CN109520803A (en) | 2019-03-26 |
| CN109520803B CN109520803B (en) | 2022-01-18 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864967A (en) * | 2012-12-11 | 2014-06-18 | 中国科学院大连化学物理研究所 | Polymer particles modified by amphoteric carrier and application thereof in pretreatment of protein sample |
| CN104031201A (en) * | 2014-05-29 | 2014-09-10 | 深圳市新产业生物医学工程股份有限公司 | Preparation method and application of magnetic microsphere for biological protein separation |
| CN105126714A (en) * | 2015-08-05 | 2015-12-09 | 上海交通大学 | Functional nano particle composite microsphere, preparation and applications thereof |
| CN106237947A (en) * | 2016-08-31 | 2016-12-21 | 上海美吉生物医药科技有限公司 | Magnetic microsphere of high density carboxyl modified and preparation method thereof |
-
2018
- 2018-12-29 CN CN201811648131.5A patent/CN109520803B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864967A (en) * | 2012-12-11 | 2014-06-18 | 中国科学院大连化学物理研究所 | Polymer particles modified by amphoteric carrier and application thereof in pretreatment of protein sample |
| CN104031201A (en) * | 2014-05-29 | 2014-09-10 | 深圳市新产业生物医学工程股份有限公司 | Preparation method and application of magnetic microsphere for biological protein separation |
| CN105126714A (en) * | 2015-08-05 | 2015-12-09 | 上海交通大学 | Functional nano particle composite microsphere, preparation and applications thereof |
| CN106237947A (en) * | 2016-08-31 | 2016-12-21 | 上海美吉生物医药科技有限公司 | Magnetic microsphere of high density carboxyl modified and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| ZAMMATTEO N 等: "Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays", 《ANALYTICAL BIOCHEMISTRY》 * |
| 文颖慧 等: "多醇法可控制备羧基化Fe3O4超顺磁性微球", 《功能材料》 * |
| 许永娟 等: "载体材料表面化学组成对蛋白质吸附行为的影响研究进展", 《化工进展》 * |
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Inventor after: Zhang Xiaoqin Inventor after: Hong Ming Inventor after: Wang Yao Inventor after: Xu Hong Inventor after: Gu Hongchen Inventor after: He Guoqing Inventor after: Wang Jufeng Inventor after: Liu Wei Inventor after: Zhou Xuyi Inventor before: Hong Ming Inventor before: Liu Wei Inventor before: Zhou Xuyi Inventor before: Wang Yao Inventor before: Du Weilin Inventor before: Xu Hong Inventor before: Liu Xiang Inventor before: Gu Hongchen Inventor before: He Guoqing Inventor before: Wang Jufeng Inventor before: Shi Lili |
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