CN106345544A - Active amino-modified substrate for microarray and preparation method of active amino-modified substrate - Google Patents
Active amino-modified substrate for microarray and preparation method of active amino-modified substrate Download PDFInfo
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- CN106345544A CN106345544A CN201610839386.4A CN201610839386A CN106345544A CN 106345544 A CN106345544 A CN 106345544A CN 201610839386 A CN201610839386 A CN 201610839386A CN 106345544 A CN106345544 A CN 106345544A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0819—Microarrays; Biochips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0822—Slides
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/12—Specific details about materials
Abstract
The invention relates to an active amino-modified substrate for a microarray and a preparation method of the active amino-modified substrate and belongs to the technical field of medical science and biology. The biological chip substrate is characterized in that amino silanization treatment is performed on the surface of a glass substrate, and polylysine coating is performed on the obtained glass substrate with rich amino on the surface to obtain the active amino-modified glass substrate. The active amino-modified substrate has the advantages that due to the fact preprocessing is performed by using concentrated sulfuric acid and hydrogen peroxide according to optimal proportion, the problem that a common substrate has many impurities is solved, polylysine coating is performed after the amino silanization, the dual combined effect of static absorption and chemical coupling is formed, the fixing rate of the substrate to a probe is evidently increased, and the problems that the substrate is low in sensitivity and poor in stability are solved; the substrate is a biological chip probe vector with excellent performance and quality and is widely applicable to biological chip preparation.
Description
Technical field
The invention belongs to medical science and biology techniques field, it is related to a kind of bio-chip substrate and preparation method thereof, especially
It is to be related to a kind of microarray active amino to modify substrate and preparation method thereof.
Background technology
Since nineteen nineties, the intension being covered is expanding biochip technology all the time, technology
Method is also perfect all the time.Because it can detect the expression of ten hundreds of genes simultaneously, therefore, it can help
The molecular mechanism of our study of disease and biological pathways, have become as in Biochemistry and Molecular Biology field one very
Important research tool.Substrate is the basis of gene chip, and the preparation of substrate is highly important link in gene chip exploitation.
The gene chip commonly used at present belongs to simple silanized substrate, carries out silylation modification in substrate surface, then carries out small molecule
Modify, add amino or aldehyde radical etc., however, these substrates are relatively low to the fixed rate of probe, sensitivity is not high, and exist steady
Qualitative poor the problems such as.
Content of the invention
The invention aims to solving existing gene chip with the glass of glass substrate or simple silanization treatment
Glass substrate is the deficiency of carrier, provides a kind of microarray active amino to modify substrate and preparation method thereof.This preparation method is grasped
Make simple, low cost, suitable industrialized production, and obtained substrate can significantly improve probe fixed efficiency, be a kind of
The remarkable micro array carrier of performance.
Since nineteen nineties, the intension being covered is expanding biochip technology all the time, technology
Method is also perfect all the time.Because it can detect the expression of ten hundreds of genes simultaneously, therefore, it can help
The molecular mechanism of our study of disease and biological pathways, have become as in Biochemistry and Molecular Biology field one very
Important research tool.Substrate is the basis of gene chip, and the preparation of substrate is highly important link in gene chip exploitation.
The gene chip commonly used at present belongs to simple silanized substrate, carries out silylation modification in substrate surface, then carries out small molecule
Modify, add amino or aldehyde radical etc., however, these substrates are relatively low to the fixed rate of probe, sensitivity is not high, and exist steady
Qualitative poor the problems such as.
The present invention realizes its purpose and adopts the following technical scheme that
A kind of microarray modifies the preparation method of substrate with active amino, and step is as follows:
Step (1), the pretreatment of glass substrate: by glass substrate in the mixed solution of concentrated sulphuric acid and hydrogen peroxide soaked overnight,
Using deionized water, the concentrated sulphuric acid remaining glass substrate surface and dioxygen water mixed liquid clean up and dry, and obtain pretreatment
Glass substrate afterwards；
Step (2), the Aminosilylation (entrance active amine groups) of glass substrate: by step (1) obtain pretreated
Glass substrate and 3- aminopropyl diethoxy silane solution reaction, introduce active amine groups, then with ethanol purge residual
3- aminopropyl diethoxy silane solution, finally dries, and obtains the glass substrate of surface amino groups silanization；
Step (3), poly-D-lysine coating, the glass substrate surface of the Aminosilylation that step (2) is obtained is coated poly and relies
Then the not coated poly-D-lysine of its excess surface is cleaned up with distilled water, then dries up, be cooled to room temperature by propylhomoserin,
Obtain microarray active amino and modify substrate.
Further preferably step (1) is entered to glass substrate as abluent using the mixed liquor of concentrated sulphuric acid and hydrogen peroxide
Row pre-treatment, concentrated sulphuric acid: hydrogen peroxide=7:3, it is placed on wherein immersion 13-18 h.
Further preferably step (1) adopts deionized water to clean the concentrated sulphuric acid of glass substrate residual and the mixed of hydrogen peroxide
When closing soak, need continuous wash 2-5 time, each 3-5 min.
Further preferably in step (2), it is 3- ammonia third that the Aminosilylation of glass substrate processes adopted reagent
Base diethoxy silane.
Further preferably in step (2), the Aminosilylation of glass substrate processes adopted 3- aminopropyl diethyl
TMOS solution, mass concentration is 1.0%-3.0%, and the response time is 2-6h.
Further preferably in step (2), after Aminosilylation is processed, glass substrate cleanout fluid uses ethanol (body
Long-pending concentration ratio is 60%-80%), clean 2-5 time, each 1-5 min.
Further preferably in step (3), the preparation method of Poly-L-Lysine Solution, using pbs as configuration poly
The solvent of lysine solution.
Further preferably in step (3), the mass concentration of Poly-L-Lysine Solution is 1.5%-3.5%, and 4 DEG C of preservations are standby
With.
Further preferably in step (3), it is 25- that the poly-D-lysine of the glass substrate of Aminosilylation is coated the time
45 min, being coated temperature is room temperature.
Further preferably in step (3), after poly-D-lysine is coated, rinsed 2-5 time using distilled water, each 1-5
Min, dries up standby.
Further preferably described microarray modifies the preparation method of substrate with active amino, comprises the steps:
Step (1), the pretreatment of glass substrate: glass substrate is placed in concentrated sulphuric acid: soak in the mixed solution of hydrogen peroxide=7:3
15 h, using deionized water, the concentrated sulphuric acid remaining glass substrate surface and dioxygen water mixed liquid clean up and dry, and obtain
Pretreated glass substrate；
Step (2), the Aminosilylation (introducing active amine groups) of glass substrate: by step (1) obtain pretreated
Glass substrate and mass concentration are that 2% 3- aminopropyl diethoxy silane solution fully reacts, and introduce active amine groups, so
Afterwards with the 3- aminopropyl diethoxy silane solution of 75% ethanol purge residual, finally dry, obtain surface amino groups silanization
Glass substrate；
Step (3), poly-D-lysine coating, the glass substrate of the Aminosilylation that step (2) is obtained is dipped in 2.5% poly and relies
Propylhomoserin pbs solution is coated, and is then cleaned up the not coated poly-D-lysine of its excess surface with distilled water, then blows
Dry, it is cooled to room temperature, obtain microarray active amino and modify substrate.
Further preferably described microarray modifies the preparation method of substrate with active amino, comprises the steps:
Step (1), the pretreatment of glass substrate: glass substrate is placed in concentrated sulphuric acid: soak in the mixed solution of hydrogen peroxide=7:3
15 h, the glass substrate of the concentrated sulphuric acid of remained on surface and dioxygen water mixed liquid is cleaned 2 times using deionized water, each 3min,
Finally dry, obtain pretreated glass substrate；
Step (2), the Aminosilylation (introducing active amine groups) of glass substrate: by step (1) obtain pretreated
Glass substrate and mass concentration are that 2.5% 3- aminopropyl diethoxy silane solution fully reacts, and introduce active amine groups,
Then with the 3- aminopropyl diethoxy silane solution of 75% ethanol purge residual, finally dry, obtain surface amino groups silanization
Glass substrate；
Step (3), poly-D-lysine coating, the glass substrate of the Aminosilylation that step (2) is obtained is dipped in 2.0% poly and relies
Propylhomoserin pbs solution is coated, and is then cleaned up the not coated poly-D-lysine of its excess surface with distilled water, then blows
Dry, it is cooled to room temperature, obtain microarray active amino and modify substrate.
The present invention also provides the microarray that the preparation method that a kind of above-mentioned microarray active amino modifies substrate obtains to use
Active amino modifies substrate.
Substrate of the present invention, is the glass substrate surface that processed of mixed liquor in suitable proportion concentrated sulphuric acid and hydrogen peroxide, adopts
With 3- aminopropyl diethoxy silane, Aminosilylation process is carried out to it, then carried out using the pbs solution of poly-D-lysine
It is coated, the final active amino obtaining is modified substrate and be can be used to fixing biomacromolecule.
Compared with prior art, its advantage is the present invention: the pre-treatment of glass substrate use concentrated sulphuric acid with
The mixed liquor of hydrogen peroxide 7:3 ratio is carried out, and has captured the more difficult problem of common substrate impure point；Aminosilylation was processed
Glass substrate carry out being coated of poly-D-lysine, significantly improve the fixed rate to probe for the substrate, fixed rate is than common ammonia
Base substrate improves 10 times, more than solves the problems, such as that substrate sensitivity is low and stability is poor, and convenient, economical and quick.Cause
This, the amino-group substrate that the present invention provides is a kind of excellent biochip probe carrier, can be widely applied to the system of biochip
Standby.
Brief description
Fig. 1 is glass substrate structural representation.
Fig. 2 is amido modified glass substrate schematic diagram.
Amido modified glass substrate schematic diagram after the process of Fig. 3 poly-D-lysine.
Specific embodiment
Following examples are used for the present invention is described, but are not limited to the scope of the present invention.
Embodiment 1
A kind of microarray modifies the preparation method of substrate with active amino, comprises the steps:
Step (1), the pretreatment of glass substrate: glass substrate is placed in concentrated sulphuric acid: soak in the mixed solution of hydrogen peroxide=7:3
13 h, the glass substrate of the concentrated sulphuric acid of remained on surface and dioxygen water mixed liquid cleans 2 times using deionized water, and every time 5
Min, finally dries, and obtains pretreated glass substrate；
Step (2), the Aminosilylation (introducing active amine groups) of glass substrate: by step (1) obtain pretreated
Glass substrate and mass concentration are that 1.0% 3- aminopropyl diethoxy silane solution fully reacts, and introduce active amine groups,
Then with the 3- aminopropyl diethoxy silane solution of 60% ethanol purge residual, finally dry, obtain surface amino groups silanization
Glass substrate；
Step (3), poly-D-lysine coating, the glass substrate of the Aminosilylation that step (2) is obtained is dipped in 1.5% poly and relies
Propylhomoserin pbs solution is coated, and is then cleaned up the not coated poly-D-lysine of its excess surface with distilled water, then blows
Dry, it is cooled to room temperature, obtain microarray active amino and modify substrate.
Embodiment 2
A kind of microarray modifies the preparation method of substrate with active amino, comprises the steps:
Step (1), the pretreatment of glass substrate: glass substrate is placed in concentrated sulphuric acid: soak in the mixed solution of hydrogen peroxide=7:3
18 h, the glass substrate of the concentrated sulphuric acid of remained on surface and dioxygen water mixed liquid cleans 5 times using deionized water, and every time 5
Min, finally dries, and obtains pretreated glass substrate；
Step (2), the Aminosilylation (introducing active amine groups) of glass substrate: by step (1) obtain pretreated
Glass substrate and mass concentration are that 3.0% 3- aminopropyl diethoxy silane solution fully reacts, and introduce active amine groups,
Then with the 3- aminopropyl diethoxy silane solution of 80% ethanol purge residual, finally dry, obtain surface amino groups silanization
Glass substrate；
Step (3), poly-D-lysine coating, the glass substrate of the Aminosilylation that step (2) is obtained is dipped in 3.5% poly and relies
Propylhomoserin pbs solution is coated, and is then cleaned up the not coated poly-D-lysine of its excess surface with distilled water, then blows
Dry, it is cooled to room temperature, obtain microarray active amino and modify substrate.
Embodiment 3
A kind of microarray modifies the preparation method of substrate with active amino, comprises the steps:
Step (1), the pretreatment of glass substrate: glass substrate is placed in concentrated sulphuric acid: soak in the mixed solution of hydrogen peroxide=7:3
15 h, the glass substrate of the concentrated sulphuric acid of remained on surface and dioxygen water mixed liquid cleans 3 times using deionized water, and every time 4
Min, finally dries, and obtains pretreated glass substrate；
Step (2), the Aminosilylation (introducing active amine groups) of glass substrate: by step (1) obtain pretreated
Glass substrate and mass concentration are that 2.5% 3- aminopropyl diethoxy silane solution fully reacts, and introduce active amine groups,
Then with the 3- aminopropyl diethoxy silane solution of 70% ethanol purge residual, finally dry, obtain surface amino groups silanization
Glass substrate；
Step (3), poly-D-lysine coating, the glass substrate of the Aminosilylation that step (2) is obtained is dipped in 2.5% poly and relies
Propylhomoserin pbs solution is coated, and is then cleaned up the not coated poly-D-lysine of its excess surface with distilled water, then blows
Dry, it is cooled to room temperature, obtain microarray active amino and modify substrate.
Performance detection
Point sample: synthesize the fluorescent probe of cy5 labelling from Invitrogen, 1:1 mixes by volume with pbs, is prepared into probe solution,
By the probe preparing according in every hole 50 μ l point sample 96 hole sample panel, by probe solution, point sample prepares in the present invention respectively
Active amino modify in base and comparison substrate and (do not carry out the common amino-group substrate of poly-D-lysine process), 60 DEG C to fix 2 little
When.
The mensure of fluorescence intensity before eluting: after completing point sample, the chip after the completion of fixation is divided using multi-function microplate reader
Do not measure its fluorescence intensity, the results are shown in Table 1.
The mensure of fluorescence intensity after eluting: after the completion of digital independent, carry out microarray eluting, elution process is: 0.2%sds
Cleaning 3 times, then cleaned with distilled water 3 times, same addition 50 μ l distilled waters, put the fluorescence after multi-function microplate reader detection is developed a film
Intensity, the results are shown in Table 1.
The calculating of data: the fluorescence intensity ratio after computing chip eluting and before eluting.
Result of calculation lists table 1 in.
Table 1 active amino modifies the fixed rate contrast of substrate and common substrate
From table 1 result, active amino modifies the probe fixed rate average 18.02% of substrate, and common ammonia substrate probe is solid
Determine rate average be only 1.59% it can be seen that active amino of the present invention modify substrate can be probe fixed rate improve 10 times with
On.
Therefore, microarray active amino of the present invention modifies substrate is a kind of remarkable micro array carrier of performance, its
Preparation method is simple, low cost, be applied to industrialization promotion produce.
Principal character and the advantage of the present invention have been shown and described above, it should be understood by those skilled in the art that
The present invention is not restricted to the described embodiments, above-described embodiment and description description merely illustrate the principles of the invention,
Without departing from the scope of the invention, the present invention also has various changes and modifications, and the improvement of these changes and technology is all
To fall in scope of the claimed invention, the scope of protection of present invention is by appending claims and its equivalent
Define.
Claims (5)
1. a kind of microarray active amino modifies the preparation method of substrate it is characterised in that comprising the steps: step (1),
The pretreatment of glass substrate: by glass substrate in the mixed solution of concentrated sulphuric acid and hydrogen peroxide soaked overnight, using deionized water
The concentrated sulphuric acid that remain glass substrate surface and dioxygen water mixed liquid clean up and dry, and obtain pretreated glass base
Piece；
Step (2), the Aminosilylation (introducing active amine groups) of glass substrate: by step (1) obtain pretreated
Glass substrate and 3- aminopropyl diethoxy silane solution reaction, introduce active amine groups, then with ethanol purge residual
3- aminopropyl diethoxy silane solution, finally dries, and obtains the glass substrate of surface amino groups silanization；
Step (3), poly-D-lysine coating, the glass substrate surface of the Aminosilylation that step (2) is obtained is coated poly and relies
Then the not coated poly-D-lysine of its excess surface is cleaned up with distilled water, then dries up, be cooled to room temperature by propylhomoserin,
Obtain microarray active amino and modify substrate.
2. microarray according to claim 1 active amino modifies the preparation method of substrate it is characterised in that step (1)
The soak that slide pretreatment is adopted is (concentrated sulphuric acid and hydrogen peroxide) and its ratio 7:3, the soak time 13-18 h of slide,
Cleaned using deionized water the concentrated sulphuric acid of glass substrate residual and hydrogen peroxide soak when, need continuous wash 2-5 time, each 3-
5 min.
3. microarray according to claim 1 active amino modifies the preparation method of substrate it is characterised in that step (2)
In, it is 3- aminopropyl diethoxy silane that the Aminosilylation of glass substrate processes adopted reagent, the amino of glass substrate
The 3- aminopropyl diethoxy silane solution that silanization treatment is adopted, mass concentration is 1.0%-3.0%, and the response time is 2-
6h, after Aminosilylation is processed, glass substrate cleanout fluid uses ethanol (volumetric concentration ratio is 60%-80%), cleans 2-5 time,
1-5 min every time.
4. microarray according to claim 1 active amino modifies the preparation method of substrate it is characterised in that step (3)
In, the preparation method of Poly-L-Lysine Solution, using pbs as the solvent configuring Poly-L-Lysine Solution, poly-D-lysine is molten
The mass concentration of liquid is 1.5%-3.5%, and 4 DEG C save backup, and the poly-D-lysine of the glass substrate of Aminosilylation is coated the time
It is 25-45 min, being coated temperature is room temperature.
5. claim 1-9 any one microarray active amino modifies the microarray activity that the preparation method of substrate is obtained
Amido modified substrate.
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| CN108855259A (en) * | 2018-06-05 | 2018-11-23 | 中国科学院苏州生物医学工程技术研究所 | A kind of surface modifying method of micro-array chip |
| WO2021185086A1 (en) * | 2020-03-19 | 2021-09-23 | 京东方科技集团股份有限公司 | Detection chip and modification method therefor |
| WO2021196918A1 (en) * | 2020-03-31 | 2021-10-07 | 京东方科技集团股份有限公司 | Quality detection method for immune chip |
| CN114075592A (en) * | 2020-08-12 | 2022-02-22 | 深圳市真迈生物科技有限公司 | Amino modified chip and preparation method and application thereof |
| CN114682309A (en) * | 2020-12-29 | 2022-07-01 | 深圳华大生命科学研究院 | Chip, method for preparing chip and use of chip |
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