CN1281953C - Method for making bio-chip - Google Patents
Method for making bio-chip Download PDFInfo
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- CN1281953C CN1281953C CN 03157324 CN03157324A CN1281953C CN 1281953 C CN1281953 C CN 1281953C CN 03157324 CN03157324 CN 03157324 CN 03157324 A CN03157324 A CN 03157324A CN 1281953 C CN1281953 C CN 1281953C
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- och
- silica gel
- polyethyleneglycol derivative
- polyethyleneglycol
- making
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Abstract
The present invention relates to a method for making a biological chip, which comprises the following steps: firstly, making a silica gel stamp; secondly, coating a polyethyleneglycol derivant A for formatting a solid surface on the surface of the silica gel stamp made in the first step, and then, covering the silica gel stamp on a spare solid surface; transferring the polyethyleneglycol derivant A according to a designed pattern on the solid surface; finally, soaking a formatted solid substrate in a solution of a polyethyleneglycol derivant B. thus, the polyethyleneglycol derivant B is combined in a white space covered by the polyethyleneglycol derivant A. Active groups on the polyethyleneglycol derivant B are activated by a general chemical method. Biosamples are put on the solid surface where the polyethyleneglycol derivant B covers by a mechanical spotting machine. After being incubated, biomolecules which are not fixed on the solid surface covalently are washed away by water to obtain a membranous layer of the biomolecules which are evenly distributed. The method for making a biological chip has the advantages of simple operation and good repetitiveness; the dots of the biological chip have even shape and size and the biomolecules in each dot are evenly distributed.
Description
Technical field
The present invention relates to a kind of biochip method for making, particularly relate to a kind of on biochip surface manufactured samples point shape size uniformity, the method for making of the biochip that biological sample is evenly distributed in a single point.
Background technology
Biochip has become field flourish in the present biotechnology.At aspects such as biological study, medical diagnosis on disease, environmental monitorings, biochip has huge potential application foreground.The making of biochip has had several different methods.At present, using the most extensively, also is that the simplest method is the mechanical deposition method, biological sample is selected with sample injector be added on the substrate, and is arranged on the substrate with the form of matrix.The point sample method can be divided into two classes again, contact and contactless point sample.Although the mechanical deposition method can accurately be controlled the biological sample amount of every bit, point can't be accomplished shape size uniformity at on-chip point.Because behind the point sample, substrate does not clean, point is in on-chip biomolecule just is deposited in a little, skewness, uneven as list of references 1:GavinMacBeath, Stuart L Schreiber, Printing proteins as microarrays for high-throughputfunction determination, Science, 289 (2000) 1760-1763 are described.If clean, will cause the pollution in zone between points, all there is biomolecule on whole surface, can't distinguish, so generally do not clean.The result of this point sample method makes present biochip only can be used for qualitative or sxemiquantitative is measured, and realizes that accurate quantification is very difficult.
Summary of the invention
The objective of the invention is to overcome present used mechanical deposition legal system and make the weak point that the method for biochip exists; Thereby provide a kind of on biochip surface manufactured samples point shape size uniformity, the method for making of the biochip that biological sample is evenly distributed in a single point.
The object of the present invention is achieved like this:
The method for making of biochip provided by the invention may further comprise the steps:
One. at first make the silica gel seal: photoresist is coated on the silicon chip, carves required pattern, just produced mould by conventional photoetching technique;
Two. the silica gel of polymethoxy siloxane (PDMS) is poured on the mould of step 1 preparation, treats to take off the silica gel seal that has just obtained having required pattern from mould again after silica gel solidifies;
Three. after the polyethyleneglycol derivative A that is used for formaing solid surface being coated in the seal surface of step 2 making, cover again on stand-by solid substrate surface, just polyethyleneglycol derivative A according to the design pattern transfer on the solid substrate surface, obtain formative solid substrate, promptly some zone is covered by polyethyleneglycol derivative A on the solid substrate surface;
Four. the formatted solid substrate of step 3 is dipped in the polyethyleneglycol derivative B solution, and polyethyleneglycol derivative B just is combined in the white space of no polyethyleneglycol derivative A covering;
Five. with the reactive group on the conventional chemical method activated polyethylene glycol derivant B, the zone that biological sample point is covered by polyethyleneglycol derivative B on solid surface by the mechanical deposition instrument; After hatching certain hour under the normal temperature, wherein incubation time length is decided according to different protein and different concentration; Water rinses out not by after the Covalent Immobilization biomolecule from the teeth outwards again, has just obtained the biologic single molecular rete that is evenly distributed.
Described solid material substrate comprises: silicon chip, glass sheet, sheet metal or plastic sheet etc. can be used for the material of chip manufacturing.
The pattern of described silica gel seal refers to that all are suitable for the pattern of biochip point sample, and pattern dimension can be as small as one micron, greatly to one millimeter.
Described polyethyleneglycol derivative A comprises: Cl
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3, HS (CH
2)
11(0CH
2CH
2)
3OH, (CH
3CH
2O)
3Si (CH
2)
11(0CH
2CH
2)
3OCH
3, and all can suppress the polyethyleneglycol derivative of biomolecule generation physisorption on solid surface.
Described polyethyleneglycol derivative B solution comprises: Cl
3Si (CH
2)
11(OCH
2CH
2)
3OCH
2COOCH
2CH
3, HS (CH
2)
11(OCH
2CH
2)
7OCH
2COOH, (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3OCHO, (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3CH
2NH
4, and all can either suppress the biological physisorption that takes place on solid surface, again can be by chemical reaction biomolecule Covalent Immobilization polyethyleneglycol derivative from the teeth outwards.
Preparation method's of the present invention advantage is:
The solid substrate material that the present invention adopts is a conventional material, and for example: silicon chip, glass sheet, sheet metal or plastic sheet etc. can be used for the material of chip manufacturing, so cost is low; Adopt the lip-deep appointed area of polyethyleneglycol derivative A sealing solid substrate, can prevent that biomolecule from the effect of physisorption taking place on the solid substrate surface, therefore cleans and can not cause surface contamination; Polyethyleneglycol derivative B end has reactive group, can be after the activation with the group generation chemical reaction on the protein molecule, thereby in the region surface that is covered by polyethyleneglycol derivative B, and each some internal protein is to distribute with the form of unimolecule rete required biomolecule Covalent Immobilization; Because the quantity of putting on the chip surface, shape and size are determined by the pattern on the silica gel seal, therefore can produce shape size homogeneous a little by this method, the biochip that biomolecule is evenly distributed in each point.This preparation method is simple, good reproducibility, and be easy to suitability for industrialized production.
The pattern of silica gel seal refers to that all are suitable for the pattern of biochip point sample among the preparation method of the present invention, and pattern dimension can be as small as one micron, greatly to one millimeter, therefore is widely used.
Description of drawings
Fig. 1 is a preparation method's of the present invention process chart
Embodiment
Embodiment 1
With reference to Fig. 1, describing a kind of preparation in detail is the method for making of the methyl polyethyleneglycol derivative biochip of substrate with the silicon chip
1. at first make the silica gel seal: photoresist is coated on the silicon chip, produces mould by conventional photoetching technique with 500 circular patterns;
2. the silica gel of commercially available PDMS is poured on the mould that step 1 makes, treat PDMS silica gel solidify after again from mould take off just obtained having circular pattern, diameter is 20 microns silica gel seal;
On the silica gel seal that step 2 is made with polyethyleneglycol derivative Cl
2Si (CH
2)
11(OCH
2CH
2)
3OCH
3Solution is coated on the silica gel seal of step 2 making, and transfer printing is on silicon chip;
4. the silica gel seal that step 3 is made is immersed in polyethyleneglycol derivative Cl again
3Si (CH
2)
11(OCH
2CH
2)
3OCH
2COOCH
2CH
3Middle modification does not have polyethyleneglycol derivative Cl
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3The zone that covers; Handle silicon chip with dilution heat of sulfuric acid, promptly conventional de-ester reaction removes carboxy protective; Re-use N-hydroxy-succinamide ester (NHS) processing carboxyl and generate active ester derivant; By point sample instrument at polyethyleneglycol derivative Cl
3Si (CH
2)
11(OCH
2CH
2)
3OCH
2COOCH
2CH
3The protein solution of the protein chip that point will prepare in the overlay area, present embodiment can be put the 1-500 kind on 500 border circular areas, after hatching 30 minutes under the normal temperature, deionized water rinsing, promptly preparing with the silicon chip is the methyl polyethyleneglycol derivative biochip of substrate.
Embodiment 2
With reference to Fig. 1, describing a kind of preparation in detail is the method for making of the hydroxyl polyethyleneglycol derivative biochip of substrate with gold-plated silicon chip
1. at first make the silica gel seal: photoresist is coated on the silicon chip, produces by conventional photoetching technique and have 10000 square pattern;
2. the silica gel of commercially available PDMS is poured on the mould that step 1 makes, treat PDMS silica gel solidify after again from mould take off just obtained having square pattern, the length of side is 100 microns silica gel seal;
On the silica gel seal that step 2 is made with polyethyleneglycol derivative HS (CH
2)
11(OCH
2CH
2)
3OH solution is coated on the silica gel seal of step 2 making, and transfer printing is on silicon chip;
4. the silica gel seal that step 3 is made is immersed in polyethyleneglycol derivative HS (CH again
2)
11(OCH
2CH
2)
7OCH
2Modification does not have polyethyleneglycol derivative HS (CH among the COOH
2)
11(OCH
2CH
2)
3The zone that OH covers; Use N-hydroxy-succinamide ester (NHS) to handle carboxyl and generate active ester derivant; By point sample instrument at polyethyleneglycol derivative HS (CH
2)
11(OCH
2CH
2)
7OCH
2The protein solution of the protein chip that point will prepare in the COOH overlay area, present embodiment can be put the 1-10000 kind on 10000 square area, after hatching 30 minutes under the normal temperature, deionized water rinsing, promptly preparing with gold-plated silicon chip is the hydroxyl polyethyleneglycol derivative biochip of substrate.
Embodiment 3
With reference to Fig. 1, describing a kind of preparation in detail is the method for making of the methyl polyethyleneglycol derivative biochip of substrate with the glass sheet
1. at first make the silica gel seal: photoresist is coated on the silicon chip, produces mould by conventional photoetching technique with 1500 circular patterns;
2. the silica gel of commercially available PDMS is poured on the mould that step 1 makes, treat PDMS silica gel solidify after again from mould take off just obtained having circular pattern, diameter is 200 microns silica gel seal;
On the silica gel seal that step 2 is made with polyethyleneglycol derivative (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3Solution is coated on the silica gel seal of step 2 making, and transfer printing is on silicon chip;
4. the silica gel seal that step 3 is made is immersed in polyethyleneglycol derivative (CH again
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3Modification does not have polyethyleneglycol derivative (CH among the OCHO
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3The zone that covers; Handle silicon chip with dilution heat of sulfuric acid, promptly conventional de-ester reaction removes carboxy protective; Re-use N-hydroxy-succinamide ester (NHS) processing carboxyl and generate active ester derivant; By point sample instrument at polyethyleneglycol derivative (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3The protein solution of the protein chip that point will prepare in the OCHO overlay area, present embodiment can be put the 1-1500 kind on 1500 border circular areas, after hatching 30 minutes under the normal temperature, deionized water rinsing, promptly preparing with the glass sheet is the methyl polyethyleneglycol derivative biochip of substrate.
Embodiment 4
With reference to Fig. 1, describe a kind of preparation in detail with the plastic sheet to be the method for making of the methyl polyethyleneglycol derivative biochip of substrate
1. at first make the silica gel seal: photoresist is coated on the silicon chip, produces mould by conventional photoetching technique with 800 square pattern;
2. the silica gel of commercially available PDMS is poured on the mould that step 1 makes, treat PDMS silica gel solidify after again from mould take off just obtained having square pattern, the length of side is 500 microns silica gel seal;
On the silica gel seal that step 2 is made with polyethyleneglycol derivative (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3Solution is coated on the silica gel seal of step 2 making, and transfer printing is on silicon chip;
4. the silica gel seal that step 3 is made is immersed in polyethyleneglycol derivative (CH again
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3CH
2NH
4Middle modification does not have polyethyleneglycol derivative (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3The zone that covers; By point sample instrument at polyethyleneglycol derivative (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3CH
2NH
4Point nucleic acid solution in the overlay area, present embodiment can be on 800 square area point 1-800 kind, hatch 30 minutes under the normal temperature after, deionized water rinsing, promptly preparing with the plastic sheet is the methyl polyethyleneglycol derivative biochip of substrate.
Claims (4)
1. the method for the making of a biochip is characterized in that: may further comprise the steps:
(1) at first makes the silica gel seal: photoresist is coated on the silicon chip, carves required pattern, just produced mould by conventional photoetching technique;
(2) a kind of silica gel of polymethoxy siloxane is poured on the mould, after the silica gel for the treatment of the polymethoxy siloxane solidifies, takes off the silica gel seal that has just obtained having required pattern from mould again;
(3) the polyethyleneglycol derivative A that is used for formaing solid surface is coated in the silica gel seal surface that step (2) makes after, cover again on stand-by solid substrate surface, just polyethyleneglycol derivative A according to the design pattern transfer on the solid substrate surface, obtain formative solid substrate; Wherein polyethyleneglycol derivative A comprises Cl
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3, HS (CH
2)
11(OCH
2CH
2)
3OH, (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3OCH
3);
(4) the formatted solid substrate of step (3) is dipped in the polyethyleneglycol derivative B solution, polyethyleneglycol derivative B just is combined in the white space of no polyethyleneglycol derivative A covering;
Wherein polyethyleneglycol derivative B comprises Cl
3Si (CH
2)
11(OCH
2CH
2)
3OCH
2COOCH
2CH
3, HS (CH
2)
11(OCH
2CH
2)
7OCH
2COOH, (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3OCHO, (CH
3CH
2O)
3Si (CH
2)
11(OCH
2CH
2)
3CH
2NH
4
(5) with the reactive group on the conventional chemical method activated polyethylene glycol derivant B, the zone that biological sample point is covered by polyethyleneglycol derivative B on solid surface by the mechanical deposition instrument; After hatching, water rinses out not by after the Covalent Immobilization biomolecule from the teeth outwards again, has just obtained the biomolecule rete that is evenly distributed.
2. press the method for the making of the described biochip of claim 1, it is characterized in that: described solid material comprises: silicon chip, glass sheet, sheet metal or plastic sheet.
3. press the method for the making of the described biochip of claim 1, it is characterized in that: the pattern of described silica gel seal refers to that all are suitable for the pattern of biochip point sample, and pattern dimension can be as small as one micron, greatly to one millimeter.
4. press the method for the making of the described biochip of claim 1, it is characterized in that: described step is hatched in (4), and wherein incubation time length decides routinely according to different protein and different concentration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03157324 CN1281953C (en) | 2003-09-18 | 2003-09-18 | Method for making bio-chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03157324 CN1281953C (en) | 2003-09-18 | 2003-09-18 | Method for making bio-chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1598575A CN1598575A (en) | 2005-03-23 |
| CN1281953C true CN1281953C (en) | 2006-10-25 |
Family
ID=34660262
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03157324 Expired - Lifetime CN1281953C (en) | 2003-09-18 | 2003-09-18 | Method for making bio-chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1281953C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5543485B2 (en) * | 2008-12-11 | 2014-07-09 | コーニンクレッカ フィリップス エヌ ヴェ | Detection device for detecting a target component in a fluid |
| CN101955595B (en) * | 2010-08-11 | 2012-01-04 | 东南大学 | Method for guiding fixed-point cell growth by preparing chemical micro-patterns on surfaces of various materials |
| CN102080072A (en) * | 2010-11-29 | 2011-06-01 | 中国科学院化学研究所 | Biochip and preparation method thereof |
| CN107305214B (en) * | 2016-04-22 | 2019-01-04 | 清华大学 | A kind of production method of hard micro-fluid chip |
| CN108020258A (en) * | 2017-11-09 | 2018-05-11 | 清华大学 | The method for preparing pattern visual under polarisation |
| CN110075766A (en) * | 2019-04-03 | 2019-08-02 | 同济大学 | A kind of preparation method of concentration gradient aerogel composite |
-
2003
- 2003-09-18 CN CN 03157324 patent/CN1281953C/en not_active Expired - Lifetime
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| Publication number | Publication date |
|---|---|
| CN1598575A (en) | 2005-03-23 |
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| C14 | Grant of patent or utility model | ||
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Application publication date: 20050323 Assignee: GUIZHOU ZHONGKE JINJIU BIOTECHNOLOGY CO.,LTD. Assignor: INSTITUTE OF MECHANICS, CHINESE ACADEMY OF SCIENCES Contract record no.: 2015990000794 Denomination of invention: Method for making bio-chip Granted publication date: 20061025 License type: Common License Record date: 20150906 |
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