CN1014901B - Method for preparing solid film of biological molecules - Google Patents
Method for preparing solid film of biological moleculesInfo
- Publication number
- CN1014901B CN1014901B CN87107256A CN87107256A CN1014901B CN 1014901 B CN1014901 B CN 1014901B CN 87107256 A CN87107256 A CN 87107256A CN 87107256 A CN87107256 A CN 87107256A CN 1014901 B CN1014901 B CN 1014901B
- Authority
- CN
- China
- Prior art keywords
- solution
- bacteriorhodopsin
- biological
- film
- soak
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
- Y02P70/50—Manufacturing or production processes characterised by the final manufactured product
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- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a method for preparing a solid film of a biological molecule, which comprises extraction and purification of a biological photosensitive molecule, surface processing of a solid substrate and fixing and filming of the biological photosensitive molecule on the surface of the solid substrate. The method provided by the present invention is simple and convenient, and the prepared solid film of the biological molecule has the advantages of high strength, good photoelectric effect and stable performance.
Description
The present invention relates to the method that a kind of preparation has the solid-state miniature thin-film of photoelectric biomolecules opto-electronic conversion.It comprises the biological photosensitive molecular of extraction and purification, the surface treatment of solid substrate, and biological photosensitive molecular is in three steps of solid substrate surface set film forming.It belongs to the optoelectronic information solid support material.
Along with rapid development of science and technology, people have predicted the hyperfine processing and the information storage of silicon, do not go out 10 years, will face its theoretical boundary.The new outlet of the essential searching of development microelectronic device.Adopt molecule or molecular aggregate imperative as the novel information solid support material.Current emerging biotechnology is permeated to microelectronics, and combination has dexterously caused making first appearance and attracting worldwide attention of biochip (Biochip).Utilize biomaterial to carry out information transmission and processing on molecular level, the comparable conventional semiconductors of its integrated level exceeds 1,000,000,000 to 1,000,000,000,000 times, and arithmetic speed can reach 10 of present robot calculator
15Doubly, and has the three-dimension device of caning be assembled into, advantage such as current consumption is low.The development of biochip at first will be looked for appropriate biologically functional molecule, also must solve biomolecules and keep its active film preparation problem.The research of relevant biomolecule activity method for manufacturing thin film worldwide, all also is in the trial stage at initial stage.Someone tests with bilayer lipoid film (BLM) method, or recommends blue Mil's Blodget (Langmuir-Blodgett) legal system film on probation, does not obtain success fully as yet.And the film made of these methods or be liquid film, or the bad mechanical strength of film, or many deficiencies such as molecular assembly arrangement difference can not satisfy the requirement of development biochip.These class methods all need specific appointed condition in addition, and the use instrument that has is quite expensive.The preparation biologically active can meet the biomolecules method for manufacturing thin film of making the biomolecular device particular requirement, demands urgently exploring and researching and solving.
The objective of the invention is to avoid above-mentioned the deficiencies in the prior art part, and provide a kind of easy, preparation to have method photoelectric, the solid-state miniature thin-film of biomolecules opto-electronic conversion.
Purpose of the present invention can reach by following measure:
1. the extraction and purification of biological photosensitive molecular bacteriorhodopsin.
The halobacterium halobium (Halobacterium hal-obium) that contains biological photosensitive molecular bacteriorhodopsin is after incubation growth is vigorous, centrifugal collecting cell, broken cell homogenate is again through centrifugation, obtain required biological photosensitive molecular bacteriorhodopsin, vacuum-drying is standby.
2. solid substrate surface treatment.
Solid substrate carries out spatter property earlier and cleans, and handles with hydrophobizing agent again, obtains having strong hydrophobic surface.
3. biological photosensitive molecular bacteriorhodopsin is in solid substrate surface set film forming,
With the above-mentioned solid substrate of handling well, place protein soln, glutaraldehyde solution to soak successively with strong-hydrophobicity surface; Wash with phosphoric acid buffer again; Place biological photosensitive molecular bacteriorhodopsin solution to soak then in order to the system film; Soak with glycine solution again; At last with urea soln washing, distilled water wash, airing.
The present invention has following advantage:
Utilize method provided by the invention, the solid film of biological molecules that makes is a dry film, has overcome the deficiency that can only obtain wet film in the prior art.Film thickness be tens dusts (
) to tens dusts, repetitive operation can obtain the different thicker film of thickness.This film dress is with electrode, and irradiation has good photovoltaic effect.This film also has physical strength height, steady performance.Do not require expensive plant and instrument with our thin films, simple and easy to do.
The present invention will now be further detailed embodiment.
Embodiment one
1. contain the cultivation of the halobacterium halobium of biological photosensitive molecular bacteriorhodopsin.
Bacterial classification halobacterium halobium (Halobacterium halobium) is inoculated into slant medium.Medium component: yeast extract paste 0.2%; Milk protein hydrolysate 0.25%; MgCl
23%; NaCl25%.Cultivated 5 days down in 37 ℃ of illumination.Culture transferring is in the liquid nutrient medium of same composition then, shaking culture under the illumination.Be transferred to again that the liquid nutrient medium with same composition carried out illumination cultivation 10 days in the tun.
2. the extraction and purification of biological photosensitive molecular bacteriorhodopsin.
Halobacterium halobium behind a large amount of cells of incubation growth, centrifugal (3500RPM, 15 minutes) collecting cell.The cell of collecting is suspended from the 25%NaCl solution, adds a small amount of deoxyribonuclease, and 0.1M NaCl solution was dialysed 48 hours at 4 ℃.The suspension of dialysing is with 60, centrifugal 40 minutes of 000g.After centrifugal, abandoning supernatant suspends the NaCl solution of throw out with 0.1M, carries out homogenate.So repeat twice with smudge cells.Centrifugal, collecting precipitation, and throw out is suspended from the 0.1M NaCl solution.Carry out the linear sucrose density gradient centrifugation (180,000g, 10 ℃, 9.5 hours) of 30-50% again.Take out the bacteriorhodopsin of separation and purification, use distilled water diluting, centrifugal (100,000g, 1 hour) washing.Receive to such an extent that precipitate through centrifugal again, vacuum-drying is preserved standby.
3. biological photosensitive molecular bacteriorhodopsin anchors at the solid substrate silicon chip surface and makes solid film.
To place 10%(W/W through the cleaning silicon wafer that cleans with the trieline preparation) dichlorodimethylsilane solution soaks a moment, makes this silicon chip have the strong-hydrophobicity surface.Then with the distillation washing.Be immersed in again and use 0.02M, in the 1mg/ml bovine serum albumin solution of pH6.8 phosphoric acid buffer preparation.Be immersed in 9 ℃ of environment and carry out, and constantly mild agitation it.Soak after 24 hours, take out.Then earlier in order to 0.02M, 2.5% glutaraldehyde solution washing of pH6.8 phosphoric acid buffer preparation is soaked in this glutaraldehyde solution again, soaks taking-up after 25 minutes.With distilled water wash it.Be soaked in again in the bacteriorhodopsin solution of the 2.7mg/ml for preparing with above-mentioned phosphoric acid buffer.Be immersed under 9 ℃ and carry out, and mild agitation.Soak after 28 hours and to take out, wash with the glycine solution of the 0.1M of same damping fluid preparation, and soaked 80 minutes.After the taking-up,, use distilled water wash again, airing with the washing of 4M urea soln.
Embodiment two
1. contain the cultivation of the bacterium of biological photosensitive molecular bacteriorhodopsin.
Bacterial classification halobacterium halobium (Halobacterium halobium) is inoculated into slant medium.Substratum composition: yeast extract 0.2%: milk protein hydrolysate 0.25%:MgCl
29H
2O3%; Table salt 25%.In 37 ℃ of illumination cultivation 4 days.Culture transferring is in the liquid nutrient medium of same composition then, shaking culture 5 days, again transferred species in the tun of same substratum, 37 ℃ of illumination cultivation 10 days.
2. the extraction and purification of biological photosensitive molecular bacteriorhodopsin.
After halobacterium halobium is cultivated, obtain the culture of a large amount of cells of growth and breeding.Culture is through centrifugal (4000RPM, 15 minutes) collecting cell.The cell of collecting is suspended from the 25% NaCl solution, adds a small amount of deoxyribonuclease, then to the NaCl solution of 0.1M at 4 ℃ of liquid of dialysing.The suspension of dialysing is through 50, centrifugal 50 minutes of 000g, and the collecting precipitation thing is suspended from the NaCl solution of 0.1M, uses the tissue homogenizer broken cell homogenate.Centrifugal collecting precipitation.With 0.1M NaCl solution washing throw out.Be suspended from again in the 0.1M NaCl solution, this suspension is added on the 35% and 43% double-deck sucrose solution, carry out sucrose gradient centrifugation.Centrifugal condition is 10 ℃, 200, and 000g, 4 hours.Behind centrifugal the finishing, from centrifuge tube, take out bacteriorhodopsin.With distilled water suspended centrifugal (10 ℃, 100,000g, 40 minutes) washing, throw out is in 37 ℃ of drying for standby.
3. biological photosensitive molecular bacteriorhodopsin anchors at the solid substrate surface of glass slide and makes solid film.
Place the 10% trimethyl silicane oil solution that is mixed with trieline to soak the cleaning slide that cleaned.Clean with distilled water then.Be dipped into again among the 1mg/ml human serum albumin solution who prepares with the phosphoric acid buffer of 0.02M pH6.8, be immersed in 5 ℃ and carry out, and the mild agitation soak solution.Soak and remove after 24 hours.To use 0.02M, 2.5% glutaraldehyde solution of pH6.8 phosphoric acid buffer preparation dashes to be washed, and is soaked in this glutaraldehyde solution again, soaks after 25 minutes and takes out then.Behind distilled water wash, be soaked in again in the bacteriorhodopsin solution of the 4mg/ml for preparing with above-mentioned damping fluid, soaked 30 hours at 5 ℃, and make the soak solution mild agitation.Immersion finishes, and takes out, and with glycine solution (using 0.02M, the preparation of the pH6.5 phosphoric acid buffer) washing of 0.1M, and is soaked in wherein, soaks after 80 minutes, takes out, and with the urea soln washing of 4M, uses the distilled water wash airing at last again.
Embodiment three
1. contain the cultivation of the halobacterium halobium of bacteriorhodopsin.
Bacterial classification halobacterium halobium (Halobaclerium halobium) is inoculated into slant medium.Medium component: yeast extract paste 0.2%; Milk protein hydrolysate 0.25%; MgCl
23%; NaCl 25%.Cultivated 5 days down in 37 ℃ of illumination.Culture transferring is in the liquid nutrient medium with sample ingredient then, shaking culture under the illumination.Be transferred to tun again and contain in the liquid nutrient medium with sample ingredient, 37 ℃ of illumination cultivation 10 days.
2. the extraction and purification of biological photosensitive molecular bacteriorhodopsin.
Halobacterium halobium is behind a large amount of cells of aforesaid method incubation growth, through centrifugal (3,000RPM, 30 minutes) collecting cell.The cell of collecting is suspended from the 25%NaCl solution.With this cell suspension to the 0.1M NaCl solution liquid (5 ℃) of dialysing.The suspension 60 of dialysing, centrifugal 40 minutes of 000g.Remove supernatant, throw out is suspended in the NaCl solution of 0.1M, carry out homogenized with tissue homogenizer.So repeat 2 times, obtain the cell homogenates thing.This cell homogenates thing is passed through 30-50% sucrose linear gradient centrifugal (180,000g, 10 ℃, 10 hours).Take out the bacteriorhodopsin band.Behind distilled water diluting, through 100, centrifugal 1 hour of 000g washs 1 time, again through centrifugal receive throw out, put 37 ℃ and wait to do.
3. biological photosensitive molecular bacteriorhodopsin anchors at the quartzy surface of glass slide of solid substrate and makes solid film.
To place 10% trimethyl silicane oil solution to soak the taking-up in a moment through the quartzy slide that cleans with the trieline preparation.Use distilled water wash then.Be dipped into again and use 0.02M, in the 1mg/ml bSA solution of pH6.8 phosphoric acid buffer preparation, 5 ℃ of immersions (mild agitation) 24 hours.Take out, earlier in order to 0.02M, the 2.5% glutaraldehyde solution washing of pH6.8 phosphoric acid buffer preparation is soaked in this glutaraldehyde solution again, soaks 25 minutes.After the taking-up, use 0.02M, the phosphoric acid buffer washing of pH7.5.Be dipped into 0.02M again, in the bacteriorhodopsin solution of the 5mg/ml of pH7.5 phosphoric acid buffer preparation, 5 ℃, mild agitation was soaked 38 hours.After the taking-up, wash earlier, be soaked in again in this solution 80 minutes with 0.1M glycine solution (use 0.02M, the preparation of pH6.5 phosphoric acid buffer).After the taking-up,, use distilled water wash again, airing with the urea soln washing of 4M.
Claims (1)
1, the method for preparing solid film of biological molecules comprises the biological photosensitive molecular of extraction and purification, and silicon chip surface is handled, and biological photosensitive molecular is characterized in that in three steps of silicon chip surface set film forming:
(1). the halobacterium halobium (Halobac-terium halobium) that contains biological photosensitive molecular bacteriorhodopsin after the artificial culture growth is vigorous, centrifugal collecting cell, broken cell homogenate carries out centrifugation again, the bacteriorhodopsin vacuum-drying that separation is obtained;
(2). silicon chip is at the hydrophobizing agent dichlorodimethylsilane, immersion treatment in the trimethyl silicane oil solution;
(3). with the silicon chip of above-mentioned processing, place protein soln successively, soak in the glutaraldehyde solution and take out, after the phosphoric acid buffer washing, place bacteriorhodopsin solution to soak again in order to the system film, soak with glycine solution at last, the urea soln washing, distilled water wash, airing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN87107256A CN1014901B (en) | 1987-12-04 | 1987-12-04 | Method for preparing solid film of biological molecules |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN87107256A CN1014901B (en) | 1987-12-04 | 1987-12-04 | Method for preparing solid film of biological molecules |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1033463A CN1033463A (en) | 1989-06-21 |
| CN1014901B true CN1014901B (en) | 1991-11-27 |
Family
ID=4816031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN87107256A Expired CN1014901B (en) | 1987-12-04 | 1987-12-04 | Method for preparing solid film of biological molecules |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1014901B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100533146C (en) * | 1999-05-19 | 2009-08-26 | 埃佩多夫阵列技术股份有限公司 | Method for the identification and/or the quantification of a target compound |
-
1987
- 1987-12-04 CN CN87107256A patent/CN1014901B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| CN1033463A (en) | 1989-06-21 |
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