CN102399290B - Method for fixing proteins on surface of solid - Google Patents
Method for fixing proteins on surface of solid Download PDFInfo
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- CN102399290B CN102399290B CN201110346431.XA CN201110346431A CN102399290B CN 102399290 B CN102399290 B CN 102399290B CN 201110346431 A CN201110346431 A CN 201110346431A CN 102399290 B CN102399290 B CN 102399290B
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Abstract
The invention discloses a method for fixing proteins on the surface of a solid, which relates to the field of a biosensor. The method comprises the following steps of: (1) cleaning the surface of the solid; (2) depositing an aluminium nitride thin film on the surface of the solid; (3) fixing the proteins, i.e. directly dropwise adding the proteins on the surface of the aluminium nitride thin film and washing after incubating. The method can be used for fixing the proteins on a biological chip and a biological probe in the biological field and has the advantages that the operation steps are simple, the cost is low, the proteins have high fixation quantity and high activity and are uniform and firm, and the graphical fixation can be realized.
Description
Technical field
The present invention relates to a kind of protein fixation method, specifically, relating to is a kind of aluminium nitride film method for immobilizing protein that utilizes, and belongs to biological detection and sensor field.
Background technology
In recent years, along with the development of biotechnology, the research of biosensor and biochip and apply increasingly extensive.How effectively proteinaceous solid to be fixed on biosensor and biochip, be the key issue in biological detection.At present, at the conventional fixing means of solid surface fixing protein, mainly contain absorption method, entrapping method, covalent coupling method etc., as (1) Hoyer-Hansen G, Hamers M J, Pedersen A N, etc.Loss of ELISA specificity due to biotinylation of monoclonal antibodies.ImmunolMethods 2000,235 (1-2): 91-9; (2) Haodan, Wayne M.Mullett and JanuszPawliszyn, Biological sample anysis with immounaffinity solid-phasemicroextraction, The Analyst, described in 2001,126,1456-1461.Although aforesaid method has also been realized the fixing of protein, also exists corresponding defect, mainly contains: the first, step is more loaded down with trivial details; The second, fixed effect is not too obvious; The 3rd, cost is higher.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of at solid surface method for immobilizing protein, make protein be fixed on efficiently solid surface, it is advantageous that step is simple, good fixing effect, cost be low.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
(1) solid surface cleans;
(2) in solid surface deposit aluminum nitride films;
(3) protein is fixing: directly protein is dripped on aluminium nitride film surface, hatch post-flush.
Described solid surface cleans, and refers to and uses successively acetone, dehydrated alcohol, deionized water supersound process 3 minutes separately, and nitrogen dries up.
Described solid material can be surface finish and the higher glass of smooth finish, silicon chip, pottery, metal, organic polymer etc.
Described aluminium nitride film adopts the method preparation of sputter, metal organic chemical vapor deposition or pulsed laser deposition.
The thickness of described aluminium nitride film is that 10 nanometers are to 100 nanometers.
Described aluminium nitride film forms various figures by etching, stripping technology, realizes the graphical fixing of protein.
Fixing of described protein, refers on aluminium nitride film surface and drips protein, after constant-temperature incubation, with phosphoric acid buffer, clean, nitrogen dries up.
Described constant-temperature incubation, refers to 37 ℃ of incubations 2 hours.
Described phosphoric acid buffer, refers to the phosphoric acid buffer of PH=7.0.
Beneficial effect of the present invention is:
1 operation steps of the present invention is simple, can omit the solid surface of protein in fixing and modify and reactivation process, has shortened experimental period.
The content of 2 fixing proteins of the present invention is high, active good, even, firm.
The preparation of AIN film that 3 the present invention use is convenient, and cost is lower.
4 the present invention can, by the method for etching aluminium nitride thin film, realize proteocrasic graphical.
The aluminium nitride film fixed surface that 5 the present invention can be prepared has higher stability, is easy to long-time preservation.
Embodiment
Below specific embodiments of the invention are elaborated: the present embodiment be take technical solution of the present invention and implemented as prerequisite, provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
The present embodiment is to implement under following implementation condition and technical qualification requirement:
The solid substrate that the present embodiment is used is for covering the silicon chip of one deck 100 nanometer thick gold membranes above.
Concrete protein fixedly process is:
(1) silicon chip is put into acetone, dehydrated alcohol, deionized water successively, ultrasonic cleaning was taken out after 3 minutes separately, and nitrogen dries up.
(2) utilizing sputter-deposition technology is the aluminium nitride film of 10 nanometers in golden film surface deposition a layer thickness.
(3) on aluminium nitride film surface, drip the human IgG of 0.2mg/mL, 37 ℃ of constant-temperature incubations 2 hours.
(4) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
(5) at aln surface, drip the bovine serum albumin confining liquid of 10mg/mL, 37 ℃ of incubations 1 hour.
(6) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
(7) at aln surface, drip the FITC mark goat anti-human igg solution of 0.2mg/mL, 37 ℃ of incubation 2h.
(8) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
By fluorescence microscope, the present embodiment finally in the proteopexy amount of solid surface apparently higher than gold surface sulfydryl self-assembly covalency fixed sample, and proteopexy layer is even, after ultrasonic 2 minutes without coming off.
Embodiment 2
The present embodiment is to implement under following implementation condition and technical qualification requirement:
The solid substrate that the present embodiment is used is glass slide.
(1) glass slide is put into acetone, dehydrated alcohol, deionized water successively, ultrasonic cleaning was taken out after 3 minutes separately, and nitrogen dries up.
(2) utilizing sputter-deposition technology is the aluminium nitride film of 50 nanometers in golden film surface deposition a layer thickness.
(3) on aluminium nitride film surface, drip the human IgG of 0.2mg/mL, 37 ℃ of constant-temperature incubations 2 hours.
(4) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
(5) at aln surface, drip the bovine serum albumin confining liquid of 10mg/mL, 37 ℃ of incubations 1 hour.
(6) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
(7) at aln surface, drip the FITC mark goat anti-human igg solution of 0.2mg/mL, 37 ℃ of incubations 2 hours.
(8) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
By fluorescence microscope, the present embodiment finally in the proteopexy amount of solid surface apparently higher than gold surface sulfydryl self-assembled film covalency fixed sample, and proteopexy layer is even, after ultrasonic 2 minutes without coming off.
Embodiment 3
The present embodiment is to implement under following implementation condition and technical qualification requirement:
The solid substrate that the present embodiment is used is silicon chip.
(1) silicon chip is put into acetone, dehydrated alcohol, deionized water successively, ultrasonic cleaning was taken out after 3 minutes separately, and nitrogen dries up.
(2) aluminium nitride film is graphical:
5 microns of spin coating photoresist materials also carry out photoetching, form the photoetching offset plate figure contrary with needed aluminium nitride figure.
Utilizing sputter-deposition technology is the aluminium nitride film of 100 nanometers in photoresist material surface deposition a layer thickness.
With acetone, soak sample 10 minutes, remove photoresist material, finally obtain aluminium nitride figure.
(3) on aluminium nitride film surface, drip 0.2mg/mL human IgG, 37 ℃ of constant-temperature incubations 2 hours.
(4) with the phosphoric acid buffer of PH=7.0, rinse aluminium nitride film surface 3 times, nitrogen dries up.
(5) at aln surface, drip the bovine serum albumin confining liquid of 10mg/mL, 37 ℃ of incubations 1 hour.
(6) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
(7) at aln surface, drip the FITC mark goat anti-human igg solution of 0.2mg/mL, 37 ℃ of incubations 2 hours.
(8) with the phosphoric acid buffer of PH=7.0, rinse 3 times, nitrogen dries up.
By fluorescence microscope, the present embodiment finally in the proteopexy amount of solid surface apparently higher than gold surface sulfydryl self-assembled film covalency fixed sample, and proteopexy layer is even, after ultrasonic 2 minutes without coming off.
As can be seen from the above embodiments, operation steps of the present invention is simple, and cost is lower, and the fixed amount of protein is high, active good, even, firm, and can realize graphical fixing, can fixing for protein on biochip in biological field and bioprobe.
Although content of the present invention has been done detailed introduction by above preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.Those skilled in the art, read after foregoing, for multiple modification of the present invention with to substitute will be all apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (8)
1. at a solid surface method for immobilizing protein, it is characterized in that comprising the steps:
(1) solid surface cleans;
(2) in solid surface deposit aluminum nitride films; The thickness of described aluminium nitride film is that 10 nanometers are to 100 nanometers;
(3) protein is fixing: directly protein is dripped on aluminium nitride film surface, hatch post-flush.
2. as claimed in claim 1 a kind of at solid surface method for immobilizing protein, it is characterized in that: described solid material is a kind of in glass, silicon chip, pottery, metal, organic polymer.
3. as claimed in claim 1 a kind of at solid surface method for immobilizing protein, it is characterized in that: described aluminium nitride film adopts the method preparation of sputter, metal organic chemical vapor deposition or pulsed laser deposition.
4. as claimed in claim 1 a kind of at solid surface method for immobilizing protein, it is characterized in that: described aluminium nitride film forms various figures by etching, stripping technology, realize the graphical fixing of protein.
5. as claimed in claim 1 a kind of at solid surface method for immobilizing protein, it is characterized in that: fixing of described protein, refer on aluminium nitride film surface and drip protein, after constant-temperature incubation, with phosphoric acid buffer, clean, nitrogen dries up.
6. as claimed in claim 5 a kind of at solid surface method for immobilizing protein, it is characterized in that: described constant-temperature incubation refers to 37 ℃ of incubations 2 hours.
7. as claimed in claim 5 a kind of at solid surface method for immobilizing protein, it is characterized in that: described phosphoric acid buffer refers to the phosphoric acid buffer of PH=7.0.
8. as claimed in claim 1 a kind of at solid surface method for immobilizing protein, it is characterized in that: described solid surface cleans, refer to and use successively acetone, dehydrated alcohol, deionized water supersound process 3 minutes separately, nitrogen dries up.
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| CN201110346431.XA CN102399290B (en) | 2011-11-04 | 2011-11-04 | Method for fixing proteins on surface of solid |
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| CN201110346431.XA CN102399290B (en) | 2011-11-04 | 2011-11-04 | Method for fixing proteins on surface of solid |
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| CN102399290A CN102399290A (en) | 2012-04-04 |
| CN102399290B true CN102399290B (en) | 2014-02-26 |
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