CN101486662A - Preparation of nitrilotriacetic acid affinity molecular layer - Google Patents
Preparation of nitrilotriacetic acid affinity molecular layer Download PDFInfo
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- CN101486662A CN101486662A CNA2009101162407A CN200910116240A CN101486662A CN 101486662 A CN101486662 A CN 101486662A CN A2009101162407 A CNA2009101162407 A CN A2009101162407A CN 200910116240 A CN200910116240 A CN 200910116240A CN 101486662 A CN101486662 A CN 101486662A
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Abstract
The invention discloses a preparation method of a nitrilotriacetic acid compatible molecular film, which is characterized in that the molecular film with carboxyl groups is placed in a mixed aqueous solution containing 0.01M to10M of N-hydroxy succinimide and 0.01M to10M of ethyl-dimethyl aminopropyl-carbodiimide for soaking for not less than one minute; the activated molecular film is placed in an aqueous solution containing 0.01mM to10M of nitrilotriacetic acid for soaking for not less than one minute; the above steps are repeated for more than one time; and the molecular film is placed in an aqueous solution containing 0.01M to10M of diethanolamine hydrochloride for soaking for not less than one minute. The preparation method can effectively strengthen the binding capability of compatible molecular films, has low preparation cost and simple and convenient operation, is applicable to various carried media, overcomes the defect that the binding capability of the nitrilotriacetic acid compatible molecular film of the current commercial chip is not high or is lowered after being used for several times and provides methods for improving and reusing of the current nitrilotriacetic acid compatible molecular film products.
Description
Technical field
The invention belongs to the affinity molecule layer preparation method technical field in the biological method, be specifically related to the preparation method of nitrilotriacetic acid (NTA) affinity molecule layer.
Background technology
Mounting medium such as affine resin, microbial film and biochip etc. with affinity molecule laminar surface, because of its can specific combination band specific label albumen or peptide sequence, be widely used in fields such as proteomics, cytobiology, microbiology, become one of biological important analysis ways and means.In numerous affinity molecule layers, the affinity molecule layer with nitrilotriacetic acid molecular structure is comparatively use always a kind of, and its can be specifically in conjunction with having 6 histidine-tagged albumen or peptide sequence.But all there is the shortcoming that affinity molecule layer binding ability is not high or repeatedly use the back binding ability to descend in most of commercial NTA affinity molecule layer product at present, major cause is the distance between the uncontrollable NTA molecule of prior preparation method, make 6 histidine-tagged can only be in limited distance in conjunction with the 1-2 in 3 sites, the result causes albumen or peptide sequence and NTA molecule bonding strength not enough, in use comes off easily." Chinese biological engineering magazine " (2009,29.1:44-49) inventor seminar that is reported before prepared the method for the affine chip of NTA, just the carboxylic group by the activation chip surface is coupled to the NTA affinity molecule layer that chip surface obtains individual layer with the NTA molecule, this method only rests on the level of the affine chip of commercialization, fails to solve the insufficient problem of affinity molecule layer binding ability.So far do not see the report that other preparation NTA affinity molecule layer method is arranged.
Summary of the invention
The present invention proposes the preparation method of a kind of nitrilotriacetic acid (NTA) affinity molecule layer, with the binding ability of effective enhancing NTA affinity molecule layer and applicable to various mounting mediums, overcome the shortcoming that existing NTA affinity molecule layer binding ability is not high or repeatedly use the back binding ability to descend.
The preparation method of nitrilotriacetic acid affinity molecular layer of the present invention is characterized in that comprising the steps:
(1) molecular layer that will have a carboxylic group places the mixed aqueous solution that contains 0.01-10M N-maloyl imines and 0.01-10M ethyl-dimethylamino-propyl-carbodiimide to soak to be no less than 1 minute;
(2) will soak the activatory molecular layer through step (1) places and contains the 0.01mM-10M nitrilotriacetic acid aqueous solution and soak and be no less than 1 minute;
(3) repeating step (1) and step (2) are no less than 1 time;
(4) molecular layer is placed contain 0.01-10M acidic alcohol amine aqueous solution and soak and to be no less than 1 minute.
The present invention utilizes a NTA molecule to have the characteristics of 3 carboxylic groups, the mode that adopts activated carboxylic reagent and NTA solution successively to soak, by secondary or activation-coupling NTA molecule repeatedly, has affinity molecule layer two-layer or multilayer NTA molecular structure thereby prepare.
Compare with the method for the existing affine chip of the disclosed NTA of preparation, because the present invention takes secondary or activation-coupling NTA molecule repeatedly, prepared NTA affinity molecule layer has higher NTA molecular density, and can be spatially and 6 histidine-tagged adapting, guaranteed that 6 Histidines can combine with the NTA molecule is whole in corresponding spatial dimension, thereby strengthened the binding ability of NTA affinity molecule layer effectively.
Description of drawings
Fig. 1 is that the affine chip of the NTA with existing commercial of embodiment 1 preparation is respectively in conjunction with the proteic surface plasma resonance signal curve of same concentrations PH;
Fig. 2 is that the affine chip of the NTA with existing commercial of embodiment 2 preparation is respectively in conjunction with the proteic surface plasma resonance signal curve of same concentrations SET.
Fig. 3 is that the affine chip of the NTA with existing commercial of embodiment 3 preparation is respectively in conjunction with the proteic surface plasma resonance signal curve of same concentrations Trmb;
Fig. 4 is that the affine chip of the NTA with existing commercial of embodiment 4 preparation is respectively in conjunction with the proteic surface plasma resonance signal curve of same concentrations CF1m25.
Embodiment
Below in conjunction with embodiment the inventive method is further described in detail.
Embodiment 1:
(1) molecular layer that will have a carboxylic group places the mixed aqueous solution that contains 0.2M N-maloyl imines and 0.8M ethyl-dimethylamino-propyl-carbodiimide to soak 12 minutes to carry out molecule activation;
(2) the activatory molecular layer is placed contain the 4mM NTA aqueous solution and soak 20 minutes with coupling NTA molecule;
(3) repeating step (1) and (2) 1 times are with re-activation-coupling NTA molecule;
(4) molecular layer is placed contain 2M acidic alcohol amine aqueous solution and soak 6 minutes with the sealing activating group, wash after the taking-up and at room temperature nitrogen dry up, just obtain nitrilotriacetic acid affinity molecular layer.
Fig. 1 is the proteic surface plasma resonance signal curve of PH of 9mg/L respectively in conjunction with concentration for the affine chip of the NTA with existing commercial (surface is nitrilotriacetic acid affinity molecular layer) that adopts the present embodiment preparation; Wherein be in top curve a for adopting the proteic surface plasma resonance signal curve of the affine chips incorporate PH of NTA of present embodiment preparation, being in following curve b is the proteic surface plasma resonance signal curve of the affine chips incorporate PH of commercial NTA.
Can prove that from Fig. 1 above-mentioned the present embodiment what prepare is nitrilotriacetic acid affinity molecular layer.
Curve from figure can be seen, adopt the affine chip of NTA of present embodiment preparation to compare with the affine chip of existing commercial NTA, the former protein-bonded amount is apparently higher than the latter, and after conjugated protein, the former curve is steady, illustrate that firm protein combination do not dissociate, and latter's curve obviously glides, illustrate that protein binding is unstable and dissociate, this shows, the affine chip of NTA that adopts present embodiment to prepare has effectively strengthened the binding ability of NTA affinity molecule layer, has overcome the existing commercial not high shortcoming of NTA affinity molecule layer binding ability.
Embodiment 2:
(1) molecular layer that will have a carboxylic group places the mixed aqueous solution that contains 0.01M N-maloyl imines and 0.01M ethyl-dimethylamino-propyl-carbodiimide to soak 15 minutes to carry out molecule activation;
(2) the activatory molecular layer is placed contain the 0.01mM NTA aqueous solution and soak 25 minutes with coupling NTA molecule;
(3) repeating step (1) and (2) 5 times are with activation-coupling NTA molecule repeatedly;
(4) molecular layer is placed contain 0.01M acidic alcohol amine aqueous solution and soak 15 minutes, wash after the taking-up and under 60 ℃ temperature, dry, just obtain nitrilotriacetic acid affinity molecular layer with the sealing activating group.
Fig. 2 is the proteic surface plasma resonance signal curve of SET of 15mg/L respectively in conjunction with concentration for the affine chip of the NTA with existing commercial (surface is nitrilotriacetic acid affinity molecular layer) that adopts the present embodiment preparation; Wherein be in top curve c for adopting the proteic surface plasma resonance signal curve of the affine chips incorporate SET of NTA of present embodiment preparation, being in following curve d is the existing commercial proteic surface plasma resonance signal curve of the affine chips incorporate SET of NTA.
Can prove that from Fig. 2 above-mentioned the present embodiment what prepare is nitrilotriacetic acid affinity molecular layer.
Curve from figure can be seen, adopt the affine chip of NTA of present embodiment preparation to compare with the affine chip of existing commercial NTA, the former protein-bonded amount is apparently higher than the latter, and after conjugated protein, the former curve is steady, illustrate that firm protein combination do not dissociate, and latter's curve obviously glides, illustrate that protein binding is unstable and dissociate, this shows, the affine chip of NTA that adopts present embodiment to prepare has effectively strengthened the binding ability of NTA affinity molecule layer, has overcome the existing commercial not high shortcoming of NTA affinity molecule layer binding ability.
Embodiment 3:
(1) molecular layer that will have a carboxylic group places the mixed aqueous solution that contains 10M N-maloyl imines and 10M ethyl-dimethylamino-propyl-carbodiimide to soak 1 minute to carry out molecule activation;
(2) the activatory molecular layer is placed contain the 10M NTA aqueous solution and soak 1 minute with coupling NTA molecule;
(3) repeating step (1) and (2) 3 times are with activation-coupling NTA molecule repeatedly;
(4) molecular layer is placed contain 10M acidic alcohol amine aqueous solution and soak 1 minute, wash after the taking-up and at room temperature dry naturally, just obtain nitrilotriacetic acid affinity molecular layer with the sealing activating group.
Fig. 3 is the proteic surface plasma resonance signal curve of Trmb of 11mg/L respectively in conjunction with concentration for the affine chip of the NTA with existing commercial (surface is nitrilotriacetic acid affinity molecular layer) that adopts the present embodiment preparation; Wherein be in top curve e for adopting the proteic surface plasma resonance signal curve of the affine chips incorporate Trmb of NTA of present embodiment preparation, being in following curve f is the existing commercial proteic surface plasma resonance signal curve of the affine chips incorporate Trmb of NTA.
Can prove that from Fig. 3 above-mentioned the present embodiment what prepare is nitrilotriacetic acid affinity molecular layer.
Curve from figure can be seen, adopt the affine chip of NTA of present embodiment preparation to compare with the affine chip of existing commercial NTA, the former protein-bonded amount is apparently higher than the latter, and after conjugated protein, the former the curve ratio latter is steady, illustrates that the former protein binding is more firm more stable, this shows, the affine chip of NTA that adopts present embodiment to prepare has effectively strengthened the binding ability of NTA affinity molecule layer, has overcome the existing commercial not high shortcoming of NTA affinity molecule layer binding ability.
Embodiment 4:
(1) molecular layer that will have a carboxylic group places the mixed aqueous solution that contains 4M N-maloyl imines and 3M ethyl-dimethylamino-propyl-carbodiimide to soak 7 minutes to carry out molecule activation;
(2) the activatory molecular layer is placed contain the 0.2M NTA aqueous solution and soak 12 minutes with coupling NTA molecule;
(3) repeating step (1) and (2) 2 times are with activation-coupling NTA molecule repeatedly;
(4) molecular layer is placed contain 0.8M acidic alcohol amine aqueous solution and soak 10 minutes with the sealing activating group, wash after the taking-up and under 25 ℃ temperature air blow drying, just obtain nitrilotriacetic acid affinity molecular layer.
Fig. 4 is the proteic surface plasma resonance signal curve of CF1m25 of 6mg/L respectively in conjunction with concentration for the affine chip of the NTA with existing commercial (surface is nitrilotriacetic acid affinity molecular layer) that adopts the present embodiment preparation; Wherein be in top curve g for adopting the proteic surface plasma resonance signal curve of the affine chips incorporate CF1m25 of NTA of present embodiment preparation, being in following curve h is the existing commercial proteic surface plasma resonance signal curve of the affine chips incorporate CF1m25 of NTA.
Can prove that from Fig. 4 above-mentioned the present embodiment what prepare is nitrilotriacetic acid affinity molecular layer.
Curve from figure can be seen, adopt the affine chip of NTA of present embodiment preparation to compare with the affine chip of existing commercial NTA, the former protein-bonded amount is apparently higher than the latter, and after conjugated protein, the former curve is steady, illustrate that firm protein combination do not dissociate, and latter's curve slightly is falling tendency, illustrate that protein binding does not have the former stable, this shows, the affine chip of NTA that adopts present embodiment to prepare has effectively strengthened the binding ability of NTA affinity molecule layer, has overcome the existing commercial not high shortcoming of NTA affinity molecule layer binding ability.
As from the foregoing: one of characteristics of the inventive method are to take secondary or activation-coupling NTA molecule repeatedly.Therefore, binding ability descends if NTA affinity molecule layer repeatedly uses the back, can reuse the inventive method and directly the affinity molecule layer that binding ability descends be activated-coupling NTA molecule, thereby recover its binding ability.As seen, the inventive method can not only overcome the existing not high shortcoming of NTA affinity molecule layer binding ability, can also solve the problem that NTA affinity molecule layer repeatedly uses the back binding ability to descend.
Also have, the inventive method is applicable to the molecular layer with carboxylic group, and operate simple and easy, reaction conditions is gentle.Therefore, the inventive method also has the various mounting mediums of carboxylic group molecular layer, as affine resin, microbial film etc. except being used for preparing the affine chip of the NTA of mounting medium in the foregoing description applicable to other.
Claims (1)
1, a kind of preparation method of nitrilotriacetic acid affinity molecular layer is characterized in that comprising the steps:
(1) molecular layer that will have a carboxylic group places the mixed aqueous solution that contains 0.01-10M N-maloyl imines and 0.01-10M ethyl-dimethylamino-propyl-carbodiimide to soak to be no less than 1 minute;
(2) will soak the activatory molecular layer through step (1) places and contains the 0.01mM-10M nitrilotriacetic acid aqueous solution and soak and be no less than 1 minute;
(3) repeating step (1) and step (2) are no less than 1 time;
(4) molecular layer is placed contain 0.01-10M acidic alcohol amine aqueous solution and soak and to be no less than 1 minute.
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| CNA2009101162407A CN101486662A (en) | 2009-02-25 | 2009-02-25 | Preparation of nitrilotriacetic acid affinity molecular layer |
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| CNA2009101162407A CN101486662A (en) | 2009-02-25 | 2009-02-25 | Preparation of nitrilotriacetic acid affinity molecular layer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197081A (en) * | 2013-04-17 | 2013-07-10 | 无锡优创生物科技有限公司 | Protein chip and preparation and detection method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197081A (en) * | 2013-04-17 | 2013-07-10 | 无锡优创生物科技有限公司 | Protein chip and preparation and detection method thereof |
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Open date: 20090722 |