CN105148867A - Graphene oxide-recombined streptococcal protein G non-covalent composite material as well as preparation method and application thereof - Google Patents
Graphene oxide-recombined streptococcal protein G non-covalent composite material as well as preparation method and application thereof Download PDFInfo
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- CN105148867A CN105148867A CN201510582222.3A CN201510582222A CN105148867A CN 105148867 A CN105148867 A CN 105148867A CN 201510582222 A CN201510582222 A CN 201510582222A CN 105148867 A CN105148867 A CN 105148867A
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Abstract
The invention relates to a graphene oxide-recombined streptococcal protein G non-covalent composite material as well as a preparation method and application thereof. A recombined streptococcal protein G is immobilized on a graphene oxide material by making use of the characteristic that graphene oxide is high in specific surface area by a non-covalent combination method so as to prepare a high-capacity antibody-enriched material with antibody adsorption bioactivity. The prepared graphene oxide-recombined streptococcal protein G non-covalent composite material can be used in the fields of antibody purification, antibody enrichment, pathogene detection, biological sample pretreatment and the like.
Description
Technical field
The invention belongs to chemosynthesis technical field, relate to graphene composite material technology of preparing and the application in protein-enriched thereof, specifically a kind of preparation based on graphene oxide-non-covalent composite of restructuring streptococcus protein G and functionalized application thereof.
Background technology
Infectious disease pathogens itself can produce corresponding special antibody as antigen in living organism, specific recognition between mostly existing detection method is by Ag-Ab detects pathogen, as Enzyme-linked Immunosorbent Assay technology, immunofluorescence and immune colloidal gold technique etc.These immunological techniques have easy and simple to handle, and result is easy to the features such as analysis, but the sensitivity of immunological detection method is relatively low, easily cause the false negative of result.In order to improve the detection sensitivity of immunological detection method to infectious disease pathogens, usually take by carrying out enrichment to sample.Effective pre-treatment is carried out to sample, the concentration of antibody in detected sample can be improved, thus indirectly improve the sensitivity of detection method.Be at present the restructuring streptococcus protein G of the immobilized adsorb antibodies of carrier mainly with agarose, trehalose for the commercial reagents of antibody enrichment, ProteinG as immobilized in agarose is about 80ug/mL, and its immobilized protein content is lower.Therefore develop the carrier large to target protein supported quantity, and by effective immobilized restructuring streptococcus protein G, antibody enrichment is significant for realizing.
Graphene is a kind of two-dimension nano materials only having a carbon atom thickness, is the flat film of the hexagonal honeycomb lattice that carbon atom forms with SP2 hybridized orbit.2004 by Novoselov etc. with adhesive tape layer by layer stripping method be separated from graphite and obtain, its theoretic throat is only 0.335nm.Single-layer graphene becomes the Novel Carbon Nanomaterials after CNT, has started huge research boom in various fields such as physics, chemistry, material, biologies.Without carbon atom disappearance in Graphene, mechanical strength is high, Stability Analysis of Structures, Heat stability is good, chemical stability are high, the large (2600m of specific area
2g
-1), and there is good biocompatibility, make its easily absorption other biological molecule, particularly graphene oxide is except having bigger serface, a large amount of oxy radicals is also there is on its surface, graphene oxide is made to be more suitable for the biomolecule of adsorbing and having more chemical group, as protein, nucleic acid etc.When grapheme material and other materials phase compound tense, utilize the relevant nature of other materials, the enrichment of biomolecule can be realized.
Summary of the invention
In view of this, the present invention utilizes the characteristic of the high-specific surface area of graphene oxide, by the method for Non-covalent binding, streptococcus protein G (PG) of recombinating is immobilized to graphene oxide (GO) material, prepares the high power capacity antibody enrichment material with antibody adsorbed bioactive.Concrete technical scheme is as follows.
The graphene oxide that the application provides-non-covalent composite of restructuring streptococcus protein G is attached most importance to the Non-covalent binding thing of group of streptococcus Protein G and graphene oxide, and described graphene oxide is 1:2.5-3 with the ratio of the mass number of restructuring streptococcus protein G.
Described in the application, the preparation method of non-covalent composite comprises the steps:
1) active oxidation Graphene (GO);
2) proportionally, active oxidation Graphene (GO) is mixed with restructuring streptococcus protein G (PG), and carries out oscillation incubation, after centrifugal, remove supernatant, material washs, and resulting materials is graphene oxide-non-covalent composite of restructuring streptococcus protein G (GOPG).
Further, above-mentioned steps 1) described in the step of active oxidation Graphene be: be resuspended in by graphene oxide in deionized water, room temperature is ultrasonic will fully disperse; Activate after adding 8-12 part N-hydroxysuccinimide (NHS) and 2-4 part 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) mixing in 1 part of graphene oxide composite material simultaneously; After detergent, obtain the graphene oxide of activation.
Further, the application of graphene oxide-non-covalent composite of restructuring streptococcus protein G in antibody purification, antibody enrichment, pathogen detection and/or biological sample pretreatment process field that provide of the application.
tool of the present invention has the following advantages:
1. the present invention optimizes the immobilized condition of graphene oxide composite material counterweight group of streptococcus albumin A, maintains the activity of albumen, and improves the supported quantity of albumen;
2. the preparation process of the method easy, be easy to promote, favorable reproducibility;
3. the method is prepared graphene oxide-restructuring streptococcal protein A composite and is easy to large-scale production, and cost is low.
Accompanying drawing explanation
Fig. 1. prepare material MALDI and detect;
Fig. 2. prepare Materials Antibodies absorption and detect.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described.
embodiment 1
(1) activation of graphene oxide (GO)
1.1mgGO is resuspended in the deionized water of 1mL, and the ultrasonic 3h of room temperature, fully disperses;
2. add 500mM2-(N-morpholinyl) ethyl sulfonic acid 4-morpholino b acid (pH6.1) (MES) 500 μ L, 16mg/mlNHS500 μ L and 4mg/mlEDC500uL while of then, mixing, room temperature rapid stirring 30min;
3. material 500mM(pH6.1) MES cyclic washing, NHS and EDC that removing is remaining, final volume is 1mL;
4. solution is the GO of activation.
(2) the immobilized restructuring streptococcus protein G of graphene oxide
1. select the ratio according to 1:2.5, material GO and the PG above-mentioned () prepared mixes, and carries out 4 DEG C of shaken overnight and hatches;
Removing supernatant after 2.16400 × g/10min is centrifugal, add 500mM(pH6.1) MES washs three times, and resulting materials is graphene oxide-restructuring streptococcus protein G (GOPG) non-covalent composite.
(3) support material property of protein detects
Obtained material carries out MALDI detection, result as shown in Figure 1: figure A is GOPG, and figure B is PG, PG molecular weight is 31KD, is PG peak at 15527 places, and all the other are peak of mixing.Albumen immobilized in illustrative material is PG.
(4) support material Function detection
By the non-covalent composite of GOPG of preparation after bovine serum albumin(BSA) is closed, the immunoglobulin G (IgG) marked with fluorescein isothiocynate (FITC) hatches 2h, observed by inverted fluorescence microscope, as shown in Figure 2, A figure is white light field, irregular material is GO-PG composite, B figure is the green fluorescence field of relevant position, visible by left and right contrast under 400 times of visuals field, GOPG non-covalent composite periphery has obvious luciferase expression, illustrate that the IgG that FITC marks is attracted on material, on the non-covalent composite of prompting GOPG, the activity of Protein G keeps good.
embodiment 2
In the activation protocol of graphene oxide (GO), NHS concentration changes 24mg/ml into, and other are with embodiment 1, can be prepared into the non-covalent composite of GOPG.
embodiment 3
In the activation protocol of graphene oxide (GO), NHS concentration changes 20mg/ml into, and other are with embodiment 1, can be prepared into the non-covalent composite of GOPG.
embodiment 4
In the activation protocol of graphene oxide (GO), EDC concentration changes 8mg/ml into, and other are with embodiment 1, can be prepared into the non-covalent composite of GOPG.
embodiment 5
In the activation protocol of graphene oxide (GO), EDC concentration changes 6mg/ml into, and other are with embodiment 1, can be prepared into the non-covalent composite of GOPG.
embodiment 6
In graphene oxide immobilized restructuring streptococcus protein G scheme, ratio is adjusted to 1:3, and other are with embodiment 1, can be prepared into the non-covalent composite of GOPG.
embodiment 7
In graphene oxide immobilized restructuring streptococcus protein G scheme, ratio is adjusted to 1:2.8, and other are with embodiment 1, can be prepared into the non-covalent composite of GOPG.
embodiment 8
GOPG material Protein G supported quantity is analyzed.Under the immobilized condition of embodiment 1, utilize BCA protein quantification principle, measure the Protein G content that rear different time supernatant is hatched in material GO and PG mixing, the supported quantity that indirectly can record material Protein G is 3.38mg/mg.
embodiment 9
GOPG material Protein G supported quantity is analyzed.Under the immobilized condition of embodiment 2-embodiment 7, utilize BCA protein quantification principle, measure the Protein G content that rear different time supernatant is hatched in material GO and PG mixing, indirectly can record the supported quantity of material Protein G between 1mg/mg-4mg/mg, compare with the immobilized ProteinG80ug/mL of commercially available reagent agarose, its supported quantity improves greatly.
The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; change can be expected easily or replace, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should described be as the criterion with the protection domain of claim.
Claims (4)
1. graphene oxide-non-covalent composite of restructuring streptococcus protein G, it is characterized in that, described composite is attached most importance to the Non-covalent binding thing of group of streptococcus Protein G and graphene oxide, and described graphene oxide is 1:2.5-3 with the ratio of the mass number of restructuring streptococcus protein G.
2. a preparation method for non-covalent composite according to claim 1, is characterized in that, comprise the steps:
1) active oxidation Graphene;
2) proportionally, mixed with restructuring streptococcus protein G by active oxidation Graphene, and carry out oscillation incubation, remove supernatant after centrifugal, material washs, and resulting materials is graphene oxide-non-covalent composite of restructuring streptococcus protein G.
3. follow the preparation method according to non-covalent composite according to claim 2, it is characterized in that, described in step 1), the step of active oxidation Graphene is: be resuspended in by graphene oxide in deionized water, and room temperature is ultrasonic will fully disperse; Activate after adding 8-12 part N-hydroxysuccinimide and the mixing of 2-4 part 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride in 1 part of graphene oxide composite material simultaneously; After detergent, obtain the graphene oxide of activation.
4. the graphene oxide according to claim 1-application of the non-covalent composite of restructuring streptococcus protein G in antibody purification, antibody enrichment, pathogen detection and/or biological sample pretreatment process.
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| CN1956779A (en) * | 2004-05-24 | 2007-05-02 | 株式会社资生堂 | Affinity particle and affinity separation method |
| WO2009010877A2 (en) * | 2007-07-17 | 2009-01-22 | Novartis Ag | Conjugate purification |
| CN103732533A (en) * | 2011-05-19 | 2014-04-16 | 纽约城市大学研究基金会 | Chemically modified graphene |
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