CN108084337A - The double imprinted materials of the paper substrate of Selective recognition protein and preparation method and application - Google Patents
The double imprinted materials of the paper substrate of Selective recognition protein and preparation method and application Download PDFInfo
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Abstract
The invention discloses double imprinted materials of a kind of paper substrate of Selective recognition protein and preparation method and application.The preparation method is fixed on filter paper material surface including graphene oxide hemin compound and its surface prepares double imprinted materials.Selective recognition and quantitative detection of the double imprinted materials of the paper substrate for protein.The double imprinted materials of the paper substrate have recycling of low cost, environmentally protective, easy to operate, easy and the characteristic to protein specific identification, particularly it possesses good colour developing, water conservation and water-wet behavior, realizes quick, the high-throughput detection of protein macromolecule.
Description
Technical field
The present invention relates to environmental analysis fields, and in particular to the double imprinted materials of the paper substrate of Selective recognition protein and its system
Preparation Method and application.
Background technology
Thyroglobulin is a kind of iodate glycoprotein in thyroid gland, is secreted by thyroid follicular cells, can be used as first
Shape parathyrine precursor substance, the iodate coupling generation thyroxine under the action of thyroid peroxidase；And thyroxine closes
Into with very important combination and transport protein in secretion process, with thyroid gland normal physiological function have it is close contact, first
Shape gland globulin levels are often as one of index of evaluation thyroid function.
Thyroid Hormone Disruptors be it is a kind of by influencing Thyroid Hormones Levels and target cell in terms of the reactivity two of hormone,
To disturb thyroxine synthesis, secretion, transhipment, combination, effect or the exotic of removing.In recent years to Thyroid Hormone Disruptors
Study it is more, but screen be identification, research and control Thyroid Hormone Disruptors pollution premise.Thyroglobulin is as first
The precursor substance of shape parathyrine, therefore, the substance of every synthesis and secretion for influencing thyroglobulin is all thyroxine interference
Object, then this kind of chaff interferent can be just screened by measuring thyroglobulin and primary dcreening operation.FRTL-5 cell lines are external
The rat thyroid follicular cells of culture, it has the function of that normal thyroid follicular cells secretes thyroglobulin, is mesh
Preceding unique functional thyroid cell strain available for Thyroid Hormone Disruptors known to in-vitro evaluation and screens unknown thyroxine
Chaff interferent.
Conventional thyroglobulin detection method is mainly radioimmunology, chemoluminescence method and immunofluorescence technique, however
These detection techniques are all there are the shortcomings of sample pre-treatments are cumbersome, of high cost, strongly professional, radioactivity, not only hazard detection people
Member's health, and be not suitable for laboratories and carry out response.Therefore, a kind of quick, convenient, sensitive, accurate high throughput of exploitation
Detection technique is highly desirable.
Paper material has the characteristics that derive from a wealth of sources, is of low cost, convenient for arbitrarily cutting/folding, it is environmentally friendly, and paper is fine
The abundant large specific surface area of dimension, is conducive to the attachment and modification of material.Paper chromatography technology is also frequently utilized for the multiple components of complex sample
Separation, and analyzed with reference to spectroscopy technology.But filter paper adsorption capacity itself is too low thus separation efficiency is poor, reaches
Less than requirement that is sensitive, accurately detecting, the needs of actual complex sample analysis can not be met.Surface imprinted technology refers in matrix
Surface obtains the polymer technology of preparing that space and binding site are exactly matched with template molecule, has conformation precordainment, identification
Specificity, the features such as environmental resistance and service life are long, the effect similar to biological antibody can be played.Research is main at present
It is in paper base material surface modification fluorescent material, and in phosphor surface Synthesis of Molecular Imprinting Polymers, by base material
Fluorescence signal carry out template molecule quick detection.But on the one hand modification paper surface fluorescent material signal response compared with
Weak, detection sensitivity is limited, and needs the equipment such as sepectrophotofluorometer or fluorescence microscope；On the other hand it is surface imprinted
Technology is often mainly that azodiisobutyronitrile, ammonium persulfate, dibenzoyl peroxide and n,N-Dimethylaniline etc. are difficult with initiator
Dissolubility toxic compounds, easily make protein denaturation.The present invention is with graphene-hemin compound, hydrogen peroxide and levulinic
Ketone forms ternary and triggers system, the generation of initiated polymerization；Graphene-hemin compound can with adsorbed proteins, and with
Protein interaction；It can also develop the color by the peroxidase sample activity of graphene-hemin compound, after colour developing
Image procossing is scanned to paper substrate, using gradation of image as detection signal, develops the nontoxic, lossless of specific recognition protein
Harmful, easy, quick, economic, sensitive analytical technology.
The content of the invention
The present invention solves the problems, such as that identification of protein technology is insufficient in popularization and secure context, provides a kind of paper substrate
Double imprinted materials and preparation method and application.
One side according to the invention provides a kind of preparation of the double imprinted materials of paper substrate of Selective recognition protein
Method, which is characterized in that include the following steps：
(1) graphene oxide-hemin compound is in the fixation on filter paper material surface
Graphene oxide-hemin compound is fixed on filter paper material surface by (1-1) with negative pressure；
(2) preparation of the double imprinted materials of paper substrate
Function monomer acrylamide, template protein and template 3,3', 5,5'- tetramethyl benzidine are dissolved in by (2-1)
In phosphate buffer solution, after abundant mixing, 20 DEG C of -37 DEG C of incubation 10min-30min；
The immobilized filter paper material for having graphene oxide-hemin compound in surface that (2-2) step (1-1) is prepared
It is added in the solution obtained by step (2-1), adds in crosslinking agent N, N- methylene-bisacrylamide, and add in catalystic converter system
Hydrogen peroxide and acetylacetone,2,4-pentanedione are protected from light polymerization 12h-24h at 20 DEG C -37 DEG C；
Filter paper material obtained by step (2-2) is washed away template protein and template 3,3', 5,5'- tetra- by (2-3) with eluent
After methyl biphenyl amine, the paper substrate mackle mark of 3,3', 5,5'- tetramethyl benzidine of Selective recognition template protein and template is obtained
Material.
Preferably, the function monomer acrylamide described in step (2-1) is used to provide ethylene double bond, amino and amide groups
Group and 3,3', 5,5'- tetramethyl benzidine polymerisation of template protein and template, form template protein and template 3,3',
The stereoeffect of 5,5'- tetramethyl benzidines.
Preferably, the graphene oxide described in step (1-1)-hemin compound has peroxidase sample activity, uses
In catalysis 3,3', the colour developing of 5,5'- tetramethyl benzidines is as detection signal.
Preferably, the function monomer acrylamide in step (2-1) is in graphene oxide-hemin compound and peroxidating
Under the initiation of hydrogen and acetylacetone,2,4-pentanedione, the fracture addition reaction of ethylene double bond occurs, and in crosslinking agent N, N- di-2-ethylhexylphosphine oxides third
Under the action of acrylamide, polymer is cross-linked to form.
Preferably, 3,3', 5,5'- tetramethyl benzidine of the acrylamide described in step (2-1), template protein and template
The ratio between the amount of substance be (7428-37141)：1：(1098-5492).
Preferably, the N described in step (2-2), the substance of N- methylene-bisacrylamides, hydrogen peroxide and acetylacetone,2,4-pentanedione
The ratio between amount is 1:(99-512):(1-12).
It is another aspect of this invention to provide that the double imprinted materials of the paper substrate for providing Selective recognition protein, it will by right
Any the methods of 1-6 is asked to be prepared.
It is another aspect of this invention to provide that the application of the double imprinted materials of the paper substrate for providing Selective recognition protein, it should
The double imprinted materials of paper substrate are used for the quantitative detection of the Selective recognition or protein of protein.
Preferably, the double imprinted materials of the paper substrate add in 3,3' into reaction system after Selective recognition protein,
5,5'- tetramethyl benzidines and hydrogen peroxide；3,3', 5,5'- tetramethyl benzidine competitive binding mackle mark remain site；Oxidation
Graphene-hemin peroxidase sample active catalytic hydrogen peroxide release free radical, 3,3', 5,5'- tetramethyl benzidine knots
It closes free radical and blueness is presented, the concentration of protein is calculated according to the gray value of color.
Preferably, the double imprinted materials of the paper substrate are used for the Selective recognition of thyroglobulin matter or thyroglobulin matter
Quantitative detection.
In general, by the above technical scheme conceived by the present invention compared with prior art, mainly possess following
Technological merit：
(1) paper material derives from a wealth of sources, is of low cost, and the double imprinted materials of paper substrate of preparation can be used as adsorbent, realize multiple
Other equipment and the auxiliary of instrument is not required in the highly selective identification extraction of thyroglobulin, operating process in miscellaneous sample, just
It can realize the recycling of material, it is relatively simple quick.
(2) on the one hand graphene oxide-hemin can act on adsorbed proteins by π π, increase the double imprinted materials of paper substrate
To the adsorption capacity of protein；Another aspect graphene oxide-hemin compound can also be made with hydrogen peroxide, acetylacetone,2,4-pentanedione
For ternary trigger system, nontoxic, easy dispersing and dissolving, can catalytic polymerization generation；Graphene oxide-the hemin is multiple simultaneously
Closing object has peroxidase sample activity, for being catalyzed the colour developing of 3,3', 5,5'- tetramethyl benzidines as detection signal.
(3) characteristic good with translucency can be cut/modified by paper material, and albumen can be completed by conventional developing technology
The quick detection of matter disclosure satisfy that portable quick analysis.It is surface imprinted compared with carbon material of the prior art, magnetic material
For polymer, the double advantages that imprinted material is also environmentally protective, hydrophily is high, cheap and easy to get of paper substrate can realize complicated life
The highly selective identification of target molecule in object sample both can be used for the selective enumeration method analysis of protein, and can be used for egg
Efficient absorption/enrichment of white matter has preferable actual application value.
Description of the drawings
Fig. 1 is the atomic force microscopy diagram of graphene oxide-hemin compound absorption thyroglobulin；
Fig. 2 is the electron microscope of paper substrate surface modification graphene oxide-hemin；
Fig. 3 is (a) and (b) figure after the generation of paper substrate polymerization reaction on the solid surface before paper substrate polymerization reaction on the solid surface occurs；
Fig. 4 is the electron microscope of the double imprinted materials (a, b) of paper substrate and paper substrate non-imprinted material (c, d)；
Fig. 5 is that the double imprinted materials of paper substrate and paper substrate non-imprinted material (blank control) imitate the absorption of thyroglobulin
Fruit；
Fig. 6 is the Selective recognition of the double imprinted materials (a) of paper substrate and paper substrate non-imprinted material (b) to various protein
Energy；
Fig. 7 is the application of the double imprinted materials (a) of paper substrate and paper substrate non-imprinted material (b) detection thyroglobulin；
Fig. 8 is the flow chart of the double imprinted material absorption of paper substrate and detection.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, it is right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
Conflict is not formed each other to can be combined with each other.
With reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
The advantage that the present invention can arbitrarily be cut/be modified using surface imprinted technology and paper material, by initiator graphite oxide
Alkene-hemin compound is immobilized on rich cellulose-containing paper material surface.Then, with template protein and 3,3', 5,5'- tetramethyls
Base benzidine is template, and acrylamide is function monomer, and under normal temperature condition, it is molten that the scraps of paper after modification are immersed in above-mentioned reaction
In liquid, hydrophily mackle mark polymeric gel is synthesized on paper material surface.Since graphene oxide-hemin compound is only immobilized
On filter paper fibre surface, thus the polymerization process of trace film also can only occur in filter paper fibre surface, realize polymerization process
Controllable preparation.After eluted template molecule, the double imprinted materials of paper substrate are obtained, highly selective identification of protein and quantitative inspection can be carried out
It surveys.
Embodiment 1
The preparation of graphene oxide-hemin compound：By graphene oxide (0.83mmol, piece footpath 50-200nm) and blood
Brilliant element (0.015mmol) is dissolved in the ethanol solution of 20ml 50%, with 1mol/L sodium hydroxide solutions by pH adjust to
10.0, for ultrasound after 30 minutes, postcooling when reflux 1 is small under the conditions of 100 DEG C forms graphene oxide-hemin compound, wherein
Graphene oxide and hemin form π π connections by physisorption；
By graphene oxide-hemin (graphene oxide 0.85mmol, hemin 0.015mmol) and thyroglobulin
(0.001mmol) is dispersed in 1ml phosphate buffered saline solutions, after abundant mixing, be protected from light incubation at room temperature 2-12 it is small when；
Fig. 1 shows the atomic force microscopy diagram of graphene oxide-hemin compound absorption thyroglobulin.
Atomic force microscopy diagram is shown, since graphene oxide-hemin composite surface has adsorbed thyroglobulin,
Height has been increased to 12nm, surface irregularity by 2nm, and the visual field fogs, and result above proves that graphene oxide-hemin is compound
Object can adsorb thyroglobulin.
Embodiment 2
(1) graphene oxide-hemin compound is in the fixation on filter paper fibre surface
By negative pressure by graphene oxide-hemin compound (graphene oxide 0.83mmol, hemin 0.015mmol)
Filter paper (40mm × 40mm) surface is fixed on, filter paper is cut into several sequins with card punch (8mm × 8mm).
Fig. 2 shows that graphene oxide-hemin compound is fixed on the electron microscope on filter paper material surface.Fig. 2 (a) is
SEM figures under the conditions of 20KV, X300 and 50 μm；Fig. 2 (b) is the SEM figures under the conditions of 20KV, X3000 and 5 μm.
(2) preparation of the double imprinted materials of paper substrate
(2-1) by function monomer acrylamide (0.056mmol), template thyroglobulin (1.515nmol) and 3,3',
5,5'- tetramethyl benzidines (0.004mmol) are dissolved in 1ml phosphate buffer solutions (10mmol/L, pH=6.8), vortex
Mixing 10 seconds is incubated at room temperature 30 minutes.
The graphene oxide that (2-2) prepares step (1)-hemin modification filter paper dick is put into above-mentioned mixed solution,
Add in crosslinking agent N, N- methylene-bisacrylamide (0.0065mmol), catalyst system and catalyzing hydrogen peroxide (1.96mmol) and levulinic
Ketone (0.035mmol), be protected from light at room temperature polymerization 10 it is small when, wash away mould with 10% acetic acid solution of 10% lauryl sodium sulfate
Plate thyroglobulin and 3,3' after 5,5'- tetramethyl benzidines, has obtained the double imprinted materials of paper substrate.Among this process,
Graphene oxide-hemin compound and hydrogen peroxide, acetylacetone,2,4-pentanedione form ternary and trigger system, and acetylacetone,2,4-pentanedione is as in organic
Mesosome so that graphene oxide-hemin catalyzing hydrogen peroxide generates release free radical, triggers paper substrate polymerization reaction on the solid surface hair
It is raw.The preparation process of paper substrate non-imprinted material (blank control) is same as above, is simply added without template thyroid gland in preparation process
Globulin and 3,3', 5,5'- tetramethyl benzidine.
Fig. 3 is shown in embodiment 2 before the generation of paper substrate polymerization reaction on the solid surface (a) and (b) after the generation of paper substrate polymerization reaction on the solid surface.
As shown in the figure, since graphene oxide-hemin compound is only fixed on paper substrate surface, when add in hydrogen peroxide and
After acetylacetone,2,4-pentanedione, graphene oxide-hemin compound and hydrogen peroxide, acetylacetone,2,4-pentanedione form ternary and trigger system, trigger paper
The generation of primary surface polymerisation.
Fig. 4 shows the Electronic Speculum of the double imprinted materials (a, b) of paper substrate prepared by embodiment 2 and paper substrate non-imprinted material (c, d)
Figure.
Fig. 4 (a, c) is the SEM figures under the conditions of 20KV, X300 and 50 μm；Fig. 4 (b, d) is 20KV, X3000 and 5 μm
Under the conditions of SEM figure.
Electron microscope is shown：Due to surface aggregate technology, double imprinted polymer films are only grown in filter paper fibre surface, still have
The net structure of standby filter paper fibre, contributes to fast mass to transfer.The film of the polymer-modified filter paper of mackle mark is comparatively dense,
It is even to be coated on filter paper fibre surface, and irregular laminated structure is presented, imply the presence of function monomer/template prepolymer.
And the electron microscope of paper substrate non-imprinted material is shown, non-trace film is implied with the equally distributed filter paper fibre surface of graininess
The presence of non-trace prepolymer.The above results also turn out that the polymerization process of trace film can only occur in filter paper fibre surface, real
The controllable preparation of existing polymerization process.
Embodiment 3
The double imprinted materials (8mm × 8mm) of paper substrate and paper substrate non-imprinted material (8mm × 8mm) prepared by embodiment 2 is respectively
It is added among the phosphate buffer solution (pH=6.8) containing thyroglobulin of 5mL, the concentration difference of thyroglobulin
For 50-500mg/L.Vibrate 10 it is small when after, will using the content of thyroglobulin in fluorescent spectrophotometer assay supernatant
The total amount of thyroglobulin subtracts the content of thyroglobulin in supernatant, calculates the double imprinted materials of paper substrate and paper substrate is non-
Imprinted material is to the adsorption capacity of thyroglobulin.The results are shown in Figure 5, and the double imprinted materials of paper substrate are to thyroglobulin
Adsorption capacity reaches 87.78mg/g, and the non-imprinted polymer as control is only to the adsorption capacity of thyroglobulin
45.37mg/g shows that the imprinted sites of specific recognition thyroglobulin have been formed.
Embodiment 4
The double imprinted materials (8mm × 8mm) of paper substrate and paper substrate non-imprinted material (8mm × 8mm) prepared by embodiment 2 is respectively
It is added among the phosphate buffer solution (pH=6.8) containing different proteins of 5mL, the concentration of protein is 300mg/L.
Vibrate 5 it is small when after, using the content of protein in fluorescence spectrophotometry supernatant, and then calculate double imprinted polymers
With non-imprinted polymer to the adsorption capacity of protein, the Selective recognition ability of the double imprinted polymers of investigation.As a result such as Fig. 6 institutes
Show, since double imprinted polymers have the functional group exactly matched with template protein molecule (thyroglobulin) distribution and sky
Between structure, thus the specific recognition capability comparable to biological antibody is shown to thyroprotein.With the double imprinted materials of paper substrate
The ratio of adsorption capacity and non-imprinted material adsorption capacity, it is alternatively that sex factor further evaluates the recognition capability of material.
The result shows that the double imprinted materials of paper substrate reach 2.61 to the selectivity factor of thyroglobulin, higher than the choosing of other protein
Select sex factor (0.55-1.46).It, being capable of highly selective identification thyroid gland ball egg in the environmental samples thus coexisted in complex matrices
In vain, you can for the selective extraction of thyroglobulin in sample, and can be used for the efficient absorption of thyroglobulin/
Enrichment.
Embodiment 5
The double imprinted materials of paper substrate prepared by embodiment 2 establish a kind of FRTL-5 cells training as solid phase extraction adsorbents
The New Method for Rapid of thyroglobulin in nutrient solution.Detailed process：Material (8mm × 8mm) is used into 10%SDS respectively first
Acetic acid (10%) solution and phosphate buffer solution (pH=6.8) impregnate, further remove remaining template molecule, keep it
Adsorption activity.Then, it is immersed in 5mLFRTL-5 cell culture fluids, realizes absorption of the thyroglobulin on the scraps of paper.
After being cleaned twice with pure water, 3,3', 5,5'- tetramethyl benzidines and hydrogen peroxide are added in.Or filter paper is directly immersed in TMB and is shown
In color liquid, 3,3', 5,5'- tetramethyl benzidines and hydrogen peroxide are contained in TMB developing solutions.As shown in figure 8,3,3', 5,5'- tetra-
Methyl biphenyl amine competitive binding mackle mark remains site, under graphene oxide-hemin peroxidase sample activity, is catalyzed
Hydrogen oxide discharges free radical, and simultaneously blueness is presented in 3,3', 5,5'- tetramethyl benzidine combination free radicals.Through scanning after scraps of paper colour developing
Instrument scans, photoshop software processing image informations, and obtains corresponding gray value, is converted into corresponding protein molecular concentration.Only
After template molecule all removes, polymer can just recombine template molecule, when there are mackle mark, during two template molecules,
Competitive binding will be generated when adsorbing template molecule, because not only molecular weight is big for protein, but also space structure is big, corresponding to print
Mark hole space is also big, trace hole specific adsorption template molecule, but specificity does not also represent uniqueness, so small point
The 3,3' of son amount, 5,5'- tetramethyl benzidines are also the moiety site that can take up thyroglobulin.It is developed the color by detecting
The amount of 3,3', 5,5'- tetramethyl benzidine can obtain the content of thyroglobulin indirectly.
The double imprinted materials of paper substrate prepared by embodiment 2 are as solid phase extraction adsorbents, by material (8mm × 8mm) first
It is impregnated respectively with acetic acid (10%) solution and phosphate buffer solution (pH=6.8) of 10%SDS, further removes remaining mould
Plate molecule keeps its adsorption activity.Then, be immersed in 5mL thyroglobulin concentration be respectively 0.1,1,10,50,80,
In the FRTL-5 cell culture fluids of 100ug/L, absorption of the thyroglobulin on the scraps of paper is realized.After being cleaned twice with pure water,
Add in 3,3', 5,5'- tetramethyl benzidines and hydrogenperoxide steam generator, 3,3', 5,5'- tetramethyl benzidine competitive binding mackle marks
Site is remained, under graphene oxide-hemin peroxidase sample activity, catalyzing hydrogen peroxide release free radical, 3,3', 5,
Simultaneously blueness is presented in 5'- tetramethyl benzidine combination free radicals, and the blank filter paper piece for being covered in paper substrate mackle mark material surface is complete
Through scanner scanning, photoshop software processing image informations after full dyeing, and obtain thyroglobulin concentration from low to high
Corresponding gray value, be followed successively by 201.95,205.19,207.45,211.93,215.86,216.52.The range of linearity of method is
1-100ug/L, detection are limited to 0.1ug/L, can realize the quick, high of thyroglobulin in actual FRTL-5 cell culture fluids
Flux detects.
Fig. 7 shows the double imprinted materials (a) of paper substrate and paper substrate non-imprinted material (b) absorption thyroglobulin and 3,3',
Colour developing figure after 5,5'- tetramethyl benzidines.
As shown in fig. 7, the double imprinted materials of paper substrate are due to there are the trace hole of 3,3', 5,5'- tetramethyl benzidines, specifically
3,3', 5,5'- tetramethyl benzidines in property adsorbent solution, scraps of paper color substantially deepens, and paper substrate non-imprinted material is not due to
There are the trace emptying aperture caves of 3,3', 5,5'- tetramethyl benzidines, almost do not have to the adsorbance of 3,3', 5,5'- tetramethyl benzidines
Have, scraps of paper color is apparently without changing, it was demonstrated that the successful preparation in 3,3', 5,5'- tetramethyl benzidine trace holes, and can
As the sensitive signal of detection thyroglobulin after being developed the color using absorption.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, all any modification, equivalent and improvement made within the spirit and principles of the invention etc., should all include
Within protection scope of the present invention.
Claims (10)
1. the preparation method of the double imprinted materials of a kind of paper substrate of Selective recognition protein, which is characterized in that include the following steps：
(1) graphene oxide-hemin compound is in the fixation on filter paper material surface
Graphene oxide-hemin compound is fixed on filter paper material surface by (1-1) with negative pressure；
(2) preparation of the double imprinted materials of paper substrate
Function monomer acrylamide, template protein and template 3,3', 5,5'- tetramethyl benzidine are dissolved in phosphoric acid by (2-1)
In salt buffer solution, after abundant mixing, 20 DEG C of -37 DEG C of incubation 10min-30min；
The immobilized filter paper material for having graphene oxide-hemin compound in surface that (2-2) step (1-1) is prepared adds in
Into the solution obtained by step (2-1), crosslinking agent N, N- methylene-bisacrylamide is added in, and adds in hydrogen peroxide and levulinic
Ketone is protected from light polymerization 12h-24h at 20 DEG C -37 DEG C；
Filter paper material obtained by step (2-2) is washed away template protein and template 3,3', 5,5'- tetramethyl by (2-3) with eluent
After benzidine, the paper substrate mackle mark material of 3,3', 5,5'- tetramethyl benzidine of Selective recognition template protein and template is obtained
Material.
2. the preparation method of the double imprinted materials of the paper substrate of Selective recognition protein as described in claim 1, which is characterized in that
Function monomer acrylamide described in step (2-1) is used to provide ethylene double bond, amino and amide group and template protein
With 3,3', 5,5'- tetramethyl benzidine polymerisation of template, 3,3', 5,5'- tetramethyl biphenyl of template protein and template is formed
The stereoeffect of amine.
3. the preparation method of the double imprinted materials of the paper substrate of Selective recognition protein as described in claim 1, which is characterized in that
Function monomer acrylamide in step (2-1) is in graphene oxide-hemin compound and hydrogen peroxide and acetylacetone,2,4-pentanedione
Initiation under, the fracture addition reaction of ethylene double bond occurs, and in crosslinking agent N, the effect of N- methylene-bisacrylamides
Under, it is cross-linked to form polymer.
4. the preparation method of the double imprinted materials of the paper substrate of Selective recognition protein as described in claim 1, which is characterized in that
Graphene oxide-hemin compound described in step (1-1) has peroxidase sample activity, for being catalyzed 3,3', 5,5'-
Tetramethyl benzidine colour developing is as detection signal.
5. the preparation method of the double imprinted materials of the paper substrate of the Selective recognition protein as described in claim 1-4 is any, special
Sign is, the substance of acrylamide, template protein and 3,3', 5,5'- tetramethyl benzidine of template described in step (2-1)
The ratio between amount is (7428-37141)：1：(1098-5492).
6. the preparation method of the double imprinted materials of the paper substrate of the Selective recognition protein as described in claim 1-4 is any, special
Sign is, the N described in step (2-2), and the ratio between amount of substance of N- methylene-bisacrylamides, hydrogen peroxide and acetylacetone,2,4-pentanedione is
1:(99-512):(1-12)。
7. the double imprinted materials of a kind of paper substrate of Selective recognition protein, which is characterized in that by either one legal system of claim 1-6
It is standby to obtain.
8. the application of the double imprinted materials of the paper substrate of Selective recognition protein as claimed in claim 7, which is characterized in that the paper
The double imprinted materials of base are used for the quantitative detection of the Selective recognition or protein of protein.
9. application as claimed in claim 8, which is characterized in that the double imprinted materials of the paper substrate Selective recognition protein it
Afterwards, 3,3', 5,5'- tetramethyl benzidines and hydrogen peroxide are added in into reaction system, 3,3', 5,5'- tetramethyl benzidines are competing
Strive and remain site with reference to mackle mark, the peroxidase sample active catalytic hydrogen peroxide of graphene oxide-hemin be released from by
Base, 3,3', 5,5'- tetramethyl benzidine combination free radicals are simultaneously presented blueness, protein are calculated according to the gray value of color
Concentration.
10. application as claimed in claim 8 or 9, which is characterized in that the double imprinted materials of the paper substrate are used for thyroglobulin matter
Selective recognition or thyroglobulin matter quantitative detection.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042050A (en) * | 2019-04-23 | 2019-07-23 | 浙江凯恩特种材料股份有限公司 | It is a kind of for plaque detection paper substrate micro-fluidic chip, the detection method and application of kit and plaque |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103063652A (en) * | 2012-12-31 | 2013-04-24 | 济南大学 | Preparation method of voltage-controllable multiplex multichannel sensing paper chip for detecting antibiotic residues by molecular imprinting electroluminescence |
| CN103551194A (en) * | 2013-11-14 | 2014-02-05 | 厦门大学 | Graphene-heme and nanogold ternary composite material, preparation method and application |
| CN103868966A (en) * | 2014-03-21 | 2014-06-18 | 上海出入境检验检疫局工业品与原材料检测技术中心 | Molecularly-imprinted polymer-graphene composite material modified electrode as well as preparation method and application thereof |
| CN104987460A (en) * | 2015-06-11 | 2015-10-21 | 衡阳师范学院 | Amino-contained antibiotic nanometer molecularly imprinted polymer and preparation method and application thereof |
-
2017
- 2017-12-06 CN CN201711275084.XA patent/CN108084337B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103063652A (en) * | 2012-12-31 | 2013-04-24 | 济南大学 | Preparation method of voltage-controllable multiplex multichannel sensing paper chip for detecting antibiotic residues by molecular imprinting electroluminescence |
| CN103551194A (en) * | 2013-11-14 | 2014-02-05 | 厦门大学 | Graphene-heme and nanogold ternary composite material, preparation method and application |
| CN103868966A (en) * | 2014-03-21 | 2014-06-18 | 上海出入境检验检疫局工业品与原材料检测技术中心 | Molecularly-imprinted polymer-graphene composite material modified electrode as well as preparation method and application thereof |
| CN104987460A (en) * | 2015-06-11 | 2015-10-21 | 衡阳师范学院 | Amino-contained antibiotic nanometer molecularly imprinted polymer and preparation method and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042050A (en) * | 2019-04-23 | 2019-07-23 | 浙江凯恩特种材料股份有限公司 | It is a kind of for plaque detection paper substrate micro-fluidic chip, the detection method and application of kit and plaque |
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