CN108084337B - The double imprinted materials of the paper base of Selective recognition protein and preparation method and application - Google Patents
The double imprinted materials of the paper base of Selective recognition protein and preparation method and application Download PDFInfo
- Publication number
- CN108084337B CN108084337B CN201711275084.XA CN201711275084A CN108084337B CN 108084337 B CN108084337 B CN 108084337B CN 201711275084 A CN201711275084 A CN 201711275084A CN 108084337 B CN108084337 B CN 108084337B
- Authority
- CN
- China
- Prior art keywords
- double
- protein
- paper base
- template
- imprinted materials
- 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.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/291—Gel sorbents
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2201/00—Foams characterised by the foaming process
- C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
- C08J2201/042—Elimination of an organic solid phase
- C08J2201/0424—Elimination of an organic solid phase containing halogen, nitrogen, sulphur or phosphorus atoms
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2333/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
- C08J2333/24—Homopolymers or copolymers of amides or imides
- C08J2333/26—Homopolymers or copolymers of acrylamide or methacrylamide
Abstract
The invention discloses double imprinted materials of the paper base of a kind of Selective recognition protein and the preparation method and application thereof.The preparation method includes that graphene oxide-hemin compound is fixed on filter paper material surface and the double imprinted materials of surface preparation.The double imprinted materials of the paper base are used for the Selective recognition and quantitative detection of protein.The double imprinted materials of the paper base have characteristic that is low in cost, environmentally protective, easy to operate, being easy recycling and identify to protein specific, especially it has 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 base of Selective recognition protein and its system
Preparation Method and application.
Background technique
Thyroglobulin is one of thyroid gland iodate glycoprotein, is secreted by thyroid follicular cells, can be used as first
Shape parathyrine precursor substance, iodate coupling generates thyroxine under the action of thyroid peroxidase;It is also that thyroxine closes
At with combination very important in secretion process and transport protein, with thyroid gland normal physiological function have it is close contact, first
Index one of of the shape gland globulin levels often as evaluation thyroid function.
Thyroid Hormone Disruptors are a kind of two aspects of reactivity by influencing Thyroid Hormones Levels and target cell to hormone,
To interfere 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 all synthesis for influencing thyroglobulin and secretion are all thyroxine interference
Object, then examination and primary dcreening operation can be carried out to this kind of chaff interferent by measuring thyroglobulin.FRTL-5 cell strain is 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 can be used for Thyroid Hormone Disruptors known to in-vitro evaluation and screen unknown thyroxine
Chaff interferent.
Conventional thyroglobulin detection method is mainly radioimmunology, chemoluminescence method and immunofluorescence technique, however
All there is the disadvantages of cumbersome sample pre-treatments, at high cost, strongly professional, radioactivity in these detection techniques, 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 is developed
Detection technique is highly desirable.
Paper material have the characteristics that it is from a wealth of sources, low in cost, convenient for arbitrarily cutting/folding, it is environmentally friendly, and paper is fine
Abundant large specific surface area is tieed up, the attachment and modification of material are conducive to.Paper chromatography technology is also frequently utilized for the multiple components of complex sample
Separation, and analyzed in conjunction with 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, it is unable to satisfy the needs of actual complex sample analysis.Surface imprinted technology refers in matrix
Surface obtains the polymer technology of preparing of space and binding site and template molecule exact matching, has conformation precordainment, identification
The features such as specificity, environmental resistance and long service life, 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 surface imprinted
Technology is often mainly that azodiisobutyronitrile, ammonium persulfate, dibenzoyl peroxide and n,N-Dimethylaniline etc. are difficult with initiator
Dissolubility toxic compounds are easy to make protein denaturation.The present invention is with graphene-hemin compound, hydrogen peroxide and levulinic
Ketone constitutes ternary and causes system, the generation of initiated polymerization;Graphene-hemin compound can with adsorbed proteins, and with
Protein interaction;It can also develop the color by graphene-hemin compound peroxidase sample activity, after colour developing
Image procossing is scanned to paper base, is detection signal with image grayscale, develops the nontoxic, lossless of specific recognition protein
Evil, easy, quick, economy, sensitive analytical technology.
Summary 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 base
Double imprinted materials and the preparation method and application thereof.
According to one aspect of the present invention, a kind of preparation of the double imprinted materials of paper base of Selective recognition protein is provided
Method, which comprises the steps of:
(1) fixation of the graphene oxide-hemin compound on filter paper material surface
Graphene oxide-hemin compound is fixed on filter paper material surface with negative pressure by (1-1);
(2) preparation of the double imprinted materials of paper base
Function monomer acrylamide, template protein and template 3,3', 5,5'- tetramethyl benzidine are dissolved in by (2-1)
In phosphate buffer solution, after mixing well, 20 DEG C of -37 DEG C of incubation 10min-30min;
(2-2) has graphene oxide-hemin compound filter paper material for surface that step (1-1) is prepared is immobilized
It is added in step (2-1) resulting solution, crosslinking agent N, N- methylene-bisacrylamide is added, and catalystic converter system is added
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- with eluent by (2-3)
After methyl biphenyl amine, the paper base mackle mark of 3,3', 5,5'- tetramethyl benzidine of Selective recognition template protein and template is obtained
Material.
Preferably, function monomer acrylamide described in step (2-1) is for providing ethylene double bond, amino and amide groups
Group and 3,3', 5,5'- tetramethyl benzidine polymerization reaction of template protein and template, form template protein and template 3,3',
The stereoeffect of 5,5'- tetramethyl benzidine.
Preferably, graphene oxide-hemin compound described in step (1-1) has peroxidase sample activity, uses
In catalysis 3,3', the colour developing of 5,5'- tetramethyl benzidine 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 oxide third
Under the action of acrylamide, it is cross-linked to form polymer.
Preferably, 3,3', 5,5'- tetramethyl benzidine of acrylamide, template protein described in step (2-1) and template
The ratio between the amount of substance be (7428-37141): 1:(1098-5492).
Preferably, N described in step (2-2), the substance of N- methylene-bisacrylamide, 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 base for providing Selective recognition protein, are wanted by right
Any the method for 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 base for providing Selective recognition protein, it should
The double imprinted materials of paper base are for the Selective recognition of protein or the quantitative detection of protein.
Preferably, 3,3' are added into reaction system after Selective recognition protein for the double imprinted materials of the paper base,
5,5'- tetramethyl benzidine and hydrogen peroxide;3,3', 5,5'- tetramethyl benzidine competitive binding mackle mark remain site;Oxidation
Graphene-hemin peroxidase sample active catalytic hydrogen peroxide discharges free radical, 3,3', 5,5'- tetramethyl benzidine knots
It closes free radical and blue is presented, the concentration of protein is calculated according to the gray value of color.
Preferably, the double imprinted materials of the paper base are used for the Selective recognition or thyroglobulin matter of thyroglobulin matter
Quantitative detection.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, mainly have below
Technological merit:
(1) paper material is from a wealth of sources, low in cost, and the double imprinted materials of the paper base of preparation can be used as adsorbent, realizes multiple
The highly selective identification extraction of thyroglobulin, operating process do not need the auxiliary of other equipment and instrument in miscellaneous sample, just
It can be realized 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 base
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 cause 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 can satisfy portable quick analysis.Carbon material, magnetic material in compared with the existing technology is surface imprinted
For polymer, there are also the advantages that environmentally protective, hydrophily is high, cheap and easy to get for the double imprinted materials of paper base, can be realized 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 practical application value.
Detailed description of the invention
Fig. 1 is graphene oxide-hemin compound absorption thyroglobulin atomic force microscopy diagram;
Fig. 2 is paper base surface modification graphene oxide-hemin electron microscope;
Fig. 3 is (a) and (b) figure after the generation of paper base polymerization reaction on the solid surface before paper base polymerization reaction on the solid surface occurs;
Fig. 4 is the electron microscope of paper base double imprinted materials (a, b) and paper base non-imprinted material (c, d);
Fig. 5 is that the double imprinted materials of paper base and paper base 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 base and paper base non-imprinted material (b) to various protein
Energy;
Fig. 7 is the application of the double imprinted materials (a) of paper base and paper base non-imprinted material (b) detection thyroglobulin;
Fig. 8 is the flow chart of paper base double imprinted material absorption and detection.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, 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
Not constituting a conflict with each other can be combined with each other.
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The advantage that the present invention utilizes surface imprinted technology and paper material that can arbitrarily cut/modify, 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, under normal temperature conditions, it is molten that the scraps of paper after modification is immersed in above-mentioned reaction
In liquid, synthesize hydrophily mackle mark polymeric gel 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 base are obtained, highly selective identification of protein and quantitative inspection can be carried out
It surveys.
Embodiment 1
Graphene oxide-hemin compound preparation: by graphene oxide (0.83mmol, piece diameter 50-200nm) and blood
Brilliant element (0.015mmol) is dissolved in the ethanol solution of 20ml 50%, is adjusted to pH with 1mol/L sodium hydroxide solution
10.0, ultrasound is cooling after flowing back 1 hour under the conditions of 100 DEG C after 30 minutes, forms graphene oxide-hemin compound, wherein
Graphene oxide and hemin form π π connection by physisorption;
By graphene oxide-hemin (graphene oxide 0.85mmol, hemin 0.015mmol) and thyroglobulin
(0.001mmol) is dispersed in 1ml phosphate buffered saline solution, after mixing well, is protected from light incubation at room temperature 2-12 hours;
Fig. 1 shows graphene oxide-hemin compound absorption thyroglobulin atomic force microscopy diagram.
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) fixation of the graphene oxide-hemin compound on filter paper fibre surface
By negative pressure by graphene oxide-hemin compound (graphene oxide 0.83mmol, hemin 0.015mmol)
It is fixed on the surface filter paper (40mm × 40mm), filter paper is cut into several sequins with punch (8mm × 8mm).
Fig. 2 shows the electron microscopes that graphene oxide-hemin compound is fixed on filter paper material surface.Fig. 2 (a) is
SEM figure under the conditions of 20KV, X300 and 50 μm;Fig. 2 (b) is the SEM figure under the conditions of 20KV, X3000 and 5 μm.
(2) preparation of the double imprinted materials of paper base
(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 solution (10mmol/L, pH=6.8), vortex
Mixing 10 seconds is incubated at room temperature 30 minutes.
Graphene oxide-hemin modification filter paper dick prepared by step (1) is put into above-mentioned mixed solution by (2-2),
Crosslinking agent N, N- methylene-bisacrylamide (0.0065mmol), catalyst system hydrogen peroxide (1.96mmol) and levulinic is added
Ketone (0.035mmol) is protected from light polymerization 10 hours at room temperature, washes 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 base.In this process,
Graphene oxide-hemin compound and hydrogen peroxide, acetylacetone,2,4-pentanedione constitute ternary and cause system, and acetylacetone,2,4-pentanedione is as in organic
Mesosome causes paper base polymerization reaction on the solid surface hair so that graphene oxide-hemin catalyzing hydrogen peroxide generates release free radical
It is raw.The preparation process of paper base non-imprinted material (blank control) is same as above, and template thyroid gland is only added without in preparation process
Globulin and 3,3', 5,5'- tetramethyl benzidine.
Fig. 3 is shown in embodiment 2 before the generation of paper base polymerization reaction on the solid surface (a) and (b) after the generation of paper base polymerization reaction on the solid surface.
As shown, since graphene oxide-hemin compound is only fixed on paper base surface, when be added hydrogen peroxide and
After acetylacetone,2,4-pentanedione, graphene oxide-hemin compound and hydrogen peroxide, acetylacetone,2,4-pentanedione constitute ternary and cause system, cause paper
The generation of primary surface polymerization reaction.
Fig. 4 shows the Electronic Speculum of the paper base double imprinted materials (a, b) and paper base non-imprinted material (c, d) of the preparation of embodiment 2
Figure.
Fig. 4 (a, c) is the SEM figure 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 reticular structure of standby filter paper fibre facilitates fast mass transmitting.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 base 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 base prepared by embodiment 2 and paper base non-imprinted material (8mm × 8mm) are respectively
It is added in the phosphate buffer solution (pH=6.8) containing thyroglobulin of 5mL, the concentration difference of thyroglobulin
For 50-500mg/L.It, will using the content of thyroglobulin in fluorescent spectrophotometer assay supernatant after oscillation 10 hours
The total amount of thyroglobulin subtracts the content of thyroglobulin in supernatant, calculates the double imprinted materials of paper base and paper base is non-
Adsorption capacity of the imprinted material to thyroglobulin.As a result as shown in figure 5, the double imprinted materials of paper base 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 base prepared by embodiment 2 and paper base non-imprinted material (8mm × 8mm) are respectively
It is added in the phosphate buffer solution (pH=6.8) containing different proteins of 5mL, the concentration of protein is 300mg/L.
After oscillation 5 hours, using the content of protein in fluorescence spectrophotometry supernatant, and then double imprinted polymers are calculated
With non-imprinted polymer to the adsorption capacity of protein, the Selective recognition ability of double imprinted polymers is investigated.As a result such as Fig. 6 institute
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 base
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 base 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 thus in the environmental samples that complex matrices coexist
It is white, it 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 base prepared by embodiment 2 establish a kind of FRTL-5 cell 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 fluid, 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 is added.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 solution.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 blue is presented in 3,3', 5,5'- tetramethyl benzidine combination free radicals.Through scanning after scraps of paper colour developing
Instrument scanning, photoshop software handles image information, 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, when 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 benzidine is also the moiety site that can take up thyroglobulin.It is developed the color by detection
The amount of 3,3', 5,5'- tetramethyl benzidine can obtain the content of thyroglobulin indirectly.
The double imprinted materials of paper base prepared by embodiment 2 are as solid phase extraction adsorbents, first by material (8mm × 8mm)
It is impregnated respectively with acetic acid (10%) solution of 10%SDS and phosphate buffer solution (pH=6.8), 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 fluid of 100ug/L, absorption of the thyroglobulin on the scraps of paper is realized.After being cleaned twice with pure water,
3,3', 5,5'- tetramethyl benzidines and hydrogenperoxide steam generator, 3,3', 5,5'- tetramethyl benzidine competitive binding mackle marks are added
Site is remained, under graphene oxide-hemin peroxidase sample activity, catalyzing hydrogen peroxide release free radical, 3,3', 5,
Simultaneously blue is presented in 5'- tetramethyl benzidine combination free radical, and the blank filter paper piece for being covered on paper base mackle mark material surface is complete
Through scanner scanning after full dyeing, photoshop software handles image information, and obtains 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 be realized the quick, high of thyroglobulin in practical FRTL-5 cell culture fluid
Flux detection.
Fig. 7 shows the double imprinted materials (a) of paper base and paper base non-imprinted material (b) absorption thyroglobulin and 3,3',
Colour developing figure after 5,5'- tetramethyl benzidine.
As shown in fig. 7, the double imprinted materials of paper base are since there are the trace holes of 3,3', 5,5'- tetramethyl benzidines, specifically
3,3', 5,5'- tetramethyl benzidines in property adsorbent solution, scraps of paper color obviously deepens, and paper base 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
Using after absorption colour developing as the sensitive signal of detection thyroglobulin.
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, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (8)
1. a kind of preparation method of the double imprinted materials of paper base of Selective recognition protein, which comprises the steps of:
(1) fixation of the graphene oxide-hemin compound on filter paper material surface
Graphene oxide-hemin compound is fixed on filter paper material surface with negative pressure by (1-1);
(2) preparation of the double imprinted materials of paper base
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 mixing well, 20 DEG C of -37 DEG C of incubation 10min-30min;The acrylamide, template protein and
The ratio between amount of substance of template 3,3', 5,5'- tetramethyl benzidine is (7428-37141): 1:(1098-5492);
(2-2) has the addition of graphene oxide-hemin compound filter paper material for surface that step (1-1) is prepared is immobilized
Into step (2-1) resulting solution, crosslinking agent N, N- methylene-bisacrylamide is added, and hydrogen peroxide and levulinic is added
Ketone is protected from light polymerization 12h-24h at 20 DEG C -37 DEG C;N,N methylene bis acrylamide, hydrogen peroxide and the acetylacetone,2,4-pentanedione
The ratio between the amount of substance be 1:(99-512): (1-12);
Filter paper material obtained by step (2-2) is washed away template protein and template 3,3', 5,5'- tetramethyl with eluent by (2-3)
After benzidine, the paper base 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 base of Selective recognition protein as described in claim 1, which is characterized in that
Function monomer acrylamide described in step (2-1) is for providing ethylene double bond, amino and amide group and template protein
With 3,3', 5,5'- tetramethyl benzidine polymerization reaction of template, template protein and 3,3', 5,5'- tetramethyl biphenyl of template are formed
The stereoeffect of amine.
3. the preparation method of the double imprinted materials of the paper base 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-bisacrylamide
Under, it is cross-linked to form polymer.
4. the preparation method of the double imprinted materials of the paper base of Selective recognition protein as described in claim 1, which is characterized in that
Graphene oxide described in step (1-1)-hemin compound has peroxidase sample activity, for being catalyzed 3,3', 5,5'-
Tetramethyl benzidine colour developing is as detection signal.
5. a kind of double imprinted materials of paper base of Selective recognition protein, which is characterized in that by either one legal system of claim 1-4
It is standby to obtain.
6. the application of the double imprinted materials of the paper base of Selective recognition protein as claimed in claim 5, which is characterized in that the paper
The double imprinted materials of base are for the Selective recognition of protein or the quantitative detection of protein.
7. application as claimed in claim 6, which is characterized in that the double imprinted materials of the paper base Selective recognition protein it
Afterwards, 3,3', 5,5'- tetramethyl benzidines and hydrogen peroxide are added into reaction system, 3,3', 5,5'- tetramethyl benzidines are competing
Strive combine mackle mark remain site, graphene oxide-hemin peroxidase sample active catalytic hydrogen peroxide be released from by
Base, 3,3', 5,5'- tetramethyl benzidine combination free radicals are simultaneously presented blue, calculate protein according to the gray value of color
Concentration.
8. application as claimed in claims 6 or 7, which is characterized in that the double imprinted materials of the paper base are used for thyroglobulin matter
Selective recognition or thyroglobulin matter quantitative detection.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711275084.XA CN108084337B (en) | 2017-12-06 | 2017-12-06 | The double imprinted materials of the paper base of Selective recognition protein and preparation method and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711275084.XA CN108084337B (en) | 2017-12-06 | 2017-12-06 | The double imprinted materials of the paper base of Selective recognition protein and preparation method and application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108084337A CN108084337A (en) | 2018-05-29 |
CN108084337B true CN108084337B (en) | 2019-06-07 |
Family
ID=62174435
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201711275084.XA Active CN108084337B (en) | 2017-12-06 | 2017-12-06 | The double imprinted materials of the paper base of Selective recognition protein and preparation method and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108084337B (en) |
Families Citing this family (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 |
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 |
Also Published As
Publication number | Publication date |
---|---|
CN108084337A (en) | 2018-05-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Luo et al. | Molecular imprinting resonance light scattering nanoprobes based on pH-responsive metal-organic framework for determination of hepatitis A virus | |
Al-Kindy et al. | Molecularly imprinted polymers and optical sensing applications | |
Tarannum et al. | Molecularly imprinted polymers as receptors for assays of antibiotics | |
CN107677656B (en) | A kind of ratio fluorescent nano probe and its application | |
JP5661848B2 (en) | Western blot immunodetection method | |
CN102268119B (en) | Preparation method of molecular imprinted polymer for detecting lung cancer tumor markers | |
CN104403051B (en) | A kind of fluorescence selects to identify the molecular engram material preparation method of Cyhalothrin | |
Zhang et al. | Determination of nereistoxin-related insecticide via quantum-dots-doped covalent organic frameworks in a molecularly imprinted network | |
Lu et al. | Rapid isolation and determination of bisphenol A in complicated matrices by magnetic molecularly imprinted electrochemical sensing | |
CN110702647A (en) | Construction and application of novel fluorescent imprinting sensor based on magnetic Metal Organic Framework (MOF) | |
CN108084337B (en) | The double imprinted materials of the paper base of Selective recognition protein and preparation method and application | |
Mamipour et al. | Molecularly imprinted polymer grafted on paper and flat sheet for selective sensing and diagnosis: a review | |
CN106568748A (en) | Method for detecting microcystin LR based on fluorescence resonance energy transfer of shell-core type up-conversion material and molybdenum disulfide | |
Fan et al. | Molecularly imprinted polymer coated Mn-doped ZnS quantum dots embedded in a metal–organic framework as a probe for selective room temperature phosphorescence detection of chlorpyrifos | |
Jiang et al. | A recyclable colorimetric sensor made of waste cotton fabric for the detection of copper ions | |
Mao et al. | Luminescent europium (III)-organic framework for visual and on-site detection of hydrogen peroxide via a tablet computer | |
US11220569B2 (en) | Porous material and methods related thereto | |
CN111534299A (en) | GOQDs @ PDA-ir-MIP molecularly imprinted fluorescence sensor and preparation method and application thereof | |
CN112979893B (en) | Preparation of magnetic fluorescent material @ molecularly imprinted particle and method for preparing composite membrane by using same | |
CN101672847A (en) | Preparation method of protein chip glass carrier | |
Zhao et al. | A visual chiroptical system with chiral assembly graphene quantum dots for D-phenylalanine detection | |
CN108459002A (en) | A kind of preparation method of magnetic blotting sensor and application | |
CN107003306A (en) | Porous membrane, its method and the purposes being grafted with polymer | |
CN112322280A (en) | Preparation method of piperazine functionalized carbon quantum dots and application of piperazine functionalized carbon quantum dots in oxytetracycline detection | |
Varyambath et al. | Hyper-cross-linked polypyrene spheres functionalized with 3-aminophenylboronic acid for the electrochemical detection of diols |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |