CN109232813A - A kind of preparation method of HRP trace hydrogel - Google Patents

A kind of preparation method of HRP trace hydrogel Download PDF

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CN109232813A
CN109232813A CN201811107118.9A CN201811107118A CN109232813A CN 109232813 A CN109232813 A CN 109232813A CN 201811107118 A CN201811107118 A CN 201811107118A CN 109232813 A CN109232813 A CN 109232813A
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CN109232813B (en
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谭娟娟
李艳霞
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Minjiang University
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    • 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
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/0052—Preparation of gels
    • B01J13/0065—Preparation of gels containing an organic phase
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • 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
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28047—Gels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
    • 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

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Abstract

The invention discloses a kind of preparation methods of HRP trace hydrogel, use acrylamide (AAm) for function monomer, methylene diacrylamide (MBAA) is crosslinking agent, ammonium persulfate (APS) is catalyst, tetramethylethylenediamine (TEMED) is initiator, horseradish peroxidase (HRP) is template molecule, the HRP trace hydrogel that in-situ polymerization is made with a thickness of 1mm according to a certain ratio, wash away template molecule, sample solution is directly added drop-wise to gel surface, through adsorbing, wash away non-binding molecule, using 3, 3', 5, 5'- tetramethyl benzidine (TMB)-H2O2System colour developing, generated color directly can be detected visually.Preparation method of the invention is simple, at low cost, and the HRP trace hydrogel of preparation is easy to detect, can repeatedly use, and can identify HRP in blood serum sample with high selectivity, be effectively removed matrix interference, has very big application value and market prospects.

Description

A kind of preparation method of HRP trace hydrogel
Technical field
The invention belongs to analytical chemistry and protein identification sensor technical field, and in particular to a kind of HRP trace water-setting The preparation method of glue.
Background technique
Molecular imprinting technology is Molecular Recognization present in simulation nature, the polymerization of preparation package template molecule The artificial receptors of object reticular structure, it is be related to the subjects such as polymer chemistry, biochemistry, materials chemistry, analytical chemistry one Advanced technology of the kind for isolating and purifying.Prepared polymer is known as molecularly imprinted polymer (molecular imprinted Polymer, MIP).Molecularly imprinted polymer is a kind of novel high polymer biomimetic material with stronger molecule distinguishability.Base There is the dual of the corrosion resistance of identification selection similar with natural antibody and high molecular material in molecularly imprinted polymer Advantage, thus just gradually it is being applied to the various fields such as analytical chemistry, bioengineering, clinical medicine, environmental monitoring, food industry, It is hopeful gradually substituting unstable conventional biosensor in the future.
Molecular engram material is the polymer that a kind of hole size, shape and functional group and template molecule match, right Template molecule has specific recognition capability, can be used for the separation, enrichment, qualitative and quantitative analysis of target molecule, it advanced Place is that its precordainment, specific recognition, extensive practicability.Protein imprinted material has to pH value of solution and environment temperature Degree etc. tolerances it is strong, it is at low cost, stability is high the advantages that, the pretreatment of sample, separation and concentration, in terms of take Obtained certain achievement, but with small molecular phase ratio, Western blotting technique is still in the stage of fumbling, especially in the kind of template molecule Preparation, selectivity in class, aqueous phase system etc. still suffer from many difficult and challenge.
Replace biomimetic sensor of the biomolecule as artificial antibody based on " molecularly imprinted polymer (MIP) ", in 21 generation It records and is increasingly becoming the research hotspot of sensor field.The common analysis method of molecular imprinting technology generally includes: ultraviolet-visible Spectrum, electrochemistry, surface plasma resonance, chromatography, fluorescence etc..However these methods need special instrument and equipment or cumbersome behaviour Make step, it is therefore desirable to establish more direct, efficient, simple detection method to improve the application value of molecular imprinting technology.
In numerous analysis methods, due to method for visualizing have many advantages, such as low cost, quickly, it is simple, can open hole detection And by Many researchers extensive concern.But research report is few in molecular imprinting technology, this may be because analysis method needs It is consistent with imprinted material, it is just achieved signal conversion, therefore need to consider imprinted material in the design of imprinted material The color of itself, and do not destroy the coloration method of imprinted material, the present invention designs a kind of HRP trace colorimetric hydrogel accordingly Preparation method, prepares HRP trace acrylamide gel using a step situ aggregation method, detects target using direct development process Object can be used for the direct analysis of object, and the gel process for preparing is simple, low in cost, reusable, colorimetric method, no Special installation is needed, directly goes out analysis as a result, can be used for the portable inspectiont of object, realizes quantitative and semi-quantitative analysis.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of HRP trace hydrogel, identification and inspection for target protein It surveys.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of preparation method of HRP trace hydrogel, comprising the following steps:
(1) prepared by A liquid: acrylamide (AAm) and methylene diacrylamide (MBAA) being completely dissolved in ultrapure water, are settled to 50 mL seal stored refrigerated, obtained A liquid.
(2) prepared by HRP trace hydrogel solution: HRP protein dissolution in A liquid and being stirred evenly, then successively addition is super Pure water, 10wt% ammonium persulfate (APS) and 4 μ L tetramethylethylenediamines (TEMED) are uniformly mixed, and obtain HRP trace water-setting peptization Liquid.
(3) preparation of HRP trace polyacrylamide hydrogel: the resulting HRP trace hydrogel solution of step (2) is placed in It in gel making device, is taken out after aggregating into gel, first with a small amount of multiple elution its template HRP molecule of eluent, then is washed, made Obtain HRP trace hydrogel.
The mass concentration of acrylamide is 10~40% in step (1), the mass ratio of acrylamide and methylene diacrylamide For 15:(0.2~0.8).
The dosage of HRP albumen is 2.0~10 mg, the volume of A liquid in step (2) are as follows: 2.0~10 mL add ultrapure water Volume are as follows: the volume of 4.0~8.0 mL, 10wt% ammonium persulfate are as follows: 20~200 μ L.
Gel making device used in step (3) is the small-sized glue-pouring device of bio-red, with a thickness of 1mm, when gel polymerisation Between be 10~60 min, eluent used be 10 wt% SDS, 1mol/L NaCl, 10 vol.% acetic acid, 10wt%SDS with 10 vol.% acetic acid are in equal volume than one of mixed solution.
A kind of application of the HRP trace hydrogel of above method preparation in Visual retrieval HRP, colorimetric determination: will HRP trace hydrogel obtained above is cut into adhesive tape, is fixed on white bottom plate, and room temperature is dried, and white bottom plate carries out point sample Label, the sample solution of the series of concentrations containing HRP are added drop-wise to corresponding position, stand, and sufficiently adsorb, and washed away with ultrapure water and be not bonded Molecule, room temperature are dried, and TMB-H is added dropwise2O2Solution records color change.
Wherein white bottom plate dry degree be it is half-dried, be just advisable without free water, the volume of sample solution is 5~20 μ L, standing adsorption time are 10~60 min, the concentration range of HRP are as follows: 0~2.0 mg/mL；
TMB-H2O2The concentration of TMB is 20~80 mmol/L, TMB and H in solution2O2Molar ratio are as follows: 5:(4~10), TMB- H2O2Mixed solution needs Fresh, and dropwise addition volume is 5~20 μ L, and developing time is 1~10 min, and developing time is too long, back Scape color burn, influences colorimetric.
The present invention uses acrylamide (AAm) for function monomer, and methylene diacrylamide (MBAA) is crosslinking agent, persulfuric acid Ammonium (APS) is catalyst, and tetramethylethylenediamine (TEMED) is initiator, and horseradish peroxidase (HRP) is template molecule, is pressed The HRP trace hydrogel with a thickness of 1mm is made according to certain proportion in-situ polymerization, washes away template molecule, sample solution is directly added dropwise It to gel surface, is adsorbed, washes away non-binding molecule, using 3,3', 5,5'- tetramethyl benzidine (TMB)-H2O2System colour developing, Generated color directly can be detected visually.
Remarkable advantage of the invention is:
1) the HRP trace hydrogel used in the present invention uses a step in-situ polymerization, and preparation is simple, low in cost, repeats It uses, it is easy to carry, it can be used for real-time online sample monitoring.
2) the hydrogel stable in physicochemical property prepared by has very big flexibility, non-breakable, convenient for saving, thoroughly Bright sexual compatibility monitors the variation of color in naked eyes.
3) the HRP trace hydrogel prepared by contains the imprinted cavity to match with HRP spatial configuration of molecules, has to HRP There is specific recognition capability.
4) it can be used for manufacturing experimently HRP quick detection kit by the gel strips that HRP trace hydrogel is cut, detection process is most It is completed in fast 10 min, for the blood serum sample of matrix complexity, can directly detect, not need preprocessing process.
Detailed description of the invention
The preparation process and colour developing schematic diagram of mechanism of Fig. 1 HRP trace hydrogel；
Fig. 2 HRP trace water-setting adhesive tape adsorbs the ratio chromatic graph of various concentration HRP standard solution, and HRP concentration is from left to right successively are as follows: 0,0.001mg/mL,0.01 mg/mL,0.05 mg/mL,0.1 mg/mL,0.5 mg/mL；
The ratio chromatic graph of Fig. 3 HRP non-trace water-setting adhesive tape absorption various concentration HRP standard solution, HRP concentration is from left to right successively Are as follows: 0,0.001mg/mL, 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL；
Fig. 4 HRP trace water-setting adhesive tape adsorbs the ratio chromatic graph of different albumen；
Fig. 5 HRP trace water-setting adhesive tape adsorbs the ratio chromatic graph of different blood serum samples, from left to right successively are as follows: pure water, 0.1mg/mL HRP, serum, the serum solution of the HRP containing 0.1mg/mL；
The non-trace water-setting adhesive tape of Fig. 6 HRP adsorbs the ratio chromatic graph of different blood serum samples, from left to right successively are as follows: pure water, 0.1mg/ ML HRP, serum, the serum solution of the HRP containing 0.1mg/mL.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, the present invention will be further described for following instance, but It is not used to limit the scope of the present invention.
Embodiment 1
Recognition effect of the HRP trace hydrogel to various concentration HRP:
(1) prepared by A liquid: 15 g AAm and 0.4 g MBAA being completely dissolved in ultrapure water, 50 mL is settled to, obtains A Liquid.
(2) prepared by HRP trace hydrogel solution: 5 mg HRP protein dissolutions are sufficiently stirred in 5 mL A liquid, then according to 4.9 mL ultrapure waters of secondary addition, 100 μ L 10 wt% APS and 4 μ L TEMED are simultaneously uniformly mixed, and obtain HRP trace hydrogel Solution.
(3) preparation of HRP trace hydrogel: above-mentioned HRP trace hydrogel solution pours into bio-rad glue-pouring device, It is taken out after aggregating into gel, first with a small amount of multiple elution its template HRP molecule of eluent, then is washed, HRP trace water is made Gel (MIPs), with a thickness of 1mm.
(4) colorimetric determination: trace hydrogel obtained above is cut into the adhesive tape that broadband is 1cm, is fixed on white On bottom plate, room temperature is dried, and white bottom plate carries out sampling point label, is respectively 0,0.001mg/mL, 0.01 by 10 μ L HRP concentration Mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL sample solution be added drop-wise to corresponding position, stand, sufficiently adsorb 30 Min, and non-binding molecule is washed away with ultrapure water, room temperature is dried.
(5) the TMB solution of 50 mmol/L of fresh configuration and the H of 80 mmol/L are taken respectively2O2Solution, the two is in will It being mixed in equal volume when colour developing, each absorption point adds 10 μ L developing solutions, and develop the color 10 min, and color developing effect figure is taken, sees Fig. 2, It can be seen that the aobvious color of institute is gradually deepened with the increase of HRP concentration, yellow is gradually become again to dark blue by light blue.
Embodiment 2
Recognition effect of the non-HRP trace hydrogel to various concentration HRP:
Step (2) in embodiment 1 is not added 5 mg HRP albumen, the non-trace of HRP is made with embodiment 1 in remaining condition and step Hydrogel (NIPs), the non-trace hydrogel of HRP can be obtained to the adsorption effect figure of various concentration HRP, as shown in Figure 3, it can be seen that With the increase of HRP concentration, shown color shows shallower color without significant change, in 0.5 region mg/mL of high concentration, Significant difference is shown with the color shown in embodiment 1, this shows the non-trace hydrogel of the HRP to HRP without specific adsorption.
Embodiment 3
Recognition capability of the HRP trace hydrogel to different albumen:
(1) HRP trace hydrogel prepared in embodiment 1 is cut into the adhesive tape that broadband is 1cm, is fixed on white bottom plate On, room temperature is dried, and white bottom plate carries out sampling point label, 10 μ L, 0.05 mg/mL difference albumen [HRP(horseradish peroxidase Enzyme), Try(trypsase), OVA(oralbumin), ConA(canavaline), BSA(bovine serum albumin), GOD(grape glycosyloxy Change enzyme)] sample solution be added drop-wise to corresponding position respectively, stand, sufficiently adsorb 30 min, and washed away with ultrapure water and not to be bonded point Son, room temperature are dried.
(2) the TMB solution of 50 mmol/L of fresh configuration and the H of 80 mmol/L are taken respectively2O2Solution mixes in equal volume, The two mixing when will develop the color, each absorption point add 10 μ L developing solutions, and develop the color 10 min, take color developing effect figure, see Fig. 4, as shown in figure 4, obviously to observe that HRP point of sample show apparent blue for naked eyes, and shown by remaining albumen point of sample Weak color has significant difference, shows that the HRP trace hydrogel of the preparation has good selectivity these types of albumen.
Embodiment 4
Application of the HRP trace hydrogel in blood serum sample:
(1) FBS pure water dilutes 20 times, and refrigerator saves backup (following to test the FBS solution that FBS used is 20 times of dilution).
(2) trace hydrogel prepared in embodiment 1 is cut into the adhesive tape that broadband is 1cm, is fixed on white bottom plate On, room temperature is dried, and white bottom plate carries out sampling point label, and by 10 μ L different solutions, (pure water, serum, contains 0.1mg/mL HRP The serum solution of 0.1mg/mL HRP) it is added drop-wise to corresponding position respectively, it stands, sufficiently adsorbs 30 min, and washed away with ultrapure water Non- binding molecule, room temperature are dried.
(3) chromogenic reaction is carried out according to step (5) in embodiment 1, takes color developing effect figure, see Fig. 5, as shown in figure 5, pure Without color change, background color is transparent, illustrates that HRP albumen is not detected in blood serum sample, works as serum solution for water and blood serum sample When middle addition 0.1mg/mL HRP, it can be seen that color burn illustrates that serum solution does not interfere the identification of HRP, further proves Prepared HRP trace hydrogel has apparent adsorption effect to target HRP albumen, shows stronger specific recognition energy Power.
Embodiment 5
Application of the non-trace hydrogel of HRP in blood serum sample:
HRP trace hydrogel in embodiment 4 is changed to the non-trace hydrogel of HRP, remaining condition and step with embodiment 4, is clapped Lower color developing effect figure, is shown in Fig. 6, as shown in fig. 6, this 4 sample spots illustrate the non-trace hydrogel of HRP to HRP without color change Without specific adsorption ability, this is because the trace that the non-trace hydrogel of HRP does not match with the space HRP and recognition site is empty Cave.
Embodiment 6
By HRP trace hydrogel prepared in embodiment 1, the HRP protein solution of 0.05mg/mL is adsorbed, by 1 step of embodiment Chromogenic reaction is carried out, color change is recorded, is eluted through 10% acetic acid, the rear HRP protein solution for repeating to adsorb 0.05 mg/mL, then Secondary carry out chromogenic reaction, photographs to record color change, and Reusability 5 times, the color no significant difference of display, it was demonstrated that the HRP Trace hydrogel has good reusability.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

Claims (9)

1. a kind of preparation method of HRP trace hydrogel, it is characterised in that: the preparation method comprises the following steps:
(1) prepared by A liquid: acrylamide and methylene diacrylamide being completely dissolved in ultrapure water, sealing is stored refrigerated, is made A liquid；
(2) prepared by HRP trace hydrogel solution: template HRP protein dissolution in A liquid and being stirred evenly, then successively addition is super Pure water, 10w% ammonium persulfate and tetramethylethylenediamine are uniformly mixed, and obtain HRP trace hydrogel solution；
(3) preparation of HRP trace polyacrylamide hydrogel: the resulting HRP trace hydrogel solution of step (2) is aggregated into solidifying After glue, its template of elution HRP protein molecular is first used, then washed, HRP trace hydrogel is made.
2. a kind of preparation method of HRP trace hydrogel according to claim 1, it is characterised in that: step (1) is resulting The mass concentration of acrylamide is 10~40% in A liquid, the mass ratio of acrylamide and methylene diacrylamide be 15:(0.2~ 0.8).
3. a kind of preparation method of HRP trace hydrogel according to claim 1, it is characterised in that: HRP in step (2) The dosage of albumen is 2.0~10 mg, the volume of A liquid are as follows: 2.0~10 mL add the volume of ultrapure water are as follows: 4.0~8.0 mL, The volume of 10w% ammonium persulfate are as follows: 20~200 μ L, the volume of tetramethylethylenediamine are 4 μ L.
4. a kind of preparation method of HRP trace hydrogel according to claim 1, it is characterised in that: gel in step (3) Polymerization time is 10~60 min.
5. a kind of preparation method of HRP trace hydrogel according to claim 1, it is characterised in that: used in step (3) Eluent is 10 wt% SDS, 1mol/L NaCl, 10 vol.% acetic acid, 10 wt%SDS compare in equal volume with 10 vol.% acetic acid One of mixed liquor.
6. a kind of HRP trace hydrogel prepared by claim 1-5 any one the method.
7. a kind of application of HRP trace hydrogel as claimed in claim 6 in Visual retrieval HRP, it is characterised in that: will HRP trace hydrogel obtained is cut into adhesive tape, is fixed on white bottom plate, and room temperature is dried, and sample solution containing HRP is added drop-wise to glue It on item, stands, sufficiently adsorbs, and wash away non-binding molecule with ultrapure water, room temperature is dried, and TMB-H is added dropwise2O2Solution observes color Variation.
8. application of the HRP trace hydrogel according to claim 7 in Visual retrieval HRP, it is characterised in that: sample The volume of solution is 5~20 μ L, and the standing adsorption time is 10~60 min.
9. application of the HRP trace hydrogel according to claim 7 in Visual retrieval HRP, it is characterised in that: described TMB-H2O2The concentration of TMB is 20~80 mmol/L, TMB and H in solution2O2Molar ratio are as follows: 5:(4~10), TMB-H2O2It is mixed Closing solution needs Fresh, and dropwise addition volume is 5~20 μ L, and developing time is 1~10 min.
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CN109970909A (en) * 2019-04-03 2019-07-05 中国石油大学(北京) The method for preparing Janus nanometer sheet using reusable cross-linked polymer microsphere template
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