CN114354554B - Preparation method and application of detection platform for full-time line biomarker - Google Patents
Preparation method and application of detection platform for full-time line biomarker Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 38
- 239000000090 biomarker Substances 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000000499 gel Substances 0.000 claims abstract description 68
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 57
- 239000000017 hydrogel Substances 0.000 claims abstract description 41
- 239000011521 glass Substances 0.000 claims abstract description 39
- 238000000059 patterning Methods 0.000 claims abstract description 24
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- PIZHFBODNLEQBL-UHFFFAOYSA-N 2,2-diethoxy-1-phenylethanone Chemical compound CCOC(OCC)C(=O)C1=CC=CC=C1 PIZHFBODNLEQBL-UHFFFAOYSA-N 0.000 claims description 21
- 230000002209 hydrophobic effect Effects 0.000 claims description 12
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- OMIGHNLMNHATMP-UHFFFAOYSA-N 2-hydroxyethyl prop-2-enoate Chemical compound OCCOC(=O)C=C OMIGHNLMNHATMP-UHFFFAOYSA-N 0.000 claims description 11
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 11
- GMSCBRSQMRDRCD-UHFFFAOYSA-N dodecyl 2-methylprop-2-enoate Chemical compound CCCCCCCCCCCCOC(=O)C(C)=C GMSCBRSQMRDRCD-UHFFFAOYSA-N 0.000 claims description 11
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 11
- HMZGPNHSPWNGEP-UHFFFAOYSA-N octadecyl 2-methylprop-2-enoate Chemical compound CCCCCCCCCCCCCCCCCCOC(=O)C(C)=C HMZGPNHSPWNGEP-UHFFFAOYSA-N 0.000 claims description 11
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- 241000196324 Embryophyta Species 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 108010081589 Becaplermin Proteins 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 229930003270 Vitamin B Natural products 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 235000019156 vitamin B Nutrition 0.000 description 2
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- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 1
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 description 1
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- 230000002745 absorbent Effects 0.000 description 1
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- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
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- XCOBTUNSZUJCDH-UHFFFAOYSA-B lithium magnesium sodium silicate Chemical compound [Li+].[Li+].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[OH-].[Na+].[Na+].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].[Mg+2].O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3.O1[Si](O2)([O-])O[Si]3([O-])O[Si]1([O-])O[Si]2([O-])O3 XCOBTUNSZUJCDH-UHFFFAOYSA-B 0.000 description 1
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- 238000006116 polymerization reaction Methods 0.000 description 1
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- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 description 1
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- 238000004393 prognosis Methods 0.000 description 1
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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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
- C08F220/10—Esters
- C08F220/12—Esters of monohydric alcohols or phenols
- C08F220/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
- C08F220/18—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms with acrylic or methacrylic acids
- C08F220/1812—C12-(meth)acrylate, e.g. lauryl (meth)acrylate
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- 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
- C08F220/10—Esters
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- C08F220/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
- C08F220/18—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms with acrylic or methacrylic acids
- C08F220/1818—C13or longer chain (meth)acrylate, e.g. stearyl (meth)acrylate
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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Abstract
The invention relates to the field of material preparation, detection and analysis, in particular to a preparation method and application of a detection platform for full-time line biomarkers, wherein the method comprises the following steps: preparing a super-hydrophilic-hydrophobic glass surface patterning template; preparing an oil-in-water emulsion; and synthesizing a surface patterning oil-water gel droplet array to obtain the detection platform for the full-time line biomarker. The beneficial effects of the invention are as follows: the detection platform takes the surface patterning oil-water gel as a substrate, has simple preparation process and good stability, and can prolong the application time of the substrate to 120min compared with the traditional detection method. Based on the principle that the surface patterning oil hydrogel controls water evaporation and water is supplied upwards, long-time moisture preservation of liquid drops can be realized.
Description
Technical Field
The invention relates to the field of material preparation, detection and analysis, in particular to a preparation method and application of a detection platform for full-time line biomarkers.
Background
Accurate and sensitive identification of biomarkers is essential from various fields of prognosis, disease diagnosis, basic bioscience, etc. However, conventional non-absorbent chips suffer from drawbacks in terms of droplet moisturization, resulting in incomplete reactions, and detection interruptions. However, the existing moisturizing strategy has no universality and can influence the detection process. Therefore, the preparation of the graphic chip with good water retention performance has important significance.
The plant leaves include a waxy coating and pores that establish a moisture-retaining steady-state structure. The stomata of the plants are used as communication channels between the plants and the external volatile environment, and play an important role in the effective utilization of moisture. The wax layer can prevent excessive water loss caused by excessive evaporation of water. Here, we report a biosensor based on surface patterned oil-water gel material, which has good water retention ability, and can be used for full time line biosensing, inspired by the water retention structure of the blade.
Disclosure of Invention
The invention discloses a preparation method and application of a detection platform for full-time line biomarkers, which are used for solving any one of the above and other potential problems in the prior art.
In order to solve the problems, the technical scheme of the invention is as follows: the preparation method of the detection platform for the full-time line biomarker specifically comprises the following steps:
s1) preparing a super-hydrophilic-hydrophobic glass surface patterning template;
s2) preparing an oil-in-water emulsion;
s3) solidifying the oil-in-water emulsion obtained in the step S2) on the surface patterning template obtained in the step S1) to synthesize a surface patterning oil-water gel droplet array, and thus obtaining the detection platform for the full-time line biomarker.
Further, the specific steps of S1) are as follows:
s1.1) selecting a template, carrying out pretreatment, then modifying the surface of the template to obtain the template with a hydrophobic layer formed by self-assembly of the surface,
s1.2) clamping the mask plate on the surface of the template obtained in the S1.1) through a pattern die, and placing the template in a plasma cleaning machine for 3-8min to obtain the super-hydrophilic-hydrophobic glass surface patterning template.
Further, the modification process in S1.1) is as follows: in a vacuum environment, the temperature is 55-65 ℃, the modification time is 5.5-6.5 hours, and the modifier is: 1H, 2H-perfluorodecyl trimethoxysilane.
Further, the specific process of S2) is as follows:
s2.1) preparing hydrogel prepolymer; the method comprises the steps of carrying out a first treatment on the surface of the Weighing deionized water, hydroxyethyl acrylate, nanoclay and 2, 2-diethoxyacetophenone, mixing and stirring for 1-1.5h to obtain an oil gel prepolymer;
s2.2) preparing an oleogel prepolymer: respectively weighing lauryl methacrylate, stearyl methacrylate, ethylene glycol dimethacrylate and 2, 2-diethoxyacetophenone, mixing and stirring for 1-1.5h to obtain an oil gel prepolymer;
s2.3) mixing the oil gel prepolymer obtained in the step S2.2) and the hydrogel prepolymer obtained in the step S2.1) according to a mass ratio of 1:1.24, adding the mixture into a cell crusher, and treating the mixture for 1 to 3 minutes under the power of 580W to form the oil-in-water emulsion with oil drops with the particle size of 2 to 10 mu m.
Further, in S2.1): the hydrogel prepolymerization solution comprises the following components in percentage by mass: deionized water: 81-86 wt% of hydroxyethyl acrylate: 10-15 wt% of nano clay: 3 to 4 weight percent, 2-diethoxyacetophenone: 0.03wt% to 0.05wt%.
Further, the oil gel prepolymer in S2.2) comprises the following components in percentage by mass: 45-50 wt% of lauryl methacrylate and octadecyl methacrylate: 45-50 wt% of ethylene glycol dimethacrylate: 3-4wt% of 2, 2-diethoxyacetophenone: 0.7wt% to 1wt%.
Further, the specific steps of S3) are as follows:
s3.1) pouring the oil-in-water emulsion obtained in the step S2) into the super-hydrophilic-hydrophobic glass surface patterning template obtained in the step S1);
s3.2) at a wavelength of 365nm, an optical power of 12mW cm -2 Irradiating with ultraviolet for 12-18min to obtain gel after polymerization.
S3.3) soaking the gel in water, removing unreacted monomers and impurities, and fully swelling to obtain a detection platform for the full-time line biomarker.
The detection platform for the full-time line biomarker prepared by the method can be applied to the field of biomarker detection in various time periods.
The detection platform for the full-time line biomarker is prepared by adopting the preparation method.
The invention also provides a detection method of the detection platform adopting the full-time line biomarker, which specifically comprises the following steps:
firstly, mixing a liquid to be tested and a mixed solution into a1 XPBS buffer solution to obtain a liquid drop containing a certain concentration of reaction liquid
The reaction solution was added to the hydrophilic region of OH-gel, water was added to a humidity of 20-60, fluorescence signal measurements were recorded under a laser confocal scanning microscope every 30 minutes, and the droplets were placed in the hydrogel region to react. And (5) photographing and recording every 20min to obtain a relationship graph of time and signals of the biomarker detection.
The beneficial effects of the invention are as follows: by adopting the technical scheme, the surface patterning oil-water gel obtained by the wetting transfer strategy has an accurately controllable hydrophobic oil gel area and a hydrophilic hydrogel area. The oil gel region on the surface of the template is hydrophobic, and can play a role in reducing the evaporation of water in the gel, the hydrogel region is hydrophilic, and the water is evaporated from the porous hydrogel region. This property of directional evaporation of moisture can provide a high humidity environment for the droplets that are immobilized on the hydrophilic areas. Meanwhile, the support structure of the gel is an internal uniform oil-water gel structure, and the continuous hydrogel phase is a three-dimensional network structure filled with water to provide a moisture source for moisture preservation. The dispersed oil gel phase and the continuous hydrogel phase form a capillary channel together, the capillary force promotes water to be transported upwards to the surface hydrogel area so as to meet the long-time detection requirement, and the moisturizing strategy can create a high-humidity environment for reaction liquid drops so as to realize long-time moisturizing, and has no influence on biological reactions. The long-time moisture preservation can be realized through the characteristic that the gel is internally provided with self-water and can be connected with an external water source. As an open droplet reaction platform, the biological sensing array can be applied to biomarker detection in various time periods and combined with various detection means, and compared with a traditional detection method, the substrate application time can be prolonged to 120min.
Description of the drawings:
FIG. 1 is a schematic illustration of a surface patterned oil hydrogel of the present invention.
FIG. 2 is a graph of time versus signal for biomarker detection compared to a common super-invasive chip (SHI/SHO) provided by the present invention.
The specific embodiment is as follows:
the technical scheme of the invention is further described below with reference to the attached drawings and specific embodiments.
As shown in fig. 1, the preparation method of the detection platform for the full-time line biomarker provided by the invention specifically comprises the following steps:
s1) preparing a super-hydrophilic-hydrophobic glass surface patterning template;
s2) preparing an oil-in-water emulsion;
s3) synthesizing a surface patterning oil-water gel drop array to obtain the detection platform for the full-time line biomarker, wherein the platform has a surface partition structure of hydrogel and oil gel and a uniform support structure of the oil-water gel.
The specific steps of S1) are as follows:
s1.1) selecting a template, carrying out pretreatment, then modifying the surface of the template to obtain the template with a hydrophobic layer formed by self-assembly of the surface,
s1.2) clamping the mask plate on the surface of the template obtained in the S1) through a pattern die, and placing the template in a plasma cleaning machine for 3-8min to obtain the super-hydrophilic-hydrophobic glass surface patterning template.
The modification process in S1.1) is as follows: in a vacuum environment, the temperature is 55-65 ℃, the modification time is 5.5-6.5 hours, and the modifier is: 1H, 2H-perfluorodecyl trimethoxysilane.
The specific process of S2) is as follows:
s2.1) preparing hydrogel prepolymer; the method comprises the steps of carrying out a first treatment on the surface of the Weighing deionized water, hydroxyethyl acrylate, nanoclay and 2, 2-diethoxyacetophenone, mixing and stirring for 1-1.5h to obtain an oil gel prepolymer;
s2.2) preparing an oleogel prepolymer: respectively weighing lauryl methacrylate, stearyl methacrylate, ethylene glycol dimethacrylate and 2, 2-diethoxyacetophenone, mixing and stirring for 1-1.5h to obtain an oil gel prepolymer;
s2.3) mixing the oil gel prepolymer obtained in the step S2.2) and the hydrogel prepolymer obtained in the step S2.1) according to a mass ratio of 1:1.24, adding into a cell crusher, and treating for 1-3min under the power of 580W to form the oil-in-water emulsion with the particle size of 2-10 mu m.
The following S2.1): the hydrogel prepolymerization solution comprises the following components in percentage by mass: deionized water: 81-86 wt% of hydroxyethyl acrylate: 10-15 wt% of nano clay: 3-4wt% of 2, 2-diethoxyacetophenone: 0.03wt% to 0.05wt%.
Further, the oil gel prepolymer in S2.2) comprises the following components in percentage by mass: 45-5-0 wt% of lauryl methacrylate and octadecyl methacrylate: 45-50 wt% of ethylene glycol dimethacrylate: 3-4wt% of 2, 2-diethoxyacetophenone: 0.7wt% to 1wt%.
The nanoclay brand Laponite XLS, ingredient [ Mg 5.34 Li 0.66 Si 8 O 20 (OH) 4 ]Na 0.66 20-30nm diameter and 1nm thickness, mw= 762.24.
The specific steps of the S3) are as follows:
s3.1) pouring the oil-in-water emulsion obtained in the step S2) into the super-hydrophilic-hydrophobic glass surface patterning template obtained in the step S1);
s3.2) at a wavelength of 365nm, an optical power of 12mW cm -2 Irradiating with ultraviolet for 12-18min to obtain polymerized gel,
s3.3) soaking the gel in water, removing unreacted monomers and impurities, and fully swelling to obtain a detection platform for the full-time line biomarker;
the surface patterned hydrogel has precisely controllable hydrophobic hydrogel regions and hydrophilic hydrogel regions. The oil gel region on the surface of the template is hydrophobic, and can play a role in reducing the evaporation of water in the gel, the hydrogel region is hydrophilic, and the water is evaporated from the porous hydrogel region. This property of directional evaporation of moisture can provide a high humidity environment for the droplets that are immobilized on the hydrophilic areas. Meanwhile, the support structure of the gel is an internal uniform oil-water gel structure, and the continuous hydrogel phase is a three-dimensional network structure filled with water to provide a moisture source for moisture preservation. The dispersed oil gel phase and the continuous hydrogel phase form a capillary channel together, the capillary force promotes water to be transported upwards to the surface hydrogel area so as to meet the long-time detection requirement, and the moisturizing strategy can create a high-humidity environment for reaction liquid drops so as to realize long-time moisturizing, and has no influence on biological reactions. The long-time moisture preservation can be realized through the characteristic that the gel is internally provided with self-water and can be connected with an external water source.
The detection platform for the full-time line biomarker prepared by the method can be applied to the field of biomarker detection in various time periods.
Example 1
10cm of glass is selected as a template, the surface of the template is cleaned, 1H, 2H-perfluoro decyl trimethoxy silane is modified for 6h in a vacuum environment of 60 ℃,1H, 2H-perfluoro decyl trimethoxy silane is self-assembled on the surface of the glass to form a hydrophobic layer, then a mask plate with 2mm diameter micropores is clamped on the surface of the glass through a dovetail clamp, the glass is placed in a plasma environment for 5min, and a glass sheet is taken out to obtain the super-hydrophilic-hydrophobic glass surface patterning template. Preparing an oil-water gel prepolymerization solution, wherein the components of the hydrogel prepolymerization solution are as follows: 60g of water, 8.55g of hydroxyethyl acrylate, 2.4g of nanoclay, 30mg of 2, 2-diethoxyacetophenone are stirred for 4h. Preparing an oil gel prepolymerization liquid: 38.1g of lauryl methacrylate, 38.1g of stearyl methacrylate, 3g of ethylene glycol dimethacrylate, 60mg of 2, 2-diethoxyacetophenone are stirred for 1h. The oleogel hydrogels were mixed in a mass ratio of 1:1.24 and treated under a cell breaker for 1min. A uniform oil-in-water emulsion is formed. Preparing a groove mold on the glass patterned surface obtained in the step (1), pouring the oil-water gel prepolymer into the mold, and irradiating for 15min under 365nm ultraviolet. The polymerized gel was removed. The gel is soaked in water to remove unreacted monomers and impurities and fully swelled.
Configuration of Fe 3+ Is a reaction liquid drop of (1): 10nM FeCl 3 The solution was added dropwise to a 60mM potassium thiocyanate solution. And 10. Mu.l of the droplets were placed in the hydrogel area for reaction. Record was photographed every 1min, and the results (shown in fig. 2).
Example 2
Selecting 10cm multiplied by 10cm glass as a template, cleaning the surface of the template, modifying the template in a vacuum environment of 1H, 2H-perfluoro decyl trimethoxy silane at 60 ℃ for 6 hours, enabling the 1H, 2H-perfluoro decyl trimethoxy silane to self-assemble into a hydrophobic layer on the surface of the glass, clamping a mask plate with micropores with the diameter of 2mm on the surface of the glass through a dovetail clamp, placing the mask plate in a plasma environment for 5 minutes, and taking out a glass sheet to obtain the super-hydrophilic-hydrophobic glass surface patterning template. Preparing an oil-water gel prepolymerization solution, wherein the components of the hydrogel prepolymerization solution are as follows: 60g of water, 8.55g of hydroxyethyl acrylate, 2.4g of nanoclay, 30mg of 2, 2-diethoxyacetophenone are stirred for 4h. Preparing an oil gel prepolymerization liquid: 38.1g of lauryl methacrylate, 38.1g of stearyl methacrylate, 3g of ethylene glycol dimethacrylate, 60mg of 2, 2-diethoxyacetophenone are stirred for 1h. The oleogel hydrogels were mixed in a mass ratio of 1:1.24 and treated under a cell breaker for 1min. A uniform oil-in-water emulsion is formed. Preparing a groove mold on the glass patterned surface obtained in the step (1), pouring the oil-water gel prepolymer into the mold, and irradiating for 15min under 365nm ultraviolet. The polymerized gel was removed. The gel is soaked in water to remove unreacted monomers and impurities and fully swelled.
Preparing reaction liquid drops of vitamin B: a10 mM vitamin B solution was added dropwise to a 60mM diazotized p-aminobenzenesulfonic acid solution. And 10. Mu.l of the droplets were placed in the hydrogel area for reaction. Record was photographed every 3min and the results (as shown in fig. 2).
Example 3
Selecting 10cm multiplied by 10cm glass as a template, cleaning the surface of the template, modifying the template in a vacuum environment of 1H, 2H-perfluoro decyl trimethoxy silane at 60 ℃ for 6 hours, enabling the 1H, 2H-perfluoro decyl trimethoxy silane to self-assemble into a hydrophobic layer on the surface of the glass, clamping a mask plate with micropores with the diameter of 2mm on the surface of the glass through a dovetail clamp, placing the mask plate in a plasma environment for 5 minutes, and taking out a glass sheet to obtain the super-hydrophilic-hydrophobic glass surface patterning template. Preparing an oil-water gel prepolymerization solution, wherein the composition table of the hydrogel prepolymerization solution is as follows: 60g of water, 8.55g of hydroxyethyl acrylate, 2.4g of nanoclay, 30mg of 2, 2-diethoxyacetophenone are stirred homogeneously for 4h. Preparing an oil gel prepolymerization liquid: 38.1g of lauryl methacrylate, 38.1g of stearyl methacrylate, 3g of ethylene glycol dimethacrylate, 60mg of 2, 2-diethoxyacetophenone are stirred homogeneously for 1h. The oleogel hydrogels were mixed in a mass ratio of 1:1.24 and treated under a cell breaker for 1min. A uniform oil-in-water emulsion is formed. Preparing a groove mold on the glass patterned surface obtained in the step (1), pouring the oil-water gel prepolymer into the mold, and irradiating for 15min under 365nm ultraviolet. The polymerized gel was removed. The gel is soaked in water to remove unreacted monomers and impurities and fully swelled.
Preparing reaction liquid drops of vitamin C: a10 mM vitamin C solution was added dropwise to a 60mM aqueous ammonium molybdate solution. And 10. Mu.l of the droplets were placed in the hydrogel area for reaction. Record was photographed every 5min and the results (as shown in fig. 2).
Example 4
Selecting 10cm multiplied by 10cm glass as a template, cleaning the surface of the template, modifying the template in a vacuum environment of 1H, 2H-perfluoro decyl trimethoxy silane at 60 ℃ for 6 hours, enabling the 1H, 2H-perfluoro decyl trimethoxy silane to self-assemble into a hydrophobic layer on the surface of the glass, clamping a mask plate with micropores with the diameter of 2mm on the surface of the glass through a dovetail clamp, placing the mask plate in a plasma environment for 5 minutes, and taking out a glass sheet to obtain the super-hydrophilic-hydrophobic glass surface patterning template. Preparing an oil-water gel prepolymerization solution, wherein the composition table of the hydrogel prepolymerization solution is as follows: 60g of water, 8.55g of hydroxyethyl acrylate, 2.4g of nanoclay, 30mg of 2, 2-diethoxyacetophenone are stirred homogeneously for 4h. Preparing an oil gel prepolymerization liquid: 38.1g of lauryl methacrylate, 38.1g of stearyl methacrylate, 3g of ethylene glycol dimethacrylate, 60mg of 2, 2-diethoxyacetophenone are stirred homogeneously for 1h. The oleogel hydrogels were mixed in a mass ratio of 1:1.24 and treated under a cell breaker for 1min. A uniform oil-in-water emulsion is formed. Preparing a groove mold on the glass patterned surface obtained in the step (1), pouring the oil-water gel prepolymer into the mold, and irradiating for 15min under 365nm ultraviolet. The polymerized gel was removed. The gel is soaked in water to remove unreacted monomers and impurities and fully swelled.
Configuration of PDGF-BB-reactive droplets: in detecting PDGF-BB, 50nM PDGF-BB was mixed with 200nM H1, DNA2 solution to a final volume of 0.1mL in 1 XPBS buffer. 10ul of the reaction solution was added to the hydrophilic region of OH-gel, and reacted at 25℃and 50% humidity. Fluorescence signal measurements were recorded under a laser confocal scanning microscope at 30 minute intervals. And the droplets are placed in the hydrogel region for reaction. Record was photographed every 20min and the results (as shown in fig. 2). The nucleic acid sequences used are shown below, and were synthesized by the company Biotechnology Co., ltd (Beijing).
Example 5
Selecting 10cm multiplied by 10cm glass as a template, cleaning the surface of the template, modifying the template in a vacuum environment of 1H, 2H-perfluoro decyl trimethoxy silane at 60 ℃ for 6 hours, enabling the 1H, 2H-perfluoro decyl trimethoxy silane to self-assemble into a hydrophobic layer on the surface of the glass, clamping a mask plate with micropores with the diameter of 2mm on the surface of the glass through a dovetail clamp, placing the mask plate in a plasma environment for 5 minutes, and taking out a glass sheet to obtain the super-hydrophilic-hydrophobic glass surface patterning template. Preparing an oil-water gel prepolymerization solution, wherein the composition table of the hydrogel prepolymerization solution is as follows: 60g of water, 8.55g of hydroxyethyl acrylate, 2.4g of nanoclay, 30mg of 2, 2-diethoxyacetophenone are stirred homogeneously for 4h. Preparing an oil gel prepolymerization liquid: 38.1g of lauryl methacrylate, 38.1g of stearyl methacrylate, 3g of ethylene glycol dimethacrylate, 60mg of 2, 2-diethoxyacetophenone are stirred homogeneously for 1h. The oleogel hydrogels were mixed in a mass ratio of 1:1.24 and treated under a cell breaker for 1min. A uniform oil-in-water emulsion is formed. Preparing a groove mold on the glass patterned surface obtained in the step (1), pouring the oil-water gel prepolymer into the mold, and irradiating for 15min under 365nm ultraviolet. The polymerized gel was removed. The gel is soaked in water to remove unreacted monomers and impurities and fully swelled.
Configuration of reaction droplets of RABV: in 1 XTBS buffer, 100nM RABV was mixed with 400nM HP-A, HP-B, HP-C solution to a final volume of 0.1mL. 10ul of the reaction solution was added to the hydrophilic region of OH-gel, and water was continuously supplied thereto, and the reaction was carried out at 25℃and 50% humidity. Fluorescence signal measurements were recorded under a laser confocal scanning microscope at 30 minute intervals (as shown in fig. 2). The nucleic acid sequences used are shown below, and were synthesized by the company Biotechnology Co., ltd (Beijing).
The preparation method and the application of the detection platform for the full-time line biomarker provided by the embodiment of the application are described in detail. The above description of embodiments is only for aiding in understanding the method of the present application and its core ideas; meanwhile, as those skilled in the art will have modifications in the specific embodiments and application scope in accordance with the ideas of the present application, the present description should not be construed as limiting the present application in view of the above.
Certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will appreciate that a hardware manufacturer may refer to the same component by different names. The description and claims do not take the form of an element differentiated by name, but rather by functionality. As referred to throughout the specification and claims, the terms "comprising," including, "and" includes "are intended to be interpreted as" including/comprising, but not limited to. By "substantially" is meant that within an acceptable error range, a person skilled in the art is able to solve the technical problem within a certain error range, substantially achieving the technical effect. The description hereinafter sets forth the preferred embodiment for carrying out the present application, but is not intended to limit the scope of the present application in general, for the purpose of illustrating the general principles of the present application. The scope of the present application is defined by the appended claims.
It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a product or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such product or system. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a commodity or system comprising such elements.
It should be understood that the term "and/or" as used herein is merely one relationship describing the association of the associated objects, meaning that there may be three relationships, e.g., a and/or B, may represent: a exists alone, A and B exist together, and B exists alone. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.
While the foregoing description illustrates and describes the preferred embodiments of the present application, it is to be understood that this application is not limited to the forms disclosed herein, but is not to be construed as an exclusive use of other embodiments, and is capable of many other combinations, modifications and environments, and adaptations within the scope of the teachings described herein, through the foregoing teachings or through the knowledge or skills of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the present invention are intended to be within the scope of the appended claims.
Claims (5)
1. The preparation method of the detection platform for the full-time line biomarker is characterized by specifically comprising the following steps of:
s1) preparing a super-hydrophilic-hydrophobic surface patterning template;
the method comprises the following specific steps:
s1.1) selecting a template, and after pretreatment, modifying the surface of the template to obtain the template with a hydrophobic layer formed by self-assembly of the surface, wherein the modification process comprises the following steps: in a vacuum environment, the temperature is 55-65 ℃, the modification time is 5.5-6.5 hours, and the modifier is: 1h,2 h-perfluorodecyl trimethoxysilane;
s1.2) clamping the mask plate on the surface of the template obtained in the S1.1) through a pattern die, and placing the template in a plasma cleaning machine for 3-8min to obtain the super-hydrophilic-hydrophobic glass surface patterning template;
s2) preparing an oil-in-water emulsion;
the specific process comprises the following steps:
s2.1) preparing hydrogel prepolymer; mixing deionized water, hydroxyethyl acrylate, nano clay and 2, 2-diethoxyacetophenone, and stirring for 1-1.5h to obtain an oil gel prepolymer;
s2.2) preparing an oleogel prepolymer: respectively weighing lauryl methacrylate, stearyl methacrylate, ethylene glycol dimethacrylate and 2, 2-diethoxyacetophenone, mixing and stirring for 1-1.5h to obtain an oil gel prepolymer;
s2.3) mixing the oil gel prepolymer liquid obtained in the step S2.2) and the hydrogel prepolymer liquid obtained in the step S2.1) according to a mass ratio of 1:1.24, adding the mixture into a cell crusher, and treating the mixture for 1 to 3 minutes under the power of 580W to form an oil-in-water emulsion with the particle size of 2 to 10 mu m;
s3) solidifying the oil-in-water emulsion obtained in the step S2) on the surface patterning template obtained in the step S1), and synthesizing a surface patterning oil-water gel droplet array to obtain a detection platform for the full-time line biomarker;
the method comprises the following specific steps:
s3.1) pouring the oil-in-water emulsion obtained in the step S2) onto the super-hydrophilic-hydrophobic surface patterning template obtained in the step S1);
s3.2) at a wavelength of 365nm, an optical power of 12mW cm -2 Irradiating with ultraviolet light for 12-18min to obtain polymerized gel,
s3.3) soaking the gel in water, removing unreacted monomers and impurities, and fully swelling to obtain a detection platform for the full-time line biomarker.
2. The preparation method according to claim 1, wherein the mass percentages of the components of the hydrogel prepolymer in S2.1) are: deionized water: 81-86 wt% of hydroxyethyl acrylate: 10-15 wt% of nano clay: 3-4wt% of 2, 2-diethoxyacetophenone: 0.03wt% to 0.05wt%.
3. The preparation method of claim 1, wherein the oil gel prepolymer in S2.2) comprises the following components in percentage by mass: 45-50 wt% of lauryl methacrylate and octadecyl methacrylate: 45-50 wt% of ethylene glycol dimethacrylate: 3-4wt% of 2, 2-diethoxyacetophenone: 0.7wt% to 1wt%.
4. A detection platform for full-time line biomarkers prepared by the method of any one of claims 1-3 applied to the field of biomarker detection for multiple time periods.
5. An assay platform for a full-time line biomarker, wherein the assay platform for a full-time line biomarker is prepared by the method of any one of claims 1-3.
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