CN111440355B - Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis - Google Patents
Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis Download PDFInfo
- Publication number
- CN111440355B CN111440355B CN202010303755.4A CN202010303755A CN111440355B CN 111440355 B CN111440355 B CN 111440355B CN 202010303755 A CN202010303755 A CN 202010303755A CN 111440355 B CN111440355 B CN 111440355B
- Authority
- CN
- China
- Prior art keywords
- bladder cancer
- microcarrier
- cancer protein
- color hydrogel
- magnetic
- 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
- 239000000017 hydrogel Substances 0.000 title claims abstract description 57
- 230000005291 magnetic effect Effects 0.000 title claims abstract description 51
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 47
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 47
- 206010005003 Bladder cancer Diseases 0.000 title claims abstract description 41
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 title claims abstract description 41
- 201000005112 urinary bladder cancer Diseases 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 238000000491 multivariate analysis Methods 0.000 title claims abstract description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 72
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 36
- 239000002105 nanoparticle Substances 0.000 claims abstract description 31
- 239000004038 photonic crystal Substances 0.000 claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 22
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 16
- 239000000499 gel Substances 0.000 claims abstract description 15
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 6
- 238000007711 solidification Methods 0.000 claims abstract description 6
- 230000008023 solidification Effects 0.000 claims abstract description 6
- 239000004005 microsphere Substances 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 16
- 238000004458 analytical method Methods 0.000 claims description 11
- 239000002202 Polyethylene glycol Substances 0.000 claims description 10
- 125000004386 diacrylate group Chemical group 0.000 claims description 10
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 9
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 9
- 102100036961 Nuclear mitotic apparatus protein 1 Human genes 0.000 claims description 8
- 108010036112 nuclear matrix protein 22 Proteins 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 5
- 238000002425 crystallisation Methods 0.000 claims description 5
- 230000008025 crystallization Effects 0.000 claims description 5
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 3
- 239000011022 opal Substances 0.000 claims description 3
- 230000007797 corrosion Effects 0.000 claims description 2
- 238000005260 corrosion Methods 0.000 claims description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 229920002545 silicone oil Polymers 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 239000007791 liquid phase Substances 0.000 abstract description 5
- 239000011148 porous material Substances 0.000 abstract description 4
- 230000008878 coupling Effects 0.000 abstract description 2
- 238000010168 coupling process Methods 0.000 abstract description 2
- 238000005859 coupling reaction Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000000379 polymerizing effect Effects 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 26
- 239000000243 solution Substances 0.000 description 8
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 239000012474 protein marker Substances 0.000 description 4
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- AMTWCFIAVKBGOD-UHFFFAOYSA-N dioxosilane;methoxy-dimethyl-trimethylsilyloxysilane Chemical compound O=[Si]=O.CO[Si](C)(C)O[Si](C)(C)C AMTWCFIAVKBGOD-UHFFFAOYSA-N 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 230000000737 periodic effect Effects 0.000 description 3
- 229940083037 simethicone Drugs 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000012864 cross contamination Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 1
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Images
Classifications
-
- 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
- C08J9/36—After-treatment
- C08J9/40—Impregnation
- C08J9/42—Impregnation with macromolecular compounds
-
- 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
- C08F2/00—Processes of polymerisation
- C08F2/46—Polymerisation initiated by wave energy or particle radiation
- C08F2/48—Polymerisation initiated by wave energy or particle radiation by ultraviolet or visible light
-
- 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
-
- 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
- C08J9/36—After-treatment
- C08J9/40—Impregnation
-
- 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
- C08J9/36—After-treatment
- C08J9/40—Impregnation
- C08J9/405—Impregnation with polymerisable compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- C08J2335/00—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 at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers
- C08J2335/02—Characterised by the use of homopolymers or copolymers of esters
-
- 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
- C08J2451/00—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers
- C08J2451/08—Characterised by the use of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives of such polymers grafted on to macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Materials Engineering (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Hospice & Palliative Care (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a preparation method and application of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis, wherein a silicon dioxide photonic crystal microstructure is taken as a template, hydrogel with good mechanical property is filled in pores of nanoparticles, and the template is removed after solidification to obtain the microcarrier with an inverse opal structure; and preparing functional pre-gel mixed with magnetic nano particles, filling the functional pre-gel into gaps of the inverse opal structure, and polymerizing by ultraviolet light to obtain the periodically ordered magnetic structure color hydrogel microcarrier similar to the template. The invention has the advantages of low preparation cost, good repeatability, simple and convenient operation and the like, the prepared magnetic structure color hydrogel microcarrier has the characteristics of resisting nonspecific protein combination, supplying probe coupling active groups, directional magnetic control effect and the like, and has the performance advantages of high sensitivity and specificity when being used for the multivariate analysis of the bladder cancer protein besides the performances of good coding stability, high flexibility and the like of a liquid phase chip.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a preparation method and application of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis.
Background
Bladder cancer is the most common malignant tumor of the urinary system, and the incidence and the fatality rate of the bladder cancer are at the front of the common tumors in the world at present, thus seriously threatening the life and health. Bladder cancer has obvious multicentric occurrence characteristics and has common relapse. Therefore, effective early diagnosis and prognostic monitoring methods are of great significance for the diagnosis and treatment of bladder cancer. The method for detecting the tumor protein marker has the potential of predicting tumor progression and evaluating the malignancy degree of tumors.
At present, the tumor protein marker quantitative determination methods commonly used in clinical laboratories include enzyme-linked immunoassay, electrochemiluminescence, and the like. However, the common disadvantages of these diagnostic techniques are that a single test is only for a single biomarker and the cost of the test is high, the throughput is low, and the clinical diagnosis and treatment requirements are difficult to meet. The existing multi-path analysis technology, such as the traditional solid phase chip and microarray, has the defects of large steric hindrance, easy cross contamination, poor repeatability and the like by fixing probe molecules on a planar substrate and coding according to the arrangement positions of the probe molecules.
To solve the above problems, photonic crystal microcarriers used as microcoded carriers have been developed. The periodic ordered nano structure of the photonic crystal can generate a photonic band gap effect, so that a bright structural color is presented, and a characteristic reflection peak of the photonic crystal is used for a coding element of multivariate analysis. The liquid phase code has excellent spectroscopic characteristics, has good stability in biological multivariate coding, and has the advantage of high flexibility of liquid phase coding.
Disclosure of Invention
The invention aims to solve the defects of large steric hindrance, easy cross contamination and poor repeatability of the traditional solid phase chip and provides a preparation method and application of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a preparation method of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis is characterized by comprising the following steps:
s1: taking the monodisperse silicon dioxide nanoparticle suspension as an internal phase and the dimethyl fluorinated silicone oil as an external phase, injecting the external phase and the internal phase into corresponding channels of a microfluidic device, collecting the external phase and the internal phase in a container through an output channel of the microfluidic device, and forming silicon dioxide photonic crystal microspheres through solidification and crystallization; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and placing the crucible in a muffle furnace for high-temperature treatment to obtain a coding microcarrier template;
s2: soaking the encoded microcarrier template prepared in the step S1 in a solution of ethylene glycol dimethacrylate, and performing photopolymerization curing, stripping and corrosion to obtain microspheres with inverse opal structures;
s3: and (2) preparing functional pre-gel mixed with magnetic nanoparticles, pouring the functional pre-gel into the microspheres with inverse opal structures prepared in the step S2, and performing photopolymerization to obtain the magnetic structure color hydrogel microcarrier, wherein the magnetic structure color hydrogel microcarrier integrates photonic crystal coding and directional magnetic control effects into a whole, so that biomolecule coding and multivariate analysis are realized.
In order to optimize the technical scheme, the specific measures adopted further comprise:
in step S1, the flow rates of the external phase and the internal phase in the corresponding channels of the microfluidic device were 3.0 mL/h and 1.5 mL/h, respectively.
In the step S1, the concentration of the silica nanoparticles in the monodisperse silica nanoparticle suspension is 20 m/v%; wherein the particle size of the silica nanoparticles includes any one of 215nm, 237nm and 247nm, and the silica nanoparticles are dispersed in ethanol. According to the particle size of the monodisperse silicon dioxide nano particles, the wavelength of the reflection peak of the prepared photonic crystal microsphere can be calculated through a Bragg diffraction equation.
In step S3, the functional pre-gel is prepared from polyethylene glycol diacrylate, acrylic acid, and Fe3O4Nano particles and a photoinitiator; wherein the concentration of the polyethylene glycol diacrylate is 10-30 v/v%, the concentration of the acrylic acid is 0-20 v/v%, and the Fe content is3O4The concentration of the nano particles is Fe3O41-8 m/v%, and the concentration of the photoinitiator is 0.1 m/v%.
And after the step S3, performing probe molecule modification on the surface of the prepared magnetic structure color hydrogel microcarrier, capturing and marking the bladder cancer protein by using a double-antibody sandwich principle, and performing multivariate analysis on the bladder cancer protein by using a fluorescence microscope. The coded signal is used for marker identification, and the fluorescence intensity is used for marker quantification, so that the multivariate analysis of the magnetic structure color hydrogel microcarrier on the bladder cancer protein is realized.
The bladder cancer protein includes any one of BTA protein molecule, NMP22 protein molecule or FDP protein molecule.
The above molecules include any one of an anti-BTA antibody, an anti-NMP22 antibody, or an anti-FDP antibody; wherein the concentration of the probe molecules is 0.001-0.5 mg/mL, and the reaction time for capturing the bladder cancer protein by the probe molecules is 20-120 min.
The invention also protects the application of the magnetic structure color hydrogel microcarrier prepared by the method in preparing a bladder cancer protein marker material.
The invention has the beneficial effects that:
1) the invention is based on the microfluidic technology, takes the silicon dioxide photonic crystal microspheres as the microcarrier template, has simple and convenient preparation method, is quick and efficient, and can regulate and control the microcarrier preparation method according to the application and functional requirements.
2) The invention applies liquid phase coding, has the advantages of good stability and high flexibility, and provides a high-efficiency method for biological multivariate analysis.
3) The magnetic structure color hydrogel microcarrier prepared by the invention has the characteristics of nonspecific protein combination resistance, probe coupling active group supply, directional magnetic control effect and the like, has the performances of good coding stability, high flexibility and the like of a liquid phase chip, and also has the performance advantages of high sensitivity and specificity when being used for the multivariate analysis of the bladder cancer protein.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the magnetic structure color hydrogel microcarrier of the invention.
FIG. 2 is a representation of the nano-microstructure of the magnetic structure color hydrogel microcarrier of the invention.
FIG. 3 is a schematic diagram showing the change of concentration of polyethylene glycol diacrylate with the intensity of the encoding signal.
FIG. 4 is a graph showing the change in acrylic acid concentration with the intensity of the coding signal.
FIG. 5 is a graph showing the dilution gradient of the antibody probe as a function of fluorescence intensity.
FIG. 6 is a graph showing the reaction time of the probe molecules to capture bladder cancer protein as a function of fluorescence intensity.
FIG. 7 is a performance characterization diagram of magnetic structure color hydrogel microcarrier for multiplex analysis of bladder cancer protein markers.
Detailed Description
The invention is further illustrated by the following figures and examples. The examples, in which specific conditions are not specified, were conducted according to conventional conditions well known in the art or conditions recommended by the manufacturer, and the apparatus or reagents used are not specified by the manufacturer, and are all conventional products commercially available.
Example 1
Preparation of magnetic structure color hydrogel microcarrier
(1) Referring to the preparation process of fig. 1, silica nanoparticles with a particle size of 215nm are respectively selected, monodisperse silica nanoparticle suspensions with a concentration of 20m/v% are prepared as an internal phase, simethicone (50 cSt) is used as an external phase, the external phase and the internal phase are injected into corresponding channels of a microfluidic device, the external phase and the internal phase pass through an output channel of the microfluidic device and are collected in a container, droplets of the silica nanoparticles are self-assembled along with water evaporation at 65 ℃, and the silica photonic crystal microspheres are formed through 12h solidification and crystallization; heating to 95 ℃ to solidify the silicon dioxide photonic crystal microspheres for 2 hours; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and slowly placing the crucible in a muffle furnace for high-temperature treatment to improve the mechanical stability of the crucible to obtain a coding microcarrier template;
(2) dehydrating and drying the prepared coding microcarrier template into milk white by alcohol, soaking the template into 100v/v% ethylene glycol dimethacrylate solution, adding 0.1m/v% photoinitiator, and curing the hydrogel by ultraviolet irradiation for 30 s; transferring to a clean culture dish, and placing under a microscope to strip the microspheres from the cured hydrogel; collecting the microspheres in an EP tube, and corroding the template by using a low-concentration hydrofluoric acid solution to obtain microspheres with an inverse opal structure;
(3) 20v/v% of polyethylene glycol diacrylate, 15 v/v% of acrylic acid and 4 m/v% of Fe3O4Adding nano particles and 0.1m/v% of photoinitiator into deionized water to prepare a functional pre-gel mixed with magnetic nano particles, soaking microspheres with an inverse opal structure into the functional pre-gel until the communicated pores of the inverse opal structure are completely filled, and curing the hydrogel by ultraviolet irradiation for 30 seconds to obtain the magnetic structure colored hydrogel microcarrier.
Referring to fig. 2, panel a is an SEM characterization of a photonic crystal microcarrier; b, image is SEM representation of inverse opal structure; the figure c is the SEM representation of the magnetic structure color hydrogel microcarrier, and the figure shows that the interior of the silicon dioxide photonic crystal microsphere presents a periodic ordered nano structure, and the hydrogel prepared by taking the periodic ordered nano structure as a template through copying has a similar highly ordered inverse opal structure; with the re-pouring of the functional pre-gel, the average refractive index of the microcarrier is changed, but the microcarrier still has periodically ordered nano-arrangement and is used as the structural basis for the photonic crystal to exert the coding effect through the photon forbidden band principle.
Example 2
Preparation of magnetic structure color hydrogel microcarrier
(1) Referring to the preparation process of fig. 1, silica nanoparticles with a particle size of 237nm are respectively selected, monodisperse silica nanoparticle suspensions with a concentration of 20m/v% are prepared as an internal phase, simethicone (50 cSt) is used as an external phase, the external phase and the internal phase are injected into corresponding channels of a microfluidic device, the external phase and the internal phase pass through an output channel of the microfluidic device and are collected in a container, droplets of the silica nanoparticles are self-assembled along with water evaporation at 65 ℃, and the silica photonic crystal microspheres are formed through 12h solidification and crystallization; heating to 95 ℃ to solidify the silicon dioxide photonic crystal microspheres for 2 hours; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and slowly placing the crucible in a muffle furnace for high-temperature treatment to improve the mechanical stability of the crucible to obtain a coding microcarrier template;
(2) dehydrating and drying the prepared coding microcarrier template into milk white by alcohol, soaking the template into 100v/v% ethylene glycol dimethacrylate solution, adding 0.1m/v% photoinitiator, and curing the hydrogel by ultraviolet irradiation for 30 s; transferring to a clean culture dish, and placing under a microscope to strip the microspheres from the cured hydrogel; collecting the microspheres in an EP tube, and corroding the template by using a low-concentration hydrofluoric acid solution to obtain microspheres with an inverse opal structure;
(3) 10v/v% of polyethylene glycol diacrylate, 20v/v% of acrylic acid and 1m/v% of Fe3O4Adding nano particles and 0.1m/v% of photoinitiator into deionized water to prepare a functional pre-gel mixed with magnetic nano particles, soaking microspheres with an inverse opal structure into the functional pre-gel until the communicated pores of the inverse opal structure are completely filled, and curing the hydrogel by ultraviolet irradiation for 30 seconds to obtain the magnetic structure colored hydrogel microcarrier.
Example 3
Preparation of magnetic structure color hydrogel microcarrier
(1) Referring to the preparation process of fig. 1, silica nanoparticles with a particle size of 247nm are respectively selected, monodisperse silica nanoparticle suspensions with a concentration of 20m/v% are prepared as an internal phase, simethicone (50 cSt) is used as an external phase, the external phase and the internal phase are injected into corresponding channels of a microfluidic device, the external phase and the internal phase pass through an output channel of the microfluidic device and are collected in a container, droplets of the silica nanoparticles are self-assembled along with water evaporation at 65 ℃, and the silica photonic crystal microspheres are formed through 12h solidification and crystallization; heating to 95 ℃ to solidify the silicon dioxide photonic crystal microspheres for 2 hours; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and slowly placing the crucible in a muffle furnace for high-temperature treatment to improve the mechanical stability of the crucible to obtain a coding microcarrier template;
(2) dehydrating and drying the prepared coding microcarrier template into milk white by alcohol, soaking the template into 100v/v% ethylene glycol dimethacrylate solution, adding 0.1m/v% photoinitiator, and curing the hydrogel by ultraviolet irradiation for 30 s; transferring to a clean culture dish, and placing under a microscope to strip the microspheres from the cured hydrogel; collecting the microspheres in an EP tube, and corroding the template by using a low-concentration hydrofluoric acid solution to obtain microspheres with an inverse opal structure;
(3) 30v/v% of polyethylene glycol diacrylate and 8 m/v% of Fe3O4Adding nano particles and 0.1m/v% of photoinitiator into deionized water to prepare a functional pre-gel mixed with magnetic nano particles, soaking microspheres with an inverse opal structure into the functional pre-gel until the communicated pores of the inverse opal structure are completely filled, and curing the hydrogel by ultraviolet irradiation for 30 seconds to obtain the magnetic structure colored hydrogel microcarrier.
Examples of the experiments
The preparation of the magnetic structure color hydrogel microcarrier influences the biocompatibility and the magnetic control effect of the magnetic structure color hydrogel microcarrier, and the bioanalysis performance of the magnetic structure color hydrogel microcarrier is optimized by comparing the working conditions in the hydrogel construction process. Referring to fig. 3, as the concentration of the polyethylene glycol diacrylate increases, the intensity of the encoded signal of the magnetic structure color hydrogel microcarrier decreases accordingly; referring to fig. 4, as the concentration of acrylic acid increases, the intensity of the encoded signal of the magnetic structure color hydrogel microcarrier increases accordingly; said Fe3O4The concentration of the nano particles can directly change the magnetic effect of the magnetic structure color hydrogel microcarrier, and the frequency of vibration of the magnetic structure color hydrogel microcarrier can be judged macroscopically by naked eyes, so that the Fe is increased within a certain range3O4The concentration ratio of the nano particles and the directional magnetic control effect of the magnetic structure color hydrogel microcarrier are correspondingly enhanced; by setting polyethylene glycol diacrylate, acrylic acid and Fe3O4Ratio of nanoparticlesFor example, the encoded signal intensity is measured as a preferred standard, combined with its diffusion efficiency and mechanical properties in perfusing an inverse opal structure, with 20v/v% PEGDA, 15 v/v% AA, and 4 m/v% Fe3O4The nano-particle is used as the preferable condition for constructing the detection platform.
Application example
And modifying the surface of the prepared magnetic structure color hydrogel microcarrier with probe molecules, capturing and marking the bladder cancer protein by a double-antibody sandwich principle, and performing multiplex analysis on the bladder cancer protein by a fluorescence microscope. Selecting BTA, NMP22 and FDP three protein molecules approved by FDA to be applied to clinical tumor markers from bladder cancer protein; the probe molecules comprise anti-BTA antibodies, anti-NMP22 antibodies and anti-FDP antibodies, and are used for capturing the bladder cancer proteins BTA, NMP22 and FDP.
After APTES amination and succinic anhydride carboxylation are carried out on the magnetic structural color hydrogel microcarrier, carboxyl is activated by NHS and EDC in MES (pH 6.0) buffer solution, and covalent reaction is carried out on the carboxyl and amino of Anti-BTA, Anti-NMP22 and Anti-FDP of target protein antibodies, so that the antibodies are modified on the microcarrier. Referring to fig. 5 and 6, setting an antibody probe dilution gradient of 0.001-0.5 mg/mL and a reaction time of probe molecules for capturing bladder cancer protein of 20-120 min, and measuring signal intensity under different experimental conditions to serve as an optimization standard. Selecting the probe antibody concentration of 0.01 mg/mL and the protein marker reaction time of 60min as the optimal condition for biological analysis of the magnetic structure color hydrogel microcarrier.
Referring to FIG. 7, under optimized conditions, the bladder cancer protein markers BTA, NMP22 and FDP were quantitatively added, the concentration gradient thereof was set to be 0.001 ng/mL-0.01 mg/mL, and the detection curve of the platform was fitted by concentration-signal intensity correlation analysis. The detection signal intensity is correspondingly increased along with the increase of the concentration of the protein markers BTA, NMP22 and FDP, and when the concentration of the protein markers BTA, NMP22 and FDP is increased from 1.0 ng/mL to 1000 ng/mL, the concentration-signal intensity correlation coefficient R is20.9970, 0.9925 and 0.9912, respectively. Meanwhile, in the multiplex analysis of the protein markers, the cross-reactivity is evaluated by increasing the concentration (0 ng/mL-100 ng/mL) of other protein markers in the detection solution,the signal intensity change amplitude of the specific marker (20 ng/mL) is analyzed to be less than or equal to 5 percent, which indicates that the cross reaction of the multivariate analysis of the system does not influence the detection specificity of the system.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (7)
1. A preparation method of a magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis is characterized by comprising the following steps:
s1: taking the monodisperse silicon dioxide nanoparticle suspension as an internal phase and the dimethyl fluorinated silicone oil as an external phase, injecting the external phase and the internal phase into corresponding channels of a microfluidic device, collecting the external phase and the internal phase in a container through an output channel of the microfluidic device, and forming silicon dioxide photonic crystal microspheres through solidification and crystallization; then collecting the silicon dioxide photonic crystal microspheres in a crucible, washing the microspheres for multiple times by using normal hexane, completely absorbing the normal hexane after the washing is finished, and placing the crucible in a muffle furnace for high-temperature treatment to obtain a coding microcarrier template;
s2: soaking the encoded microcarrier template prepared in the step S1 in a solution of ethylene glycol dimethacrylate, and performing photopolymerization curing, stripping and corrosion to obtain microspheres with inverse opal structures;
s3: preparing functional pre-gel mixed with magnetic nano particles, pouring the functional pre-gel into the microspheres with the inverse opal structure prepared in the step S2, and performing photopolymerization to obtain the magnetic structure color hydrogel microcarrier, wherein the functional pre-gel is prepared from polyethylene glycol diacrylate, acrylic acid and Fe3O4Nano particles and a photoinitiator; wherein the concentration of the polyethylene glycol diacrylate is 10-30 v/v%, the concentration of the acrylic acid is 0-20 v/v%, and the Fe content is3O4The concentration of the nano particles is Fe3O41-8 m/v%, and the concentration of the photoinitiator is 0.1 m/v%.
2. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 1, wherein the method comprises the following steps: in step S1, the flow rates of the external phase and the internal phase in the corresponding channels of the microfluidic device are 3.0 mL/h and 0.5 mL/h, respectively.
3. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 1, wherein the method comprises the following steps: in the step S1, the concentration of the silica nanoparticles in the monodisperse silica nanoparticle suspension is 20 m/v%; wherein the particle size of the silica nanoparticles includes any one of 215nm, 237nm and 247nm, and the silica nanoparticles are dispersed in ethanol.
4. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 1, wherein the method comprises the following steps: and step S3, performing probe molecule modification on the surface of the prepared magnetic structure color hydrogel microcarrier, capturing and marking the bladder cancer protein by using a double-antibody sandwich principle, and performing multivariate analysis on the bladder cancer protein by using a fluorescence microscope.
5. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 4, wherein the method comprises the following steps: the bladder cancer protein comprises any one of BTA protein molecules, NMP22 protein molecules or FDP protein molecules.
6. The method for preparing the magnetic structural color hydrogel microcarrier for the multiplex analysis of bladder cancer protein according to claim 5, wherein the method comprises the following steps: the probe molecule comprises any one of an anti-BTA antibody, an anti-NMP22 antibody or an anti-FDP antibody; wherein the concentration of the probe molecules is 0.001-0.5 mg/mL, and the reaction time for capturing the bladder cancer protein by the probe molecules is 20-120 min.
7. Use of the magnetic structure color hydrogel microcarrier prepared by the method of claim 1 in preparation of a material for bladder cancer protein labeling, wherein the surface of the magnetic structure color hydrogel microcarrier is modified with probe molecules, the bladder cancer protein is captured and labeled by a double antibody sandwich principle, and the bladder cancer protein is subjected to multiplex analysis by a fluorescence microscope, wherein the probe molecules comprise any one of anti-BTA antibody, anti-NMP22 antibody or anti-FDP antibody; the bladder cancer protein comprises any one of BTA protein molecules, NMP22 protein molecules or FDP protein molecules, wherein the concentration of the probe molecules is 0.001-0.5 mg/mL, and the reaction time for capturing the bladder cancer protein by the probe molecules is 20-120 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010303755.4A CN111440355B (en) | 2020-04-17 | 2020-04-17 | Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010303755.4A CN111440355B (en) | 2020-04-17 | 2020-04-17 | Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111440355A CN111440355A (en) | 2020-07-24 |
| CN111440355B true CN111440355B (en) | 2020-12-08 |
Family
ID=71653221
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010303755.4A Active CN111440355B (en) | 2020-04-17 | 2020-04-17 | Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111440355B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112842333A (en) * | 2020-12-31 | 2021-05-28 | 华中科技大学 | Visual glucose concentration detection microneedle patch, preparation method and application |
| CN113358618A (en) * | 2021-06-04 | 2021-09-07 | 南京鼓楼医院 | Exosome capturing method based on surface multi-thorn coding microspheres |
| CN113607682A (en) * | 2021-07-30 | 2021-11-05 | 复旦大学 | Coding microcarrier material and application thereof |
| CN114767618B (en) * | 2022-05-17 | 2023-02-24 | 南京鼓楼医院 | Inverse opal structure microneedle array with structural color and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004170447A (en) * | 2002-11-15 | 2004-06-17 | Mitsubishi Gas Chem Co Inc | Photonic crystal using compound having sulfide group |
| CN103143303A (en) * | 2013-03-01 | 2013-06-12 | 东南大学 | Wide-visual-angle colloid crystal film and preparation method thereof |
| CN103316615A (en) * | 2013-06-17 | 2013-09-25 | 东南大学 | Preparation and detection method of magnetic microspheres with visible glucose detection function |
| CN108373518A (en) * | 2018-05-14 | 2018-08-07 | 东南大学 | A kind of mixing photonic crystal and the preparation method and application thereof |
| CN109856300A (en) * | 2018-11-22 | 2019-06-07 | 天津大学 | A kind of preparation method of silica inverse opal hydrogel photonic crystal microballoon |
| CN110055307A (en) * | 2019-04-23 | 2019-07-26 | 东南大学 | A kind of ultra trace multi-element biologic big Molecular Detection method |
-
2020
- 2020-04-17 CN CN202010303755.4A patent/CN111440355B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004170447A (en) * | 2002-11-15 | 2004-06-17 | Mitsubishi Gas Chem Co Inc | Photonic crystal using compound having sulfide group |
| CN103143303A (en) * | 2013-03-01 | 2013-06-12 | 东南大学 | Wide-visual-angle colloid crystal film and preparation method thereof |
| CN103316615A (en) * | 2013-06-17 | 2013-09-25 | 东南大学 | Preparation and detection method of magnetic microspheres with visible glucose detection function |
| CN108373518A (en) * | 2018-05-14 | 2018-08-07 | 东南大学 | A kind of mixing photonic crystal and the preparation method and application thereof |
| CN109856300A (en) * | 2018-11-22 | 2019-06-07 | 天津大学 | A kind of preparation method of silica inverse opal hydrogel photonic crystal microballoon |
| CN110055307A (en) * | 2019-04-23 | 2019-07-26 | 东南大学 | A kind of ultra trace multi-element biologic big Molecular Detection method |
Non-Patent Citations (1)
| Title |
|---|
| Multiplexed Detection Strategy for Bladder Cancer MicroRNAs Based on Photonic Crystal Barcodes;Xiaowei Wei et al;《ANALYTICAL CHEMISTRY》;20200331;第92卷(第8期);第6121-6127页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111440355A (en) | 2020-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111440355B (en) | Preparation method and application of magnetic structure color hydrogel microcarrier for bladder cancer protein multivariate analysis | |
| Fathi et al. | Photonic crystal based biosensors: Emerging inverse opals for biomarker detection | |
| Abbas et al. | Hot spot‐localized artificial antibodies for label‐free plasmonic biosensing | |
| Jarockyte et al. | Multiplexed nanobiosensors: current trends in early diagnostics | |
| Zhao et al. | Encoded porous beads for label‐free multiplex detection of tumor markers | |
| US8565475B2 (en) | Optical system and method for reading encoded microbeads | |
| KR100962290B1 (en) | Method of detecting bioproducts using localized surface plasmon resonance sensor of gold nanoparticles | |
| US10241110B2 (en) | Plasmonic biosensors with built-in artificial antibodies | |
| WO2016187588A1 (en) | Plasmonic nanoparticles and lspr-based assays | |
| JP2003329682A (en) | Method of optically fixing microscopic object, carrier for fixing microscopic object, and method of observing microscopic object | |
| CN107694649A (en) | Microarray, its preparation method and application based on coding chip | |
| Wei et al. | Multiplex assays of bladder cancer protein markers with magnetic structural color hydrogel microcarriers based on microfluidics | |
| Zhang et al. | Preparation of graphene oxide-based surface plasmon resonance biosensor with Au bipyramid nanoparticles as sensitivity enhancer | |
| Roh et al. | Microfluidic fabrication of encoded hydrogel microparticles for application in multiplex immunoassay | |
| Wei et al. | Fabrication and evaluation of sulfanilamide-imprinted composite sensors by developing a custom-tailored strategy | |
| Lard et al. | Biosensing using arrays of vertical semiconductor nanowires: Mechanosensing and biomarker detection | |
| CN101988898A (en) | Liquid phase chip of quantum dot coding microspheres | |
| Lu et al. | Multifunctional nanocone array as solid immunoassay plate and SERS substrate for the early diagnosis of prostate cancer on microfluidic chip | |
| Chen et al. | The visual detection of anesthetics in fish based on an inverse opal photonic crystal sensor | |
| CN113189181A (en) | Single-cell protein quantitative analysis method based on electrophoresis technology | |
| CN108896526A (en) | The detection method and device of the liquid phase biochip of Raman spectrum coding | |
| Zhang et al. | Fluorimetric identification of sulfonamides by carbon dots embedded photonic crystal molecularly imprinted sensor array | |
| Yang et al. | Handy, rapid and multiplex detection of tumor markers based on encoded silica–hydrogel hybrid beads array chip | |
| Sankova et al. | Spectrally encoded microspheres for immunofluorescence analysis | |
| TWI390202B (en) | The sensing method and system of using nanometer aggregated particles |
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 |