CN116559467A - Colorimetric method for rapidly detecting mucin 1 based on aptamer - Google Patents
Colorimetric method for rapidly detecting mucin 1 based on aptamer Download PDFInfo
- Publication number
- CN116559467A CN116559467A CN202310569116.6A CN202310569116A CN116559467A CN 116559467 A CN116559467 A CN 116559467A CN 202310569116 A CN202310569116 A CN 202310569116A CN 116559467 A CN116559467 A CN 116559467A
- Authority
- CN
- China
- Prior art keywords
- mucin
- aptamer
- muc1
- apt
- aunps
- 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.)
- Pending
Links
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 35
- 108010008707 Mucin-1 Proteins 0.000 title claims abstract description 35
- 238000004737 colorimetric analysis Methods 0.000 title claims abstract description 16
- 102000007298 Mucin-1 Human genes 0.000 title claims abstract 16
- 102100034256 Mucin-1 Human genes 0.000 claims abstract description 57
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 31
- 239000011324 bead Substances 0.000 claims abstract description 27
- 239000002299 complementary DNA Substances 0.000 claims abstract description 16
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 7
- 230000000295 complement effect Effects 0.000 claims abstract description 6
- 239000010931 gold Substances 0.000 claims abstract description 6
- 229910052737 gold Inorganic materials 0.000 claims abstract description 6
- 238000007885 magnetic separation Methods 0.000 claims abstract description 5
- 239000000872 buffer Substances 0.000 claims description 18
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 238000002835 absorbance Methods 0.000 claims description 5
- 230000027455 binding Effects 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 108010090804 Streptavidin Proteins 0.000 claims description 4
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000003638 chemical reducing agent Substances 0.000 claims description 3
- 239000000523 sample Substances 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- 230000009870 specific binding Effects 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
- 229940038773 trisodium citrate Drugs 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 102000003992 Peroxidases Human genes 0.000 claims 2
- 238000009835 boiling Methods 0.000 claims 1
- 238000002156 mixing Methods 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 230000009471 action Effects 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 239000002105 nanoparticle Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000007853 buffer solution Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 4
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 4
- 102000008100 Human Serum Albumin Human genes 0.000 description 4
- 108091006905 Human Serum Albumin Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000002086 nanomaterial Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000002122 magnetic nanoparticle Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 108091008104 nucleic acid aptamers Proteins 0.000 description 3
- 102000013415 peroxidase activity proteins Human genes 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- QZPSXPBJTPJTSZ-UHFFFAOYSA-N aqua regia Chemical compound Cl.O[N+]([O-])=O QZPSXPBJTPJTSZ-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 101100346932 Mus musculus Muc1 gene Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000012295 fluorescence in situ hybridization assay Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
-
- 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
- G01N33/57488—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 involving compounds identifable in body fluids
-
- 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
- G01N33/57492—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 involving compounds localized on the membrane of tumor or cancer cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4725—Mucins, e.g. human intestinal mucin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a colorimetric method for rapidly detecting mucin 1 based on an aptamer, and belongs to the technical field of analytical chemistry and biology. The method comprises three steps of preparation of AuNPs@ (MUC 1-Apt), preparation of functionalized magnetic beads and colorimetric detection of mucin 1. According to the method, an AuNPs-Apt conjugate modified by HRP is connected to the surface of a magnetic bead through biotin-streptavidin action and a base complementary pairing principle, when MUC1 exists, specific recognition and combination are carried out between mucin 1 and an aptamer, competition is formed between the mucin 1 and cDNA, and the gold nanoparticle conjugate AuNPs@ (MUC 1-Apt) -HRP is separated from the surface of the magnetic bead and enters supernatant. After magnetic separation, TMB is added into the clear liquid, HRP catalyzes TMB to oxidize, and the color of the solution changes from colorless to blue, so that colorimetric detection of mucin 1 is realized.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry and biology, and particularly relates to a colorimetric method for rapidly detecting mucin 1 based on an aptamer.
Background
Mucin 1 (MUC 1, recombinant Mucin 1) is an important protein biomarker in cancer, and MUC1 has been detected in various tumor tissues such as colon cancer, breast cancer, ovarian cancer, lung cancer and pancreatic cancer. Therefore, if the MUC1 can be accurately and quantitatively detected with high sensitivity, the method has great significance for early and timely detection of canceration.
In general, serum concentrations of MUC1 in healthy humans below 30U/mL, a 100-fold increase in concentration indicates a greater likelihood of cancer, indicating that MUC1 has potential to evaluate tumor treatment as a serological or histochemical diagnostic marker. In routine clinical practice, immunoassay assays based on antigen-antibody interactions have been the primary method to meet the increasing demand for biomarker detection. The sensing and diagnostic market is primarily antibodies because antibodies can capture target proteins with high specificity from complex backgrounds. In the traditional technology, the techniques such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, fluorescence in situ hybridization and radioimmunoassay are regarded as gold standards for biomarker detection, but the problems such as long antibody preparation period, large difference between antibody batches, high detection cost and the like still exist, and the application of the method is limited to a certain extent.
The aptamer (Apt) is a short oligonucleotide sequence, either DNA or RNA, synthesized by exponential enrichment of ligand system evolution (SELEX). The aptamer binds to the target with high specificity and high affinity. Compared with antibodies, the aptamer has the advantages of easy synthesis, high stability, low cost, easy modification and the like. Aptamer sensors are of a wide variety including electrochemiluminescence, colorimetry, fluorescence and electrochemical sensors. Among these aptamer sensors, colorimetric methods are low-cost, simple and practical, achieve visual observation of color changes without any precise instrument to obtain qualitative or semi-quantitative results, and can achieve on-site analytical detection by combining a portable ultraviolet detector.
In order to further improve the detection sensitivity and reduce the detection limit, various nano materials are increasingly used as platforms for constructing sensing interfaces or as signal amplifying elements, mainly because of the large specific surface area of the nano materials, which is favorable for combining with more signal molecules. Colorimetric sensors based on nanomaterials have the advantages of simplicity and rapidness in analysis, low cost, convenience in operation, visible signals and the like, and are widely focused on. Nanogold (AuNPs) is easy to prepare and has excellent optical properties, so it is increasingly used for biosensing. Magnetic Beads (MBs), also known as magnetic nanoparticles or magnetic microspheres, are generally uniform, spherical, and superparamagnetic particles. After a certain treatment, certain biomolecules can be fixed on the surfaces of the magnetic beads, the complex is generated by utilizing the interaction of the biomolecules and the targets, and the magnetic beads become carriers of the complex. Under the suction action of the magnet, mechanical movement occurs to separate the compound from other substances, thereby achieving the purposes of separating and extracting the target object and minimizing the matrix effect.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides a colorimetric method for rapidly detecting mucin 1 based on an aptamer. The method has the advantages of simple operation process, strong specificity and lower cost, and can be used for on-site rapid detection.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a colorimetric method for rapidly detecting mucin 1 based on an aptamer: binding magnetic beads of complementary cDNA of the mucin 1 aptamer with a nano gold-mucin 1 aptamer-horseradish peroxidase (HRP) (AuNPs@MUC 1-Apt) -HRP) complex; when MUC1 exists, the specific binding force of MUC1 and MUC1-Apt is stronger than the binding force of MUC1-Apt and cDNA, auNPs@ (MUC 1-Apt) -HRP breaks away from the surface of magnetic beads and enters supernatant, after 3,3', 5' -tetramethyl benzidine (TMB) is added, the HRP in the AuNPs@ (MUC 1-Apt) -HRP complex catalyzes TMB to generate a chromogenic reaction, and the solution changes from colorless to blue, so that colorimetric detection of mucin 1 is realized.
A colorimetric method for rapidly detecting mucin 1 based on an aptamer comprises the following specific steps:
(1) Preparation of AuNPs@ (MUC 1-Apt)
(1) Synthetic nanogold (AuNPs): all glassware in aqua regia (HCl: HNO) 3 =3:1) rinsing with ultrapure water after soakingDrying before use. Placing 5 mL trisodium citrate (1 wt%) into brown conical flask, adding 90 mL ultrapure water, heating to boil, and adding 5 mL of HAuCl 4 (0.2 wt%) when the color is changed from light yellow to wine red, cooling to room temperature for standby;
(2) Preparation of a nano gold-mucin 1 aptamer-horseradish peroxidase (AuNPs@ (MUC 1-Apt) -HRP) complex: 30. Mu.L of 10. Mu.M MUC1-Apt was taken, activated 1 h by adding the reducing agent TCEP at room temperature, followed by addition of 600. Mu.L of AuNPs solution, shaking incubation of 16 h (150 rpm), addition of 50. Mu.L of 100 mM PB buffer pH7.4 in five portions (5. Mu.L, 10. Mu.L, 20. Mu.L), shaking incubation of 24 h followed by centrifugation for 30 min (13500 rpm) and removal of the supernatant; subsequently, 200. Mu.L of 10 mM PBS buffer pH7.4 was added to wash three times, and then resuspended in 200. Mu. LpH 7.4 PBST buffer;
(3) Preparation of functionalized magnetic beads: mu.L of 0.4. 0.4 mg/mL streptavidin-coated magnetic beads (Avidin-MBs) and 100. Mu.L of complementary cDNA of mucin 1 aptamer of nM were mixed in 5. Mu.L of PBST and incubated at 25℃for 30 min. Blocking was performed by adding 50. Mu.L of 1. Mu.M 9A and incubating at 25℃for 30 min; adding 1 mu L of 1 mu M HRP, 5 mu L of 100 nM MUC1-Apt and 4 mu L of PBST mixed solution with pH of 7.4 which are reacted at 25 ℃, incubating 1 h at 37 ℃, and washing with 200 mu L of PBST with pH of 7.4 to obtain functionalized magnetic beads for later use;
(4) Colorimetric detection of mucin 1
Dispersing the functionalized magnetic beads, adding MUC1 solutions with different concentrations prepared by PBST with pH of 7.4, and incubating at 37 ℃ for 45 min; TMB was added to the collected supernatant after magnetic separation, color was observed after 10 min, and absorbance was measured at 650 nm.
The nucleotide sequence of the MUC1 Aptamer Aptamer in the step (2) is as follows: 5' -biotin-GCAGTTGATCCTTTGGATACCCTGG- (CH) 2 ) 6 -SH-3′。
The nucleotide sequence of the cDNA probe in the above step (3) is: 5'-CCAGGGTATCCAATTTTT-biotin-3'.
The nucleotide sequence of 9A in the step (3) is as follows: 5'-biotin-AAAAAAAAA-3'.
The method comprises the following steps:
the 100 mM pH7.4 PB buffer solution formula was: 100 mM Na 2 HPO 4 ,100 mM NaH 2 PO 4 3M NaCl, pH was adjusted to 7.4.
The PBS buffer solution with the pH of 7.4 comprises the following components: 137 mM NaCl,10 mM Na 2 HPO 4 ,1.4 mM KH 2 PO 4 2.7 mM KCl, pH was adjusted to 7.4.
The 10 μm MUC1 Aptamer was formulated with PBS buffer at pH 7.4.
The pH7.4 PBST buffer solution comprises the following components: prepared from PBS buffer at pH7.4 above plus 0.05% (v/v) Tween-20.
In the above method, the concentration of the MUC1 solution is: 75-500 mug/mL.
The colorimetric method for detecting mucin 1 based on the aptamer is applied to mucin 1 detection.
The technical principle of the invention is as follows:
the present invention links streptavidin-functionalized magnetic beads (avidins-MBs) to biotin-modified cDNA to form MBs-cDNA. One end of MUC1-Apt is modified with biotin, and can be combined with streptavidin modified horseradish peroxidase (Avidin-HRP); the other end is modified with sulfhydryl group, which can be combined with gold nanoparticle AuNPs; the AuNPs@ (MUC 1-Apt) -HRP complex was prepared. According to the design of DNA sequences, MUC1-Apt and cDNA are coupled to functionalized magnetic beads MBs-cDNA by base complementary pairing. Theoretically, when MUC1 was not present, the supernatant after magnetic separation was substantially free of AuNPs@ (MUC 1-Apt) -HRP, and after addition of TMB chromogenic solution, the supernatant was colorless. When MUC1 is present, the complex AuNPs@ (MUC 1-Apt) -HRP is released from the surface of the beads into the supernatant due to the specific binding of MUC1 to MUC1-Apt, which is stronger than the binding of MUC1-Apt to cDNA. After adding TMB color development solution, HRP in the compound can catalyze H 2 O 2 The TMB was oxidized to change the color of the solution from colorless to blue. The schematic diagram is shown in fig. 1.
In this method, the nucleic acid aptamer of MUC1 is used instead of the antibody, ensuring the specificity and stability of the detection method. The aptamer is a sequence obtained by screening through an exponential enrichment evolution technique (SELEX)Columns, typically composed of tens to tens of nucleotides, may be DNA or RNA. SELEX screening was from 10 15 The process of specifically recognizing and binding the nucleic acid sequence of the target molecule is obtained through 6-20 rounds of repeated screening in a random sequence library of different oligonucleotides. Nucleic acid aptamers can specifically bind to their targets by hydrogen bonding, electrostatic interactions, van der waals forces, or shape complementarity, which function similarly to antibodies, and thus, nucleic acid aptamers are known as "chemical antibodies". However, compared with antibodies, the aptamer has the advantages of low cost, repeatable synthesis, easy modification, long-term stability, low immunogenicity, structural conversion capability and the like, and has been widely used for research of various biosensor platforms. Therefore, the introduction of the aptamer capable of specifically recognizing and combining with MUC1 to replace the antibody is a guarantee of the specificity and stability of the detection method, and meanwhile, the method has the advantages of short preparation period, higher thermal stability, lower cost and the like.
In the method, the applied magnetic nano particles have the effect of a signal method, and more importantly, the magnetic nano particles can quickly and effectively separate the target from the complex matrix, so that the matrix effect is reduced to the greatest extent. The nano gold is easy to prepare, has large specific surface area, and can carry more HRP, so that signals are amplified more effectively, and the sensitivity of the method is improved.
The invention has the advantages that:
1. according to the invention, the MUC1 specific aptamer is used as an identification element, so that the stability and selectivity of a detection method are ensured.
2. The invention introduces two nano materials of magnetic beads and nano gold to amplify signals, thereby further improving the sensitivity of the colorimetric detection method.
3. The invention realizes high specificity detection of MUC1, and has simple operation and low cost.
4. The method belongs to a signal-on type detection method, and can reduce the influence of false positive results of a signal-off type method.
Drawings
FIG. 1 is a schematic diagram of detection of MUC1 based on a magnetic bead and nanogold colorimetric aptamer sensing method.
FIG. 2 results of colorimetric detection of MUC1 at various concentrations by the method of the present invention.
FIG. 3A standard operating curve established when the method of the present invention detects MUC1 at different concentrations.
FIG. 4 shows a specific assay for detecting MUC1 by the method of the present invention.
Detailed Description
In order to make the contents of the present invention easier to understand, the technical solutions of the present invention will be further described with reference to the specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
Example 1
A colorimetric method for rapidly detecting mucin 1 based on an aptamer comprises the following specific steps:
(1) Synthetic nano gold particles (AuNPs): all glassware in aqua regia (HCl: HNO) 3 =3:1) after soaking, washed with ultrapure water and dried before use. Placing 5 mL trisodium citrate (1 wt%) into brown conical flask, adding 90 mL ultrapure water, heating to boil, and adding 5 mL of HAuCl 4 (0.2 wt%) when the color changed from pale yellow to reddish wine, it was cooled to room temperature for use.
(2) Preparation of a nano gold-mucin 1 aptamer-horseradish peroxidase (AuNPs@ (MUC 1-Apt) -HRP) complex: mu.L of 10. Mu.M MUC1 Aptamer Aptamer (MUC 1-Apt) was taken, activated 1 h at room temperature by adding the reducing agent TCEP, and then 600. Mu.L of AuNPs solution prepared in step (1) was added thereto, followed by shaking incubation of 16 h (150 rpm). 50. mu.L of 100 mM PB buffer pH7.4 was added in five portions (5. Mu.L, 10. Mu.L, 20. Mu.L), and after incubation with shaking 24 h, centrifuged for 30 min (13500 rpm) and the supernatant removed. Then 200. Mu.L of 10 mM PBS buffer at pH7.4 was added for three washes and resuspended in 200. Mu.L of PBST buffer at pH 7.4.
(3) Preparation of functionalized magnetic beads
5. Mu.L of 0.4 mg/mL streptavidin-labeled magnetic beads (Avidin-MBs washed 3 times with 50. Mu. L W & B buffer) and 100 nM cDNA probes washed 3 times with 50. Mu. L W & B buffer were mixed in 5. Mu. LpH 7.4 PBST buffer and incubated at 25℃for 30 min. Blocking was performed by adding 50. Mu.L of 1. Mu.M 9A and incubating at 25℃for 30 min. 1. Mu.L of 1. Mu.M HRP, 5. Mu.L of 100 nM MUC1-Apt, 4. Mu.L of a pH7.4 PBST mixture previously reacted at 25℃was added, 1 h was incubated at 37℃and washed with 200. Mu.L of pH7.4 PBST to obtain functionalized magnetic beads for use.
(4) Colorimetric detection of mucin 1
The functionalized magnetic beads are added into MUC1 solutions with different concentrations prepared by PBST, and incubated for 45 min at 37 ℃. TMB color development was added to the collected supernatant after magnetic separation, the color was observed after 10 min, and the absorbance was detected at 650 nm.
The nucleotide sequence of the MUC1 Aptamer Aptamer in the step (2) is as follows: 5' -biotin-GCAGTTGATCCTTTGGATACCCTGG- (CH) 2 ) 6 -SH-3′。
The nucleotide sequence of the cDNA probe in the above step (3) is: 5'-CCAGGGTATCCAATTTTT-biotin-3'.
The nucleotide sequence of 9A in the step (3) is as follows: 5'-biotin-AAAAAAAAA-3'.
The method comprises the following steps:
the W & B buffer: prepared with 10 mM Tris-HCl, 1 mM EDTA, 2M NaCl (pH 7.5);
the 100 mM pH7.4 PB buffer solution formula was: 100 mM Na 2 HPO 4 ,100 mM NaH 2 PO 4 3M NaCl, pH was adjusted to 7.4.
The PBS buffer with pH of 7.4 has the formula: 137 mM NaCl,10 mM Na 2 HPO 4 ,1.4 mM KH 2 PO 4 2.7 mM KCl, pH was adjusted to 7.4.
The 10. Mu.M MUC1 Aptamer Aptamer was formulated with PBS buffer pH 7.4.
The pH7.4 PBST buffer solution comprises the following components: prepared from PBS buffer at pH7.4 above+0.05% (v/v) Tween-20.
Example 2
The concentrations of the MUC1 solution added in step (4) of example 1 were set as follows: 0. mu.g/mL, 75. Mu.g/mL, 150. Mu.g/mL, 250. Mu.g/mL, 350. Mu.g/mL, 500. Mu.g/mL, and the rest of the procedure was as in example 1. The colorimetric detection of mucin 1 with different concentrations is realized. The detection results are shown in FIG. 2. It can be seen that as the concentration of mucin 1 increases, the blue color of the solution also becomes deeper and the absorbance value becomes progressively greater.
Quantitative detection of mucin 1 was achieved by analyzing the relationship between absorbance at 650 nm and MUC1 concentration, creating a standard curve (fig. 3). The detection Limit (LOD) of the method is calculated through 3 sigma/S (wherein sigma represents the standard deviation of signals measured by a blank liquid to be measured for a plurality of times, S is the slope of a fitting linear equation), and the theoretical detection limit of the method is 41.95 mug/mL.
Example 3 specificity verification
The specificity of the method of the present invention was verified by substituting MUC1 solution in step (3) of example 1 with Human Serum Albumin (HSA), carcinoembryonic antigen (CEA) alone or with a mixture of MUC1 and both proteins at a concentration of 250. Mu.g/mL, and by the remaining steps as in example 1, with a mucin 1 concentration of 250. Mu.g/mL. The detection results are shown in FIG. 4. It can be seen that when CEA and HSA are detected, A 650 Almost the same as the blank values, whereas the mixed solution containing MUC1, CEA and HSA was slightly lower than the detection values for pure MUC1. The result shows that the method established by the invention has good specificity for MUC1 detection.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (6)
1. A colorimetric method for rapidly detecting mucin 1 based on an aptamer is characterized by comprising the following steps of: combining magnetic beads connected with complementary cDNA of mucin 1 aptamer with a nano gold-mucin 1 aptamer-horseradish peroxidase AuNPs@ (MUC 1-Apt) -HRP complex; when MUC1 exists, the specific binding force of MUC1 and MUC1-Apt is stronger than the binding force of MUC1-Apt and cDNA, the AuNPs@ (MUC 1-Apt) -HRP complex breaks away from the surface of the magnetic beads and enters the supernatant, after 3,3', 5' -tetramethyl benzidine is added, the HRP in the AuNPs@ (MUC 1-Apt) -HRP complex catalyzes TMB to generate a chromogenic reaction, and the solution changes from colorless to blue, so that colorimetric detection of mucin 1 is realized.
2. The method according to claim 1, wherein: the method comprises the following specific steps:
(1) Synthesizing nano gold AuNPs: 5 mL of 1wt% trisodium citrate was placed in a brown conical flask, then 90 mL ultra pure water was added thereto, heated to boiling, and then 5 mL of 0.2wt% HAuCl was added thereto 4 When the color is changed from light yellow to wine red, cooling to room temperature for standby;
(2) Preparation of a nano gold-mucin 1 aptamer-horseradish peroxidase AuNPs@ (MUC 1-Apt) -HRP complex: 30. Mu.L of 10. Mu.M MUC1-Apt was taken, activated by adding the reducing agent TCEP at room temperature for 1 h, and then added with 600. Mu.L of AuNPs solution and incubated at 150 rpm with shaking for 16 h; 50. mu.L 100 mM PB buffer pH7.4 was added in five portions, and after incubation with shaking 24 h, centrifuged at 13500 rpm for 30 min, the supernatant was removed; subsequently, 200. Mu.L of 10 mM PBS buffer pH7.4 was added for three washes, and resuspended in 200. Mu.L of PBST buffer pH 7.4;
(3) Preparation of functionalized magnetic beads: mixing 5 mu L of 0.4 mg/mL streptavidin-coated magnetic beads and 100 nM complementary cDNA of mucin 1 aptamer into 5 mu L of PBST with pH7.4, incubating at 25 ℃ for 30 min, adding 50 mu L of 1 mu M9A, incubating at 25 ℃ for 30 min for blocking, adding 1 mu L of 1 mu M HRP, 5 mu L of 100 nM MUC1-Apt, 4 mu L of PBST with pH7.4 reacted at 25 ℃ in advance, incubating at 37 ℃ for 1 h, and washing with PBST with pH7.4 to obtain functionalized magnetic beads for later use;
(4) Colorimetric detection of mucin 1
The functionalized magnetic beads were added to a MUC1 solution prepared with PBST at pH7.4, incubated at 37℃for 45 min, TMB was added to the collected supernatant after magnetic separation, color was observed after 10 min, and absorbance was detected at 650 nm.
3. The aptamer-based colorimetric method for rapid detection of mucin 1 according to claim 2, wherein: the nucleotide sequence of the MUC1 Aptamer Aptamer in the step (2) is as follows: 5' -biotin-GCAGTTGATCCTTTGGATACCCTGG- (CH) 2 ) 6 -SH-3′。
4. The aptamer-based colorimetric method for rapid detection of mucin 1 according to claim 2, wherein: the nucleotide sequence of the cDNA probe in the step (3) is as follows: 5'-CCAGGGTATCCAATTTTT-biotin-3', the nucleotide sequence of 9A is as follows: 5'-biotin-AAAAAAAAA-3'.
5. The aptamer-based colorimetric method for rapid detection of mucin 1 according to claim 2, wherein: the concentration of the MUC1 solution in step (4) is: 75-500 mug/mL.
6. The use of an aptamer-based colorimetric method for rapid detection of mucin 1 according to claim 1 in mucin 1 detection.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310569116.6A CN116559467A (en) | 2023-05-19 | 2023-05-19 | Colorimetric method for rapidly detecting mucin 1 based on aptamer |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310569116.6A CN116559467A (en) | 2023-05-19 | 2023-05-19 | Colorimetric method for rapidly detecting mucin 1 based on aptamer |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116559467A true CN116559467A (en) | 2023-08-08 |
Family
ID=87497932
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310569116.6A Pending CN116559467A (en) | 2023-05-19 | 2023-05-19 | Colorimetric method for rapidly detecting mucin 1 based on aptamer |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116559467A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN118006733A (en) * | 2024-04-09 | 2024-05-10 | 艾特生物科技(深圳)有限公司 | Nucleic acid chemiluminescence detection method based on Cas12a and streptavidin aptamer cascade |
-
2023
- 2023-05-19 CN CN202310569116.6A patent/CN116559467A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN118006733A (en) * | 2024-04-09 | 2024-05-10 | 艾特生物科技(深圳)有限公司 | Nucleic acid chemiluminescence detection method based on Cas12a and streptavidin aptamer cascade |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Peng et al. | Emerging ELISA derived technologies for in vitro diagnostics | |
Chandra et al. | Protein microarrays and novel detection platforms | |
Oh et al. | A fluorophore‐based bio‐barcode amplification assay for proteins | |
Wang et al. | Advanced on-site and in vitro signal amplification biosensors for biomolecule analysis | |
JP2007536528A (en) | Aptamer nanoparticle conjugates and methods of use for the detection of target analytes | |
CN101144815A (en) | Preparation method of liquid phase protein chip | |
CN106980022B (en) | Homogeneous immunoassay method based on target protein induced DNase circulation generation | |
CN112725343B (en) | Protein marker detection kit combining gold nanoprobe and CRISPR-Cas and detection method | |
Thaxton et al. | Optically and chemically encoded nanoparticle materials for DNA and protein detection | |
Gao et al. | Rolling circle amplification integrated with suspension bead array for ultrasensitive multiplex immunodetection of tumor markers | |
Zhao et al. | Aptamer-based chemiluminescent optical fiber immunosensor with enhanced signal amplification for ultrasensitive detection of tumor biomarkers | |
CN116559467A (en) | Colorimetric method for rapidly detecting mucin 1 based on aptamer | |
Zhou et al. | A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality | |
Song et al. | In vitro diagnostic technologies for the detection of extracellular vesicles: current status and future directions | |
Nishat et al. | Nanoparticle-based point of care immunoassays for in vitro biomedical diagnostics | |
Wang et al. | Novel competitive chemiluminescence DNA assay based on Fe 3 O 4@ SiO 2@ Au-functionalized magnetic nanoparticles for sensitive detection of p53 tumor suppressor gene | |
Chen et al. | Ps− Pt nanozyme-based synergistic signal amplification biosensor for highly sensitive colorimetric detection of protein | |
Chang et al. | A novel ultrasensitive and fast aptamer biosensor of SEB based on AuNPs-assisted metal-enhanced fluorescence | |
Wu et al. | Recent advances in analysis technology for detection of prostate cancer biomarkers | |
Tong et al. | Hybridization chain reaction engineered DNA nanopolylinker for amplified electrochemical sensing of biomarkers | |
Ge et al. | Cocaine detection in blood serum using aptamer biosensor on gold nanoparticles and progressive dilution | |
Zhang et al. | A micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads and tyramide signal amplification strategy | |
CN110553991B (en) | Biological/chemical detection reagent and detection method based on hollow gold nanoparticle-DNA compound | |
Zhang et al. | Magnetic immunoassay for tumor clinical diagnosis based on rolling circular amplification (RCA) coupled with ICP-MS | |
Wang et al. | Magnetic particles-integrated CRISPR/Cas systems for biosensing |
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 |