CN113740398B - 一种比率型生物传感器及用于检测muc1的方法 - Google Patents
一种比率型生物传感器及用于检测muc1的方法 Download PDFInfo
- Publication number
- CN113740398B CN113740398B CN202110933652.0A CN202110933652A CN113740398B CN 113740398 B CN113740398 B CN 113740398B CN 202110933652 A CN202110933652 A CN 202110933652A CN 113740398 B CN113740398 B CN 113740398B
- Authority
- CN
- China
- Prior art keywords
- electrode
- muc1
- gce
- ratio
- dna
- 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
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 title claims abstract description 57
- 102100034256 Mucin-1 Human genes 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 13
- 239000000523 sample Substances 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 21
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000012921 cobalt-based metal-organic framework Substances 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000001903 differential pulse voltammetry Methods 0.000 claims description 12
- 239000006185 dispersion Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 229910001868 water Inorganic materials 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 239000012621 metal-organic framework Substances 0.000 claims description 7
- 230000009871 nonspecific binding Effects 0.000 claims description 7
- 229920001661 Chitosan Polymers 0.000 claims description 6
- KUUVQVSHGLHAKZ-UHFFFAOYSA-N thionine Chemical compound C=1C=CC=CSC=CC=1 KUUVQVSHGLHAKZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000005498 polishing Methods 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 239000003792 electrolyte Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 238000007605 air drying Methods 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- UGZAJZLUKVKCBM-UHFFFAOYSA-N 6-sulfanylhexan-1-ol Chemical compound OCCCCCCS UGZAJZLUKVKCBM-UHFFFAOYSA-N 0.000 claims description 2
- 229910018072 Al 2 O 3 Inorganic materials 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 2
- 108091023037 Aptamer Proteins 0.000 abstract description 15
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 239000000126 substance Substances 0.000 abstract description 7
- 230000011664 signaling Effects 0.000 abstract description 5
- 238000012986 modification Methods 0.000 abstract description 4
- 230000004048 modification Effects 0.000 abstract description 4
- 238000001179 sorption measurement Methods 0.000 abstract description 4
- 239000008151 electrolyte solution Substances 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 abstract 1
- 239000010931 gold Substances 0.000 description 39
- 239000000463 material Substances 0.000 description 20
- 230000004044 response Effects 0.000 description 20
- 238000011534 incubation Methods 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 8
- 239000002086 nanomaterial Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 7
- 229910052737 gold Inorganic materials 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 6
- 102000007066 Prostate-Specific Antigen Human genes 0.000 description 6
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 108010074051 C-Reactive Protein Proteins 0.000 description 5
- 102100032752 C-reactive protein Human genes 0.000 description 5
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 5
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 5
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 description 5
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 5
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 5
- 239000002105 nanoparticle Substances 0.000 description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 description 4
- 108090000190 Thrombin Proteins 0.000 description 4
- 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 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 229960004072 thrombin Drugs 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000002484 cyclic voltammetry Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000157 electrochemical-induced impedance spectroscopy Methods 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- MWVTWFVJZLCBMC-UHFFFAOYSA-N 4,4'-bipyridine Chemical compound C1=NC=CC(C=2C=CN=CC=2)=C1 MWVTWFVJZLCBMC-UHFFFAOYSA-N 0.000 description 2
- 108010063954 Mucins Proteins 0.000 description 2
- 102000015728 Mucins Human genes 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 2
- QGUAJWGNOXCYJF-UHFFFAOYSA-N cobalt dinitrate hexahydrate Chemical compound O.O.O.O.O.O.[Co+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O QGUAJWGNOXCYJF-UHFFFAOYSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000002848 electrochemical method Methods 0.000 description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000002073 nanorod Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- YCGAZNXXGKTASZ-UHFFFAOYSA-N thiophene-2,5-dicarboxylic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)S1 YCGAZNXXGKTASZ-UHFFFAOYSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- YCIHPQHVWDULOY-FMZCEJRJSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide;hydrochloride Chemical group Cl.C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O YCIHPQHVWDULOY-FMZCEJRJSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 108091008102 DNA aptamers Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- -1 HCG Proteins 0.000 description 1
- 101001133081 Homo sapiens Mucin-2 Proteins 0.000 description 1
- 108010008707 Mucin-1 Proteins 0.000 description 1
- 102100034263 Mucin-2 Human genes 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
- 240000001846 Pinus cembra Species 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000010411 electrocatalyst Substances 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000004070 electrodeposition Methods 0.000 description 1
- 238000003891 environmental analysis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000001453 impedance spectrum Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 108091008819 oncoproteins Proteins 0.000 description 1
- 102000027450 oncoproteins Human genes 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 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 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
一种比率型生物传感器及用于检测MUC1的方法,属生物传感器检测技术领域。构建了一种基于DNA四面体的比率型电化学适体传感器并用于真实样品中MUC1的定量测定。以电解质溶液硫堇作为参比探针(IIR),Co‑MOFs标记的适配体作为信号探针(ISP),构建比率型电化学传感器。传感器具有较强的抗干扰能力,仅需要与一种电活性物质结合。修改过程可以大大简化。具有很高的稳定性,即使在一个电极上进行十次重复扫描后,电流比(ISP/IIR)仍保持不变,极大地减少了随机误差。此外,DNA NTH有效消除了非特异性吸附,提高了检测精度。具有较大比表面积和良好电导率的黑磷被用作传感界面,进一步提高检测灵敏度,检测极限为1.34fM。
Description
技术领域
本发明属于比率型生物传感器检测技术领域,具体为一种用基于DNA四面体的比率型传感器和检测MUC1的方法。
背景技术
近年来,癌症已成为威胁世界各地人们健康的最严重疾病之一。癌症的早期诊断对于及时治疗和降低死亡率至关重要。生物标志物的存在可以指示某些疾病或感染的存在,在临床筛查癌症疾病中已变得越来越重要。MUC1是一种跨膜糖蛋白,具有超过200KD的高分子量[Florea A,Taleat Z,Cristea C,et al.Label free MUC1 aptasensors basedon electrodeposition of gold nanoparticles on screen printed electrodes[J].Electrochemistry Communications,2013,33:127-130.]。它已被用于癌症的早期诊断和生物治疗,因为它通常在恶性肿瘤组织的上皮细胞上异常表达,例如乳腺癌[Jo H H,JinH,Ban C.Dual aptamer-functionalized silica nanoparticles for the highlysensitive detection of breast cancer[J].Biosensors&Bioelectronics,2015,71:129-136.],肺[Raina D,Kosugi M,Ahmad R,et al.Dependence on the MUC1-COncoprotein in Non–Small Cell Lung Cancer Cells[J].Molecular CancerTherapeutics,2011,10(5):806-816.],胰腺癌,卵巢癌,前列腺癌,膀胱癌,结肠癌和胃癌[Baldus S E,Zirbes T K,Engel S,et al.Correlation of the immunohistochemicalreactivity of mucin peptide cores MUC1 and MUC2 with the histopathologicalsubtype and prognosis of gastric carcinomas[J].International Journal ofCancer,1998,79(2):133–138.]。由于细胞癌变的早期阶段MUC1的含量低不易检测,因此MUC1的超灵敏检测对于癌症,尤其是乳腺癌的早期诊断非常重要。
电化学生物传感器已广泛应用于环境分析、食品加工和医学诊断等各个领域。因其仪器简单、灵敏度高等特点受到了广泛关注。近年来,比率型传感器已成为提高传感系统实际应用的一种智能选择,可用于DNA和蛋白质之间的特异性检验。比率型传感器通过内部参考探针(IR),信号探针(SP)之间的比率响应用于内置校正,可以消除DNA负载浓度不同、温度变化和非靶诱导的DNA解离所产生的影响,提供更准确的信号。目前,常见的比率电化学DNA传感器有两种工作类型。第一种类型将信号分子固定在电极上。例如谢等人设计了一种基于双信号发夹DNA的比率测定策略用于粘蛋白1的检测[Deng C Y,Pi X M,Qian P, etal.High-Performance Ratiometric Electrochemical Method Based on theCombination of Signal Probe and Inner Reference Probe in One Hairpin-Structured DNA[J].Analytical chemistry,2017,89(1):966-973.]。艾灵顿等人开发了比率式电化学DNA传感器,用于检测单核苷酸多态性(SNP)[Du Y,Lim B J,Li B L,etal.ratiometric electrochemical DNA sensors with improved robustness andreproducibility[J].Analytical chemistry,2016,86(15):8010-8016.]。一般需要两个信号标签来标记核酸。一个用作IR,另一个用作SP。通常,二茂铁和亚甲蓝被用作比率式DNA电化学传感器的信号标签。第二种是在电解质溶液中加入 IR分子。与第一种类型相比,只需要一种电活性物质与检测探针(如核酸)偶联。因此,第二种类型更适合构建比率电化学生物传感器。
DNA作为遗传信息的载体存在于细胞核内大量存在。根据DNA特殊的空间结构,可根据碱基互补配对的原则进行自组装,与此同时空间结构具有较高的可控度和精密度,故易于组装成多种形态的DNA纳米材料。相比于传统材料,DNA纳米材料表现出易穿透带负电的细胞膜,低毒性,能抵抗核酶,具有高稳定性,能根据需要进行丰富的功能性位点修饰等优点。DNA四面体是由4条相互配对的单链DNA构成的DNA纳米结构。它通常需设计 4条单链DNA的碱基序列,根据碱基互补配对的原则,将合成的4条单链DNA等量加入到缓冲液中,通过一步退火操作,4条单链即可自动互补杂交形成具有四面体形状的三维DNA 结构[Chen XQ,Zhou G B,Song P,et al.Ultrasensitive electrochemical detection of prostate-specific antigen by using antibodies anchored on a DNA nanostructuralscaffold[J].Analytical chemistry,2014,86(15):7337-7342.]。DNA四面体可以有效消除对 DNA和蛋白质的非特异性吸附,具有很强的抗干扰能力。具有三维(3D)支架的DNA四面体也可以调节探针的空间需求,从而方便识别探针的可及性,同时提高信号标签的负载量。并且可在它单链的5'或3'端修饰特异性功能分子,实现DNA四面体功能化。
适配体是单链RNA或DNA寡核苷酸,能够特异性地有效地结合一系列蛋白质和细胞[Tuerk C,Gold L.Systematic Evolution of Ligands by Exponential Enrichment:RNALigands to Bacteriophage T4 DNA Polymerase[J].Science,1990,249(4968):505-510.]。与抗体相比,适配体更稳定,适应性更强,修饰更灵活。因此,开发一种新型的基于aptasensor 的高灵敏度和选择性定量MUC1是可取的。
发明内容
本发明的目的:为了克服现有技术的缺点,提供一种比率型生物传感器及用于检测MUC1 的方法,以提高MUC1检测的灵敏性、重现性,准确性和稳定性。
本发明产品比率型生物传感器是基于DNA四面体的传感器,其特征在于按以下步骤制得:
1、合成DNA四面体、制备Au NPs、Co-MOFs、BP无水乙醇分散液。
2、将玻碳电极GCE依次进行抛光,表面洁净化处理和干燥;最好是在麂皮绒上用Al2O3粉末进行不少于3次的抛光,然后依次用V水:V硝酸=1:1的HNO3溶液洗涤、无水乙醇洗涤,超纯水超声2.5~3.5min,晾干。
3、将9~11μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干。
4、将合成的四面体DNA滴于步骤3冲洗好的GCE电极上,于25~38℃下培育过夜后用PBS缓冲溶液冲洗;最好是于37℃下培育过夜。
5、将浓度为0.9~1.1mM,体积为9~11μL的MCH滴加到步骤4制备的电极表面,于36~38℃下培育8~12min,封闭非特异性结合位点;最好是将浓度为1mM的10μL MCH 滴加到步骤4制备的电极表面,于37℃下培育10min,封闭非特异性结合位点。
6、向步骤5制备的电极表面滴加9~11μL浓度为0.004pM~400pM的MUC1稀释液,于36~38℃下培育0.8~1.2h;最好是向步骤5制备的电极表面依次滴加10μL浓度为0.004pM;0.02pM;0.04pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM的 MUC1稀释液,于37℃下培育1h。
7、向步骤6制备的电极表面滴加9~11μL标记有信号探针的Au NPs@Co-MOFs,于36~38℃培育0.8~1.2h,最后得到基于DNA四面体的比率型生物传感器。最好是向步骤6制备的电极表面滴加10μL标记有信号探针的Au NPs@Co-MOFs于37℃培育1h。
合成DNA四面体、制备Au NPs、Co-MOFs、BP无水乙醇分散液的方法为在此之前公开文献报道的已有技术。
本发明产品比率型生物传感器用于检测MUC1的方法是:将制备的传感器在硫堇电解质中通过差分脉冲伏安法DPV检测剩余在电极上的标记物中Co-MOFs的电流。
本发明构建了一种基于DNA四面体的比率型电化学适体传感器并用于真实样品中MUC1的定量测定。本发明以电解质溶液硫堇作为参比探针(IIR),Co-MOFs标记的适配体作为信号探针(ISP),构建比率型电化学传感器。比率型电化学适体传感器具有较强的抗干扰能力,可以有效地消除由于不同DNA负载密度、环境影响和仪器效率等因素造成的干扰。与常规的比率型适体传感器相比,该DNA适体传感器仅需要与一种电活性物质结合。修改过程可以大大简化。所构造的比例式适体传感器具有很高的稳定性,即使在一个电极上进行十次重复扫描后,电流比(ISP/IIR)仍保持不变。因此,极大地减少了随机误差。此外, DNANTH有效消除了非特异性吸附,提高了检测精度。具有较大比表面积和良好电导率的黑磷被用作传感界面,以进一步提高检测灵敏度。该生物传感器具有高灵敏度,检测极限为 1.34fM。由于这些优越的性能,这种比例适体传感器实现了高重现性,准确性,稳定性和灵敏性。这种通用比例传感器可用于根据适体的诱导变化检测其他目标分析物。
本发明的优点:与传统的茎环或线性DNA探针相比,本发明利用DNA四面体,可以增加信号物质的装载量,有效消除DNA和蛋白质的非特异性吸附。当引入H1/H2/DNA2三条引物扩增链,生物传感器的灵敏度显著地提高,这一特性可以用于构建新型的双信号放大型的生物传感器。实验结果表明,与传统的方法相比较,该生物传感提高了灵敏性、重现性,准确性和稳定性,并且操作简单。
附图说明
图1为实施例纳米材料的透射电镜图,其中的(A)为Co-MOFs材料,15000X;(B)为Au NPs@Co-MOFs材料,15000X;(C)为BP材料,20000X;(D)为Au NPs@BP材料, 15000X;(E)为DNA四面体。
图2为实施例的的传感体系不同修饰电极的电化学阻抗谱图,a:bare GCE;b:AuNPs@BP/GCE;c:NTH/Au NPs@BP/GCE;d:MCH/NTH/Au NPs@BP/GCE;e:MUC1/MCH/NTH/Au NPs@BP/GCE;f:DNA1-Au NPs@Co-MOFs/MUC1/MCH/NTH/ Au NPs@BP/GCE)。
图3为实施例的传感体系不同修饰电极的循环伏安法谱图,a:bare GCE;b:AuNPs@BP/GCE;c:NTH/Au NPs@BP/GCE;d:MCH/NTH/Au NPs@BP/GCE;e: MUC1/MCH/NTH/AuNPs@BP/GCE;f:DNA1-Au NPs@Co-MOFs/MUC1/MCH/NTH/Au NPs@BP/GCE)。.
图4显示实施例不同的pH值条件对传感性能的影响。
图5显示实施例MUC1的孵育时间对传感性能的影响。
图6为实施例无H1/H2/DNA2三条引物扩增链时,加入不同浓度的MUC1之后传感器的DPV响应,自下到上的8条曲线(a,b,c,d,e,f,g,h)MUC1的浓度分别为0pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM。
图7为实施例ICo-MOFs-SP和IThi-IR分别与MUC1浓度的对数关系。
图8为实施例电流比值ICo-MOFs-SP/IThi-IR随MUC1浓度增加的响应关系。
图9为实施例电流比值ICo-MOFs-SP/IThi-IR响应和目标浓度对数的关系。
图10为实施例有H1/H2/DNA2三条引物扩增链时,加入不同浓度的MUC1之后传感器的DPV响应,自下到上的11条曲线(a,b,c,d,e,f,g,h,I,j,k)MUC1的浓度分别为0pM;0.004pM; 0.02pM;0.04pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM。
图11为实施例ICo-MOFs-SP和IThi-IR分别与MUC1浓度的对数关系。
图12为实施例电流比值ICo-MOFs-SP/IThi-IR随MUC1浓度增加的响应关系。
图13为实施例电流比值ICo-MOFs-SP/IThi-IR响应和目标浓度对数的关系。
图14为实施例检测方法的特异性,Blank为背景实验,AFP为甲胎蛋白,CEA为癌胚抗原,PSA为前列腺特异抗原,HCG为人绒毛膜促性腺激素,CRP为C反应蛋白,Thrombin 为凝血酶,MUC1为粘蛋白。
图15为实施例检测方法的重复性。
图16为实施例检测方法的重现性,三个不同电极的电化学信号响应曲线。
图17为非比率电化学传感器的电化学信号响应比值。
图18为比率电化学传感器的电化学信号响应比值。
实施例:见图1~18,表1,表2及表3。
1.仪器和试剂
N,N-二甲基甲酰胺(DMF)均购于西陇化工有限公司(中国,广州);无水乙醇(CH3CH2OH)购于成都格雷西亚化学技术有限公司(中国,成都);6-巯基己醇(MCH, 97%),凝血酶,MUC1,癌胚抗原(CEA),C反应蛋白(CRP),前列腺特异抗原(PSA),人绒毛膜促性腺激素(HCG),甲胎蛋白(AFP),壳聚糖(CHIT),1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC),磷酸盐缓冲液(PBS,0.01mol/L,0.138mol/L NaCl, pH 7.4)和N-羟丁二酰亚胺(NHC)购于美国Sigma公司(美国);黑磷(BP)购于先丰纳米材料技术有限公司(中国,南京);氯金酸(HAuCl4·4H2O)和硝酸钴六水合物(Co (NO3)2·6H2O)购于国药集团化学试剂有限公司(中国,上海);柠檬酸三钠和4,4'-联吡啶(Bpy)购于上海阿拉丁生化技术有限公司(中国,上海);2,5-噻吩二甲酸(Tdc) 购于上海麦克林生物化学有限公司(中国,上海);硫堇购于大连美伦生物技术有限公司(中国,大连);本研究中使用的DNA样本购自上海生工生物工程股份有限公司(中国,上海)。表1给出了所有寡核苷酸的序列。所有其他试剂均为分析纯试剂,可直接使用。
TGL16离心机为长沙湘智离心机仪器有限公司产品;PHS-29A型pH计为上海精科雷磁产品;真空干燥箱为上海博迅实业有限公司产品;K-Alpha+型X-射线光电子能谱仪为美国赛默飞世尔科技公司;TEM2100透射电镜为日本电子株式会社产品;ST2200HP超声波清洗器为上海科导超声仪器有限公司产品;CHI650E电化学工作站为上海辰华仪器公司产品。
2.材料的合成
2.1四面体DNA(DNA NTH)材料的合成
将购买的Tetra-A,Tetra-B,Tetra-C和Tetra-D链分别溶于一定量的缓冲溶液(20mM tris,50mM MgCl2,pH=8.0)中。然后,将四条DNA链均匀混合,在95℃下加热2min后,在4℃下保存30min。最后将合成的DNA NTH与缓冲液以1:1的比例混合(混合后浓度为 2.5μM)[参照文献:Li J,Fan C,Pei H,et al.Smart Drug Delivery Nanocarriers withSelf-Assembled DNA Nanostructures[J].Advanced Materials,2013,25:4386-4396.]
2.2 Au NPS@Co-MOFs的合成
根据文献报道采用柠檬酸盐还原四氯金酸的方法制备金纳米颗粒[例如文献:Huang M F, Kuo Y C,Huang C C,et al.Separation of Long Double-Stranded DNA byNanoparticle-Filled Capillary Electrophoresis[J].Analytical Chemistry,2004,76(1):192-196.],量取50mL水和 500μL HAuCI4·4H2O于100mL的圆底烧瓶中,在搅拌下小火加热至混合液沸腾后,迅速加入1%的柠檬酸钠溶液1.75mL。待溶液变为酒红色后停止加热,冷却至室温就得到了金溶胶。
金属有机框架材料MOFs的合成按现有技术[例如文献:Hau H J,Bai Z Y,Wang XB, et al.Highly dispersed Co nanoparticles inlayed in S,N-doped hierarchicalcarbon nanoprisms derived from Co-MOFs as efficient electrocatalysts foroxygen reduction reaction[J].Catalysis Today,2018,318:126-131.]称取0.582g六水合硝酸钴于100mL烧杯中,溶于40mL N,N-二甲基甲酰胺(VH2O:VDMF=1:1)得到溶液A;再称取0.344g 2,5-噻吩二羧酸和0.312g 4,4’-联吡啶倒入另一烧杯中,同时缓慢加入溶液A得到溶液B。把溶液B转移至反应釜中,105℃下反应72h后,过滤得到粉红色粉末,乙醇洗3次,于45℃真空干燥 12h,即制得Co-MOFs材料。
称取50mg Co-MOFs粉末溶于25mL灭菌水中,在搅拌下缓慢滴加上述合成的金溶胶25mL,常温下搅拌24h,然后在常温下离心5min,用灭菌水清洗3次,于60℃下真空烘干24h。
2.3 Au NPs@BP复合材料的合成
称取0.0120g黑磷分散于200mL无水乙醇中,解离超声4h,离心,取上清液,即制得黑磷的无水乙醇分散液。[参照文献:Tiouitchi G,Ali M A,Benyoussef A,et al.An easyroute to synthesis high-quality black phosphorus from amorphous redphosphorus[J]. Materials Letters,2018,236:56-59.]将体积为20mL黑磷的无水乙醇分散液和Au NPs以 1:1比例混合反应搅拌24h,离心(6000rpm),用灭菌水清洗3次,分散在水中(2mg/mL)。
2.3 Au NPs@Co-MOFs标记的信号探针DNA1
将10mg Au NPs@Co-MOFs溶解在500μL H2O中。200μL NHS(100mM)和200μL EDC(400mM)转移到上述混合物中,室温下振荡2h;离心,将沉淀物分散在200μL蒸馏水中,将200μL链霉亲和素(0.5mg/L)添加到该混合溶液中于4℃下振荡4h;紧接着滴加250μL DNA1(10μM)反应2h。最后,将1mL巯基乙醇(1mM)加入上述混合物中封闭非特异性结合位点,并在4℃下摇动1h,离心(10000rpm),并用PBS冲洗。最后,将DNA1标记的Au NPs@Co-MOFs分散在500μL无菌水中,在4℃下保存备用。
2.4 Au NPs@Co-MOFs标记的信号探针H1/H2
将10mg Au NPs@Co-MOFs溶解在500μL H2O中。200μL NHS(100mM)和200μL EDC(400mM)转移到上述混合物中,室温下振荡2h;离心,将沉淀物分散在200μL蒸馏水中,将200μL链霉亲和素(0.5mg/L)添加到该混合溶液中于4℃下振荡4h;紧接着滴加250μL H1/H2(10μM)反应2h。最后,将1mL巯基乙醇(1mM)加入上述混合物中封闭非特异性结合位点,并在4℃下摇动1h,离心(10000rpm),并用PBS冲洗。最后,将DNA2标记的Au NPs@Co-MOFs分散在500μL无菌水中,在4℃下保存备用。
3.检测方法
具体操作如下:
Ⅰ.将玻碳电极(GCE)在麂皮绒上用不同粒径的Al2O3粉末进行抛光,依次用HNO3溶液(V水:V硝酸=1:1)、无水乙醇和超纯水超声3min后晾干。
Ⅱ.10μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干。
Ⅲ.将合成的四面体DNA滴于上述冲洗好的GCE电极上,于37℃下培育过夜后用PBS缓冲溶液冲洗。
Ⅴ.加入10μL MCH(1mM)于37℃下培育10min,封闭非特异性结合位点。
Ⅵ.加入10μL的不同浓度的MUC1稀释液,于37℃下培育1h。
Ⅶ.加入10μL Au NPs@Co-MOFs/DNA1(信号未扩大)Au NPs@ Co-MOFs/DNA2/H1/H2(信号扩大),于37℃下培育1h。
Ⅷ.最后冲洗电极后在硫堇电解质中通过差分脉冲伏安法(DPV)检测剩余在电极上的标记物中Co-MOFs的电流,硫堇电解质的电流变化不大,Co-MOFs的电流增大,从而制备了一个比率型的适体传感器。详细的原理如图1所示。
4.结果与分析
4.1.材料表征
本实施例选取BP和Co-MOFs材料作为纳米框架材料的研究对象。利用高倍透射电镜 (TEM)确定本实施例中的纳米材料是否合成成功,并观察纳米颗粒的微观形貌特征。由图1(A)可知中Co-MOFs材料为纳米棒结构。由图1(B)可知大量的Au NPs密集分布在纳米棒材料上;BP材料的结构如图1(C)所示具有清晰的晶格条纹;当金颗粒负载至BP材料上后,从图1(D)中可观察到许多小黑点在BP材料上,说明金颗粒已负载成功,利于和DNA 四面体形成稳定的Au-S键。以上结果证明纳米材料是合成成功的。
4.2修饰电极的电化学表征
电化学阻抗谱(EIS)和循环伏安法(CV)技术用于研究修饰的电极在5.0mM[Fe(CN)6]3-/4-溶液中的固定步骤。图2显示了EIS结果,其对应于GCE表面的每个制备过程步骤。在裸电极上测出一个小的半圆(图2,曲线a,Ret=280Ω)。当Au NPs@BP沉积在GCE的表面上时,观察到较小的半圆直径(图2,曲线b,Ret=340Ω)。DNA四面体修饰修饰后,Ret (曲线c)增加到2200Ω,原因是带负电荷的寡核苷酸与带负电荷的氧化还原探针[Fe(CN)6]3-/4-之间产生静电排斥。由于MCH的存在会阻碍电子的转移,因此MCH的固定化后可能导致 Ret值增加到(图2,曲线d,Ret=2500Ω)。与MUC1连接后,Ret显着增强(图2,曲线e, Ret=3300Ω),MUC1作为生物大分子而没有优异的导电性。当标记为Au NPs@Co-MOFs 的信号探针与修饰电极特异性结合时,Ret进一步增加(图2,曲线f,Ret=4200Ω)。
图3显示了[Fe(CN)6]3-/4-在电极不同表面上的电子转移的循环伏安(CV)曲线。如图3 所示,裸露的GCE电极显示出具有优良峰形的氧化还原峰(曲线a)。当在裸露的GCE上涂覆Au NPs@BP时(图3,曲线b),氧化还原峰值电流几乎恒定,显示出这些材料的优异电导率。与DNA NTH孵育后(图3,曲线c),氧化还原峰值电流降低。然后固定了MCH,并屏蔽了活性位点后,氧化还原峰值电流被明显抑制(图3,曲线d)。当修饰的电极表面与MUC1 结合时,氧化还原峰值电流进一步降低(图3,曲线e)。将信号探针插入电极后,氧化还原峰值电流的信号再次降低(图3,曲线f)。
4.3不同pH值条件对传感性能的影响
为了达到最佳的传感性能,本实施例进行不同的pH值条件下的优化。实验结果表明随着pH值的升高,体系的信号值增大。但是当pH值超过7.5时,体系的信号值逐渐减小,当pH值为7.5条件时为传感系统提供了最大的信号与背景比值(见图4)。因此,使用 7.5的pH值进行以后的实验。
4.4.MUC1的孵育时间对传感性能的影响
为了达到最佳的传感性能,体系对MUC1的孵育时间进行了优化。实验结果表明随着 MUC1的孵育时间的增加,体系的信号值增大。但是当MUC1的孵育时间达到60min后,体系的信号值逐渐减小(见图5)。因此,使用MUC1的孵育时间为60min进行以后的实验。
4.5.分析性能
为了考察该本实施例的比率型生物传感器可以用于生物分子的定量分析,在最佳的反应条件下,传感体系检测了一系列不同浓度的MUC1。结果如图6所示当目标MUC1浓度从0.4 pM增加到400pM时,所提出的比率式适体传感器的DPV响应逐渐增加。从图7可见,当MUC1的浓度增加时,Co-MOFs-SP的电化学信号逐渐增加,但Thi-IR的DPV响应几乎相同。因此,比例响应ICo-MOFs-SP/IThi-IR随着MUC1浓度的增加而逐渐增加(图8)。图9 所示的线性回归方程为ICo-MOFs-SP/IThi-IR=1.4881lgC+1.4186,相关系数(R2)为0.997。当信噪比为3时,检测限(LOD)计算为0.134pM。
除此之外,我们引入了H1/H2/DNA2三条引物扩增链进一步降低该生物传感器的检测下限。如图10所示随着MUC1的浓度从0.004pM增加到400pM,所提出的比率式适体传感器的DPV响应逐渐增加;从图11可见,当MUC1的浓度增加时,Co-MOFs-SP的电化学信号逐渐增加,但Thi-IR的DPV响应几乎相同。因此,比例响应ICo-MOFs-SP/IThi-IR随着MUC1浓度的增加而逐渐增加(图12)。图13所示的线性回归方程为ICo-MOFs-SP/IThi-IR=1.289lgC+3.5462,相关系数(R2)为0.992。当信噪比为3时,检测限(LOD)计算为1.34fM。比未引入扩增链的放大体系的灵敏度降了2个数量级。这些结果表明引入 H1/H2/DNA2三条引物扩增链显著提高了生物传感器的灵敏度。
4.6.电化学生物传感器的选择性和重复性
特异性是一个成功的实验体系的另一个关键因素,选择了2pM的AFP,CEA,PSA,HCG, CRP和凝血酶作为干扰物质,并用于研究特异性。如图14所示,与干扰物质相比,即使当MUC1浓度低10倍时,靶物的电流响应率仍比干扰物质大得多。测量结果表明所提出的比率生物传感平台具有优异的选择性。当MUC1浓度(2pM)时,在十次重复扫描下单电极的电化学信号响应率没有明显变化,图15显示了十次重复扫描下单电极的DPV曲线,以及相对标准偏差(RSD)为2.8683%。
4.7.电化学生物传感器的重现性
为了研究该本实施例的电化学生物传感器的重现性,在相同条件下,使用十个工作电极和30个测量值来检测MUC1(2pM)。如图16所示,来自三个不同电极的电化学信号响应曲线,每个电极表面上电流信号比值几乎相同。从图17中,在30个测量值中,非比率电化学传感器的电化学信号响应变化较大,平均值为19.98μA,但在我们提出的比率电化学生物传感器中,这种变化被显著降低,30个试验的相对标准偏差(RSD)为3.18%,平均值为3.710(图 18)。上述结果表明,与非比率测量方法相比,比率电化学生物传感器更加可靠,可重现和可重复(表2)。采用比例双信号策略,可以减少背景干扰并获得出色的灵敏度。测量结果表明比率型电化学生物传感器具有优异的重现性。
4.8.电化学生物传感器的回收率
为了评价该本实施例在实际样品中的应用,采用标准加入法,将该传感器应用于人血清样品中MUC1的检测。结果见表3,三种血清样品中的MUC1(0.04μM、0.4μM和4.0μM),回收率为97.0%~104.4%,相对标准偏差为1.219%~3.569%。这些结果表明我们提出的比率生物传感器可以用于实际样品的分析。
表1为所有寡核苷酸的序列。
表2比率和非比率比率电化学传感器的重现性对比。
表3为10倍稀释人血清样品中MUC1的回收。
表1:所有寡核苷酸的序列。
表2:比率和非比率比率电化学传感器的重现性对比。
表3:10倍稀释人血清样品中MUC1的回收。
Claims (3)
1.一种比率型生物传感器,其特征在于按以下步骤制得:
(1)、合成DNA四面体、制备Au NPs、Co-MOFs、黑磷BP无水乙醇分散液;
(2)、将玻碳电极GCE依次进行抛光,表面洁净化处理和干燥;
(3)、将9~11μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干;
(4)、将合成的四面体DNA滴于步骤(3)冲洗好的GCE电极上,于25~38℃下培育过夜后用PBS缓冲溶液冲洗;
(5)、将浓度为0.9~1.1mM,体积为9~11μL的6-巯基己醇MCH滴加到步骤(4)制备的电极表面,于36~38℃下培育8~12min,封闭非特异性结合位点;
(6)、向步骤(5)制备的电极表面滴加9~11μL浓度为0.004pM~400pM的粘蛋白MUC1稀释液,于36~38℃下培育0.8~1.2h;
(7)、向步骤(6)制备的电极表面滴加9~11μL标记有信号探针的Au NPs@Co-MOFs,于36~38℃培育0.8~1.2h,最后得到基于DNA四面体的比率型生物传感器。
2.如权利要求1所述的一种比率型生物传感器,其特征在于按以下步骤制得:
(1)、合成DNA四面体、制备Au NPs、Co-MOFs、BP无水乙醇分散液;
(2)、将玻碳电极GCE在麂皮绒上用Al2O3粉末进行不少于3次的抛光,然后依次用V水:V硝酸=1:1的HNO3溶液洗涤、无水乙醇洗涤,超纯水超声2.5~3.5min,晾干;
(3)、将9~11μL等体积混合的Au NPs@BP分散液与壳聚糖溶液滴加到GCE表面,室温下晾干;
(4)、将合成的四面体DNA滴于步骤(3)冲洗好的GCE电极上,于37℃下培育过夜后用PBS缓冲溶液冲洗;
(5)、将浓度为1mM的10μL MCH滴加到步骤(4)制备的电极表面,于37℃下培育10min,封闭非特异性结合位点;
(6)、向步骤(5)制备的电极表面依次滴加10μL浓度为0.004pM;0.02pM;0.04pM;0.4pM;2pM;4pM;20pM;40pM;200pM;400pM的MUC1稀释液,于37℃下培育1h;
(7)、向步骤(6)制备的电极表面滴加10μL标记有信号探针的Au NPs@Co-MOFs于37℃培育1h,最后得到基于DNA四面体的比率型生物传感器。
3.如权利要求1所述的比率型生物传感器用于制备MUC1检测试剂的方法,其特征在于:将传感器在硫堇电解质中通过差分脉冲伏安法DPV检测剩余在电极上的标记物中Co-MOFs的电流。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110933652.0A CN113740398B (zh) | 2021-08-14 | 2021-08-14 | 一种比率型生物传感器及用于检测muc1的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110933652.0A CN113740398B (zh) | 2021-08-14 | 2021-08-14 | 一种比率型生物传感器及用于检测muc1的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113740398A CN113740398A (zh) | 2021-12-03 |
CN113740398B true CN113740398B (zh) | 2023-12-22 |
Family
ID=78731117
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110933652.0A Active CN113740398B (zh) | 2021-08-14 | 2021-08-14 | 一种比率型生物传感器及用于检测muc1的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113740398B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115266894B (zh) * | 2022-06-14 | 2024-03-01 | 广州大学 | 一种糖轭合物的比率型电化学检测方法 |
CN115950938B (zh) * | 2023-03-14 | 2023-06-02 | 武汉理工大学 | 一种电化学生物传感器的制作方法及电化学检测仪 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108490053A (zh) * | 2018-03-08 | 2018-09-04 | 清华大学 | 一种三维石墨烯基比例型信号放大适体传感器及其制备方法与应用 |
CN110346436A (zh) * | 2019-06-18 | 2019-10-18 | 山东大学 | 检测尿嘧啶-dna糖基化酶的、基于非酶纳米材料信号放大的无底物电化学生物传感器 |
CN110687182A (zh) * | 2019-10-30 | 2020-01-14 | 云南大学 | 一种检测前列腺特异性抗原的电化学免疫传感器的制备方法 |
CN110849867A (zh) * | 2019-12-06 | 2020-02-28 | 东北师范大学 | 基于双发夹结构的电致发光传感器及其构建方法、用途 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110455896B (zh) * | 2019-08-27 | 2020-02-11 | 青岛大学 | 金属有机骨架复合物比率电化学miR3123适体传感器的制备方法 |
-
2021
- 2021-08-14 CN CN202110933652.0A patent/CN113740398B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108490053A (zh) * | 2018-03-08 | 2018-09-04 | 清华大学 | 一种三维石墨烯基比例型信号放大适体传感器及其制备方法与应用 |
CN110346436A (zh) * | 2019-06-18 | 2019-10-18 | 山东大学 | 检测尿嘧啶-dna糖基化酶的、基于非酶纳米材料信号放大的无底物电化学生物传感器 |
CN110687182A (zh) * | 2019-10-30 | 2020-01-14 | 云南大学 | 一种检测前列腺特异性抗原的电化学免疫传感器的制备方法 |
CN110849867A (zh) * | 2019-12-06 | 2020-02-28 | 东北师范大学 | 基于双发夹结构的电致发光传感器及其构建方法、用途 |
Non-Patent Citations (4)
Title |
---|
A Sensitive Electrochemical MUC1 Sensing Platform Based on Electroactive Cu-MOFs Decorated by AuPt Nanoparticles;Ya Li et al.;《Journal of The Electrochemical Society》;第167卷;第087502页 * |
Fe-MOFs as signal probes coupling with DNA tetrahedral nanostructures for construction of ratiometric electrochemical aptasensor;Fa-Ting Xie et al.;《Analytica Chimica Acta》;第1135卷;第123-131页 * |
High-Performance Ratiometric Electrochemical Method Based on the Combination of Signal Probe and Inner Reference Probe in One Hairpin-Structured DNA;Chunyan Deng et al.;《Anal. Chem.》;第89卷;第966-973页 * |
Paper-Based Constant Potential Electrochemiluminescence Sensing Platform with Black Phosphorus as a Luminophore Enabled by a Perovskite Solar Cell;Chaomin Gao et al.;《Anal. Chem.》;第92卷;第6822-6826页 * |
Also Published As
Publication number | Publication date |
---|---|
CN113740398A (zh) | 2021-12-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yang et al. | Precise capture and direct quantification of tumor exosomes via a highly efficient dual-aptamer recognition-assisted ratiometric immobilization-free electrochemical strategy | |
Wei et al. | Graphene nanocomposites modified electrochemical aptamer sensor for rapid and highly sensitive detection of prostate specific antigen | |
Pastucha et al. | Magnetic nanoparticles for smart electrochemical immunoassays: a review on recent developments | |
Wang et al. | RNA aptamer-based electrochemical aptasensor for C-reactive protein detection using functionalized silica microspheres as immunoprobes | |
Jin et al. | Fabrication strategies, sensing modes and analytical applications of ratiometric electrochemical biosensors | |
Topkaya et al. | Electrochemical biosensors for cancer biomarkers detection: recent advances and challenges | |
Zhang et al. | Application of electrochemical biosensors in tumor cell detection | |
Hasanzadeh et al. | Iron and iron-oxide magnetic nanoparticles as signal-amplification elements in electrochemical biosensing | |
Guo et al. | Electrochemical immunoassay for the protein biomarker mucin 1 and for MCF-7 cancer cells based on signal enhancement by silver nanoclusters | |
Meirinho et al. | Voltammetric aptasensors for protein disease biomarkers detection: A review | |
Zong et al. | Chemiluminescence imaging immunoassay of multiple tumor markers for cancer screening | |
Chen et al. | Electrochemical aptasensor for mucin 1 based on dual signal amplification of poly (o-phenylenediamine) carrier and functionalized carbon nanotubes tracing tag | |
Daneshpour et al. | A novel electrochemical nanobiosensor for the ultrasensitive and specific detection of femtomolar-level gastric cancer biomarker miRNA-106a | |
Yáñez-Sedeño et al. | Pushing the limits of electrochemistry toward challenging applications in clinical diagnosis, prognosis, and therapeutic action | |
Yin | Functional nucleic acids for electrochemical and electrochemiluminescent sensing applications | |
Wei et al. | Simultaneous electrochemical determination of ochratoxin A and fumonisin B1 with an aptasensor based on the use of a Y-shaped DNA structure on gold nanorods | |
CN113740398B (zh) | 一种比率型生物传感器及用于检测muc1的方法 | |
Quan et al. | Electrochemical detection of carcinoembryonic antigen based on silver nanocluster/horseradish peroxidase nanocomposite as signal probe | |
CN107841527B (zh) | 一种利用核酸适配体和磁性材料检测凝血酶的荧光检测方法 | |
Dong et al. | Graphene/aptamer probes for small molecule detection: from in vitro test to in situ imaging | |
Guan et al. | A novel method for detection of ochratoxin A in foods—Co-MOFs based dual signal ratiometric electrochemical aptamer sensor coupled with DNA walker | |
Zhu et al. | Colorimetric detection of immunomagnetically captured rare number CTCs using mDNA-wrapped single-walled carbon nanotubes | |
Zhong et al. | Expanding the scope of chemiluminescence in bioanalysis with functional nanomaterials | |
Zhang et al. | A novel electrochemical aptasensor for serum dopamine detection based on methylene blue-integrated m-PdNFs signal material | |
Zheng et al. | Boronic acid functionalized magnetic composites with sandwich-like nanostructures as a novel matrix for PDGF detection |
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 |