CN115594843B - Synthesis method of electronic mediator, biosensor and preparation method of biosensor - Google Patents
Synthesis method of electronic mediator, biosensor and preparation method of biosensor Download PDFInfo
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- CN115594843B CN115594843B CN202211378027.5A CN202211378027A CN115594843B CN 115594843 B CN115594843 B CN 115594843B CN 202211378027 A CN202211378027 A CN 202211378027A CN 115594843 B CN115594843 B CN 115594843B
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- 238000001308 synthesis method Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 44
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 44
- 229920000642 polymer Polymers 0.000 claims abstract description 41
- 229910052723 transition metal Inorganic materials 0.000 claims abstract description 23
- 150000003624 transition metals Chemical class 0.000 claims abstract description 23
- 229920002873 Polyethylenimine Polymers 0.000 claims abstract description 15
- GJAWHXHKYYXBSV-UHFFFAOYSA-N pyridinedicarboxylic acid Natural products OC(=O)C1=CC=CN=C1C(O)=O GJAWHXHKYYXBSV-UHFFFAOYSA-N 0.000 claims abstract description 13
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 claims abstract description 12
- 150000005045 1,10-phenanthrolines Chemical class 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 10
- LVPMIMZXDYBCDF-UHFFFAOYSA-N isocinchomeronic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)N=C1 LVPMIMZXDYBCDF-UHFFFAOYSA-N 0.000 claims abstract description 7
- SIOXPEMLGUPBBT-UHFFFAOYSA-N picolinic acid Chemical compound OC(=O)C1=CC=CC=N1 SIOXPEMLGUPBBT-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910021381 transition metal chloride Inorganic materials 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 100
- 238000006243 chemical reaction Methods 0.000 claims description 60
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 39
- 239000003960 organic solvent Substances 0.000 claims description 35
- 108090000790 Enzymes Proteins 0.000 claims description 32
- 102000004190 Enzymes Human genes 0.000 claims description 32
- 229940088598 enzyme Drugs 0.000 claims description 32
- 239000011259 mixed solution Substances 0.000 claims description 32
- 238000010992 reflux Methods 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- WJJMNDUMQPNECX-UHFFFAOYSA-N dipicolinic acid Chemical compound OC(=O)C1=CC=CC(C(O)=O)=N1 WJJMNDUMQPNECX-UHFFFAOYSA-N 0.000 claims description 18
- 238000010438 heat treatment Methods 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 238000001816 cooling Methods 0.000 claims description 14
- 239000007787 solid Substances 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 11
- 239000003431 cross linking reagent Substances 0.000 claims description 11
- 229940024606 amino acid Drugs 0.000 claims description 10
- MBOIBXSDCWRKJR-UHFFFAOYSA-N 1,10-phenanthroline-4,7-dicarboxylic acid Chemical compound C1=CC2=C(C(O)=O)C=CN=C2C2=C1C(C(=O)O)=CC=N2 MBOIBXSDCWRKJR-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- -1 amino acid salt Chemical class 0.000 claims description 9
- 238000004132 cross linking Methods 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000003248 enzyme activator Substances 0.000 claims description 8
- 239000003223 protective agent Substances 0.000 claims description 8
- 238000003786 synthesis reaction Methods 0.000 claims description 8
- AOBIOSPNXBMOAT-UHFFFAOYSA-N 2-[2-(oxiran-2-ylmethoxy)ethoxymethyl]oxirane Chemical compound C1OC1COCCOCC1CO1 AOBIOSPNXBMOAT-UHFFFAOYSA-N 0.000 claims description 7
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 7
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 7
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 claims description 7
- 238000007650 screen-printing Methods 0.000 claims description 7
- 239000000600 sorbitol Substances 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-M Aminoacetate Chemical compound NCC([O-])=O DHMQDGOQFOQNFH-UHFFFAOYSA-M 0.000 claims description 6
- 108010015776 Glucose oxidase Proteins 0.000 claims description 6
- 239000004366 Glucose oxidase Substances 0.000 claims description 6
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000013078 crystal Substances 0.000 claims description 6
- 229940116332 glucose oxidase Drugs 0.000 claims description 6
- 235000019420 glucose oxidase Nutrition 0.000 claims description 6
- 239000011261 inert gas Substances 0.000 claims description 6
- 229910017604 nitric acid Inorganic materials 0.000 claims description 6
- 150000005846 sugar alcohols Polymers 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000004094 surface-active agent Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 239000002390 adhesive tape Substances 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- NWZBFJYXRGSRGD-UHFFFAOYSA-M sodium;octadecyl sulfate Chemical compound [Na+].CCCCCCCCCCCCCCCCCCOS([O-])(=O)=O NWZBFJYXRGSRGD-UHFFFAOYSA-M 0.000 claims description 4
- 239000000230 xanthan gum Substances 0.000 claims description 4
- 229920001285 xanthan gum Polymers 0.000 claims description 4
- 229940082509 xanthan gum Drugs 0.000 claims description 4
- 235000010493 xanthan gum Nutrition 0.000 claims description 4
- MNXMBMNXSPNINS-UHFFFAOYSA-N 1,10-phenanthroline-5,6-diamine Chemical compound C1=CC=C2C(N)=C(N)C3=CC=CN=C3C2=N1 MNXMBMNXSPNINS-UHFFFAOYSA-N 0.000 claims description 3
- JIVLDFFWTQYGSR-UHFFFAOYSA-N 4,7-dimethyl-[1,10]phenanthroline Chemical compound C1=CC2=C(C)C=CN=C2C2=C1C(C)=CC=N2 JIVLDFFWTQYGSR-UHFFFAOYSA-N 0.000 claims description 3
- IYUMNONNHYADBU-UHFFFAOYSA-N 4-chloropyridine-2,6-dicarboxylic acid Chemical compound OC(=O)C1=CC(Cl)=CC(C(O)=O)=N1 IYUMNONNHYADBU-UHFFFAOYSA-N 0.000 claims description 3
- CMMURVLHXMNTHY-UHFFFAOYSA-N 4-methylpyridine-2-carboxylic acid Chemical compound CC1=CC=NC(C(O)=O)=C1 CMMURVLHXMNTHY-UHFFFAOYSA-N 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- 108010089254 Cholesterol oxidase Proteins 0.000 claims description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 claims description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 3
- 239000007993 MOPS buffer Substances 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 239000007990 PIPES buffer Substances 0.000 claims description 3
- 108010092464 Urate Oxidase Proteins 0.000 claims description 3
- 229940009098 aspartate Drugs 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 229950005499 carbon tetrachloride Drugs 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 3
- MPFLRYZEEAQMLQ-UHFFFAOYSA-N dinicotinic acid Chemical compound OC(=O)C1=CN=CC(C(O)=O)=C1 MPFLRYZEEAQMLQ-UHFFFAOYSA-N 0.000 claims description 3
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 claims description 3
- 150000002411 histidines Chemical class 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 229920001451 polypropylene glycol Polymers 0.000 claims description 3
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 claims description 3
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 3
- 229940005267 urate oxidase Drugs 0.000 claims description 3
- GUXKBDHITFXEJG-UHFFFAOYSA-N 5-methoxycarbonylpyridine-3-carboxylic acid Chemical compound COC(=O)C1=CN=CC(C(O)=O)=C1 GUXKBDHITFXEJG-UHFFFAOYSA-N 0.000 claims description 2
- 229920005862 polyol Polymers 0.000 claims description 2
- 150000003077 polyols Chemical class 0.000 claims description 2
- 229910052703 rhodium Inorganic materials 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000008901 benefit Effects 0.000 abstract description 5
- 230000002194 synthesizing effect Effects 0.000 abstract description 4
- 230000005284 excitation Effects 0.000 abstract description 3
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 22
- 229910052707 ruthenium Inorganic materials 0.000 description 22
- 230000000052 comparative effect Effects 0.000 description 16
- 230000004044 response Effects 0.000 description 7
- 238000002484 cyclic voltammetry Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 230000027756 respiratory electron transport chain Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- YBCAZPLXEGKKFM-UHFFFAOYSA-K ruthenium(iii) chloride Chemical compound [Cl-].[Cl-].[Cl-].[Ru+3] YBCAZPLXEGKKFM-UHFFFAOYSA-K 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000012065 filter cake Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000033116 oxidation-reduction process Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- UIGBPUBMNDUKPL-UHFFFAOYSA-N 2-methylpyridine-3,5-dicarboxylic acid Chemical class CC1=NC=C(C(O)=O)C=C1C(O)=O UIGBPUBMNDUKPL-UHFFFAOYSA-N 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 241000282320 Panthera leo Species 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000000970 chrono-amperometry Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- QKIUAMUSENSFQQ-UHFFFAOYSA-N dimethylazanide Chemical compound C[N-]C QKIUAMUSENSFQQ-UHFFFAOYSA-N 0.000 description 1
- 238000003411 electrode reaction Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 1
- AACACXATQSKRQG-UHFFFAOYSA-L magnesium;2-aminoacetate Chemical class [Mg+2].NCC([O-])=O.NCC([O-])=O AACACXATQSKRQG-UHFFFAOYSA-L 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- GZWNUORNEQHOAW-UHFFFAOYSA-M potassium;2-aminoacetate Chemical class [K+].NCC([O-])=O GZWNUORNEQHOAW-UHFFFAOYSA-M 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 150000003303 ruthenium Chemical class 0.000 description 1
- 238000003980 solgel method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229910001428 transition metal ion Inorganic materials 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/0206—Polyalkylene(poly)amines
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/004—Enzyme electrodes mediator-assisted
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
-
- 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
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- 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
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
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- 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
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The application provides a synthesis method of an electronic mediator, a biosensor and a preparation method thereof, wherein the synthesis method of the electronic mediator comprises the following steps: synthesizing a first intermediate: mixing and reacting the 1, 10-phenanthroline derivative with potassium bromide to obtain a first intermediate; synthesizing a second intermediate: mixing and reacting the first intermediate with polyethyleneimine to obtain a second intermediate; synthetic transition metal polymer electron mediator: a transition metal chloride is mixed with a pyridine carboxylic acid or pyridine dicarboxylic acid homologue to react, and a transition metal polymer electron mediator is used. The biosensor contains the electron mediator synthesized by the synthesis method, and the preparation method comprises the following steps: preparing an electron mediator solution and an oxidase solution, fixing the oxidase solution and preparing a biosensor. The electronic mediator has the advantages that the electronic mediator is applied to the biosensor, so that the excitation voltage of the biosensor can be reduced, the anti-interference performance of the biosensor is improved, and the biosensor has high sensitivity and high stability.
Description
Technical Field
The application relates to the technical field of biosensors, in particular to a synthesis method of an electronic mediator, a biosensor and a preparation method of the biosensor.
Background
The amperometric enzyme biosensor has high sensitivity, good selectivity, quick response, simple and convenient operation, small sample requirement, microminiaturization and low price, is widely applied to medical detection, and has the quantitative detection principle that: after the sample to be tested enters the surface of the enzyme electrode, the enzyme catalyzes the substrate in the sample to be tested and reacts with the substrate electrochemically, and electron transfer is generated on the upper surface of the electrode, so that response current is generated, and the magnitude of the response current and the concentration of the substrate are in a linear relation. However, since the active center of the enzyme is generally inside the protein and the enzyme is easily deformed after being adsorbed on the electrode surface, electron transfer between the enzyme and the electrode is very difficult. At this time, there is an urgent need for an electron mediator capable of transferring electrons generated during an enzyme reaction from a reaction center of the enzyme to a surface of an electrode, thereby causing the electrode to generate a corresponding current change. Thus, the search for a suitable electron mediator is critical for amperometric enzyme biosensors.
In the prior art, the first generation amperometric enzyme biosensor uses O 2 Is an electron mediator, but such sensors have poor response characteristics and are susceptible to the effects of oxygen concentration in the environment and interference from other electroactive species; the second-generation current type enzyme biosensor uses an artificially synthesized electron mediator to replace oxygen to transfer electrons between enzyme and electrode, so that direct electron transmission between enzyme and electrode is enhanced, electrode reaction is accelerated, and the influence of oxygen concentration in the environment on detection is eliminated. In the second-generation amperometric enzyme biosensors, electron mediators are mostly ruthenium complexes, but such complexes have high oxidation potentials and their application is limited to a certain extent. In addition, the ruthenium polymer obtained by polymer grafting is used as an electron mediator, and the problems of too low amount of grafted metal ruthenium, large steric hindrance of a molecular group and unstable connection between enzyme and the electron mediator exist, so that the current type enzyme biosensor has low sensitivity, short response time, large noise and inaccurate concentration of detected substrate.
In view of the foregoing, there is a need for an electron mediator that can be used in a biosensor to solve the problems of the prior art.
Disclosure of Invention
The application aims to provide a method for synthesizing an electron mediator, which can be applied to a biosensor, can improve the sensitivity and the linear range of the biosensor, improve the stability of the biosensor, reduce the detection noise of the biosensor and obtain longer response time, and comprises the following specific technical scheme:
the synthesis method of the electron mediator is characterized in that the electron mediator is a transition metal polymer electron mediator and comprises the following steps:
step S1.1: the first intermediate is synthesized, specifically: 1, 10-phenanthroline derivative and potassium bromide are mixed according to the following ratio of 1-3: mixing the materials according to the mass ratio of 7-10; sequentially adding concentrated sulfuric acid and concentrated nitric acid to obtain a first mixed solution; reacting the first mixed solution to obtain a first intermediate;
step S1.2: the synthesis of the second intermediate, in particular: mixing the first intermediate with polyethylenimine according to the ratio of 0.5-1.5: adding and mixing the materials according to the mass ratio of 5-8 to obtain a second mixed solution; the second mixed solution reacts to obtain a second reaction solution; separating out solid from the second reaction solution to obtain a second intermediate;
step S1.3: the synthetic transition metal polymer electron mediator is specifically: the transition metal chloride and pyridine carboxylic acid or pyridine dicarboxylic acid homologue are mixed according to the following ratio of 2-4: mixing the materials according to the mass ratio of 1 to 3 to obtain a third mixed solution; the third mixed solution reacts to obtain a third reaction solution; and adding 0.2-0.4 g of the second intermediate into the third reaction solution with the solvent removed to react, thereby obtaining the transition metal polymer electron mediator.
Preferably, the synthesizing the first intermediate in the step S1.1 specifically includes: 1, 10-phenanthroline derivative and potassium bromide are mixed according to the following ratio of 1-3: 7-10 mass percent of the catalyst is added into a reaction vessel; sequentially adding concentrated sulfuric acid and concentrated nitric acid into a reaction container under ice bath condition to obtain a first mixed solution; turning into water bath, heating the first mixed solution to 65-90 ℃, and carrying out first reflux for 4-8 h to obtain a first reaction solution; adjusting the pH value of the first reaction solution to 6-8; treating with a first organic solvent to obtain a first intermediate;
the synthesis of the second intermediate in the step S1.2 specifically comprises the following steps: mixing the first intermediate with polyethylenimine according to the ratio of 0.5-1.5: adding the mixture into a second organic solvent according to the mass ratio of 5-8 to dissolve the mixture to obtain a second mixed solution; heating the second mixed solution to 50-70 ℃, and carrying out secondary reflux for 3-6 h to obtain a second reaction solution; cooling the second reaction solution to 5-25 ℃ to precipitate solid; treating with a third organic solvent to obtain a second intermediate;
the synthetic transition metal polymer electron mediator in the step S1.3 is specifically: the transition metal chloride and pyridine carboxylic acid or pyridine dicarboxylic acid homologue are mixed according to the following ratio of 2-4: adding the mixture into a fourth organic solvent according to the mass ratio of 1-3 to obtain a third mixed solution; reacting in an inert gas environment, and carrying out third reflux for 3-6 h to obtain a third reaction solution; cooling the third reaction solution to 10-30 ℃ and removing the solvent; adding 0.2-0.4 g of a second intermediate, heating to 90-130 ℃, and reacting for 18-36 h; cooling to separate out solid crystal; and (3) treating the mixture by a fifth organic solvent to obtain the transition metal polymer electron mediator.
Preferably, the first organic solvent is any one or more of chloroform, tetrachloromethane or diethyl ether; the second organic solvent is one or more of methanol, ethanol or diethyl ether; the third organic solvent is any one of ethanol, acetone or toluene; the fourth organic solvent is any one or more of methanol, ethanol and diethyl ether; the fifth organic solvent is any one or more of methanol, ethanol and diethyl ether; the 1, 10-phenanthroline derivative is at least one of 1, 10-phenanthroline-4, 7-dicarboxylic acid, 5, 6-diamino-1, 10-phenanthroline and 4, 7-dimethyl-1, 10-phenanthroline; the pyridine carboxylic acid or pyridine dicarboxylic acid homologue is at least one of 4-methylpyridine-carboxylic acid, pyridine-2, 6-dicarboxylic acid, 4-chloro-2, 6-pyridine dicarboxylic acid, 2, 3-pyridine dicarboxylic acid, 3, 5-pyridine dicarboxylic acid methyl ester and pyridine-3, 5-dicarboxylic acid; the transition metal chloride is RuCl 3 ·3H 2 O、OsCl 3 ·3H 2 O、Cl 3 Rh and Cl 3 At least one of Ir.
Preferably, in step S1.1: under the water bath condition, heating the first mixed solution to 70-80 ℃, wherein the heating time is 0.5-1.5 h, and the reflux time is 4-5 h; in the step S1.2, the second mixed solution is heated to 50-60 ℃, the reflux time is 3-4 h, and the cooling temperature is 15-25 ℃; in step S1.3: the inert gas is nitrogen, and the reflux time is 3-4 h.
Preferably, in step S1.1: the specific operation of adjusting the pH of the first reaction solution is: after the reflux is finished, adding water into the reaction container to dilute the first reaction solution; adding carbonate or bicarbonate into the diluted first reaction solution until the solution is neutral.
Compared with the existing electron mediator, the transition metal polymer electron mediator synthesized by the synthesis method of the electron mediator can reduce the excitation voltage of the biosensor and is beneficial to improving the anti-interference performance of the biosensor when being applied to the biosensor, and the mechanism is that the transition metal ion is immobilized by adopting 1, 10-phenanthroline derivative and polyethyleneimine to coordinate the chemical bond in the synthesis method; meanwhile, the transition metal polymer electron mediator increases the content of grafted transition metal through modification of the 1, 10-phenanthroline derivative and polyethyleneimine, thereby realizing obvious left shift of oxidation-reduction peaks and obviously improving peak intensity.
The application also provides a biosensor and a preparation method thereof, wherein the biosensor has high sensitivity and good stability, and the specific technical scheme is as follows:
a biosensor comprising an electron mediator synthesized according to the above synthesis method.
The preparation method of the biosensor comprises the following steps:
step S2.1: preparing an electron mediator solution and an oxidase solution, specifically: adding a transition metal polymer electron mediator into deionized water to obtain an electron mediator solution; wherein, in the electron mediator solution, the concentration of the electron mediator is 2-20 g/L;
adding an organic buffer solution, bovine serum albumin, amino acid salt, polyalcohol, an enzyme activator, xanthan gum and a surfactant into deionized water to obtain an enzyme protective agent; adding oxidase into an enzyme protecting agent to obtain an oxidase solution; wherein, in the oxidase solution, the concentration of the oxidase is 10-40 g/L;
step S2.2: the immobilized oxidase solution is specifically: the electron mediator solution, oxidase solution and cross-linking agent are mixed according to the proportion of 11-15: 4-8: 3-7, adding the mixture into a reaction container, and performing crosslinking reaction for 18-36 h to obtain a fixed oxidase solution;
step S2.3: the preparation method of the biosensor specifically comprises the following steps: taking 0.035-0.200 mu L of immobilized oxidase solution, dripping the oxidase solution on a screen printing electrode, drying, and attaching a hydrophilic film and double faced adhesive tape to obtain the biosensor.
Preferably, the organic buffer solution is any one or more of PIPES buffer solution, MOPS buffer solution and Bis-Tris buffer solution; the amino acid salt is any one or more of glycinate, histidine salt and aspartate; the polyalcohol is any one or more of mannitol, sorbitol and glycerol; the enzyme activator is any one or two of sodium cholate and chaps; the surfactant comprises sodium stearyl sulfate.
Preferably, the organic buffer is Bis-Tris buffer, the amino acid salt is glycinate, the polyol is sorbitol, and the enzyme activator is chaps.
Preferably, the oxidase is any one of glucose oxidase, urate oxidase or cholesterol oxidase; the cross-linking agent is any one or more of polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether and genipin.
Preferably, in step S2.2: the time of the crosslinking reaction is 18-24 hours; in step S2.3: the drying temperature is 30-60 ℃.
Compared with the existing biosensor, the biosensor prepared by the preparation method of the biosensor provided by the application has the advantages of high sensitivity and strong stability, and the mechanism is as follows: compared with the electron mediator synthesized by embedding or sol-gel method immobilized oxidase in the traditional method, the transition metal polymer electron mediator can improve the electron transmission efficiency and enzyme stability: in the electron mediator synthesized by the traditional method, a large number of active sites are hidden, so that a current response signal is low; in the transition metal polymer electron mediator adopted by the application, the transition metal mediator is coordinated through the 1, 10-phenanthroline derivative, so that the polymer with a modified functional group can be attached to the transition metal polymer electron mediator while the water solubility is good; meanwhile, the enzyme and the electron mediator form a 'wiring enzyme' through the chemical bond crosslinking of the crosslinking agent, so that the electron transmission efficiency and the enzyme stability are greatly improved.
In addition to the objects, features and advantages described above, the present application has other objects, features and advantages. The present application will be described in further detail with reference to the drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a schematic diagram of the synthesis of an electron mediator using ruthenium trichloride as an example in the preferred embodiment 1 of the present application;
FIG. 2 is a graph showing the results of infrared spectroscopic analysis of the ruthenium polymer electron mediator and 1.10-phenanthroline-4.7-dicarboxylic acid obtained in preferred example 1 of the present application;
FIG. 3 is a cyclic voltammogram of example 1 and comparative examples 1-4 when performing cyclic voltammetry characterization of electrodes;
FIG. 4 is a graph showing the current versus concentration at 4s when the chronoamperometric characterization of the electrodes was performed for example 1 and comparative examples 1-4.
Detailed Description
Embodiments of the application are described in detail below with reference to the attached drawings, but the application can be implemented in a number of different ways, which are defined and covered by the claims.
Example 1:
the embodiment provides a synthesis method of an electron mediator, wherein the electron mediator is a transition metal polymer electron mediator, and specifically comprises the following steps:
step S1.1: the first intermediate is synthesized, specifically: 1, 10-phenanthroline derivative and potassium bromide are mixed according to the following ratio of 1-3: 7-10 mass percent of the catalyst is added into a reaction vessel; sequentially adding concentrated sulfuric acid and concentrated nitric acid into a reaction container under ice bath condition to obtain a first mixed solution;
turning into water bath, heating the first mixed solution to 65-90 ℃, and carrying out first reflux for 4-8 h to obtain a first reaction solution; preferably, the first mixed solution is heated to 70-80 ℃, the heating time is 0.5-1.5 h, and the reflux time is 4-5 h;
adjusting the pH value of the first reaction solution to 6-8; preferably, the specific operation of adjusting the pH of the first reaction solution is: after the reflux is finished, adding water into the reaction container to dilute the first reaction solution; adding carbonate or bicarbonate into the diluted first reaction solution until the solution is neutral;
the method comprises the steps of treating by a first organic solvent, specifically: extracting with a first organic solvent, drying, recrystallizing, filtering, and drying to obtain a first intermediate; wherein the first organic solvent is any one or more of chloroform, tetrachloromethane or diethyl ether.
Step S1.2: the synthesis of the second intermediate, in particular: mixing the first intermediate with polyethylenimine according to the ratio of 0.5-1.5: adding the mixture into a second organic solvent according to the mass ratio of 5-8 to dissolve the mixture to obtain a second mixed solution; wherein the second organic solvent is any one or more of methanol, ethanol or diethyl ether;
heating the second mixed solution to 50-70 ℃, and carrying out secondary reflux for 3-6 h to obtain a second reaction solution; preferably, the second mixed solution is heated to 50-60 ℃ and the reflux time is 3-4 hours; cooling the second reaction solution to 5-25 ℃ to precipitate solid, and carrying out suction filtration; preferably, the cooling temperature is 15-25 ℃;
the third organic solvent treatment is carried out, specifically: washing the obtained solid with a third organic solvent, and vacuum drying to obtain a second intermediate; wherein the third organic solvent is any one of ethanol, acetone or toluene.
Step S1.3: the synthetic transition metal polymer electron mediator is specifically: the transition metal chloride and pyridine carboxylic acid or pyridine dicarboxylic acid homologue are mixed according to the following ratio of 2-4: adding the mixture into a fourth organic solvent according to the mass ratio of 1-3 to obtain a third mixed solution; wherein the fourth organic solvent is any one or more of methanol, ethanol or diethyl ether;
reacting in an inert gas environment, and carrying out third reflux for 3-6 h to obtain a third reaction solution; preferably, the inert gas is nitrogen, and the reflux time is 3-4 hours; cooling the third reaction solution to 10-30 ℃, and distilling under reduced pressure to remove the solvent; adding 0.2-0.4 g of a second intermediate, heating to 90-130 ℃, and reacting for 18-36 h to obtain a fourth reaction solution;
cooling to precipitate solid crystals, specifically: adding an organic solvent into the cooled fourth reaction solution, precipitating solid crystals, and filtering; wherein the organic solvent is any one of diethyl ether or acetone;
the treatment by a fifth organic solvent is specifically as follows: washing the resulting solid crystals with a fifth organic solvent, drying, and passing the transition metal polymer electron mediator; wherein the fifth organic solvent is one or more of methanol, ethanol or diethyl ether.
The embodiment also provides a biosensor and a preparation method thereof, wherein the biosensor contains the electronic mediator synthesized according to the synthesis method, and the preparation method specifically comprises the following steps:
step S2.1: preparing an electron mediator solution and an oxidase solution, specifically: adding a transition metal polymer electron mediator into deionized water to obtain an electron mediator solution; wherein, in the electron mediator solution, the concentration of the electron mediator is 2-20 g/L, preferably 2-6 g/L;
adding an organic buffer solution, bovine serum albumin, amino acid salt, polyalcohol, an enzyme activator, xanthan gum and a surfactant into deionized water to obtain an enzyme protective agent; adding oxidase into an enzyme protecting agent to obtain an oxidase solution; wherein, in the oxidase solution, the concentration of the oxidase is 10-40 g/L, preferably 15-30 g/L;
wherein the organic buffer solution is any one or more of PIPES buffer solution, MOPS buffer solution and Bis-Tris buffer solution, preferably Bis-Tris buffer solution; the amino acid salt is any one or more of glycinate, histidine salt and aspartate, preferably glycinate, including amino acid magnesium salt and amino acid potassium salt; the polyalcohol is one or more of mannitol, sorbitol and glycerol, preferably sorbitol; the enzyme activator is any one or two of sodium cholate and chaps, preferably chaps; the surfactant comprises sodium stearyl sulfate.
Step S2.2: the immobilized oxidase solution is specifically: the electron mediator solution, oxidase solution and cross-linking agent are mixed according to the proportion of 11-15: 4-8: 3-7, and crosslinking for 18-36 h, preferably 18-24 h to obtain immobilized oxidase solution; wherein the oxidase is any one of glucose oxidase, urate oxidase or cholesterol oxidase, preferably glucose oxidase; the cross-linking agent is any one or more of polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether and genipin.
Step S2.3: the preparation method of the biosensor specifically comprises the following steps: taking 0.035-0.200 mu L of immobilized oxidase solution, dripping the oxidase solution on a screen printing electrode, drying, preferably at 30-60 ℃, and attaching a hydrophilic film and a double-sided adhesive tape to obtain the biosensor.
The electron mediator is synthesized by taking ruthenium trichloride as an example, the synthesis principle is shown in figure 1 (R is-OOH group; n is 150-600; x is 100-300; y is 100-300 in figure 1), and the specific steps are as follows:
(1) 2.24g of 1, 10-phenanthroline-4, 7-dicarboxylic acid and 8.93g of potassium bromide were weighed and added to a 100mL round bottom flask; measuring 20mL of 98% concentrated sulfuric acid and 10mL of 68% concentrated nitric acid, sequentially adding the two materials into a round-bottom flask under ice bath conditions (in the embodiment, ice water bath is adopted, the temperature is 0 ℃), and stirring uniformly; after the concentrated sulfuric acid and the concentrated nitric acid are added in sequence, a first mixed solution is obtained and is converted into a water bath condition; slowly heating the first mixed solution to 80 ℃ for about 1h, and carrying out first reflux for 4h; after the reflux is finished, obtaining a first reaction solution; pouring the first reaction solution into a 500mL beaker, and adding water to dilute to 400mL; adding calcium carbonate solid into the diluted first reaction solution until the pH value of the solution is 6.8-7.2; extracting with chloroform, and drying with anhydrous magnesium sulfate; recrystallizing with methanol, filtering, and oven drying to obtain a first intermediate; wherein the ratio of the actual yield to the theoretical yield of the first intermediate is 81%.
(2) 12.44g of polyethyleneimine are weighed and dissolved in 50mL of methanol; wherein the molecular weight of the polyethyleneimine is 10000-50000; taking 1.75g of the first intermediate, and dissolving the first intermediate in a methanol solution containing polyethylene imine to obtain a second mixed solution; the dissolution order of the polyethyleneimine and the first intermediate in methanol is not limited, but the polyethyleneimine is relatively insoluble, so that the polyethyleneimine is dissolved in methanol before the first intermediate is added; heating the second mixed solution to 50 ℃ and carrying out second reflux for 3 hours; after the reflux is finished, obtaining a second reaction solution; cooling the second reaction solution to 25 ℃, precipitating solid, and carrying out suction filtration; washing the filter cake with ethanol for multiple times until the filtrate is colorless and transparent; vacuum drying the filter cake to obtain a second intermediate; wherein the ratio of the actual yield to the theoretical yield of the second intermediate is 85%.
(3) To a 100mL round bottom flask was added 12mL of methanol followed by 0.1572g of RuCl 3 ·3H 2 O, 18mL of a methanol solution containing 0.234g of pyridine-2, 6-dicarboxylic acid is added to obtain a third mixed solution; here is not against RuCl 3 ·3H 2 The dissolution order of O and pyridine-2, 6-dicarboxylic acid is defined; carrying out third reflux for 3h under the environment of nitrogen protection; after the reflux is finished, obtaining a third reaction solution; cooling the third reaction solution to 25 ℃, and distilling under reduced pressure to remove the solvent; adding 0.312g of a second intermediate, uniformly mixing, dissolving in 300mL of N, N-dimethylamide, heating to 100 ℃, and reacting for 24 hours; after the reaction is finished, the temperature is reduced to 25 ℃, 200mL of diethyl ether is added, solid crystals are separated out, and the filtration is carried out; washing the solid with diethyl ether for multiple times, and drying to obtain the ruthenium polymer electron mediator; wherein the ratio of the actual yield to the theoretical yield of the ruthenium polymer electron mediator is 76%.
Carrying out infrared spectrum analysis on the obtained ruthenium polymer electron mediator and 1, 10-phenanthroline-4, 7-dicarboxylic acid, wherein the analysis result is shown in figure 2; as can be seen from FIG. 2, the IR spectrum of the ruthenium polymer electron mediator was 470cm compared to that of 1, 10-phenanthroline-4, 7-dicarboxylic acid -1 Has weaker Ru-O bond absorption peak at 1550cm -1 And 1620cm -1 Has strong pyridine ring C=N stretching vibration absorption peak and at the same time at 1720cm -1 、2920cm -1 New absorption peaks of ruthenium polymer electron mediator occur, and surface ruthenium polymer electron mediator has been generated.
Taking the ruthenium polymer electron mediator as an example, a biosensor comprising the ruthenium polymer electron mediator synthesized according to the above synthesis method is prepared as follows:
(1) 0.4914g of Bis-Tris buffer, 0.5g of bovine serum albumin, 0.35g of magnesium glycinate salt, 0.35g of potassium glycinate salt, 0.2g of sorbitol, 0.6g of Chaps, 0.25g of xanthan gum and 0.15g of sodium stearyl sulfate were added to 200mL of deionized water to obtain an enzyme protecting agent.
(2) Dissolving ruthenium polymer electron mediator in deionized water to prepare electron mediator solution; wherein, in the electron mediator solution, the concentration of the electron mediator is 5g/L; dissolving glucose oxidase in an enzyme protective agent to prepare oxidase solution; wherein, in the oxidase solution, the concentration of the oxidase is 20g/L.
(3) The electron mediator solution and oxidase solution are combined with a cross-linking agent (preferably polyethylene glycol diglycidyl ether) according to 14:6:5, adding the mixture into a reaction container, and performing crosslinking reaction for 21 hours at room temperature to obtain the immobilized oxidase solution.
(4) Taking 0.1500 mu L of the immobilized oxidase solution, dripping the oxidase solution on a screen printing electrode, drying the electrode in a drying oven at 45 ℃ for 10min, and attaching a hydrophilic film and a double faced adhesive tape to obtain the biosensor.
Examples 2 to 5:
synthesis of electron mediator using ruthenium trichloride as an example in example 1, preparation of biosensor using ruthenium polymer electron mediator, partial material composition and parameters were modified, see table 1:
TABLE 1 partial composition of matter and parameters
Example 6:
the substitution of 5, 6-diamino-1, 10-phenanthroline for 1, 10-phenanthroline-4, 7-dicarboxylic acid was performed in accordance with the procedure, parameters and conditions described in example 1.
Example 7:
the substitution of 4, 7-dimethyl-1, 10-phenanthroline for 1, 10-phenanthroline-4, 7-dicarboxylic acid was performed in accordance with the procedure, parameters and conditions described in example 1.
Example 8:
the pyridine-2, 6-dicarboxylic acid was replaced with 4-methylpyridine-carboxylic acid, and the other steps, parameters and conditions were the same as in example 1.
Example 9:
the substitution of 4-chloro-2, 6-pyridinedicarboxylic acid for pyridine-2, 6-dicarboxylic acid was carried out in accordance with the procedure, parameters and conditions described in example 1.
Example 10:
the substitution of 2, 3-pyridinedicarboxylic acid for pyridine-2, 6-dicarboxylic acid was carried out in accordance with the procedure, parameters and conditions described in example 1.
Example 11:
the substitution of methyl 3, 5-pyridinedicarboxylic acid for pyridine-2, 6-dicarboxylic acid was carried out in the same manner as in example 1.
Example 12:
the pyridine-2, 6-dicarboxylic acid was replaced with pyridine-3, 5-dicarboxylic acid, and the other steps, parameters and conditions were the same as those in example 1.
Example 13:
with OsCl 3 ·3H 2 O replaces RuCl 3 ·3H 2 O, other steps, parameters and conditions were the same as in example 1.
Example 14:
with Cl 3 Rh substituted RuCl 3 ·3H 2 O, other steps, parameters and conditionsThe agreement was found in example 1.
Example 15:
with Cl 3 Ir substituted RuCl 3 ·3H 2 O, other steps, parameters and conditions were the same as in example 1.
Comparative example 1:
by conventional RuCl 3 The electron mediator replaced the ruthenium polymer electron mediator, and the other steps, parameters and conditions were the same as in example 1.
Comparative example 2:
the step of adding 1, 10-phenanthroline-4, 7-dicarboxylic acid was omitted, and other steps, parameters and conditions were the same as those in example 1.
Comparative example 3:
the step of adding polyethyleneimine was omitted, and the other steps, parameters and conditions were the same as those in example 1.
Comparative example 4:
the step of adding the crosslinking agent to carry out the crosslinking reaction was omitted, and the other steps, parameters and conditions were the same as those in example 1.
The biosensors obtained in example 1 and comparative examples 1 to 4 were characterized by using example 1 as an example, and specifically as follows:
performance characterization one: the cyclic voltammetry of the electrode is characterized as follows:
through an electrochemical workstation, a three-electrode system cyclic voltammetry is adopted for testing, screen printing electrodes in the biosensor are connected, 1 mu L of PBS buffer solution is dripped into a reaction area, and testing is started, wherein the testing result is shown in figure 3. The parameter settings are shown in table 2:
TABLE 2 parameter set-up table for cyclic voltammetry
| InitE(V)＝-0.4V | HighE(V)＝0.6V | LowE(V)＝-0.4V | Quiettime(s)＝4 |
| ScanRate(V/s)＝0.1 | Segment＝2 | Smplinterval(V)＝0.001 | Senstivity(A/V)＝10-4 |
By performing cyclic voltammetry test on the screen printed electrodes in the biosensors obtained in example 1 and comparative examples 1 to 4, referring to fig. 3, it is known that in example 1, the redox peak positions of the screen printed electrodes are symmetrical, the peak intensities are uniform, the oxidation peak is at-0.2V, and the reduction peak position is about 0.1V; example 1 was compared with comparative examples 1 to 4, the peak intensity was 3 times that in comparative example 1 and the oxidized peak was shifted to the left by about 0.2V, the peak intensity was 2 times that in comparative example 2, the peak intensity was 1.74 times that in comparative example 3, and the peak intensity was 1.22 times that in comparative example 4. Thus, the following can be concluded: the electron mediator synthesized by example 1 has an effect of efficiently transporting electrons, and its mechanism is that: the ruthenium polymer electron mediator of example 1 has been crosslinked with glucose oxidase; meanwhile, the ruthenium is modified by adopting the 1, 10-phenanthroline-4, 7-dicarboxylic acid and the polyethyleneimine, so that the oxidation-reduction peak position is obviously reduced, the excitation voltage of a screen printing electrode can be greatly reduced, the interference of a reduced substance on a biosensor is reduced, and the anti-interference performance of the biosensor is improved.
Performance characterization two: the chronoamperometric characterization of the electrodes is specifically as follows:
taking fresh venous blood samples, preparing 6 blood glucose solution samples with different concentration gradients, wherein the blood glucose solution samples are respectively S 1 、S 2 、S 3 、S 4 、S 5 S and S 6 By biochemical treatmentThe instrument scales all samples, and the concentration is as follows: 2.31mmol/L, 5.66mmol/L, 9.74mmol/L, 13.9mmol/L, 18.7mmol/L and 22.6mmol/L; connecting screen printing electrodes in the biosensor by a timing current method of an electrochemical workstation, sequentially adding corresponding samples, and starting a test; the parameter settings are shown in table 3:
TABLE 3 parameter set-up table for chronoamperometry
| InitE(V)＝0.25V | SmplIntvl(V)＝0.1 |
| Runtime(s)＝4 | Quiettime(s)＝0 |
Wherein: the calculation formula of the sensitivity is:specifically, S 1 And I 1,t Respectively represent blood sugar concentration S 1 S at the time of t and sample of (2) 1 A corresponding current value of the concentration; s is S x And I x,t Blood glucose concentration S 2 、S 3 、S 4 、S 5 S and S 6 S at t-time and any one of the samples x A corresponding current value of the concentration.
See table 4 for current versus concentration and time for different samples:
TABLE 4 Table 4 Current versus concentration versus time for various embodiments
Taking the 4 th s of the test as an example, the linear relation of the current with the concentration is shown in fig. 4;
referring to fig. 4 and table 4, the current intensity in example 1 is significantly higher than that in comparative example 4, so as to demonstrate that the electron transfer efficiency of the ruthenium polymer and oxidase formed into "wiring enzyme" by the crosslinking agent is significantly higher than that of the conventional ruthenium electron mediator, and the mechanism is that: the electron mediator used in example 1 forms linear molecules by directly forming a new substance with the enzyme, and can break the space barrier of the traditional ruthenium electron mediator in the electron transfer process, and the electron transfer resistance is smaller, so that the efficiency is high.
Referring to fig. 4, the current and concentration in example 1 and comparative examples 1 to 4 show a better linear relationship with respect to glucose in the range of 2.31 to 22.6, and referring specifically to table 5:
table 5 linear relationship tables of different embodiments
Referring to Table 5, the sensitivity of example 1 is significantly higher than that of comparative examples 1 to 4, and the mechanism is: in the ruthenium polymer electron mediator used in example 1, the 1, 10-phenanthroline derivative coordinates ruthenium, so that the ruthenium polymer electron mediator has good water solubility and is attached with a polymer with a modified functional group; meanwhile, the enzyme and the electron mediator form a 'wiring enzyme' through the chemical bond crosslinking of the crosslinking agent, so that the electron transmission efficiency and the enzyme stability are greatly improved.
The effects of examples 2 to 15 are similar to those of example 1, and thus it is demonstrated that the electronic mediator synthesized by the synthesis method and the biosensor prepared by the preparation method provided by the present application have advantages.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
1. The synthesis method of the electron mediator is characterized in that the electron mediator is a transition metal polymer electron mediator, and specifically comprises the following steps:
step S1.1: the first intermediate is synthesized, specifically: 1, 10-phenanthroline derivative and potassium bromide are mixed according to the following ratio of 1-3: 7-10 mass percent of the catalyst is added into a reaction vessel; sequentially adding concentrated sulfuric acid and concentrated nitric acid into a reaction container under ice bath condition to obtain a first mixed solution; turning into water bath, heating the first mixed solution to 65-90 ℃, and carrying out first reflux for 4-8 h to obtain a first reaction solution; adjusting the pH value of the first reaction solution to 6-8; treating with a first organic solvent to obtain a first intermediate;
step S1.2: the synthesis of the second intermediate, in particular: mixing the first intermediate with polyethylenimine according to the ratio of 0.5-1.5: adding the mixture into a second organic solvent according to the mass ratio of 5-8 to dissolve the mixture to obtain a second mixed solution; heating the second mixed solution to 50-70 ℃, and carrying out secondary reflux for 3-6 h to obtain a second reaction solution; cooling the second reaction solution to 5-25 ℃ to precipitate solid; treating with a third organic solvent to obtain a second intermediate;
step S1.3: the synthetic transition metal polymer electron mediator is specifically: the transition metal chloride and pyridine carboxylic acid or pyridine dicarboxylic acid homologue are mixed according to the following ratio of 2-4: adding the mixture into a fourth organic solvent according to the mass ratio of 1-3 to obtain a third mixed solution; reacting in an inert gas environment, and carrying out third reflux for 3-6 h to obtain a third reaction solution; cooling the third reaction solution to 10-30 ℃ and removing the solvent; adding 0.2-0.4 g of a second intermediate, heating to 90-130 ℃ and reacting for 18-36 h; cooling to separate out solid crystal; and (3) treating the mixture by a fifth organic solvent to obtain the transition metal polymer electron mediator.
2. The method of synthesis according to claim 1, wherein the firstThe organic solvent is any one or more of chloroform, tetrachloromethane or diethyl ether; the second organic solvent is one or more of methanol, ethanol or diethyl ether; the third organic solvent is any one of ethanol, acetone or toluene; the fourth organic solvent is any one or more of methanol, ethanol and diethyl ether; the fifth organic solvent is any one or more of methanol, ethanol and diethyl ether; the 1, 10-phenanthroline derivative is at least one of 1, 10-phenanthroline-4, 7-dicarboxylic acid, 5, 6-diamino-1, 10-phenanthroline and 4, 7-dimethyl-1, 10-phenanthroline; the pyridine carboxylic acid or pyridine dicarboxylic acid homologue is at least one of 4-methylpyridine-carboxylic acid, pyridine-2, 6-dicarboxylic acid, 4-chloro-2, 6-pyridine dicarboxylic acid, 2, 3-pyridine dicarboxylic acid, 3, 5-pyridine dicarboxylic acid methyl ester and pyridine-3, 5-dicarboxylic acid; the transition metal chloride is RuCl 3 ·3H 2 O、OsCl 3 ·3H 2 O、Cl 3 Rh and Cl 3 At least one of Ir.
3. The synthesis method according to claim 1, wherein in step S1.1: the specific operation of adjusting the pH of the first reaction solution is: after the reflux is finished, adding water into the reaction container to dilute the first reaction solution; adding carbonate or bicarbonate into the diluted first reaction solution until the solution is neutral; under the water bath condition, heating the first mixed solution to 70-80 ℃, wherein the heating time is 0.5-1.5 h, and the reflux time is 4-5 h;
in the step S1.2, the second mixed solution is heated to 50-60 ℃, the reflux time is 3-4 h, and the cooling temperature is 15-25 ℃;
in step S1.3: the inert gas is nitrogen, and the reflux time is 3-4 h.
4. A biosensor comprising an electron mediator synthesized according to the synthesis method of any one of claims 1 to 3.
5. A method for preparing a biosensor, comprising the steps of:
step S2.1: preparing an electron mediator solution and an oxidase solution, specifically: adding the transition metal polymer electron mediator obtained by the synthesis method according to any one of claims 1 to 3 into deionized water to obtain an electron mediator solution; wherein, in the electron mediator solution, the concentration of the electron mediator is 2-20 g/L;
adding an organic buffer solution, bovine serum albumin, amino acid salt, polyalcohol, an enzyme activator, xanthan gum and a surfactant into deionized water to obtain an enzyme protective agent; adding oxidase into an enzyme protecting agent to obtain an oxidase solution; wherein, in the oxidase solution, the concentration of the oxidase is 10-40 g/L;
step S2.2: the immobilized oxidase solution is specifically: the electron mediator solution, oxidase solution and cross-linking agent are mixed according to the proportion of 11-15: 4-8: 3-7, adding the mixture into a reaction container, and performing crosslinking reaction for 18-36 h to obtain a fixed oxidase solution;
step S2.3: the preparation method of the biosensor specifically comprises the following steps: taking 0.035-0.200 mu L of immobilized oxidase solution, dripping the oxidase solution on a screen printing electrode, drying, and attaching a hydrophilic film and double faced adhesive tape to obtain the biosensor.
6. The preparation method according to claim 5, wherein the organic buffer is any one or more of PIPES buffer, MOPS buffer and Bis-Tris buffer; the amino acid salt is any one or more of glycinate, histidine salt and aspartate; the polyalcohol is any one or more of mannitol, sorbitol and glycerol; the enzyme activator is any one or two of sodium cholate and chaps; the surfactant comprises sodium stearyl sulfate.
7. The method of claim 6, wherein the organic buffer is Bis-Tris buffer, the amino acid salt is glycinate, the polyol is sorbitol, and the enzyme activator is chaps.
8. The method according to claim 5, wherein the oxidase is any one of glucose oxidase, urate oxidase and cholesterol oxidase; the cross-linking agent is any one or more of polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether and genipin.
9. The method according to claim 5, wherein in step S2.2: the time of the crosslinking reaction is 18-24 hours; in step S2.3: the drying temperature is 30-60 ℃.
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| JP2008059805A (en) * | 2006-08-29 | 2008-03-13 | Toyota Motor Corp | Enzyme electrode and biological fuel cell and biosensor provided with the same |
| JP2008096352A (en) * | 2006-10-13 | 2008-04-24 | Toyota Motor Corp | Manufacturing method for enzyme electrode |
| CN113087901A (en) * | 2021-04-10 | 2021-07-09 | 可孚医疗科技股份有限公司 | Method for preparing electronic mediator |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2008059805A (en) * | 2006-08-29 | 2008-03-13 | Toyota Motor Corp | Enzyme electrode and biological fuel cell and biosensor provided with the same |
| JP2008096352A (en) * | 2006-10-13 | 2008-04-24 | Toyota Motor Corp | Manufacturing method for enzyme electrode |
| CN113087901A (en) * | 2021-04-10 | 2021-07-09 | 可孚医疗科技股份有限公司 | Method for preparing electronic mediator |
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