CN1082186C - Method for preparing biosensor by using organic hydrogel immobilized enzyme - Google Patents
Method for preparing biosensor by using organic hydrogel immobilized enzyme Download PDFInfo
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- CN1082186C CN1082186C CN 95119390 CN95119390A CN1082186C CN 1082186 C CN1082186 C CN 1082186C CN 95119390 CN95119390 CN 95119390 CN 95119390 A CN95119390 A CN 95119390A CN 1082186 C CN1082186 C CN 1082186C
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 title claims abstract description 10
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims abstract description 36
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 17
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000004094 surface-active agent Substances 0.000 claims abstract description 10
- 108010025899 gelatin film Proteins 0.000 claims description 7
- LJCNDNBULVLKSG-UHFFFAOYSA-N 2-aminoacetic acid;butane Chemical compound CCCC.CCCC.NCC(O)=O LJCNDNBULVLKSG-UHFFFAOYSA-N 0.000 claims description 4
- 108010015428 Bilirubin oxidase Proteins 0.000 claims description 4
- 108010089254 Cholesterol oxidase Proteins 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 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 claims description 3
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims description 2
- 108010025188 Alcohol oxidase Proteins 0.000 claims description 2
- 101100206191 Arabidopsis thaliana TCP21 gene Proteins 0.000 claims description 2
- 108010000659 Choline oxidase Proteins 0.000 claims description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 2
- 108010029541 Laccase Proteins 0.000 claims description 2
- 102000004882 Lipase Human genes 0.000 claims description 2
- 108090001060 Lipase Proteins 0.000 claims description 2
- 239000004367 Lipase Substances 0.000 claims description 2
- 102000003992 Peroxidases Human genes 0.000 claims description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims description 2
- 235000019421 lipase Nutrition 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 abstract description 10
- 239000007864 aqueous solution Substances 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000011259 mixed solution Substances 0.000 abstract description 2
- 239000000499 gel Substances 0.000 abstract 1
- 239000012046 mixed solvent Substances 0.000 abstract 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 8
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 6
- 239000012074 organic phase Substances 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 150000002825 nitriles Chemical class 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000003425 Tyrosinase Human genes 0.000 description 3
- 108060008724 Tyrosinase Proteins 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000011067 equilibration Methods 0.000 description 3
- 150000002989 phenols Chemical class 0.000 description 3
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 229920003026 Acene Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical class C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- LSXWFXONGKSEMY-UHFFFAOYSA-N di-tert-butyl peroxide Chemical compound CC(C)(C)OOC(C)(C)C LSXWFXONGKSEMY-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 150000001451 organic peroxides Chemical class 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- -1 tert-butyl peroxide Chemical class 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention belongs to a method for preparing a biosensor by using organic hydrogel immobilized enzyme, wherein when the biosensor is prepared, enzyme, a mediator, 0.1-5% of surfactant and 6-20% of aqueous solution of polyvinyl alcohol are uniformly mixed, 60-85% of dimethylformamide is added, the mixed solution is dripped on the surface of an electrode, and gel forming is carried out for 5-20 minutes at room temperature; the preparation process is rapid, and the prepared biosensor has excellent performance: has higher detection sensitivity to water-soluble and water-insoluble substances, and is suitable for biological detection in various media such as completely anhydrous organic solvents, oil-water mixed solvents, water and the like.
Description
The invention belongs to organic hydrogel immobilized enzyme and prepare the method for biology sensor.
Organic phase biologic sensor, especially enzyme electrode have greatly been expanded Study on Biosensor and range of application, have a wide range of applications in fields such as organic chemical industry, bioengineering, medicine analysis, environmental protection.1988, people's reported first such as Hall organic phase enzyme electrode [G.F.Hall, D.I.Best, A.P.F.Tumer, EnzymeMicrob.Technol., 10 (1988) 543].Thenceforth so far, reported the organic phase enzyme electrode of many kinds, but all adopted conventional in the past water biology sensor process for fixation preparation, and must in organic solvent, add a certain amount of water, to guarantee the catalytic activity of enzyme.For example, J.Wang etc. are with Eastman AQ fixedly horseradish peroxidase and Tyrosinase, measure hydrogen peroxide and phenol in the nitrile, must add 4% water enzyme electrode and just have response [J.Wang, Y.Lin, L.Chen, Analyst, 118 (1993) 277] in nitrile.
Shaojun Dong, Yizhu Guo, Anal.Chem., 66 (1994) 3895; Shaojun Dong, Yizhu Guo, J.Chem.Soc., Chem.Commun., (1995) 483 report organic phase biologic sensor prepared from low temperature hydrogel enzymes, because carrier itself can provide little water environment for immobilised enzymes, need not to add water in medium, has realized the biological detection of hydrogen peroxide, phenolic compound in the real complete anhydrous organic solvent.But to water-insoluble compound, this method is powerless.
The objective of the invention is to add the organic solvent dimethyl formamide in the aqueous solution of surfactant and polyvinyl alcohol (PVA), gelation takes place at room temperature 5-20 minute in this mixed solution, prepares organic hydrogel.Preparation is during organic phase biologic sensor, with the aqueous solution of enzyme, mediator and surfactant and polyvinyl alcohol (PVA) even after, the dimethyl formamide of adding 60-75%, gel film forming under the room temperature.Preparation process is rapid, and it is short that the biology sensor of preparing reaches equilibration time, and response is fast.
The present invention is with percentage by weight: add the 60-75% dimethyl formamide in 0.5-5% surfactant and the 8-15% polyvinyl alcohol water solution, placed 5-20 minute under the room temperature, prepare organic hydrogel; During the preparation biology sensor, with enzyme, comprise peroxidase, cholesterol oxidase, Tyrosine oxidase, alcohol oxidase, alcohol dehydrogenase, laccase, CHE, choline oxidase, bilirubin oxidase, lipase etc., also can the aqueous solution of mediator iron hydrofining, ferrocene and derivant, organic dyestuff and surfactant and polyvinyl alcohol (PVA) is even, add dimethyl formamide, should mix drop and be coated onto the basal electrode surface, gel film forming under the room temperature; This biology sensor all has biocatalysis at chloroform, chlorobenzene, ethylene dichloride, dioxan, in the organic solvents such as nitrile, acetone, methyl alcohol, ethanol, normal butyl alcohol, n-octyl alcohol, cyclohexanone, tetrahydrofuran.
Owing to comprise " alembroth " dimethyl formamide and water in organic hydrogel of the present invention's preparation, it is to water-soluble substances, as hydrogen peroxide, phenolic compound etc., and water-insoluble materials, as organic superoxide, cholerythrin? cholesterol etc. all have bigger partition factor, thereby water-soluble and non-water soluble substance are all had bigger detection sensitivity.
The biology sensor of the present invention's preparation is compared with the various process for fixation of reporting in the past, and have following characteristic: (1) carrier can provide little water environment for immobilised enzymes; (2), comprise water and water-containing organic solvent at various media? stable existence in the complete anhydrous organic solvent; (3) all kinds of analytes all had bigger partition factor and detection sensitivity; (4) preparation process is rapid, need not specific installation.Therefore, this biology sensor wide adaptability both can also can be worked in absolute anhydrous organic phase in water or organic water phase.
Embodiment provided by the invention is as follows:
Embodiment 1: the horseradish peroxidase electrode.3 milligrams of horseradish peroxidases are dissolved in the aqueous solution of 50ml5% surfactant and 15% polyvinyl alcohol (PVA), add the dimethyl formamide of 100ml again, after mixing, this is mixed drop be coated in graphite electrode surface, following about 10 minutes gel film forming of room temperature.This enzyme electrode is to organic peroxides such as tert-butyl peroxide, peroxidating-2-butanone, the different acenes of hydrogen peroxide, and in the presence of the medialess body, all have sensitive response, and have excellent response performance: it is short to reach equilibration time, within 10 minutes; Response time is fast, within 1 minute; Measurement range is wide, 0-5mM.Can in organic solvents such as chloroform, chlorobenzene, alcohols, work.
Embodiment 2: the Tyrosinase electrode.1.5 milligrams of Tyrosinases are dissolved in the aqueous solution of 50ml 2% surfactant and 12% polyvinyl alcohol (PVA), add the 75ml dimethyl formamide again, after stirring, drip and to be coated onto the glass-carbon electrode surface, gel film forming about 15 minutes.This electrode performance: equilibration time, within 5 minutes; Response time, 0.5-1 minute.This enzyme electrode is measured chloroform, ethylene dichloride, the phenolic compound in the organic solvent such as nitrile.
Embodiment 3: the bilirubin oxidase electrode.2 milligrams of bilirubin oxidases are dissolved in the aqueous solution of 50ml 0.5% surfactant and 8% polyvinyl alcohol (PVA), add the 150ml dimethyl formamide again, after mixing, drip and to be coated onto the glass-carbon electrode surface, gel film forming about 8 minutes.This electrode is a mediator with the carboxylic acid ferrocene, measures the cholerythrin in chloroform, the dimethyl formamide.
Embodiment 4: the cholesterol oxidase electrode.4 milligrams of cholesterol oxidases are dissolved in the aqueous solution of 50ml 1% surfactant and 10% polyvinyl alcohol (PVA), add the 150ml dimethyl formamide again, after mixing, drip the glass-carbon electrode surface that is coated onto pre-modification platinum particulate, gel film forming about 8 minutes.This enzyme electrode is used for measuring the cholesterol of organic solvents such as chloroform, chlorobenzene.
Claims (3)
1. an organic hydrogel immobilized enzyme prepares the method for biology sensor, it is characterized in that with percentage by weight: in 0.5~5% surfactant and 8~15% polyvinyl alcohol water solutions, add enzyme or enzyme and mediator, after mixing, add the 60-75% dimethyl formamide, should mix drop and be coated onto the basal electrode surface, place the gel film forming under the room temperature 5~20 minutes.
2. organic hydrogel immobilized enzyme as claimed in claim 1 prepares method for biosensor, it is characterized in that described enzyme is peroxidase, cholesterol oxidase, Tyrosine oxidase, alcohol oxidase, alcohol dehydrogenase, laccase, CHE, choline oxidase, bilirubin oxidase or lipase.
3. organic according to claim 1 hydrogel immobilized enzyme prepares method for biosensor, it is characterized in that described mediator is the carboxylic acid ferrocene.
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CN 95119390 CN1082186C (en) | 1995-12-22 | 1995-12-22 | Method for preparing biosensor by using organic hydrogel immobilized enzyme |
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CN 95119390 CN1082186C (en) | 1995-12-22 | 1995-12-22 | Method for preparing biosensor by using organic hydrogel immobilized enzyme |
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CN1067430C (en) * | 1997-09-15 | 2001-06-20 | 中国科学院长春应用化学研究所 | Method for preparing bio-sensor with embedding enzyme of collosol-gel |
BRPI0619833A2 (en) * | 2005-12-16 | 2011-10-18 | Bayer Healthcare Llc | transdermal patch sensor assembly and methods of use |
JP5572016B2 (en) * | 2009-08-04 | 2014-08-13 | シスメックス株式会社 | Tissue fluid extraction device, manufacturing method thereof, and tissue fluid analysis method using the device |
CN105651841B (en) * | 2016-01-05 | 2018-03-06 | 宁夏医科大学 | The preparation method of the multiple stimulation response type hydrogel of multi-factor structure component film layer by layer |
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