CN101231260B - Method for preparing biosensor using ionic liquid as green medium - Google Patents
Method for preparing biosensor using ionic liquid as green medium Download PDFInfo
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- CN101231260B CN101231260B CN2008100184974A CN200810018497A CN101231260B CN 101231260 B CN101231260 B CN 101231260B CN 2008100184974 A CN2008100184974 A CN 2008100184974A CN 200810018497 A CN200810018497 A CN 200810018497A CN 101231260 B CN101231260 B CN 101231260B
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- ionic liquid
- enzyme
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- ion liquid
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Abstract
The invention discloses a preparation method of a biological sensor which uses ion liquid as green medium. The method has the main steps as follows: (1) the ion liquid is purified, the ion liquid crude product is dissolved in 200 ml acetone, and inorganic salt is filtered and removed. (2) enzyme is embedded, 300 mg enzyme is added into 100 ml phosphate buffer solution, and the enzyme is caused to be completely dissolved by sufficiently stirring. 10 mL oil liquid purified according to the method (1) is added into the solution, and the solution is again acutely stirred for 2 hours (2500 turn/minute), positioned stationarily to cause lamination to appear, then the water phase is removed and the ion liquid of the lower layer is collected. (3) the biological sensor is prepared, 0.1 ml ion liquid embedding fluid prepared according to the method (2) is injected into a polypropylene tube. The lower end of the tube is sealed by a modified fiber film. A platinum thread is taken out of the upper end to a 0.5 cm position at the bottom. The upper end of the tube is sealed by conductive glue to make the biological sensor. The preparing method of the invention is simple. The width of the electrochemical window of the obtained biological sensor is more than 4 V, and the peak current of the basic body is only few nA. The electrochemistry respond is fast and the stability is good.
Description
Technical field
The present invention relates to a kind of preparation method of sensor, refer in particular to a kind of method for preparing biology sensor with ionic liquid as green medium.
Background technology
Biology sensor is to be sensitive material with immobilized biotic component (enzyme, protein, DNA, antibody, antigen, biological membrane etc.) or biosome itself (cell, microorganism, tissue etc.), combines with suitable chemical transducer to produce the device of a kind of fast detecting physics, chemistry and biomass.The application prospect that biology sensor is tempting makes it become the research focus of modern analysis chemistry rapidly, and bringing into play more and more important effect in the fast detecting in fields such as environment, biology, medical science and food security.
The core work of making biology sensor is an enzyme immobilization, and traditional process for fixation has absorption method, and investment, cross-linking method and covalent bond method, the shortcoming of their maximums are the decline significantly that causes enzymatic activity.In order to overcome this deficiency, in recent years people attempt adopting some one's own thing materials do carrier as poly-L-tyrosine (thank and be lost, Yuan if, Chai Yaqin; Gao Fengxian, Tang Mingyu, assay office; 1), peroxidating polypyrrole film (Jiang Xiaohua, Liu Weiqiang 2007,26 (9):; Chen Jianjun, SCI, 2007; 450) and tygon (Jiang Y, Wang AY, Kan JQ 28 (3):; Sensors and Actuators B, 2007,124:529) etc.Although these one's own thing materials have played certain effect to improving electrode stability and electric conductivity, they are difficult to provide best microenvironment for the catalytic action of enzyme.
In recent years, a kind of novel green solvent-ionic liquid at room temperature causes people's very big interest, obtains widespread use (AndersonJL, Armstrong DW, Wei GT, Analytical Chemistry, 2007,79 (11): 4247 at analytical chemistry; Rizvi SAA, Shamsi SA, Analytical Chemistry, 2006,78 (19): 7061).A host of facts show that ionic liquid and enzyme, substrate and other one's own thing material compatibilities are good, can significantly improve the active and stable of enzyme, these characteristics are that ionic liquid substitutes conventional adhesive or macromolecule village material has been established solid foundation (Lee JK as the biology sensor medium, Kim MJ, Journal of OrganicChemistry, 2002,67:6845; Maminska R, Dybko A, Wroblewski W, Sensors andActuators B, 2006,115:552; Lesniewski A, Niedziolka J, Palys B, Rizzi C, Gaillon L, Opallo M, Electrochemistry Communication, 2007,9:2580).Recently, Liu Yang of Changchun Inst. of Applied Chemistry, Chinese Academy of Sciences etc. discloses the method (Chinese patent, application number 200410011340.0 and 200610017201.8) of relevant ion liquid sol-gel compound membrane buried enzyme biologic sensor.Yet traditional 1-alkyl-3-methylimidazole class ionic liquid imidazole ring 2-position hydrogen atom is comparatively active, easily oxidized, causes electrochemical window to narrow down, and has limited their widespread uses in biology sensor and association area thereof.
Summary of the invention
One object of the present invention is to provide a kind of method for preparing biology sensor with ionic liquid as green medium.Resulting biology sensor institute matrix peak current is little, the fast and good stability of response.The enzyme of embedding does not have loss in the electrode, has than the long life.
Technical scheme provided by the invention is as follows:
(1) ion liquid purifying, 50～100g ionic liquid crude product is dissolved in 200～1000mL acetone, removes by filter inorganic salts; Handle with the decolouring of 50～250g super fine activated carbon and chromatographic grade aluminium oxide respectively, filter colourless solution; Filtrate is removed acetone, water and Organic Ingredients through decompression distillation and is prepared colourless, transparent oily liquids;
(2) embedding of enzyme in 100～200mL phosphate buffer solution (0.1～0.5mol/L, pH7～7.5), adds 30～50mg enzyme, fully stirs it is dissolved fully; In above-mentioned solution, add the ionic liquid of 10～15mL by (1) method purifying, vigorous stirring 2 hours (2500 rev/mins) again, standing demix then, aqueous phase discarded is collected the ionic liquid of lower floor;
(3) biology sensor preparation, the ionic liquid embedding liquid that 0.1～0.2mL is made by (2) method inject polypropylene tube (
In 2～5mm), (aperture 0.1～0.15 μ m) sealed with the modified cellulose film in the pipe lower end, insert from the upper end platinum filament (
1～1.2mm) to 0.2-0.5cm place bottom polypropylene tube, and biology sensor is made with conducting resinl envelope position in the upper end.
When embedding, it is in 7.0～7.5 the phosphate buffer solution that enzyme is dissolved in pH earlier, by extraction process it evenly is embedded among the ionic liquid again.
Described enzyme is a kind of in hydrogen peroxidase, horseradish peroxidase, glucose oxidase, dehydrogenasa or the polyphenol oxidase.
The ionic liquid that is adopted is to have 1-alkyl-2, and the ionic liquid of 3-methylimidazole structure, the alkyl in its kation comprise the various alkyl of carbon atom between 2～12, and negative ion comprises PF
6 -And N (CF
3)
2 -
Do not have tangible absorption peak when the ionic liquid behind the purifying is carried out spectral scan between 400-800nm, show that the colored compound in the ionic liquid is removed; Again with ionic liquid 250～300 ℃, be lower than under the vacuum condition of 0.01MPa and heated 1～2 hour, take the analysis of 1200L GC/MS-MS GC-MS to float on the composition of ionic liquid top, the result does not have obvious low-boiling compound to detect.
Because 1-alkyl-2, imidazole ring 2-position hydrogen atom is replaced by the methyl of inertia in the ionic liquid of 3-methylimidazole structure, improved ion liquid electrochemical stability, make prepared electrode have wide electrochemical window, thereby eliminated in testing process the interference that the oxidation-reduction because of electrode material itself causes, enlarged the range of application of electrode.Yet electrochemical research has strict demand to institute's materials used purity, can not satisfy the demand by the synthetic ionic liquid of existing method.Main impurity component comes from the oxidation in the not pure and mild course of reaction of raw material and produces colored compound in the ionic liquid.The former can select high pure raw material for use and heavily steam before use and solve, and the latter is big because of viscosity of il, adopts conventional discoloration method to be difficult to remove, become difficult point (the Earle MJ in the ionic liquid purifying, Gordon CM, Plechkova NV, Seddon KR, Welton T, Analytical Chemistry, 2007,79 (11): 4247), reduce its viscosity significantly but add suitable thinning agent at ionic liquid, the problems referred to above just solve easily.
The advantage that the present invention compared with prior art has is: 1, electrochemical window is wide, the 1-alkyl-2 that is adopted, the ionic liquid of 3-methylimidazole structure, 2-position hydrogen atom has fundamentally been eliminated the ionization or the oxidation of this hydrogen atom by methyl substituted on its kation imidazole ring; Anionicsite adopts the PF of galvanochemistry inertia
6 -And N (CF
3)
2 -, this type of ionic liquid has very desirable stability, and electrochemical window is all greater than 4V.Above character has not only been avoided the interference that causes because of medium generation redox reaction in testing process, and has expanded the range of application of biology sensor greatly.2, the matrix electric current is little, because ionic liquid itself conducts electricity, can be used as conducting medium.The ionic liquid that the present invention adopts has good stable, and its charging current is (see figure 1) in number nA scope only, much smaller than the charcoal paste electrode (in the mA level) that generally adopts now.The matrix electric current is little, and the matrix that helps reducing in the testing process disturbs and detection limit.3, long service life, the ionic liquid that is adopted have good one's own rerum natura, have improved the active and stable of enzyme greatly.The biology sensor of made of the present invention keeps measuring its electrochemical properties after 1 year, and its data are constant substantially.
Description of drawings
Fig. 1 is a 1-amyl group-2, the blank electrode cyclic voltammogram of 3-methylimidazole hexafluorophosphate ionic liquid film.
Embodiment
(1) ion liquid purifying
50g1-amyl group-2,3-methylimidazole class ionic liquid crude product is dissolved in 200mL acetone, removes by filter inorganic salts.Handled 4 hours with the decolouring of 50g super fine activated carbon and chromatographic grade aluminium oxide respectively, filter colourless solution.Filtrate is removed acetone, water and Organic Ingredients through decompression distillation and is prepared colourless, transparent oily liquids.
(2) catalatic embedding
The 100mL phosphate buffer solution (0.1mol/L, pH7) in, add the 30mg hydrogen peroxidase, fully stir and make its complete molten angle.The ionic liquid that in above-mentioned solution, adds the 10mL purifying, vigorous stirring 2 hours (2500 rev/mins) again, then, and standing demix, aqueous phase discarded is collected the ionic liquid of lower floor.
(3) preparation of hydrogen peroxide biology sensor
With 0.1mL ionic liquid embedding liquid inject polypropylene tube (
2mm), (aperture 0.1 μ m) sealed with the modified cellulose film in the pipe lower end, from upper end suction one platinum filament (
1mm) extremely from bottom 0.5cm place, then, biology sensor is made with conducting resinl envelope position in the upper end.
(4) electrochemical detection method
Adopt three-electrode system (the hydrogen peroxide biology sensor is a working electrode, and Pt silk electrode is that auxiliary electrode and Ag/AgCl normal electrode are contrast electrode), with the Na of 0.1mol/L
2HPO
412H
2O-KH
2PO
4Buffer solution (pH7) is test end liquid, feeds high pure nitrogen and removes dissolved oxygen, measures the cyclic voltammogram (CV) of system on electrochemical workstation.CV reduction peak current value is linear the minimizing with the increase of concentration of hydrogen peroxide, carries out the mensuration of concentration of hydrogen peroxide with this.
(5) analytical characteristics
With 1-amyl group-2,3-methylimidazole hexafluorophosphate/enzyme electrode is a working electrode, carries out CV by experimental technique and analyzes.We can clearly be seen that reduction peak current increases with the increase of concentration of hydrogen peroxide in the end liquid from CV figure, and linear in 3.17～12.4 μ mol/L scopes when hydrogen peroxide concentration, and its linear equation is y=-1.6776x-1.5577, R
2=0.9979.Wherein, y is electrode reduction peak current value (μ A), and x is concentration of hydrogen peroxide in the end liquid (mol/L), detects and is limited to 1.1 * 10
-6Mol/L.The response current that this enzyme electrode is measured hydrogen peroxide reaches stable at 5s with interior.After 4 ℃ of enzyme electrodes are preserved 30 days, measure its chemical property and do not have significant change.In addition, enzyme electrode has selectivity preferably.In measuring water 1 * 10
-5During the mol/L hydrogen peroxide, the Ca more than 1000 times
2+And Mg
2+, and the ascorbic acid of 5 times of concentration, glucose, citric acid, ethanol, acetate and dopamine do not produce interference, this may be relevant with ion liquid selective extraction.
(6) mensuration of trace amount hydrogen peroxide in the environmental water sample
The hydrogen peroxide biology sensor has been applied to determination of peroxide in the environmental water sample, the results are shown in table 1.The recovery of hydrogen peroxide is in the 95.3-100.8% scope, and this display packing has accuracy preferably.
Table 1: determination of peroxide in the environmental water sample
| Sample | Added (10 -5mol/L -1) | /Found (10 -5mol/L -1) | Recovery (％) |
| River underground water lake water sewage | 0.00 2.28 0.00 5.72 0.00 7.84 0.00 10.79 | 0.00 2.30 0.00 5.68 0.00 8.23 0.82 11.19 | 100.8 99.3 95.3 96.2 |
Claims (3)
1. one kind prepares the method for biology sensor with ionic liquid as green medium, and its key step is:
(1) ion liquid purifying, 50～100g ionic liquid crude product is dissolved in 200～1000mL acetone, removes by filter inorganic salts; Handle with the decolouring of 50～250g super fine activated carbon and chromatographic grade aluminium oxide respectively, filter colourless solution; Filtrate is removed acetone, water and Organic Ingredients through decompression distillation and is prepared colourless, transparent oily liquids;
(2) embedding of enzyme in 100～200mL phosphate buffer solution, adds 30～50mg enzyme, fully stirs it is dissolved fully; Add the ionic liquid of 10～15mL by (1) method purifying in above-mentioned solution, vigorous stirring is 2 hours again, and the rotating speed during stirring is 2500 rev/mins; Standing demix then, aqueous phase discarded is collected the ionic liquid of lower floor; The concentration of described phosphate buffer solution is 0.1～0.5mol/L, pH7～7.5;
(3) preparation of biology sensor with 0.1～0.2mL by the ionic liquid embedding liquid injection diameter that (2) method makes is
Polypropylene tube in, the pipe lower end is that the modified cellulose film of 0.1～0.15 μ m is sealed with the aperture, inserts a diameter from the upper end be
Platinum filament to from polypropylene tube bottom 0.2-0.5cm place, biology sensor is made with the conducting resinl envelope in the upper end;
The ionic liquid that is adopted is to have 1-alkyl-2, and the ionic liquid of 3-methylimidazole structure, the alkyl in its kation comprise the various alkyl of carbon atom between 2～12, and negative ion comprises PF
6 -And N (CF
3)
2 -
2. prepare the method for biology sensor with ionic liquid as green medium according to claim 1, it is characterized in that, described enzyme is a kind of in hydrogen peroxidase, horseradish peroxidase, glucose oxidase, dehydrogenasa or the polyphenol oxidase.
3. prepare the method for biology sensor with ionic liquid as green medium according to claim 1, it is characterized in that not having tangible absorption peak when the ionic liquid behind the purifying is carried out spectral scan between 400～800nm; Again with ionic liquid 250～300 ℃, be lower than under the vacuum condition of 0.01MPa and heated 1～2 hour, take the analysis of 1200L GC/MS-MS GC-MS to float on the composition of ionic liquid top, the result does not have obvious low-boiling compound to detect.
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| DE102008061866B9 (en) * | 2008-12-15 | 2012-12-20 | Forschungszentrum Jülich GmbH | Use of ionic liquids |
| CN103543187A (en) * | 2012-07-12 | 2014-01-29 | 西北师范大学 | Hydrophobic ionic liquid fixed cholesterol enzyme/Prussian blue modified electrode for detecting cholesterol |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661043A (en) * | 2004-12-10 | 2005-08-31 | 中国科学院长春应用化学研究所 | Biosensor prepared from embedding enzyme of sol-gel composite membrane of ion liquid |
| WO2007001355A2 (en) * | 2004-09-08 | 2007-01-04 | Rensselaer Polytechnic Institute | Enhanced stability of proteins immobilized on nanoparticles |
| CN1928554A (en) * | 2006-09-22 | 2007-03-14 | 中国科学院长春应用化学研究所 | Process for preparing ion liquid sol-gel compound membrane buried enzyme biologic sensor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007001355A2 (en) * | 2004-09-08 | 2007-01-04 | Rensselaer Polytechnic Institute | Enhanced stability of proteins immobilized on nanoparticles |
| CN1661043A (en) * | 2004-12-10 | 2005-08-31 | 中国科学院长春应用化学研究所 | Biosensor prepared from embedding enzyme of sol-gel composite membrane of ion liquid |
| CN1928554A (en) * | 2006-09-22 | 2007-03-14 | 中国科学院长春应用化学研究所 | Process for preparing ion liquid sol-gel compound membrane buried enzyme biologic sensor |
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