CN101105471A - Preparation method of enzyme electrode for detecting glucose - Google Patents
Preparation method of enzyme electrode for detecting glucose Download PDFInfo
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- CN101105471A CN101105471A CNA2007100253236A CN200710025323A CN101105471A CN 101105471 A CN101105471 A CN 101105471A CN A2007100253236 A CNA2007100253236 A CN A2007100253236A CN 200710025323 A CN200710025323 A CN 200710025323A CN 101105471 A CN101105471 A CN 101105471A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 23
- 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 title claims abstract description 19
- 239000008103 glucose Substances 0.000 title claims abstract description 19
- 108010010803 Gelatin Proteins 0.000 claims abstract description 38
- 239000008273 gelatin Substances 0.000 claims abstract description 38
- 229920000159 gelatin Polymers 0.000 claims abstract description 38
- 235000019322 gelatine Nutrition 0.000 claims abstract description 38
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 38
- 229940088598 enzyme Drugs 0.000 claims abstract description 22
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 19
- 239000004366 Glucose oxidase Substances 0.000 claims abstract description 19
- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 19
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 19
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229960003351 prussian blue Drugs 0.000 claims abstract description 16
- 239000013225 prussian blue Substances 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 65
- 239000007864 aqueous solution Substances 0.000 claims description 26
- 239000003792 electrolyte Substances 0.000 claims description 18
- 229920000447 polyanionic polymer Polymers 0.000 claims description 13
- 229910019931 (NH4)2Fe(SO4)2 Inorganic materials 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 229920000867 polyelectrolyte Polymers 0.000 claims description 8
- 240000002853 Nelumbo nucifera Species 0.000 claims description 6
- 235000006508 Nelumbo nucifera Nutrition 0.000 claims description 6
- 235000006510 Nelumbo pentapetala Nutrition 0.000 claims description 6
- -1 dextran sulfate Chemical compound 0.000 claims description 6
- 229920001467 poly(styrenesulfonates) Polymers 0.000 claims description 6
- 238000011896 sensitive detection Methods 0.000 claims description 5
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 4
- 229940059939 kayexalate Drugs 0.000 claims description 4
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 4
- 229920002554 vinyl polymer Polymers 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 229920001495 poly(sodium acrylate) polymer Polymers 0.000 claims description 3
- NNMHYFLPFNGQFZ-UHFFFAOYSA-M sodium polyacrylate Chemical compound [Na+].[O-]C(=O)C=C NNMHYFLPFNGQFZ-UHFFFAOYSA-M 0.000 claims description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 2
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 2
- 102000008186 Collagen Human genes 0.000 claims description 2
- 108010035532 Collagen Proteins 0.000 claims description 2
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 claims description 2
- 229920002971 Heparan sulfate Polymers 0.000 claims description 2
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 2
- 229920001436 collagen Polymers 0.000 claims description 2
- 229960000633 dextran sulfate Drugs 0.000 claims description 2
- 229920000669 heparin Polymers 0.000 claims description 2
- 229960002897 heparin Drugs 0.000 claims description 2
- 238000007654 immersion Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 235000010413 sodium alginate Nutrition 0.000 claims description 2
- 229940005550 sodium alginate Drugs 0.000 claims description 2
- 239000000661 sodium alginate Substances 0.000 claims description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 12
- 230000004044 response Effects 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000003068 static effect Effects 0.000 abstract description 5
- 108090000854 Oxidoreductases Proteins 0.000 abstract description 3
- 102000004316 Oxidoreductases Human genes 0.000 abstract description 3
- 238000001338 self-assembly Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 238000005498 polishing Methods 0.000 description 8
- 125000000217 alkyl group Chemical group 0.000 description 6
- IQDGSYLLQPDQDV-UHFFFAOYSA-N dimethylazanium;chloride Chemical compound Cl.CNC IQDGSYLLQPDQDV-UHFFFAOYSA-N 0.000 description 6
- PYGSKMBEVAICCR-UHFFFAOYSA-N hexa-1,5-diene Chemical group C=CCCC=C PYGSKMBEVAICCR-UHFFFAOYSA-N 0.000 description 6
- 229910052697 platinum Inorganic materials 0.000 description 5
- 238000004506 ultrasonic cleaning Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a preparation method of enzyme electrode which has high sensitivity and is used to detect glucose; a prussian blue film modified electrode prepared by static self-assembly technique is a base; a double-layer gelatin imbedment fixes a glucose oxidase. As the number of layers of the static self-assembly can be controlled and double layers of gelatin imbedment in different densities can guarantee the enzyme not to be leaked as well as guarantee the oxidase be in sound activity, the enzyme electrode has relatively high sensitivity and swift response performance; furthermore, the number of assembly layers can be changed according to the actual demand and forms the enzyme electrode in different sensitivity.
Description
Technical field
The present invention relates to a kind of preparation method of enzyme electrode, relate in particular to a kind of preparation method of enzyme electrode of highly sensitive detection glucose, prepared electrode can be applied to the detection of low concentration glucose in food or the medical and health.
Background technology
Reported based on measuring O from Clark etc.
2[Clark L C.[P] USA:3529455 since the biology sensor of reduction current, 1970], this field has obtained extensive, deep research, and the amperometric biosensor that dissimilar oxidase is formed in conjunction with platinum electrode is widely used in practice.Mostly these biology sensors are to utilize the material of Direct Electrochemistry method detection of enzymatic reactions or product and can not detect the analyte of low concentration, in addition because when measuring the reduction current of oxygen, the size of electric current is subjected to the influence of Pt electrode (or other noble metal electrode) surface state deeply, therefore the poor reproducibility that makes measurement measures H based on the platinum electrode method in the biology sensor of reporting at present
2O
2The application of the enzyme sensor of oxidation current is more extensive.Oxidation or the inorganic amboceptor of galvanochemistry of reduction such as the assembling that Prussian blue (PB) is used for the oxidase biology sensor that can catalyzing hydrogen peroxide can make and use electromotive force and descend greatly, can avoid many electrochemical interference thereupon.First example based on the glucose sensor that uses the PB modified electrode is by [KaryakinA.A. such as Karyakin, Gitelmacher O., Karyakina E.E..AAnal.Lett..1994,27 (15): 2861-2869.] report, it continues to have optimized this sensor [Karyakin AA, Gitelmacher O V, Karyakina E E.Anal.Chem..1995 again in nineteen ninety-five, 67:2419-2423], this biology sensor has the low detectability and the wide range of linearity.At present, one of them the main problem of research based on the sensor of PB is exactly an insufficient sensitivity.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of enzyme electrode of highly sensitive detection glucose is applicable to the detection of low concentration glucose in food or the medical and health, and method is simple, has degree of controllability, and higher sensitivity is arranged.
Technical scheme of the present invention is: a kind of preparation method who detects the enzyme electrode of glucose, and its concrete preparation process is as follows:
1) polycation or polyanion electrolyte and acid solution are mixed with deionized water solution, strut body electrode is carried out pre-service, be immersed in then in the polycation electrolyte, make the strut body electrode surface lotus that becomes positively charged; Perhaps pretreated supporter is immersed in the polyanion electrolyte, then negative charge is with on the strut body electrode surface;
2) the lotus strut body electrode that will become positively charged after above-mentioned steps is handled immerses earlier K
4Fe (CN)
6Or K
3Fe (CN)
6With the aqueous solution of KCl, use N
2Dry up, immerse the FeCl of same concentrations again
3Or (NH
4)
2Fe (SO
4)
2In the KCl aqueous solution, use N
2Dry up; Or be with the negative charge strut body electrode to immerse FeCl earlier after will handling
3Perhaps (NH
4)
2Fe (SO
4)
2In the aqueous solution of KCl, use N
2Dry up the K that immerses same concentrations again
4Fe (CN)
6Perhaps K
3Fe (CN)
6With the aqueous solution of KCl, use N
2Dry up;
3) repeating step 2) 1-80 time, obtain polyelectrolyte/Prussian blue membrane electrode;
4) prepare the gelatin solution of glucose oxidase solution and two kinds of variable concentrations respectively, the concentration of gelatin solution 2 is greater than the concentration of gelatin solution 1; Get glucose oxidase solution and gelatin solution 1 mixes; Pipette above-mentioned mixed solution and evenly drip on the electrode that step 3) makes, place refrigerator; And then the electrode surface after will handling is impregnated in the gelatin solution 2 back and takes out, and places the refrigerator back to take out, the enzyme electrode of highly sensitive detection glucose.
Wherein said polyanion electrolyte is a kind of or wherein any two kinds potpourri in kayexalate, polyvinyl sulfonate, dextran sulfate, chondroitin sulfate, sodium alginate, sodium polyacrylate, polymethylacrylic acid, heparin or the heparin sulfate; Described polycation electrolyte is a kind of or wherein any two kinds potpourri in PAH salt, poly-dipropenyl dimethyl ammonium chloride, polyvinyl alcohol (PVA), collagen, shitosan, poly-D-lysine or the polyvinyl inferior amine salt hydrochlorate.Polyanion or polycation electrolyte and acid solution are mixed with in the deionized water solution, and polyanion or polycation electrolyte concentration are 0.005-0.05mol/L, and the pH value of solion is controlled at 1-4.
Wherein said strut body electrode is metal electrode or glass electrode.
Step 2 wherein) K in the aqueous solution
4Fe (CN)
6, K
3Fe (CN)
6, FeCl
3Perhaps (NH
4)
2Fe (SO
4)
2Concentration be 1-500mM, KCl concentration is 0.1-0.5M.Preferred repeating step 2) number of times is 5-40 time, obtains polyelectrolyte/Prussian blue membrane electrode.Preferred steps 2) strut body electrode in is at K
4Fe (CN)
6Or K
3Fe (CN)
6With the aqueous solution of KCl, perhaps at the FeCl of same concentrations
3Or (NH
4)
2Fe (SO
4)
2Be respectively 20s-2min with the immersion time in the KCl aqueous solution.
Preferred above-mentioned steps 4) concentration of preparation glucose oxidase solution is 100-10000IU/ml in, and the mass percentage concentration of gelatin solution 1 is 0.1-1.5%, and the mass percentage concentration of gelatin solution 2 is 2-4%.
Wherein pipette glucose oxidase solution and gelatin solution 1 volume ratio in the step 4) and be and mixed in 1: 10～1: 1000.Pipette mixed solution and evenly drip on the electrode that step 3) makes, placed refrigerator 2-24 hour; And then the electrode surface after will handling is impregnated in the gelatin solution 2 and takes out behind the 2-60s, place after refrigerator 2-24 hour and take out, the enzyme electrode of highly sensitive detection glucose.
The preprocess method of strut body electrode is with reference to common methods, and the preferred following method of the pre-service of metal electrode: abrasive paper for metallograph polishing electrode surface, polishing is to smooth no special reflective.Polish electrode surface with burnishing powder.The H of dose volume than 3: 7 again
2O
2, H
2SO
4Mixed solution is put into electrode and is wherein flooded 2-24h.Taking-up is with deionized water rinsing and at 60 ℃ of following ultrasonic cleaning 10-30min.
With the preparation glucose oxidase electrode as working electrode, with the Ag/AgCl electrode is contrast electrode, platinum electrode is for to construct three-electrode system to electrode, single compartment electrolytic cell continues to stir, use the time current curve after CHI660 (Shanghai occasion China) electrochemical workstation detects the glucose that adds variable concentrations, calculate its sensitivity and can reach 60mA/cm
2About M, the response time is less than 20s, in this performance well beyond present same type of sensor aspect two.
Beneficial effect:
The present invention is a kind of based on the enzyme electrode preparation technology of static self-assembling technique in conjunction with double-deck gelatin embedding enzyme immobilization method.With the Prussian blue modified electrode of static self-assembling technique preparation as substrate, double-deck gelatin embedding fixing glucose oxidase.Because static self-assembling technique film forming driving force derives from the electrostatic forcing between positive and negative charge, interosculate with ionic link between them, acting force is stronger, can control thickness by the number of times that Control Circulation is alternately filmed, and really reaches from the molecular level the control to membrane material and membrane structure; The gelatin embedding of double-deck variable concentrations had both guaranteed that enzyme can not leak, guarantee that again enzyme has good activity, so prepared enzyme electrode can be applied to the detection of food industry and pharmaceutical sanitary field low concentration glucose, and higher sensitivity and response performance are fast arranged, and the number of plies that can change self assembly PB film changes the sensitivity of enzyme electrode to adapt to different needs.The inventive method technology simple economy, good reproducibility, controllable degree height, response performance good (as depicted in figs. 1 and 2)
Description of drawings
Fig. 1 is the response curve of prepared enzyme electrode to variable concentrations glucose, and wherein transverse axis is represented concentration of glucose (unit is 1 * 10
-6M), vertical axis represents current response intensity (unit 1 * 10
-7A).
Fig. 2 is based on the response curve of the Prussian blue enzyme electrode of the different numbers of plies to glucose, and wherein transverse axis is represented concentration of glucose (unit is 1 * 10
-6M), vertical axis represents current response intensity (unit 1 * 10
-7A); ■ is 15 layers, ● be 10 layers, ▲ be 5 layers.
Embodiment
Embodiment 1
1) will gather in the diallyl alkyl dimethyl ammonium chloride and add hydrochloric acid, and be mixed with said polycation solution, wherein poly-diallyl alkyl dimethyl ammonium chloride concentration is 0.005mol/L, and pH is 0.1mol/L;
2) with platinum electrode abrasive paper for metallograph polishing electrode surface, polishing is to smooth no special reflective.Polish electrode surface with burnishing powder.The H of dose volume than 3: 7 again
2O
2, H
2SO
4Mixed solution is put into electrode and is wherein flooded 2h.Taking-up is with deionized water rinsing and at 60 ℃ of following ultrasonic cleaning 10min.Be immersed in 10min in the poly-diallyl alkyl dimethyl ammonium chloride electrolyte, make the supporting body surface lotus that becomes positively charged;
3) prepare FeCl respectively
3, K
4Fe (CN)
6With the KCl aqueous solution, FeCl wherein
3, K
4Fe (CN)
6Concentration be 1mM, the concentration of KCl is 0.1M;
4) electrode after will handling immerses K earlier
4Fe (CN)
6KCl aqueous solution 20s, use N
2Dry up and immerse FeCl again
3The KCl aqueous solution in 20s, use N
2Dry up;
5) repeating step 4) 20 times, obtain polyelectrolyte/Prussian blue membrane electrode;
6) glucose oxidase solution of preparation 100IU/ml, massfraction is 0.1% gelatin solution 1 and 2% gelatin solution 2;
7) get 1 μ l glucose oxidase solution and 50 μ l gelatin solutions 1 fully mix.Pipette above-mentioned solution 5 μ l evenly drip in step 5) make on the electrode, placed refrigerator 12 hours;
8) electrode surface after step 7) is handled is impregnated in the gelatin solution 2 and takes out behind the 60s, places refrigerator to take out after 12 hours.
Embodiment 2
1) with adding hydrochloric acid in the kayexalate (PSS), be configured to polyanion solution, wherein kayexalate concentration is 0.01mol/L, and pH is 0.01mol/L;
2) with gold electrode with the abrasive paper for metallograph electrode surface of polishing, polishing is to smooth no special reflective.Polish electrode surface with burnishing powder.The H of dose volume than 3: 7 again
2O
2, H
2SO
4Mixed solution is put into electrode and is wherein flooded 2h.Taking-up is with deionized water rinsing and at 60 ℃ of following ultrasonic cleaning 10min.Be immersed in 10min in the PSS electrolyte, make negative charge on the supporting body surface band;
3) prepare (NH respectively
4)
2Fe (SO
4)
2, K
3Fe (CN)
6With the aqueous solution of KCl, wherein (NH
4)
2Fe (SO
4)
2, K
3Fe (CN)
6Concentration be 20mM, the concentration of KCl is 0.2M;
4) electrode after will handling immerses (NH earlier
4)
2Fe (SO
4)
2With 40s in the KCl aqueous solution, dry up with N2 and to immerse K again
3Fe (CN)
6With 40s in the aqueous solution of KCl, use N
2Dry up;
5) repeating step 4) 10 times, obtain polyelectrolyte/Prussian blue membrane electrode;
6) glucose oxidase solution of preparation 5000IU/ml, massfraction is 0.5% gelatin solution 1 and 2.5% gelatin solution 2;
7) get 1 μ l glucose oxidase solution and 500 μ l gelatin solutions 1 fully mix.Pipette above-mentioned solution 2 μ l evenly drip in step 5) make on the electrode, placed refrigerator 24 hours;
8) electrode surface after step 7) is handled is impregnated in the gelatin solution 2 and takes out behind the 20s, places refrigerator to take out after 24 hours.
Embodiment 3
1) add hydrochloric acid in the potpourri with PAA, polymethylacrylic acid, be configured to polyanion solution, wherein the total concentration of sodium polyacrylate, polymethylacrylic acid is 0.05mol/L, and pH is 0.001mol/L;
2) the ito glass electrode is at first used acetone ultrasonic cleaning 30min, the H of dose volume again than 3: 7
2O
2, H
2SO
4Mixed solution is put into electrode and is wherein flooded 24h.Take out the back with the ultrasonic 30min of deionized water, be immersed in 10min in the polyanion electrolyte, make negative charge on the supporting body surface band;
3) prepare (NH respectively
4)
2Fe (SO
4)
2, K
3Fe (CN)
6With KCl aqueous solution, wherein (NH
4)
2Fe (SO
4)
2, K
3Fe (CN)
6Concentration be 100mM, the concentration of KCl is 0.5M;
4) electrode after will handling immerses (NH earlier
4)
2Fe (SO
4)
2Aqueous solution 40s with KCl uses N
2Dry up and immerse K again
3Fe (CN)
6With 40s in the aqueous solution of KCl, use N
2Dry up;
5) repeating step 4) 5 times, obtain polyelectrolyte/Prussian blue membrane electrode;
6) glucose oxidase solution of preparation 10000IU/ml, massfraction is 1.5% gelatin solution 1 and 4% gelatin solution 2;
7) get 1 μ l glucose oxidase solution and 1000 μ l gelatin solutions 1 fully mix.Pipette above-mentioned solution 2.5 μ l evenly drip in step 5) make on the electrode, placed refrigerator 24 hours;
8) electrode surface after step 7) is handled is impregnated in the gelatin solution 2 and takes out behind the 2s, places refrigerator to take out after 24 hours.
Embodiment 4
1) will gather in the diallyl alkyl dimethyl ammonium chloride and add hydrochloric acid, and be mixed with said polycation solution, wherein poly-diallyl alkyl dimethyl ammonium chloride concentration is 0.005mol/L, and pH is 0.1mol/L;
2) with platinum electrode abrasive paper for metallograph polishing electrode surface, polishing is to smooth no special reflective.Polish electrode surface with burnishing powder.The H of dose volume than 3: 7 again
2O
2, H
2SO
4Mixed solution is put into electrode and is wherein flooded 2h.Taking-up is with deionized water rinsing and at 60 ℃ of following ultrasonic cleaning 10min.Be immersed in 10min in the poly-diallyl alkyl dimethyl ammonium chloride electrolyte, make the supporting body surface lotus that becomes positively charged;
3) prepare FeCl respectively
3, K
4Fe (CN)
6With the aqueous solution of KCl, wherein FeCl
3, K
4Fe (CN)
6Concentration be 500mM, the concentration of KCl is 0.5M;
4) electrode after will handling immerses K earlier
4Fe (CN)
6With 2min in the aqueous solution of KCl, use N
2Dry up and immerse FeCl again
3With 2min in the aqueous solution of KCl, use N
2Dry up;
5) repeating step 4) 60 times, obtain polyelectrolyte/Prussian blue membrane electrode;
6) glucose oxidase solution of preparation 10000IU/ml, massfraction is 1.5% gelatin solution 1 and 4% gelatin solution 2;
7) get 1 μ l glucose oxidase solution and 1000 μ l gelatin solutions 1 fully mix.Pipette above-mentioned solution 5 μ l evenly drip in step 5) make on the electrode, placed refrigerator 12 hours;
8) electrode surface after step 7) is handled is impregnated in the gelatin solution 2 and takes out behind the 2s, places refrigerator to take out after 24 hours.
Claims (9)
1. preparation method who detects the enzyme electrode of glucose, its concrete preparation process is as follows:
1) polycation or polyanion electrolyte and acid solution are mixed with deionized water solution, strut body electrode is carried out pre-service, be immersed in then in the polycation electrolyte, make the strut body electrode surface lotus that becomes positively charged; Perhaps pretreated supporter is immersed in the polyanion electrolyte, then makes the strut body electrode surface be with negative charge;
2) the lotus strut body electrode that will become positively charged after above-mentioned steps is handled immerses earlier K
4Fe (CN)
6Or K
3Fe (CN)
6In the aqueous solution of KCl, use N
2Dry up, immerse the FeCl of same concentrations again
3Or (NH
4)
2Fe (SO
4)
2In the aqueous solution of KCl, use N
2Dry up; Or be with the negative charge strut body electrode to immerse FeCl earlier after will handling
3Perhaps (NH
4)
2Fe (SO
4)
2In the aqueous solution of KCl, use N
2Dry up the K that immerses same concentrations again
4Fe (CN)
6Perhaps K
3Fe (CN)
6With the aqueous solution of KCl, use N
2Dry up;
3) repeating step 2) 1-80 time, obtain polyelectrolyte/Prussian blue membrane electrode;
4) prepare the gelatin solution of glucose oxidase solution and two kinds of variable concentrations respectively, the concentration of gelatin solution 2 is greater than the concentration of gelatin solution 1; Get glucose oxidase solution and gelatin solution 1 mixes; Pipette above-mentioned mixed solution and evenly drip on the electrode that step 3) makes, place refrigerator; And then the electrode surface after will handling is impregnated in the gelatin solution 2 back and takes out, and places the refrigerator back to take out, the enzyme electrode of highly sensitive detection glucose.
2. preparation method according to claim 1 is characterized in that described polyanion electrolyte is a kind of or wherein any two kinds potpourri in kayexalate, polyvinyl sulfonate, dextran sulfate, chondroitin sulfate, sodium alginate, sodium polyacrylate, polymethylacrylic acid, heparin or the heparin sulfate; Described polycation electrolyte is a kind of or wherein any two kinds potpourri in PAH salt, poly-dipropenyl dimethyl ammonium chloride, polyvinyl alcohol (PVA), collagen, shitosan, poly-D-lysine or the polyvinyl inferior amine salt hydrochlorate.
3. preparation method according to claim 1, it is characterized in that polyanion or polycation electrolyte and acid solution are mixed with in the deionized water solution, the concentration of polyanion or polycation electrolyte is 0.005-0.05mol/L, and the pH value of solion is controlled at 1-4.
4. preparation method according to claim 1 is characterized in that described strut body electrode is metal electrode or glass electrode.
5. preparation method according to claim 1 is characterized in that repeating step 2) number of times is 5-40 time, obtains polyelectrolyte/Prussian blue membrane electrode.
6. preparation method according to claim 1 is characterized in that step 2) aqueous solution in K
4Fe (CN)
6, K
3Fe (CN)
6, FeCl
3Perhaps (NH
4)
2Fe (SO
4)
2Concentration is 1-500mM, and KCl concentration is 0.1-0.5M.
7. preparation method according to claim 1 is characterized in that step 2) in strut body electrode at K
4Fe (CN)
6Or K
3Fe (CN)
6With the aqueous solution of KCl, perhaps at the FeCl of same concentrations
3Or (NH
4)
2Fe (SO
4)
2Be respectively 20s-2min with the immersion time in the KCl aqueous solution.
8. preparation method according to claim 1 is characterized in that the concentration 100-10000IU/ml of glucose oxidase solution in the step 4), and the mass percentage concentration of gelatin solution 1 is 0.1-1.5%, and the mass percentage concentration of gelatin solution 2 is 2-4%'s.
9. preparation method according to claim 1 is characterized in that pipetting in the step 4) glucose oxidase solution and gelatin solution 1 volume ratio and is 1: 10～1: 1000 and mixes.
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