CN101191782A - Lactase film preparation method - Google Patents
Lactase film preparation method Download PDFInfo
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- CN101191782A CN101191782A CNA2007101511930A CN200710151193A CN101191782A CN 101191782 A CN101191782 A CN 101191782A CN A2007101511930 A CNA2007101511930 A CN A2007101511930A CN 200710151193 A CN200710151193 A CN 200710151193A CN 101191782 A CN101191782 A CN 101191782A
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Abstract
The invention discloses a preparation method of lactase membrane, and aims to provide a core component - nucleopore immobilized enzyme membrane which can be matched with different redox electrodes, and is fit for lactase online monitoring. The method includes the following steps: beta-cyclodextrin is weighed and dissolved in low concentration sodium hydroxide solution with glutaric dialdehyde, and is placed in room temperature, so that the two are crosslinked into solid state, a precipitated crystal is washed with distilled water, and milled after being dried away from light, aldehyde group beta-cyclodextrin microsphere is prepared; the immobilized material is taken and sufficiently mixed with hybrid enzyme solution, and placed for 8 minutes at 4 degrees centigrade; immobilized enzyme is taken and uniformly applied on two polycarbonate microporous membranes, and dripped with sodium alginate solution; and an enzyme membrane with a sandwich structure is prepared, taken out after being dipped into CaCl2 solution, and washed by buffer solution to be prepared into lactase membrane. The nucleopore immobilized enzyme membrane prepared by the method of the invention can be flexibly matched with other redox electrodes, and has general applicability.
Description
Technical field
The present invention relates to field of biosensors, in particular, relate to a kind of preparation method of lactase film.
Background technology
The assay method of lactose content mainly contains: reducing sugar test method (iodimetric titration), polarimetry, near infrared spectroscopy, nuclear magnetic resonance method and high performance liquid chromatography, supercritical ultrasonics technology etc., these methods are consuming time, loaded down with trivial details, detect not only time-consuming but also require great effort.Therefore, now generally the determination method of good enzyme is a lactose biology sensor method, and this method has characteristics such as sensitivity, quick, easy, high specificity, and sample is used any pre-service hardly.
The development of lactose sensor provides favourable means for the field quick detection of lactose.Occurred various biology sensors at present, wherein the enzyme sensor of current mode still is that research is maximum, also be the most successful sensor type of commercialization, and the core parts of this class sensor is exactly fixed enzyme membrane.The quality of enzyme membrane performance depends mainly on the selection with fixing means chosen of immobilization material.
The method of immobilised enzymes mainly contains absorption method, covalent bond method, cross-linking method, polymkeric substance investment, combined method etc. on electrode at present.
For realizing lactose mensuration, invented some enzyme electrodes abroad, these enzyme electrodes mainly are the technology by immobilised enzymes.By with two kinds of enzyme: β-GAL and GOD, be fixed on the Clark-type oxygen electrode and business-like hydrogen-oxygen type sensor or platinum (Pt) electrode on, make the lactose biology sensor.On this class sensor, directly measure electrochemical reaction by measuring superoxide.Enzyme acts on the electrode and a mediators in the other electrode, for example benzoquinones combination, and this is the solution of another kind of type.Enzyme also can be fixed on the electrode together with mediators, for example Tetrathiafulvalinium, Tetracyanoquinodimethanide (TCNQ) and peroxidase (GOD).Mediators has increased enzyme ' s reaction speeding, thereby has amplified electric signal (Ferda Gurtas, 1997).Jan Tka, et al. (2000) adopt a kind of ammeter sensor design to go out the lactose biology sensor, are suitable for the mensuration of fresh feed Ruzhong lactose.This sensor adopts improvement glassy carbon electrode (GC), arranges three kinds of enzyme: β-GAL in the above, and GOD and horseradish peroxidase (POD) carry out immobilization.Be that enzyme directly is fixed on the electrode in the said method, must use the electrode that is complementary, thereby not have general applicability.
Summary of the invention
The present invention directly is fixed to enzyme on the electrode in order to overcome in the prior art, thereby do not have weak points such as general applicability, providing a kind of can be used with different oxidation-reduction electrodes, and is suitable for a core component-nuclear micropore fixed enzyme membrane of lactose on-line monitoring.
The present invention is achieved through the following technical solutions:
A kind of preparation method of lactase film is characterized in that, comprises the steps:
(1) taking by weighing beta-schardinger dextrin-is dissolved in the low-concentration sodium hydroxide solution that contains glutaraldehyde, room temperature is placed, and makes it to be cross-linked into solid state, and the crystal of separating out with distilled water flushing is neutral up to the pH of washing fluid value, lucifuge is milled after drying, and just makes aldehyde radical beta-schardinger dextrin-microballoon; Wherein beta-schardinger dextrin-is 1: 5 with the mass volume ratio that contains the low-concentration sodium hydroxide solution of glutaraldehyde;
(2) get above-mentioned aldehyde radical beta-schardinger dextrin-microballoon as immobilization material with after mixed enzyme solution fully mixes, place down for 4 degrees centigrade and made enzyme immobilization in 8 minutes; The enzyme of getting the 0.1g said fixingization evenly is applied on two polycarbonate microporous barriers, drips 1 2% sodium alginate soln again, behind the enzyme membrane of the structure that sandwiches, immerses CaCl
2Take out behind the solution, more than 3 times, make lactase film with the damping fluid flushing; Wherein the mass volume ratio of immobilization material and mixed enzyme solution is 10: 1, and described mixed enzyme solution is mixed by glucose oxidase, beta galactosidase, bovine serum albumin(BSA).
Wherein, contain the glutaraldehyde that percent by volume is 0.5-1.5% in the low-concentration sodium hydroxide solution.Described CaCl
2The solution weight percent concentration is 5-7%.The particle diameter of milling after drying is about 1nm.Described mixed enzyme solution is mixed by 0.003-0.007g glucose oxidase, 1ML beta galactosidase, 0.01g bovine serum albumin(BSA).
The present invention has following technique effect:
The immobilised enzymes method that is suitable for nuclear pore membrane that employing of the present invention is different with foreign study is made the nuclear pore membrane of immobilised enzymes, rather than directly be fixed on the electrode, thereby be suitable for the SBA-40C bio-sensing analyser that the Shandong Province academy sciences Biology Research Institute produces, also can be used with other oxidation-reduction electrode neatly, have general applicability.This lactose electrode has good linear relationship in 0.2-2% and 4-10%, response time 40s, half life period 15-20 days.The range of linearity 0-1% of glucose electrode, resolution reaches 0.001%, response time 20s, the half life period was greater than 60 days.This system is the content of lactose and glucose in the working sample simultaneously, and the glucose of lactose electrode disturbs and eliminates with method of difference.
Embodiment
Below in conjunction with specific embodiment to the detailed description of the invention.
Embodiment 1
Take by weighing beta-schardinger dextrin-20g, being added to 100ml contains in the 0.2M sodium hydroxide solution that percent by volume is 1% glutaraldehyde heating for dissolving and stirred 20 minutes, room temperature was placed 12 hours, carry out cross-linking reaction, the crystal of separating out with distilled water flushing drops to about 7.0 up to the pH of washing fluid value afterwards, after lucifuge is dried it is fully milled, the particle diameter of milling is about 1nm, just makes aldehyde radical beta-schardinger dextrin-microballoon.Pack into and keep in Dark Place in 1.5 milliliters the centrifuge tube.
After getting immobilization material 0.2g and the mixed enzyme solution of 20 microlitres fully mixing, placed 8 minutes down for 4 degrees centigrade, the enzyme of getting the 0.1g said fixingization evenly is applied on two polycarbonate microporous barriers, drip 2% sodium alginate soln again, behind the enzyme membrane of the structure that sandwiches, the immersion weight percent concentration is 6% CaCl
2Solution took out after 10 seconds, more than 3 times, made lactase film with the damping fluid flushing, can install on the SBA-40C bio-sensing analyser to be used for analytical test.Wherein mixed enzyme solution is mixed by 0.005g glucose oxidase, 1ML beta galactosidase and 0.01g bovine serum albumin(BSA).
Condition of storage: drip glycerine on film, 4 degree are preserved.
Embodiment 2
Take by weighing beta-schardinger dextrin-20g, being added to 100ml contains in the 0.2M sodium hydroxide solution that percent by volume is 1.5% glutaraldehyde heating for dissolving and stirred 20 minutes, room temperature was placed 12 hours, carry out cross-linking reaction, the crystal of separating out with distilled water flushing drops to about 7.0 up to the pH of washing fluid value afterwards, after lucifuge is dried it is fully milled, the particle diameter of milling is about 1nm, just makes aldehyde radical beta-schardinger dextrin-microballoon.Pack into and keep in Dark Place in 1.5 milliliters the centrifuge tube.
After getting immobilization material 0.2g and the mixed enzyme solution of 20 microlitres fully mixing, placed 8 minutes down for 4 degrees centigrade, the enzyme of getting the 0.1g said fixingization evenly is applied on two polycarbonate microporous barriers, drip 2% sodium alginate soln again, behind the enzyme membrane of the structure that sandwiches, the immersion weight percent concentration is 7% CaCl
2Solution took out after 10 seconds, more than 3 times, made lactase film with the damping fluid flushing, can install on the SBA-40C bio-sensing analyser to be used for analytical test.Wherein mixed enzyme solution is mixed by 0.007g glucose oxidase, 1ML beta galactosidase and 0.01g bovine serum albumin(BSA).
Condition of storage: drip glycerine on film, 4 degree are preserved.
Embodiment 3
Take by weighing beta-schardinger dextrin-20g, being added to 100ml contains in the 0.2M sodium hydroxide solution that percent by volume is 0.5% glutaraldehyde heating for dissolving and stirred 20 minutes, room temperature was placed 12 hours, carry out cross-linking reaction, the crystal of separating out with distilled water flushing drops to about 7.0 up to the pH of washing fluid value afterwards, after lucifuge is dried it is fully milled, the particle diameter of milling is about 1nm, just makes aldehyde radical beta-schardinger dextrin-microballoon.Pack into and keep in Dark Place in 1.5 milliliters the centrifuge tube.
After getting immobilization material 0.2g and the mixed enzyme solution of 20 microlitres fully mixing, placed 8 minutes down for 4 degrees centigrade, the enzyme of getting the 0.1g said fixingization evenly is applied on two polycarbonate microporous barriers, drip 2% sodium alginate soln again, behind the enzyme membrane of the structure that sandwiches, the immersion weight percent concentration is 5% CaCl
2Solution took out after 10 seconds, more than 3 times, made lactase film with the damping fluid flushing, can install on the SBA-40C bio-sensing analyser to be used for analytical test.Wherein mixed enzyme solution is mixed by 0.003g glucose oxidase, 1ML beta galactosidase and 0.01g bovine serum albumin(BSA).
Condition of storage: drip glycerine on film, 4 degree are preserved.
Measuring principle:
Lactose is hydrolyzed to β-D-glucose and β-D-galactose under the catalysis of beta galactosidase, β wherein-D-glucose generates gluconic acid and H under the catalysis of glucose oxidase
2O
2, the Chemical Measurement signal of its redox reaction shows by the electric signal of electrode, thereby reaches the purpose of lactose being carried out quantitative measurement.
The present invention utilizes starch, beta-schardinger dextrin-and sodium alginate functional for the lactase film of Mixed Stationary material, and it is 0.0005% that minimum lactose detects concentration, and 20 seconds response times, 0%-2% scope internal linear concerns R
2Reach more than 0.995, every day, test sample was 40 times, and enzyme membrane is active after 1 week does not obviously descend.
Claims (5)
1. the preparation method of a lactase film is characterized in that, comprises the steps:
(1) taking by weighing beta-schardinger dextrin-is dissolved in the low-concentration sodium hydroxide solution that contains glutaraldehyde, room temperature is placed, and makes it to be cross-linked into solid state, and the crystal of separating out with distilled water flushing is neutral up to the pH of washing fluid value, lucifuge is milled after drying, and just makes aldehyde radical beta-schardinger dextrin-microballoon; Wherein beta-schardinger dextrin-is 1: 5 with the mass volume ratio that contains the low-concentration sodium hydroxide solution of glutaraldehyde;
(2) get above-mentioned aldehyde radical beta-schardinger dextrin-microballoon as immobilization material with after mixed enzyme solution fully mixes, place down for 4 degrees centigrade and made enzyme immobilization in 8 minutes; The enzyme of getting the 0.1g said fixingization evenly is applied on two polycarbonate microporous barriers, drips 1 2% sodium alginate soln again, behind the enzyme membrane of the structure that sandwiches, immerses CaCl
2Solution took out after 10 seconds, more than 3 times, made lactase film with the damping fluid flushing; Wherein the mass volume ratio of immobilization material and mixed enzyme solution is 10: 1, and described mixed enzyme solution is mixed by glucose oxidase, beta galactosidase, bovine serum albumin(BSA).
2. the preparation method of lactase film according to claim 1 is characterized in that, contains the glutaraldehyde that percent by volume is 0.5-1.5% in the low-concentration sodium hydroxide solution.
3. the preparation method of lactase film according to claim 1 is characterized in that, described CaCl
2The solution weight percent concentration is 5-7%.
4. the preparation method of lactase film according to claim 1 is characterized in that, the particle diameter of milling after drying is about 1nm.
5. the preparation method of lactase film according to claim 1 is characterized in that, described mixed enzyme solution is mixed by 0.003-0.007g glucose oxidase, 1ML beta galactosidase, 0.01g bovine serum albumin(BSA).
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| CN2007101511930A CN101191782B (en) | 2007-12-21 | 2007-12-21 | Lactase film preparation method |
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| CN2007101511930A CN101191782B (en) | 2007-12-21 | 2007-12-21 | Lactase film preparation method |
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| CN101191782B CN101191782B (en) | 2010-12-08 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463349B (en) * | 2009-01-06 | 2012-05-30 | 天津商业大学 | Method for preparing saccharose biosensor nuclear micropore enzyme membrane using beta-galactosidase |
| CN104330448A (en) * | 2014-10-31 | 2015-02-04 | 桂林中辉科技发展有限公司 | High-sensitivity electrode type uric acid test paper and manufacturing method thereof |
| CN104483363A (en) * | 2015-01-05 | 2015-04-01 | 天津商业大学 | Method for manufacturing electrochemical biosensor for measuring antibiotic |
| CN113138220A (en) * | 2021-04-23 | 2021-07-20 | 广州万孚生物技术股份有限公司 | Electrochemical biosensor and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427940C (en) * | 2006-07-04 | 2008-10-22 | 北京化工大学 | Sensor enzyme film containing cation type bio-compatibile polymer and preparation method thereof |
-
2007
- 2007-12-21 CN CN2007101511930A patent/CN101191782B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463349B (en) * | 2009-01-06 | 2012-05-30 | 天津商业大学 | Method for preparing saccharose biosensor nuclear micropore enzyme membrane using beta-galactosidase |
| CN104330448A (en) * | 2014-10-31 | 2015-02-04 | 桂林中辉科技发展有限公司 | High-sensitivity electrode type uric acid test paper and manufacturing method thereof |
| CN104483363A (en) * | 2015-01-05 | 2015-04-01 | 天津商业大学 | Method for manufacturing electrochemical biosensor for measuring antibiotic |
| CN113138220A (en) * | 2021-04-23 | 2021-07-20 | 广州万孚生物技术股份有限公司 | Electrochemical biosensor and preparation method thereof |
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| CN101191782B (en) | 2010-12-08 |
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