CN102175731A - Horse radish peroxidase-attapulgite clay nano composite material, preparation method thereof and biosensor based on composite material - Google Patents
Horse radish peroxidase-attapulgite clay nano composite material, preparation method thereof and biosensor based on composite material Download PDFInfo
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- CN102175731A CN102175731A CN2011100640536A CN201110064053A CN102175731A CN 102175731 A CN102175731 A CN 102175731A CN 2011100640536 A CN2011100640536 A CN 2011100640536A CN 201110064053 A CN201110064053 A CN 201110064053A CN 102175731 A CN102175731 A CN 102175731A
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Abstract
The invention relates to a horse radish peroxidase-attapulgite clay nano composite material and a preparation method thereof. The nano composite material provided by the invention is characterized in that purified attapulgite clay is utilized as a vector; horse radish peroxidase is absorbed on the surface of the attapulgite clay; the infrared spectrum of the nano composite material has absorption peaks in the positions of 1653cm<-1> and 1640cm<-1>; the horse radish peroxidase maintains a natural structure and bioactivity; the ultrasonic dispersion is carried out on the purified attapulgite clay; the pH value of a dispersion solution is controlled; the dispersion solution and the horse radish peroxidase are mixed; and the horse radish peroxidase is absorbed to the surface of the attapulgite clay so as to prepare the nano composite material. The invention also discloses a biosensor based on the nano composite material, and the biosensor comprises a glassy carbon electrode, wherein the surface of the electrode is modified with the horse radish peroxidase-attapulgite clay nano composite material. In the invention, the nano composite material and the electrochemical performance of the biosensor are evaluated to indicate that the composite material has good electrocatalysis capability on H2O2 reduction, can be used for effectively detecting the H2O2, especially the H2O2 in cells.
Description
Technical field
The invention belongs to nano material preparation, bio-sensing and cell technical field of electrochemistry, relate to recessed native nano composite material of a kind of La root Guo oxide Mei – and preparation method thereof, and based on the biology sensor of described compound substance.
Background technology
Cell in metabolic process, constantly produce various active oxygens (reactive oxygen species, ROS), ultra-oxygen anion free radical (O for example
2 -), hydroxy radical (OH), alicyclic diradical (ROO), hydrogen peroxide (H
2O
2) etc.Hydrogen peroxide is wherein stable a kind of, and its concentration and O
2 -, other ROS molecules such as OH are closely related, the up-to-date hydrogen peroxide of discovering is being played the part of important regulatory role in the cellular process of life system.Hydrogen peroxide can be used as the startup that signaling molecule or second messenger participate in multiple factor cell biology effect, but also can react simultaneously with intracellular biomacromolecule, thereby directly cause the peroxidating of cell membrane lipid, intracellular protein and enzyme sex change, nucleic acid DNA damage etc., finally cause the generation of multiple diseases such as cell death, tissue damage, angiocardiopathy, tumour and neuronal degeneration.Therefore, the method that develops hydrogen peroxide in a kind of reliable, sensitive detection cell has extremely important meaning at physiology and Pathological Physiology field.
The method that detects at present hydrogen peroxide mainly contains fluorescence method, chemoluminescence method, high performance liquid chromatography, electron paramagnetic resonance method and based on the electrochemical method of enzyme sensing etc.Yet, the H of cellular level
2O
2Detect and still to be subjected to more multifactor restriction, the half life period of superoxide radical is very short in, the born of the same parents little as the cell yardstick, Css is extremely low and lack H
2O
2The probe of effectively catching etc., and some traditional analytical approachs exist deficiencies such as sample volume is big, quality testing limit for height, analysis time is long, instrument costs an arm and a leg.In numerous detection methods, based on enzyme sensing electrochemical method since its have highly sensitive, response is fast, the sample consumption is little, selectivity is strong, be easy to advantage such as preparation and miniaturization and extremely people pay close attention to.
Based on the electrochemical method of enzyme sensing is that the protein (enzyme) that will have redox characteristic is immobilized to certain material and it is modified electrode surface be built into electrochemical sensor, and the electrocatalysis characteristic by protein (enzyme) detects the substrate hydrogen peroxide.Horseradish peroxidase is known owing to its structure, wide material sources become research catalysis H
2O
2One of important models enzyme molecule of redox reaction.The key factor for preparing high performance horseradish peroxidase sensor is the selection of carrier and fixing means, to reduce zymoprotein sex change and inactivation equally likely possibility in the sensor preparation process to greatest extent.
Yet selected protein carrier mostly is all kinds of material with carbon elements (as graphite, carbon nano-tube, carbon nano-fiber, orderly carbon mesoporous material and Graphene etc.), metal oxide (Woelm Alumina, zirconia, tri-iron tetroxide etc.), macromolecule (as shitosan, sodium alginate, starch, polystyrene, tygon and methyl methacrylate MMA) etc. at present.These immobilization carriers part that all comes with some shortcomings, though have excellent conducting performance, stronger advantages such as adsorptive power as material with carbon element, thereby whether can to produce the biologically active that toxic action has influence on biomacromolecule to biomacromolecule still be not too clear at present for it; Though and metal oxide has good stability, physical strength height, low cost and other advantages, but biomacromolecule has lost original conformation on its surface, produces sex change, causes being fixed on its surperficial protein (enzyme) and loses biologically active; The characteristics of natural polymer carrier maximum are free of toxic effects, but they exist that intensity is lower, poor electric conductivity, under anaerobic easily decomposed by microorganism and shortcoming such as serviceable life is low; Synthetic high-molecular organic material is owing to its chemistry, physical property all have very big changeability, the carrier that can take on any enzyme in theory, and than the intensity height of natural polymer, but the shortcoming that but has poor electric conductivity, this is one of maximum unfavorable factor of electrochemical research.
Recessed soil (full name Concave-convex clay rod) is crystalloid hydrous magnesium aluminium silicate mineral, has unique layer chain structure feature, because the crystal structure of attapulgite uniqueness, make it to have many special materializations and shop characteristic, mainly comprise kation interchangeability, water absorptivity, adsorption bleaching, big specific surface area (9.6～36m
2/ g) etc.Attapulgite clay mineral has the attribute of nano material, it is natural nano structure mineral material with nanochannel structure, because they have very large specific surface area and certain ion exchangeable, therefore be widely used as adsorbent, catalyst support and antiseptic carrier etc.
The present invention with recessed soil be carrier fixedly horseradish peroxidase prepare horseradish peroxidase-recessed native composite nano materials, the natural structure and the biologically active that can well keep horseradish peroxidase, the recessed native nano composite material of La root Guo oxide Mei – is modified the glass-carbon electrode surface obtain biology sensor, hydrogen peroxide is had good electro catalytic activity; Can be applicable to detect the content of hydrogen peroxide in the hydrogen peroxide, particularly cell.The recessed native nano composite material of the resulting La root of the present invention Guo oxide Mei – not only helps to develop and widen the high-valued range of application of recessed soil, and has great significance in fields such as bioelectricity analytical chemistry, cytology and physiopathologies.
Summary of the invention
The objective of the invention is to develop a kind of enzyme-recessed native composite material with bio-sensing function, recessed native nano composite material of a kind of La root Guo oxide Mei – and preparation method thereof is provided, resulting nano composite material can keep the natural structure and the biologically active of horseradish peroxidase well, the biology sensor of making based on described nano composite material has good electro catalytic activity to hydrogen peroxide, can be applicable to the detection of hydrogen peroxide.
Another purpose of the present invention provides a kind of biology sensor based on horseradish peroxidase-recessed native nano composite material.
The technical scheme of finishing the foregoing invention task is:
The recessed native nano composite material of a kind of La root Guo oxide Mei – is characterized in that the recessed soil with purifying is carrier, and horseradish peroxidase is adsorbed on recessed soil surface.
On the infrared spectrogram of the recessed native nano composite material of described La root Guo oxide Mei –, 1653 cm
– 1With 1640 cm
– 1There is absorption peak in the place, and its position is compared substantially with horseradish peroxidase with shape and do not changed.Show that being adsorbed on the lip-deep horseradish peroxidase of recessed soil keeps the secondary structure of itself substantially, can well keep its enzymatic activity.
The preparation method of the recessed native nano composite material of described La root Guo oxide Mei – may further comprise the steps:
1) the recessed soil that will purify later joins in 0.1 mol/LPBS (phosphate buffer) solution, and control pH value scope is 7.5 of 7 –, and ultrasonic dispersion 0.5 – 2 hours obtains the recessed native dispersion liquid of 0.5 –, 5 mg/mL;
2) recessed native dispersion liquid is mixed with 1 –, 10 mg/mL horseradish peroxidase PBS solution (pH 7 – 7.5), 4 ° of C continuous stirring, 0.5 – 2 hours, electrostatic attraction effect by positive and negative charge, horseradish peroxidase is adsorbed onto recessed soil surface, forms the suspension of La root Guo oxide Mei – attapulgite composite material;
3) with La root Guo oxide Mei – attapulgite composite material suspension centrifugal treating 15-30 minute, and use deionized water wash, remove the loose enzyme molecule in surface, in vacuum dryer, dry, obtain La root Guo oxide Mei – attapulgite composite material (HRP – Attapulgite).
The present invention disperses the recessed soil of purifying, make its surface electronegative by the pH value of controlling recessed native dispersion liquid, utilize the horseradish peroxidase molecular adsorption that electrostatic interaction can be positively charged with the surface to arrive recessed soil surface, form described La root Guo oxide Mei – attapulgite composite material, its stereoscan photograph is seen Fig. 1.
The present invention is a carrier with the recessed soil of purifying, and recessed soil is a kind of native compound of nano-scale, has the surface adsorption effect, an effect that concentrates, absorption directionality effect and chemical activity effect etc.; A large amount of energy and protein (enzyme) reaction De – OH group is contained on recessed native surface, can improve greatly and protein (enzyme) between adhesion; Because " duct of zeolite type " structure of recessed soil, recessed soil has big specific surface area and porous structure, is easy to and stability that the crosslinked raising of protein (enzyme) is fixing; Recessed soil has excellent biological compatibility and electrochemical stability, excellent mechanical intensity, thermal stability, anti-biodegradability and to the characteristics such as high affinity of protein (enzyme), these all are the advantage that carrier had of recessed soil as horseradish peroxidase.
The purifying of described recessed soil can adopt method of the prior art, and the purification process that adopts in the specific embodiment is: after recessed soil is ground, add ultrasonic dispersion in the ultrapure water, supercentrifuge is centrifugal, cleans the recessed soil of sedimentation with deionized water; Repeat ultrasonic dispersion, centrifugally operated three times, the recessed soil of washes clean is dried 12 h at 80 ° of C, the recessed soil that obtains purifying.
The invention still further relates to a kind of biology sensor, comprise glass-carbon electrode, it is characterized in that the recessed native nano composite material of described glass-carbon electrode finishing La root Guo oxide Mei – based on the recessed native nano composite material of described La root Guo oxide Mei –.
The preparation method of described biology sensor based on the recessed native nano composite material of La root Guo oxide Mei – is: glass-carbon electrode is polished to minute surface, dries after the ultrasonic cleaning in ultrapure water then; Described La root Guo oxide Mei – attapulgite composite material is put into the PBS solution (pH 7 – 7.5) of 0.1 mol/L, obtain the equal phase dispersant liquid of 1 mg HRP – Attapulgite/mL, this dispersant liquid drop of 2 –, 10 μ L is added to the glass-carbon electrode surface, treat promptly to obtain after the solvent evaporates biology sensor, be labeled as HRP – Attapulgite/GC electrode based on horseradish peroxidase-attapulgite composite material.
Resulting electrode do not preserve under 4 ° of C by the time spent.
Horseradish peroxidase-attapulgite composite material of the present invention characterizes proof through infrared spectrum (FTIR), ultraviolet spectrum (Uv-vis) and circular dichroism spectra (CD), and the enzyme molecule after the absorption still can keep its natural secondary structure and biologically active.On the infrared spectrum of horseradish peroxidase-recessed native nano composite material (Fig. 2), can observe the horseradish peroxidase that is fixed on recessed soil surface, its characteristic peak acid amides I (1653 cm
– 1) and acid amides II(1640 cm
– 1) shape and position and pure horseradish peroxidase (acid amides I 1653 cm
– 1With acid amides II 1640 cm
– 1) unanimity, illustrate that horseradish peroxidase does not have sex change in adsorption process, still well kept its natural space conformation and enzymatic activity.
Horseradish peroxidase-attapulgite composite material and estimate: adopt cyclic voltammetry to investigate the chemical property of HRP – Attapulgite/GC electrode based on the biology sensor chemical property of described compound substance, experimental result shows, has good electrochemical properties, electrocatalysis characteristic and stability based on the sensor of La root Guo oxide Mei – attapulgite composite material.Immerse HRP – Attapulgite/GC electrode according to the present invention in the PBS solution (pH 7.4) of 0.1 mol/L as working electrode, with saturated calomel electrode (SCE) as contrast electrode, the conduct of spiral Pt silk is to electrode, carry out cyclic voltammetry scan, research HRP molecule is in the Direct Electrochemistry characteristic on recessed soil surface and the stability of electrode.On the cyclic voltammetry curve that scanning obtains, the good reversible redox peak of a pair of peak shape has appearred, illustrate that the HRP that loads on recessed soil surface can realize that its direct electron shifts; With the continuous sweep under these conditions of HRP – attapulgite/GC electrode, the shape at redox peak remains unchanged, and this electrode is after preserving for two weeks under 4 ° of C, and the peak current size does not almost change, and illustrates that HRP – attapulgite/GC electrode has stability preferably.Described electrode put into contain 0.2 mmol/LH
2O
2PBS solution in (0.1 mol/L, pH 7.4) carry out cyclic voltammetry scan, find that tangible electrical catalyze reduction peak has appearred in Zai – 400 mV places, illustrate that La root Guo oxide Mei – attapulgite composite material that the present invention prepares is to H
2O
2Electrochemical reduction possess good catalytic action, can effectively be applied to H based on the sensor of this compound preparation
2O
2Electrochemical sensing.
The present invention has the following advantages: preparation method of the present invention is simply effective, and prepared nano composite material is because the recessed soil of carrier self has stronger absorption property and zeolite cavity characteristic makes more horseradish peroxidase molecule can be adsorbed onto recessed soil surface and have more catalytic reduction reaction site; Recessed soil has excellent biological compatibility, and the enzyme molecule after the absorption still can keep its natural secondary structure and biologically active; The biology sensor based on La root Guo oxide Mei – attapulgite composite material of preparation has good electrochemical properties, electrocatalysis characteristic and stability, and hydrogen peroxide is had good electro catalytic activity, can effectively detect the H in the solution
2O
2The present invention has not only widened the high-valued application of recessed soil, and can set up H in the cell based on the biology sensor of described compound substance
2O
2The electrochemical method that detects based on the enzyme sensing, significant in physiology, pathological research.
Description of drawings
The sem photograph of the recessed native nano composite material of Fig. 1 La root Guo oxide Mei –.
Fig. 2 horseradish peroxidase (curve a), the infrared spectrogram of recessed soil (curve b) and the La root Guo oxide recessed native nano composite material of Mei – (curve c).
The recessed native nano composite material of Fig. 3 La root Guo oxide Mei – is to H
2O
2Electro-catalysis figure: HRP – attapulgite/GC electrode is not containing H
2O
2PBS solution in cyclic voltammogram (curve a); HRP – attapulgite/GC electrode is containing H
2O
2Cyclic voltammogram in the PBS solution of (0.2 mmol/L) (curve b).PBS solution (0.1 mol/L, pH 7.4), sweep speed: 100 mV/s.
Embodiment
Embodiment one
Grinding machine for grinding 2 hours, rotating speed was 800 rev/mins with the recessed soil of 10 g; Take by weighing the recessed soil that 1g ground, put it in the 100 mL ultrapure waters ultrasonic dispersion after 2 hours with supercentrifuge centrifugal (5000 rev/mins), clean the recessed soil of sedimentation with deionized water; Repeat ultrasonic dispersion, centrifugally operated three times, the recessed soil of washes clean is dried 12 h at 80 ° of C, the recessed soil that obtains purifying.
The recessed soil that 0.5 mg was purified joins 1 mL, 0.1 mol/LPBS(pH 7.4) in the solution, ultrasonic dispersion 0.5 – 2 hours obtains the recessed native dispersion liquid of 0.5mg/mL; The PBS solution (1 mg/mL, pH 7.4) of this recessed native suspending liquid and isopyknic HRP is mixed, 4 ° of C continuous stirring 0.5 hour.With mixed liquor centrifugal 30 min under 18000 rpm, and, remove the unstable HRP molecule of absorption, this material is dried in vacuum dryer obtain HRP – Attapulgite compound with the thoroughly washing 3 times of secondary water.
Glass-carbon electrode (GC, diameter are 3 mm) is used sand paper, 0.3 μ m and 0.05 μ m Al respectively No. 6
2O
3Be polished to minute surface, ultrasonic cleaning 30 seconds in ultrapure water is then dried stand-by.Take by weighing 1mg HRP – Attapulgite compound and be scattered in the PBS(pH 7.4 of 1 mL, 0.1 mol/L) in the solution, get 2 μ L dispersant liquid drops with microsyringe and be coated in pretreated glass-carbon electrode surface, it is dry that electrode is put into exsiccator, treats promptly to obtain HRP – Attapulgite/GC electrode after the solvent evaporates of electrode surface.
Embodiment two
Grinding machine for grinding 2 hours, rotating speed was 800 rev/mins with the recessed soil of 10 g; Take by weighing the recessed soil that 1g ground, put it in the 100 mL ultrapure waters ultrasonic dispersion after 2 hours with supercentrifuge centrifugal (5000 rev/mins), clean the recessed soil of sedimentation with deionized water; Repeat ultrasonic dispersion, centrifugally operated three times, the recessed soil of washes clean is dried 12 h at 80 ° of C, the recessed soil that obtains purifying.
2 mg purified, and recessed soil adds 1 mL, 0.1 mol/LPBS(pH 7.4) in the solution, ultrasonic dispersion 0.5 – 2 hours obtains the recessed native dispersion liquid of 2mg/mL; The PBS solution (5 mg/mL, pH 7.4) of this recessed native suspending liquid and isopyknic HRP is mixed, 4 ° of C continuous stirring 1 hour.With mixed liquor centrifugal 20 min under 10000 rpm, and, remove the unstable HRP molecule of absorption, this material is dried in vacuum dryer obtain HRP – Attapulgite compound with the thoroughly washing 3 times of secondary water.
Glass-carbon electrode (GC, diameter are 3 mm) is used sand paper, 0.3 μ m and 0.05 μ m Al respectively No. 6
2O
3Be polished to minute surface, ultrasonic cleaning 30 seconds in ultrapure water is then dried stand-by.Take by weighing 1mg HRP – Attapulgite compound and be scattered in the PBS(pH 7.4 of 1 mL 0.1mol/L) in the solution, get 2 μ L dispersant liquid drops with microsyringe and be coated in pretreated glass-carbon electrode surface, it is dry that electrode is put into exsiccator, treats promptly to obtain HRP – Attapulgite/GC electrode after the solvent evaporates of electrode surface.
Embodiment three
Grinding machine for grinding 2 hours, rotating speed was 800 rev/mins with the recessed soil of 10 g; Take by weighing the recessed soil that 1g ground, put it in the 100 mL ultrapure waters ultrasonic dispersion after 2 hours with supercentrifuge centrifugal (5000 rev/mins), clean the recessed soil of sedimentation with deionized water; Repeat ultrasonic dispersion, centrifugally operated three times, the recessed soil of washes clean is dried 12 h at 80 ° of C, the recessed soil that obtains purifying.
The recessed soil that 5 mg were purified adds 1 mL, 0.1 mol/L PBS(pH 7.4) in the solution, ultrasonic dispersion 0.5 – 2 hours obtains the recessed native dispersion liquid of 5mg/mL; The PBS solution (10 mg/mL, pH 7.4) of this recessed native suspending liquid and isopyknic HRP is mixed, 4 ° of C continuous stirring 1 hour.With mixed liquor centrifugal 15 min under 5000 rpm, and, remove the unstable HRP molecule of absorption, this material is dried in vacuum dryer obtain HRP – Attapulgite compound with the thoroughly washing 3 times of secondary water.
Glass-carbon electrode (GC, diameter are 3 mm) is used sand paper, 0.3 μ m and 0.05 μ m Al respectively No. 6
2O
3Be polished to minute surface, ultrasonic cleaning 30 seconds in ultrapure water is then dried stand-by.Take by weighing 1mg HRP – Attapulgite compound and be scattered in the PBS(pH 7.4 of 1 mL 0.1mol/L) in the solution, get 2 μ L dispersant liquid drops with microsyringe and be coated in pretreated glass-carbon electrode surface, it is dry that electrode is put into exsiccator, treats promptly to obtain HRP – Attapulgite/GC electrode after the solvent evaporates of electrode surface.
Embodiment four
Among the embodiment four, be coated in pretreated glass-carbon electrode surface except that getting 5 μ L dispersant liquid drops with microsyringe, other operations are all identical with example one.
Embodiment five
Among the embodiment five, be coated in pretreated glass-carbon electrode surface except that getting 10 μ L dispersant liquid drops with microsyringe, other operations are all identical with example one.
Embodiment six
Among the embodiment six, be coated in pretreated glass-carbon electrode surface except that getting 5 μ L dispersant liquid drops with microsyringe, other operations are all identical with example two.
Embodiment seven
Among the embodiment seven, be coated in pretreated glass-carbon electrode surface except that getting 10 μ L dispersant liquid drops with microsyringe, other operations are all identical with example two.
Embodiment eight
Among the embodiment eight, be coated in pretreated glass-carbon electrode surface except that getting 5 μ L dispersant liquid drops with microsyringe, other operations are all identical with example three.
Embodiment nine
Among the embodiment nine, be coated in pretreated glass-carbon electrode surface except that getting 10 μ L dispersant liquid drops with microsyringe, other operations are all identical with example three.
Embodiment ten
Utilize cyclic voltammetry to estimate to horseradish peroxidase-attapulgite composite material and based on the performance of the electrochemica biological sensor of described compound substance, experimental result shows, has good electrochemical properties, electrocatalysis characteristic and stability based on the sensor of La root Guo oxide Mei – attapulgite composite material.
The PBS solution (pH 7.4) of the HRP – Attapulgite/GC electrode of embodiment six preparations being put into 0.1 mol/L carries out cyclic voltammetry scan, contrast electrode is a saturated calomel electrode, to electrode is spiral Pt silk, (curve a) among Fig. 3 on the cyclic voltammetry curve that scanning obtains, the good reversible redox peak of a pair of peak shape has appearred, its oxidation peak and reduction peak current potential Fen other Wei – 300 He – 350 mV, this is that iron porphyrin generation redox reaction by the protoheme at HRP molecular activity center causes, illustrates that the HRP that loads on recessed soil surface can realize that its direct electron shifts; With HRP – attapulgite/GC electrode continuous sweep under these conditions (10 mV/s) 50 circles, investigate its stability, experimental result shows that the shape at redox peak remains unchanged, and this electrode is after preserving for two weeks under 4 ° of C, the peak current size does not almost change, and illustrates that HRP – attapulgite/GC electrode has stability preferably.
This electrode put into contain 0.2 mmol/LH
2O
2PBS solution in (0.1 mol/L, pH 7.4) carry out cyclic voltammetry scan, great variety Zai – 400 mV places take place and a big reduction peak occurred in cyclic voltammetry curve (curve b among Fig. 3), and oxidation peak complete obiteration, this is typical electrocatalytic reaction feature, illustrates that La root Guo oxide Mei – attapulgite composite material is to H
2O
2Electrochemical reduction possess good catalytic action, can effectively be applied to H based on the sensor of this compound preparation
2O
2Electrochemical sensing.
The peppery sensor of crossing the recessed native nano composite material of oxide enzyme – for preparing among other embodiment put under identical condition and do not contained H
2O
2PBS solution in (0.1 mol/L, pH 7.4) and contain 0.02mmol/L H
2O
2PBS solution in scanning in (0.1 mol/L, pH 7.4), experimental result shows that they are to H
2O
2Electrochemical reduction produce similar substantially catalytic action.
Claims (6)
1. cross the recessed native nano composite material of oxide enzyme – for one kind peppery, it is characterized in that described nano composite material is a carrier with the recessed soil of purifying, horseradish peroxidase is adsorbed on recessed soil surface.
2. the recessed native nano composite material of La root Guo oxide Mei – according to claim 1 is characterized in that on the infrared spectrum of the recessed native nano composite material of described La root Guo oxide Mei – 1653 cm
– 1With 1640 cm
– 1There is characteristic absorption peak in the place.
3. the described peppery preparation method who crosses the recessed native nano composite material of oxide enzyme – of a claim 1 may further comprise the steps:
1) the recessed soil that will purify later joins in the 0.1 mol/L PBS solution, and control pH value scope is 7.5 of 7 –, and ultrasonic dispersion 0.5 – 2 hours obtains the recessed native dispersion liquid of 0.5 –, 5 mg/mL;
2) the PBS solution of recessed native dispersion liquid with pH 7 – 7.5,1 – 10 mg/mL horseradish peroxidases is mixed, 4 ° of C continuous stirring, 0.5 – 2 hours, electrostatic attraction effect by positive and negative charge, horseradish peroxidase is adsorbed onto recessed soil surface, forms the suspension of La root Guo oxide Mei – attapulgite composite material;
3) cross oxide enzyme – attapulgite composite material suspension centrifugal treating 15-30 minute with peppery, and use deionized water wash, remove the loose enzyme molecule in surface, in vacuum dryer, dry, obtain peppery and cross oxide enzyme – attapulgite composite material.
4. the preparation method of the recessed native nano composite material of La root Guo oxide Mei – according to claim 3, the purification process that it is characterized in that described recessed soil is: after recessed soil is ground, add ultrasonic dispersion in the ultrapure water, supercentrifuge is centrifugal, cleans the recessed soil of sedimentation with deionized water; Repeat ultrasonic dispersion, centrifugally operated three times, the recessed soil of washes clean is dried 12 h at 80 ° of C, the recessed soil that obtains purifying.
5. the biology sensor based on the described peppery recessed native nano composite material of mistake oxide enzyme – of claim 1 comprises glass-carbon electrode, it is characterized in that the recessed native nano composite material of described glass-carbon electrode finishing peppery mistake oxide enzyme –.
6. biology sensor according to claim 5 is characterized in that the preparation method of described biology sensor is: glass-carbon electrode is polished to minute surface, dries after the ultrasonic cleaning then in ultrapure water; Described La root Guo oxide Mei – attapulgite composite material is added in the PBS solution of pH 7 – 7.5,0.1 mol/L, obtain the equal phase dispersant liquid of 1 mg/mL, this dispersant liquid drop of 2 –, 10 μ L is added to the glass-carbon electrode surface, treats promptly to obtain after the solvent evaporates biology sensor based on horseradish peroxidase-attapulgite composite material.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952792A (en) * | 2012-11-12 | 2013-03-06 | 淮阴工学院 | Preparation method of beta-glucosidase immobilized by sodium alginate loaded with attapulgite |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5227042A (en) * | 1992-05-15 | 1993-07-13 | The United States Of America As Represented By The United States Department Of Energy | Catalyzed enzyme electrodes |
| CN101344522A (en) * | 2008-08-21 | 2009-01-14 | 淮阴师范学院 | Preparation method for biologic sensor and modified electrode based on attapulgite clay soil as substrate |
| CN101805044A (en) * | 2010-03-12 | 2010-08-18 | 江苏工业学院 | Clay/carbon material-based composite electrode and production method and application thereof |
-
2011
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5227042A (en) * | 1992-05-15 | 1993-07-13 | The United States Of America As Represented By The United States Department Of Energy | Catalyzed enzyme electrodes |
| CN101344522A (en) * | 2008-08-21 | 2009-01-14 | 淮阴师范学院 | Preparation method for biologic sensor and modified electrode based on attapulgite clay soil as substrate |
| CN101805044A (en) * | 2010-03-12 | 2010-08-18 | 江苏工业学院 | Clay/carbon material-based composite electrode and production method and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952792A (en) * | 2012-11-12 | 2013-03-06 | 淮阴工学院 | Preparation method of beta-glucosidase immobilized by sodium alginate loaded with attapulgite |
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