CN110218720A - A kind of immobilised enzymes method and application improving horseradish peroxidase stability - Google Patents

A kind of immobilised enzymes method and application improving horseradish peroxidase stability Download PDF

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CN110218720A
CN110218720A CN201910397760.3A CN201910397760A CN110218720A CN 110218720 A CN110218720 A CN 110218720A CN 201910397760 A CN201910397760 A CN 201910397760A CN 110218720 A CN110218720 A CN 110218720A
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赵慧敏
谭冰
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Dalian University of Technology
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Abstract

The invention belongs to environmental monitoring technology fields, provide a kind of immobilised enzymes method and application for improving horseradish peroxidase stability.At normal temperatures and pressures, using the method for co-precipitation, the Biocomposite material of -8/ natural horseradish peroxidase of graphene immobilization of synthetic zeolite imidazate skeleton in water phase.The embedding of ZIF-8/GO composite material retains the excellent Catalyzed Synthesis By Peroxidase activity of HRP, while enhancing the stability of HRP.For individual ZIF-8 mineralising HRP material, the introducing of GO increases HRP to the tolerance of the complex environments such as resistant to high temperatures, organic solvent and denaturant, while enhancing the storage stability and recycling ability of HRP.Using the Catalyzed Synthesis By Peroxidase activity of DNA molecular regulation HRP@ZIF-8/GO Biocomposite material, unmarked colorimetric sensing method is established, the specific detection of kanamycins is realized.

Description

A kind of immobilised enzymes method and application improving horseradish peroxidase stability
Technical field
The invention belongs to environmental monitoring technology fields, are related to a kind of immobilised enzymes for improving horseradish peroxidase stability Method and application.
Background technique
Native enzyme be generated by living cells, have to its substrate high degree of specificity and high catalytic efficiency protein or RNA.Native enzyme has many advantages, such as that from a wealth of sources, many kinds of, substrate affinity is good, high catalytic efficiency, is widely used in and urges Change the various aspects of the production and living such as analysis, medical diagnosis on disease, clinical treatment.But as a kind of typical biomolecule, naturally The stability of enzyme is relatively poor, has strict requirements vulnerable to the influence of external environment, and to storage condition.Such as native enzyme pole Partial function is lost vulnerable to the extremely destruction of external condition such as high temperature, strong acid, highly basic or even loses original activity.With receiving The rapid development of rice technology in conjunction with the stability of nano material, utilized the biology point such as nano material immobilization native enzyme in recent years Son has become new research hotspot (Small, 2017,13 (32): 1700880).Immobilised enzymes refers to that enzyme is physically limited It makes or is positioned in certain area of space, catalytic activity remains unchanged.Enzyme immobilization technology can more effectively control reaction Process, the storage capacity and the stability under extreme operating condition for improving enzyme.In addition, the multilevel structure of immobilised enzymes makes it more It can be easily separated, reduce pollution to the maximum extent, recycling etc. is realized, to reduce production cost.Currently, common nano material Immobilised enzymes means have sol-gel matrix, hydrogel, organic fine particles, mesoporous silicon oxide, metal organic framework (MOFs) material Material etc., these porous materials have biggish surface area and voidage, are the ideal material (Materials of enzyme immobilization Horizons,2017,4(1):55-63；Physical Chemistry Chemical Physics,2018,20(13): 8822-8831；Nature Catalysis,2018,1(9):689-695).But then, the catalytic of these materials itself Can be poor, after immobilized enzyme, often inhibit amount of activated (the Journal of the American Chemical of native enzyme Society,2017,139(19):6530-6533；Journal of the American Chemical Society,2015, 137(13):4276-4279)。
Porous structure and graphene (GO) material that zeolitic imidazolate framework material (ZIFs) is combined in the present invention are excellent Stability, realize natural horseradish peroxidase using -8/ graphene composite material (ZIF-8/GO) of zeolite imidazole ester skeleton The fixation (HRP@ZIF-8/GO) of enzyme (HRP).The one kind of zeolite imidazole ester skeleton structure (ZIFs) material as MOFs material is Connect by transition metal atoms with imidazoles or imdazole derivatives and generate one kind it is novel, with the porous of zeolite topological Material provides for immobilization native enzyme and is effectively combined site.ZIFs not only has the advantages that MOFs, but also with MOFs material It compares, there is more excellent thermal stability and aqueous stability.Grapheme material not only has rich as typical carbon material Rich oxygen-containing functional group and pi-electron conjugation region, environmental stability also with higher, surface functional group abundant can mention For the reaction site covalently or non-covalently combined, lay a good foundation for building multilevel structure.Based on -8/ stone of zeolite imidazole ester skeleton There is the porous structure being cross-linked with each other inside the multilevel structure of black alkene composite material building, more knots can be provided for native enzyme Coincidence point enhances the ability of native enzyme anti-shock loading, further promotes the environmental stability of native enzyme.In addition, passing through utilization DNA molecular regulates and controls the Catalyzed Synthesis By Peroxidase activity of HRP@ZIF-8/GO Biocomposite material, establishes a kind of universal ratio Colour analysis method is used for the detection of kanamycins (KAN).
Summary of the invention
The present invention solves the defects of stability existing for native enzyme is insufficient, passes through -8/ graphene of zeolite imidazole ester skeleton Composite material (ZIF-8/GO) realizes the fixation (HRP@ZIF-8/GO) of natural horseradish peroxidase, and is applied to card The colorimetric detection of that mycin (KAN).
In the present invention, the biological composite wood of ZIF-8/GO nanocomposite immobilized HRP has been synthesized at normal temperatures and pressures Material.The technological means of ZIF-8/GO mineralising can remain the original Catalyzed Synthesis By Peroxidase activity of HRP.Relative to ZIF-8 mine Change for HRP or free HRP, ZIF-8/GO protective layer increases HRP confrontation extreme environment such as hydrolysising protease, high temperature and has The tolerance of solvent etc. can also effectively improve the storage stability of native enzyme and recycle performance, this protection Performance is gradually increased with the increase of GO content in composite material, absolutely proves that the introducing of GO can make up to a certain extent The disadvantage of ZIF-8 skeleton structure stability deficiency, further enhances the stability of native enzyme.In addition, can be adjusted using DNA molecular This feature of HRP@ZIF-8/GO catalytic activity is controlled, the unmarked colorimetric based on HRP@ZIF-8/GO Biocomposite material is established Method for sensing, the absorbance change of reaction system are positively correlated with the concentration of kanamycins in a certain range, to be antibiotic Quantitative analysis provide foundation.
Technical solution of the present invention:
A kind of immobilised enzymes method improving horseradish peroxidase stability, steps are as follows:
(1) prepare graphene oxide: graphene oxide is prepared using improved Hummers chemical method.Steps are as follows: taking A certain amount of concentrated sulfuric acid (98%) is slowly added into graphite powder (concentrated sulfuric acid volume: graphite powder quality=23:1), is sufficiently stirred Afterwards, KMnO is slowly added in 0 DEG C of ice-water bath4(KMnO4It is 3:1 with graphite powder mass ratio), it is sufficiently stirred simultaneously.Then it will mix After closing object continuous ultrasound 5-7h, deep brown solution is obtained.High purity water is slowly added into deep brown solution, 5- is boiled in heating After 15min, the hydrogen peroxide (H of high purity water and 30% is sequentially added2O2) reaction is terminated, obtain the graphene oxide of glassy yellow Aqueous solution.After centrifuge separation, with dilute hydrochloric acid under conditions of 8000-10000r/min 2-3 removal impurity of centrifuge washing, then With high purity water under conditions of 8000-10000r/min 5-7 removal impurity of centrifuge washing.The graphite oxygen of purifying is taken out after washing Compound is packed into bag filter (MW=14000) and dialyses 5-7 days further to remove impurity, and finally freeze-drying obtains solid oxidation Graphene.
(2) -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton (HRP@ZIF-8/ is prepared GO): graphene oxide (GO) and zinc nitrate (Zn (NO3)2) abundant ultrasonic mixing in water, form mixed liquor A；HRP and 2- methyl Imidazoles is soluble in water, forms mixed liquid B；Later, mixed liquid B is quickly poured into mixed liquor A, obtained mixed liquor is at room temperature Stir 12-24h；Wherein the mass ratio of GO and HRP is in 0:50-1:1, HRP mass, Zn (NO3)2Mole and 2-methylimidazole Mole proportions be 5g:100mol:7000mol；By obtained mixture with high purity water eccentric cleaning 3-5 times, freezing Powdered substance is obtained after dry 12-24h, is ground freezen protective under conditions of being placed in -4~-20 DEG C；Obtained difference The product of GO content is named as HRP@ZIF-8/GO-x, and wherein x represents quality of the GO being added in final product.
A kind of application for the immobilised enzymes improving horseradish peroxidase stability, the quantitative detection of kanamycins (KAN): Under the conditions of 25-35 DEG C of temperature, the KAN solution that concentration is 0-50 μ g/L is adapted to liquid solution with KAN and is placed in HEPES buffer solution Hatch 20-30min in (10-25mM, pH=7-8)；Then to addition HRP@ZIF-8/GO-x compound in mixed liquor in 25-35 Continue to hatch 20-30min at DEG C, wherein the quality of HRP@ZIF-8/GO-x compound and the ratio of KAN aptamers mole are 14×106g:214mol；And then TMB (3,3', 5,5'- tetramethyl benzidine) and H is added2O2, wherein KAN aptamers, TMB And H2O2The molar ratio 214:50:11 of three.After mixture reacts 10-30min at 25-37 DEG C, it is transferred to quartz colorimetric utensil In, system absorbance is recorded with the change curve of absorbing wavelength.
The aptamers sequence is
5'-TGG GGG TTG AGG CTA AGC CGA-3'。
Beneficial effects of the present invention:
(1) when 4% that the GO ratio of addition is final product quality, adaptation bulk concentration is 2.14 μM, HRP ZIF-8/GO Complex concentration is 0.14mg/mL, hatching and temperature be respectively 20min and 25 DEG C, reaction time and temperature be respectively 10min and At 37 DEG C, the reaction system of -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton to card that The linear detection range of mycin is limited to 0.02 μ g/L in 1-25 μ g/L, detection.
(2) present invention in -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton method It is carried out at normal temperatures and pressures by simple physical agitation, does not need the synthesis condition of the complexity such as high pressure, high temperature.
(3) the colorimetric detection body of -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton System can realize the detection of other objects by the type of replacement aptamers, that is, have the function of versatility detection.
(4) present invention in -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton method The tolerance for increasing HRP confrontation extreme environment such as hydrolysising protease, high temperature and organic solvent etc., can also effectively improve day The storage stability and recycling performance, this protective value of right enzyme gradually increase with the increase of GO content in composite material By force, it absolutely proves that the introducing of GO can make up the disadvantage of ZIF-8 skeleton structure stability deficiency to a certain extent, further increases The stability of strong native enzyme.
Detailed description of the invention
Fig. 1 is of the present invention based on -8/ graphene composite material immobilization horseradish peroxidating of zeolite imidazole ester skeleton The preparation process and detection mechanism schematic diagram of object enzyme.
Fig. 2 is -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton that the present invention obtains Through extreme condition (protein hydrolytic reagent, denaturant, high temperature, organic solvent) relative catalytic activity that treated.
Fig. 3 is -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton that the present invention obtains Through different storage number of days treated relative catalytic activity.
Fig. 4 is -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton that the present invention obtains Relative catalytic activity after being collected by centrifugation and recycling.
Fig. 5 is -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton that the present invention obtains Standard working curve applied to kanamycins detection.
Fig. 6 is -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton that the present invention obtains Normal linearity curve applied to kanamycins detection.
In figure:
Specific embodiment
A specific embodiment of the invention is illustrated below in conjunction with technical solution.
Embodiment 1
Configure the measurement of kanamycins content in water sample:
(1) preparation of graphene oxide:
Graphene oxide is prepared using improved Hummers chemical method.Specific step is as follows: it is slow to weigh 1.0g graphite powder Slowly it is added in the 23mL concentrated sulfuric acid, after being sufficiently stirred, is slowly added to 3g KMnO in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously Divide stirring, then after continuous ultrasound 7h, obtain deep brown solution, be then slowly added into 46mL deionized water, 15min is boiled in heating Afterwards, it sequentially adds 140mL high purity water and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide aqueous solution of glassy yellow. After centrifuge separation, with 5% 2 removal impurity of dilute hydrochloric acid 8000r/min centrifuge washing, then with high purity water 8000r/min from The heart washs 3 removal impurity.Take out the graphite oxide of purifying after washing, be packed into bag filter (MW=14000) dialyse one week with Impurity is further removed, finally freeze-drying obtains solid oxidation graphene.
(2) -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton (HRP@ZIF-8/ is prepared GO): the GO aqueous solution (10mL) of 1mg/mL and the Zn (NO of 10mL3)2·6H2O (0.10M) sufficiently ultrasonic mixing (mixed liquor A). 50mg HRP is taken to be dissolved in 2-methylimidazole (3.46M) aqueous solution of 20mL (mixed liquid B).Later, mixed liquid B is quickly poured into In mixed liquor A, 12h is stirred at room temperature in obtained mixed liquor.Later, mixture is through high purity water eccentric cleaning 3 times, freeze-drying For 24 hours, the powder mull that will be obtained, freezen protective at -20 DEG C.The product of obtained different GO contents is named as HRP@ZIF-8/ GO-x, wherein x represents mass percent of the GO of addition in final product.Therefore the product obtained is HRP@ZIF-8/ GO-4。
(3) -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton (HRP@ZIF-8/GO) Extreme condition stability study: take HRP@ZIF-8 and HRP the@ZIF-8/GO-x compound and equal substances of various concentration respectively The HRP aqueous solution of amount and after the trypsase of 1mg/mL or Proteinase K hatch 1h at 37 DEG C, measure its anti-proteolytic enzyme Ability.Influence for measurement urea or EDTA to enzymatic activity, takes the HRP@ZIF-8 and HRP@ZIF-8/ of various concentration respectively GO-x compound (equivalent HRP) hatches 1h with EDTA the or 6M urea of 1wt% at room temperature.High temperature is resistant to for test multienzyme complex With the ability of organic solvent, HRP@ZIF-8 and HRP@ZIF-8/GO-x compound (the equivalent HRP, HRP of various concentration are taken respectively Amount 0.14mg/mL) hatching 10-30min and 50% organic solvent (methanol, acetone and N,N-dimethylformamide) exist in boiling water Hatch 30 minutes at room temperature.Horseradish peroxidase and its multienzyme complex utilize after above-mentioned different extreme condition processing PBS (10mM, pH=7.40) is centrifuged (8000r/min) and washs 3 times.Later, typical TMB-H is utilized2O2Reaction system measurement is peppery The variation of root peroxidase and its multienzyme complex catalytic activity before and after the processing, takes HRP, HRP@ZIF-8 and HRP@ZIF- respectively 8/GO-x aqueous nancomposite dispersion liquid (equivalent HRP), the H with the TMB and 14.28mM of 0.75mM2O2It is slow in the acetic acid of 0.20M In fliud flushing after (pH=4.00) incubation 10min (37 DEG C), it is transferred in quartz colorimetric utensil, measurement system exists at room temperature Absorbance at 652nm, and compared with its untreated preceding original absorbance, relative catalytic activity (Fig. 2) is calculated.
(4) quantitative detection of kanamycins (KAN):
The KAN solution (0-50 μ g/L) of various concentration is taken to mix with KAN aptamers (2.14 μM), at room temperature 25mM's HEPES buffer solution (100mM NaCl, 2mM MgCl2, pH=7.60) in hatch 30min.Then to being added in mixed liquor The HRP@ZIF-8/GO-4 compound of 0.14mg/mL continues to hatch 20min.Add the H of the TMB and 0.11mM of 0.50mM2O2。
(5) detection method: after the mixture in step (4) reacts 10min at 37 DEG C, being transferred in quartz colorimetric utensil, Record system absorbance with absorbing wavelength (500-800nm) change curve (Fig. 5).
(5) drafting of standard working curve
With the increase of kanamycins concentration in sample in step (4), absorbance of the reaction system at 652nm constantly increases Add, within the scope of 0-50 μ g/L, the absorbance and kanamycins concentration of reaction system have good linear relationship, linear correlation system Number R2=0.99 (Fig. 6).
(6) measurement of kanamycins content in water sample is configured:
The water sample for being 0.02 μ g/L with HEPES buffer solution configuration kanamycins concentration.Sample is used for step (4) method It is detected, the standard working curve that testing result is obtained with step (5) compares, and calculates the concentration of kanamycins.Experiment knot Fruit measures 0.0225 μ g/L of kanamycins content, the rate of recovery 113%.Relative standard deviation RSD is 2.35% (n=5).
Embodiment 2
Configure the measurement of kanamycins content in water sample:
(1) preparation of graphene oxide:
Graphene oxide is prepared using improved Hummers chemical method.Specific step is as follows: it is slow to weigh 1.0g graphite powder Slowly it is added in the 23mL concentrated sulfuric acid, after being sufficiently stirred, is slowly added to 3g KMnO in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously Divide stirring, then after continuous ultrasound 6h, obtain deep brown solution, be then slowly added into 46mL deionized water, 15min is boiled in heating Afterwards, it sequentially adds 140mL high purity water and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide aqueous solution of glassy yellow. After centrifuge separation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washing, high purity water 10000r/min is then used 5 removal impurity of centrifuge washing.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=14000) and is dialysed one week Further to remove impurity, finally freeze-drying obtains solid oxidation graphene.
(2) -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton (HRP@ZIF-8/ is prepared GO): the GO aqueous solution (10mL) of 1mg/mL and the Zn (NO of 10mL3)2·6H2O (0.10M) sufficiently ultrasonic mixing (mixed liquor A). 50mg HRP is taken to be dissolved in 2-methylimidazole (3.46M) aqueous solution of 20mL (mixed liquid B).Later, mixed liquid B is quickly poured into In mixed liquor A, 12h is stirred at room temperature in obtained mixed liquor.Later, mixture is through high purity water eccentric cleaning 3 times, freeze-drying For 24 hours, the powder mull that will be obtained, freezen protective at -20 DEG C.The product of obtained different GO contents is named as HRP@ZIF-8/ GO-x, wherein x represents mass percent of the GO of addition in final product.Therefore the product obtained is HRP@ZIF-8/ GO-4。
(3) -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton (HRP@ZIF-8/GO) Stable storage Journal of Sex Research: for measure multienzyme complex storage stability, take the HRP ZIF-8 and HRP of various concentration respectively The PBS dispersion liquid (10mM, pH=7.40) of ZIF-8/GO-x (equivalent HRP) utilizes allusion quotation after storing the different time at 4 DEG C The TMB-H of type2O2Reaction system measures the change of catalytic activity in horseradish peroxidase and its multienzyme complex difference storage time Change, takes HRP, HRP@ZIF-8 and HRP@ZIF-8/GO-x aqueous nancomposite dispersion liquid (equivalent HRP, HRP amount 0.14mg/ respectively ML), with the H of the TMB of 0.75mM and 14.28mM2O2(pH=4.00) the incubation 10min in the acetate buffer of 0.20M (37 DEG C) afterwards, are transferred in quartz colorimetric utensil, at room temperature absorbance of the measurement system at 652nm, and with its original absorbance Comparison, is calculated relative catalytic activity (Fig. 3).
(4) quantitative detection of kanamycins (KAN):
The KAN solution (0-50 μ g/L) of various concentration is taken to mix with KAN aptamers (2.14 μM), at room temperature 25mM's HEPES buffer solution (100mM NaCl, 2mM MgCl2, pH=7.60) in hatch 30min.Then to being added in mixed liquor The HRP@ZIF-8/GO-4 compound of 0.14mg/mL continues to hatch 20min.Add the H of the TMB and 0.11mM of 0.50mM2O2。
(5) detection method: after the mixture in step (4) reacts 10min at 37 DEG C, being transferred in quartz colorimetric utensil, Record system absorbance with absorbing wavelength (500-800nm) change curve (Fig. 5).
(6) drafting of standard working curve
With the increase of kanamycins concentration in sample in step (5), absorbance of the reaction system at 652nm constantly increases Add, within the scope of 0.01-0.50 μ g/L, the absorbance and kanamycins concentration of reaction system have good linear relationship, linearly Coefficient R2=0.99 (Fig. 6).
(7) measurement of kanamycins content in water sample is configured:
The water sample for being 0.30 μ g/L with HEPES buffer solution configuration kanamycins concentration.Sample is used for step (4) method It is detected, the standard working curve that testing result is obtained with step (5) compares, and calculates the concentration of kanamycins.Experiment knot Fruit measures 0.35 μ g/L of kanamycins content, the rate of recovery 117%.Relative standard deviation RSD is 1.35% (n=5).
Embodiment 3
Originally in water sample kanamycins content measurement:
(1) preparation of graphene oxide:
Graphene oxide is prepared using improved Hummers chemical method.Specific step is as follows: it is slow to weigh 1.0g graphite powder Slowly it is added in the 23mL concentrated sulfuric acid, after being sufficiently stirred, is slowly added to 3g KMnO in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously Divide stirring, then after continuous ultrasound 5h, obtain deep brown solution, be then slowly added into 46mL deionized water, 15min is boiled in heating Afterwards, it sequentially adds 140mL high purity water and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide aqueous solution of glassy yellow. After centrifuge separation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washing, high purity water 10000r/min is then used 5 removal impurity of centrifuge washing.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=14000) and is dialysed one week Further to remove impurity, finally freeze-drying obtains solid oxidation graphene.
(2) -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton (HRP@ZIF-8/ is prepared GO): the GO aqueous solution (10mL) of 1mg/mL and the Zn (NO of 10mL3)2·6H2O (0.10M) sufficiently ultrasonic mixing (mixed liquor A). 50mg HRP is taken to be dissolved in 2-methylimidazole (3.46M) aqueous solution of 20mL (mixed liquid B).Later, mixed liquid B is quickly poured into In mixed liquor A, 12h is stirred at room temperature in obtained mixed liquor.Later, mixture is through high purity water eccentric cleaning 3 times, freeze-drying For 24 hours, the powder mull that will be obtained, freezen protective at -20 DEG C.The product of obtained different GO contents is named as HRP@ZIF-8/ GO-x, wherein x represents mass percent of the GO of addition in final product.Therefore the product obtained is HRP@ZIF-8/ GO-4。
(3) -8/ graphene composite material immobilized HRP of zeolite imidazole ester skeleton (HRP@ZIF-8/GO) Cycle utilize stability study: for the power of regeneration of the enzyme composite material of test preparation, in the TMB containing 0.75mM and 14.28mM H2O20.2M acetate buffer system (pH=4.00) in certain density HRP, HRP@ZIF-8 or HRP@is added ZIF-8/GO-x composite material (equivalent HRP).Mixed liquor measures supernatant absorbance after 37 DEG C of reaction 10min.Mixture exists It is centrifuged 10min under 13000rpm, to collect sediment, carries out enzymic catalytic reaction next time.The process is repeated 4 times to measure enzyme and urge The recycling ability of agent.Utilize typical TMB-H2O2Reaction system measurement horseradish peroxidase and its multienzyme complex follow Ring utilizes the variation of front and back catalytic activity, takes HRP, HRP@ZIF-8 and HRP@ZIF-8/GO-x aqueous nancomposite dispersion liquid respectively (equivalent HRP, HRP amount 0.14mg/mL), the H with the TMB and 14.28mM of 0.75mM2O2In the acetate buffer of 0.20M (pH=4.00) (37 DEG C) after incubation 10min it, is transferred in quartz colorimetric utensil, measurement system is at 652nm at room temperature Absorbance, and compared with its original absorbance, relative catalytic activity (Fig. 4) is calculated.
(4) quantitative detection of kanamycins (KAN):
The KAN solution (0-50 μ g/L) of various concentration is taken to mix with KAN aptamers (2.14 μM), at room temperature 25mM's HEPES buffer solution (100mM NaCl, 2mM MgCl2, pH=7.60) in hatch 30min.Then to being added in mixed liquor The HRP@ZIF-8/GO-4 compound of 0.14mg/mL continues to hatch 20min.Add the H of the TMB and 0.11mM of 0.50mM2O2。
(5) detection method: after the mixture in step (4) reacts 10min at 37 DEG C, being transferred in quartz colorimetric utensil, Record system absorbance with absorbing wavelength (500-800nm) change curve (Fig. 5).
(6) drafting of standard working curve
With the increase of kanamycins concentration in sample in step (5), absorbance of the reaction system at 652nm constantly increases Add, within the scope of 0.01-0.50 μ g/L, the absorbance and kanamycins concentration of reaction system have good linear relationship, linearly Coefficient R2=0.99 (Fig. 6).
(6) originally in water sample kanamycins content measurement:
Due to being free of kanamycins in the sample of municipal tap water, therefore use recovery testu.With the water of originally water sample Sample prepares kanamycins solution, and concentration is 0.25 μ g/L.Sample is used to step (4) method to detect, testing result and step Suddenly the standard working curve comparison that (6) obtain, calculates the concentration of kanamycins.Experimental result measures kanamycins content 0.30 μ g/L, the rate of recovery 120%.Relative standard deviation RSD is 2.50% (n=5).

Claims (2)

1. a kind of immobilised enzymes method for improving horseradish peroxidase stability, which is characterized in that steps are as follows:
(1) prepare graphene oxide: graphene oxide is prepared using improved Hummers chemical method；
(2) -8/ graphene composite material immobilized HRP HRP@ZIF-8/GO:GO of zeolite imidazole ester skeleton is prepared With Zn (NO3)2Abundant ultrasonic mixing in water forms mixed liquor A；HRP and 2-methylimidazole are soluble in water, form mixed liquid B； Later, mixed liquid B is quickly poured into mixed liquor A, 12-24h is stirred at room temperature in obtained mixed liquor；Wherein GO and HRP Mass ratio is 0:50-1:1, HRP mass, Zn (NO3)2Mole and 2-methylimidazole mole proportions be 5g:100mol: 7000mol；By obtained mixture with high purity water eccentric cleaning 3-5 times, powdered substance is obtained after being freeze-dried 12-24h, it will Freezen protective under conditions of its grinding is placed in -4~-20 DEG C；The product of obtained different GO contents is named as HRP@ZIF-8/ GO-x, wherein x represents mass percent of the GO being added in final product.
2. a kind of application for the immobilised enzymes for improving horseradish peroxidase stability, which is characterized in that kanamycins KAN's determines Amount detection: under the conditions of 25-35 DEG C of temperature, the KAN solution that concentration is 0-50 μ g/L is adapted to liquid solution with KAN and is placed in HEPES Hatch 20-30min in buffer；Wherein, the concentration of HEPES buffer solution is 10-25mM, pH=7-8；Add then in mixed liquor Enter HRP@ZIF-8/GO-x compound to continue to hatch 20-30min at 25-35 DEG C, wherein HRP@ZIF-8/GO-x compound The ratio of quality and KAN aptamers mole is 14 × 106g:214mol；And then 3,3', 5,5'- tetramethyl biphenyls are added Amine TMB and H2O2, wherein KAN aptamers, TMB and H2O2The molar ratio 214:50:11 of three；Mixture reacts at 25-37 DEG C It after 10-30min, is transferred in quartz colorimetric utensil, records system absorbance with the change curve of absorbing wavelength；
The aptamers sequence is 5'-TGG GGG TTG AGG CTA AGC CGA-3'.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110492088A (en) * 2019-09-16 2019-11-22 安徽师范大学 A kind of ZIF-8@redox graphene sulfur loaded composite material and preparation method and lithium-sulphur cell positive electrode and lithium-sulfur cell
CN111320697A (en) * 2020-02-13 2020-06-23 江苏大学 Enzyme-labeled secondary antibody HRP/goat anti-mouse IgG @ ZIF-L and preparation method and application thereof
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WO2020228291A1 (en) * 2019-05-14 2020-11-19 大连理工大学 Immobilised enzyme method for improving the stability of horseradish peroxidase, and application therefor
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107446915A (en) * 2017-08-30 2017-12-08 江苏大学 Magnetic oxygenated graphene composite material immobilized HRP and its preparation method and application
CN107497377A (en) * 2017-10-19 2017-12-22 山东大学 A kind of preparation method of the homogeneous metal organic framework compound of pattern/graphene oxide complex microsphere
CN107803119A (en) * 2017-11-03 2018-03-16 宁夏大学 A kind of preparation method of composite film and the method using residual tetracycline in its removal pharmacy waste water
CN108008128A (en) * 2017-12-21 2018-05-08 江南大学 Aptamer identifies and the kanamycins quick detection test paper of functionalized magnetic microsphere separation and culture and its preparation and application

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103290124B (en) * 2013-05-29 2014-12-17 华南农业大学 Detection method for kanamycin A enzyme-linked aptamer and application of kanamycin A enzyme-linked aptamer
CN105734038A (en) * 2016-01-22 2016-07-06 南京工业大学 Enzyme-GO-MOFs nano composite catalyst and preparation method thereof
CN110218720B (en) * 2019-05-14 2022-07-19 大连理工大学 Immobilized enzyme method for improving stability of horseradish peroxidase and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107446915A (en) * 2017-08-30 2017-12-08 江苏大学 Magnetic oxygenated graphene composite material immobilized HRP and its preparation method and application
CN107497377A (en) * 2017-10-19 2017-12-22 山东大学 A kind of preparation method of the homogeneous metal organic framework compound of pattern/graphene oxide complex microsphere
CN107803119A (en) * 2017-11-03 2018-03-16 宁夏大学 A kind of preparation method of composite film and the method using residual tetracycline in its removal pharmacy waste water
CN108008128A (en) * 2017-12-21 2018-05-08 江南大学 Aptamer identifies and the kanamycins quick detection test paper of functionalized magnetic microsphere separation and culture and its preparation and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
QIANQIAN ZHU等: "《Nano-biocatalysts of Cyt c@ZIF-8/GO composites with high recyclability via a de novo approach》", 《ACS APPLIED MATERIALS & INTERFACES》 *
YONG PAN等: "《Zeolitic imidazolate framework-based biosensor for detection of HIV-1 DNA》", 《ANALYTICAL BIOCHEMISTRY》 *
田运齐等: "《辣根过氧化物酶在MOF上的固定化研究》", 《辽宁师范大学学报(自然科学版)》 *

Cited By (11)

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