CN110441250B - Preparation method of double-enzyme co-immobilized copper nanoflower material and application of double-enzyme co-immobilized copper nanoflower material in glucose detection - Google Patents
Preparation method of double-enzyme co-immobilized copper nanoflower material and application of double-enzyme co-immobilized copper nanoflower material in glucose detection Download PDFInfo
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- 239000008103 glucose Substances 0.000 title claims abstract description 60
- 239000002057 nanoflower Substances 0.000 title claims abstract description 54
- 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 53
- 229910052802 copper Inorganic materials 0.000 title claims abstract description 29
- 239000010949 copper Substances 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 239000000463 material Substances 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 41
- 239000000243 solution Substances 0.000 claims abstract description 35
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- 239000007864 aqueous solution Substances 0.000 claims abstract description 22
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- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 20
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 20
- 239000002244 precipitate Substances 0.000 claims abstract description 18
- 239000006228 supernatant Substances 0.000 claims abstract description 14
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- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 10
- 239000012498 ultrapure water Substances 0.000 claims abstract description 10
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- 238000010438 heat treatment Methods 0.000 claims abstract description 7
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- 239000012467 final product Substances 0.000 abstract 1
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- 238000004458 analytical method Methods 0.000 description 5
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- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
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- 239000004472 Lysine Substances 0.000 description 1
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention provides a preparation method of a double-enzyme co-immobilized copper nano flower material and application of the double-enzyme co-immobilized copper nano flower material in glucose detection, wherein the preparation method of the copper nano flower material comprises the following steps: (1) adding a phosphate buffer solution in which a deuterohemin short peptide compound and glucose oxidase are dissolved into a reaction container, and placing the reaction container at room temperature; (2) slowly dropwise adding CuSO4Aqueous solution, so that CuSO is in solution4The final concentration of (A) is 0.5-1 mmol/L; (3) standing; (4) centrifuging, removing supernatant, and keeping precipitate; (5) sequentially adding ultrapure water and absolute ethyl alcohol to clean the precipitate; (6) placing the precipitate in a reaction container, heating in water bath until the absolute ethyl alcohol is completely evaporated to obtain the final product; the novel hybrid nano prepared by the invention simultaneously fixes glucose oxidase and deuterohemin short peptide compound, realizes the construction of a glucose detection double-enzyme system, and has the activity of both the glucose oxidase and the peroxidase, thereby realizing the cascade detection of glucose.
Description
Technical Field
The invention relates to the technical field of glucose detection, in particular to a preparation method of a double-enzyme co-immobilized copper nanoflower material and application of the double-enzyme co-immobilized copper nanoflower material in glucose detection.
Background
Glucose detection has been attracting attention as an important research field in bioanalysis because of its important application value in biology, clinical medicine, and food production.
The current glucose detection method based on glucose oxidase and peroxidase, which is commonly used for analysis, is divided into two steps: first, glucose is oxidized by glucose oxidase, which can be in the presence of oxygenIn the presence of a catalyst, the oxidation of glucose to gluconic acid and H2O2. Then, H obtained in the previous step2O2Oxidizing 3,3',5,5' -Tetramethylbenzidine (TMB) under the catalysis of peroxidase to generate a blue oxidized TMB polymer (oxTMB), wherein the oxTMB has the maximum light absorption value at 652nm, and the concentration of the oxTMB can be detected by a spectrophotometric method, so that the concentration of glucose in a sample to be detected is calculated.
However, due to the fact that the enzymes catalyzing the two reaction steps need different optimal temperatures and pH values, the existing glucose concentration detection needs multi-step operation, the operation is time-consuming and tedious, hydrogen peroxide generated by glucose oxidation can be decomposed in the two-step enzymatic reaction process, the detection sensitivity is affected, and further development of glucose detection is restricted.
Disclosure of Invention
The invention aims to provide a preparation method of a double-enzyme co-immobilized copper nanoflower material and application of the double-enzyme co-immobilized copper nanoflower material in glucose detection, the prepared novel hybrid nano simultaneously fixes glucose oxidase and deuterohemin short peptide compounds, and construction of a glucose detection double-enzyme system is realized, so that the double-enzyme co-immobilized copper nanoflower material has the activity of both the glucose oxidase and peroxidase, and thus cascade detection of glucose is realized.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a preparation method of a double-enzyme co-immobilized copper nanoflower material comprises the following steps:
(1) preparing an enzyme solution: adding a phosphate buffer solution in which a deuterohemin short peptide compound and glucose oxidase are dissolved into a reaction container, and placing the reaction container at room temperature;
(2) preparing the nanoflower: slowly dripping CuSO into the solution obtained in the step (1)4Aqueous solution, so that CuSO is in solution4The final concentration of (A) is 0.5-1 mmol/L;
(3) self-assembly: standing at 15-45 deg.C for 36-108 hr;
(4) and (3) nano-flower treatment: centrifuging for 5-10min, discarding supernatant, and collecting precipitate;
(5) sequentially adding ultrapure water and absolute ethyl alcohol to clean the precipitate;
(6) and (5) placing the precipitate obtained by the treatment in the step (5) into a reaction container, heating in a water bath until the absolute ethyl alcohol is completely evaporated, and obtaining the product.
Preferably, in step (1), the deuterohemin short peptide compound comprises: dh- β -Ala-His-Glu; dh- β -Ala-His-Lys; dh- β -Ala-His-Asp; dh- β -Ala-His-Thr-Val-Glu-Lys. Wherein Dh is deuterohemin, beta-Ala is beta-substituted alanine, His is histidine, Lys is lysine, Glu is glutamic acid, Asp is aspartic acid; thr is threonine; val is valine.
Preferably, in the step (1), the concentration of the deuterohemin short peptide compound is 1 mg/mL; the concentration of the glucose oxidase is 1 mg/mL; the concentration of the phosphate buffer was 50mmol/L, and the pH was 7.4.
Preferably, in step (2), CuSO4The concentration of the aqueous solution is 120 mmol/L; CuSO in solution4The final concentration of (A) was 0.8 mmol/L.
Preferably, in the step (5), the specific steps are as follows: in the step (5), the concrete steps are as follows: adding ultrapure water to 2/3 volume of the reaction container, performing ultrasonic treatment in an ultrasonic cleaner for 1-3min, and repeating the step (4) for 2-4 times; then adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, carrying out ultrasonic treatment in an ultrasonic cleaner for 1-3min, and repeating the step (4) for 1-2 times; adding anhydrous ethanol to 1/3 volume of the reaction vessel, performing ultrasonic treatment in an ultrasonic cleaner for 5-8min, and repeating the step (4) for 1-2 times.
Preferably, in step (6), the temperature of the water bath is 40-60 ℃.
The double-enzyme co-immobilized copper nanoflower material prepared by the invention is applied to glucose detection.
The application specifically comprises the following steps:
(1) preparing a reaction system: adding 800 μ L of 50mmol/L pH7.0 phosphate buffer solution and 100 μ L of 10mmol/L TMB aqueous solution into multiple EP tubes containing 0.25mg of co-immobilized hybrid nanoflower, and adding 100 μ L of 50mmol/L pH7.0 phosphate buffer solution and 100 μ L of glucose aqueous solutions with different concentrations into the different EP tubes to make final glucose concentrations in the EP tubes respectively 0 μmol/L, 10 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, 500 μmol/L, 1000 μmol/L and 1500 μmol/L; after the addition is finished, the mixture is swirled for 10 to 30 seconds to be fully and uniformly mixed;
(2) and (3) carrying out a nanoflower catalytic cascade reaction: standing the EP tube in a water bath at 37 ℃ for 15min to fully react;
(3) centrifuging the system: placing the EP pipe in a centrifuge to process for 2min at the rotating speed of 6000rpm, so that the nanoflower is separated from the system solution; then taking 800 mu L of supernatant for ultraviolet detection;
(4) ultraviolet detection: detecting the absorbance value of the supernatant at the wavelength of 652nm of a spectrophotometer by taking the concentration of glucose as an abscissa, A652nmFor the ordinate, a regression curve was constructed, the regression equation was fitted, the glucose concentration and A652nmA linear relationship;
(5) detecting glucose with unknown concentration by using nanoflower: repeating the steps (1) to (4) to obtain the absorbance value A652nmSubstituting into regression equation to obtain ordinate value, and obtaining abscissa value as glucose concentration.
The invention has the beneficial effects that:
the invention designs and synthesizes the His-containing peroxidase mimic enzyme deuterohemin short peptide compound according to the structure of the peroxidase taking heme as a prosthetic group, has very high peroxidase activity, the specific activity can reach 93 percent of that of natural peroxidase MP-11, and especially the optimum pH of the room catalytic reaction is close to that of glucose oxidase and is about pH 7.0.
The novel hybrid nano prepared by the invention simultaneously fixes glucose oxidase and deuterohemin short peptide compound, realizes the construction of a glucose detection double-enzyme system, and ensures that the hybrid nano has the activity of both the glucose oxidase and the peroxidase, thereby realizing the cascade detection of glucose. The process that the glucose is decomposed by the glucose oxidase and the substrate is catalyzed by the deuterohemin short peptide can be generated by adding the glucose and the TMB, so that the color of the solution in the system is obviously changed, other complex pretreatment processes are not needed in the detection process, and one-step detection can be realized. And because the optimal temperature and the optimal pH of the glucose oxidase and the deuterohemin short peptide compound are similar, the efficiency and the sensitivity of the glucose detection can be improved by the detector.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is an HPLC analytical map of Dh- β -Ala-His-Glu;
FIG. 2 is an HPLC analysis of Dh- β -Ala-His-Lys;
FIG. 3 is an HPLC analysis of Dh- β -Ala-His-Asp;
FIG. 4 is a mass spectrum of Dh- β -Ala-His-Glu;
FIG. 5 is a mass spectrum of Dh- β -Ala-His-Lys;
FIG. 6 is a mass spectrum of Dh- β -Ala-His-Asp;
FIG. 7 is a mass spectrum of Dh- β -Ala-His-Thr-Val-Glu-Lys;
FIG. 8 is a scanning electron microscope image of a Dh-beta-Ala-His-Glu-glucose oxidase-copper phosphate co-immobilized hybrid nanoflower, wherein a, b and c are prepared nanoflowers;
FIG. 9 is a regression plot of glucose measurements.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: solid phase synthesis of Dh-beta-Ala-His-Glu
(1) Swelling resin: weighing 0.1mmol Rink-NH2Resin, adding dichloromethane reagent to fully soak the resin, swelling for 1h, filtering to remove dichloromethane, and washing the resin with Dimethylformamide (DMF) for 6 times.
(2) Deprotection: the Rink-NH treated in the step (1)2Adding DEP (20% piperidine in DMF) solution into the resin, shaking at room temperature for 15-30min, filtering, washing the resin with DMF for 6 times, detecting the deprotection condition of amino group with color developer (A: 5% ninhydrin ethanol solution B: 80% phenol ethanol solution), and when all resin particles are changed into bluish purple, the deprotection is complete.
(3) Connecting amino acids: Fmoc-Glu (otBu) -OH, PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HOBT (1-Hydroxybenzotriazole), NMM (N-methylmorpholine) and Rink-NH2Adding the resin into a reactor in sequence according to a molar ratio of 3:3:3:6:1, adding about 2/3 dimethylformamide in the volume of the container, oscillating at room temperature for 1.5-2h, performing suction filtration, washing the resin for 6 times by using the dimethylformamide, detecting the connection condition of the amino acid by using a color developing agent, and completely reacting when all resin particles are colorless.
(4) Repeating steps (2) and (3), and connecting the subsequent Fmoc-His (Trt) -OH and Fmoc-beta-Ala-OH.
(5) Connecting deuterohemin: repeating the step (2), and mixing deuterohemin, PyBOP, HOBT, NMM and Rink-NH2Adding the resin into the reactor in sequence according to the mol ratio of 0.8:1.2:1.5:2: 1; the reaction was stirred at room temperature for 8h by adding about 2/3 volumes of dichloromethane, and washed with dichloromethane and dry methanol 8-10 times until the solution was colorless.
(6) And (3) drying: the reactor was dried in bulk on a vacuum drying pump for one day to remove the organic reagents.
(7) Cutting: adding 7ml of cutting reagent (95% trifluoroacetic acid, 2.5% triisopropylsilane and 2.5% ultrapure water), reacting at room temperature for 1-1.5h, collecting liquid by a centrifuge (containing 30ml of anhydrous ether stored at-20 ℃) in an ice bath environment, controlling the flow rate at 1-2 drops/s, sealing the centrifuge, and placing in a refrigerator at-20 ℃ for standing for 1 day.
(8) And (3) precipitation: centrifuging the precipitate (4 deg.C, 6000rpm, 5min), discarding the supernatant, repeating for 2 times, and drying in vacuum drying pump for 1h to obtain crude heme tripeptide.
(9) And (3) analysis: the obtained crude deuterohemin tripeptide product is detected by reversed phase preparative liquid chromatography, and the result is shown in figure 1, and the main peak of Dh-beta-Ala-His-Glu appears in about 10 min. The main peak was subjected to MS detection, and the molecular weight was determined to be 900.24, which was consistent with the theoretical molecular weight. The HPLC analysis pattern of Dh-beta-Ala-His-Glu is shown in FIG. 1, and the mass spectrum is shown in FIG. 4.
Example 2: solid phase Synthesis of Dh-beta-Ala-His-Lys
(1) Example 1, step (1) was repeated.
(2) Example 1 step (2) was repeated.
(3) Connecting amino acids: Fmoc-Lys (Boc) -OH, PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HOBT (1-Hydroxybenzotriazol), NMM (N-methylmorpholine) and Rink-NH2Adding the resin into a reactor in sequence according to a molar ratio of 3:3:3:6:1, adding about 2/3 dimethylformamide in the volume of the container, oscillating at room temperature for 1.5-2h, performing suction filtration, washing the resin for 6 times by using the dimethylformamide, detecting the connection condition of the amino acid by using a color developing agent, and completely reacting when all resin particles are colorless.
(4) Example 1 step (4) was repeated.
(5) Example 1 step (5) was repeated.
(6) Example 1 step (6) was repeated.
(7) Example 1 step (7) was repeated.
(8) Example 1 step (8) was repeated.
(9) And (3) analysis: the obtained crude deuterohemin tripeptide product is detected by reversed phase preparative liquid chromatography, and the result is shown in figure 2, and the main peak of Dh-beta-Ala-His-Lys appears at about 12 min. The main peak was subjected to MS detection, and the molecular weight was determined to be 899.41, which was consistent with the theoretical molecular weight. The HPLC analysis pattern of Dh-beta-Ala-His-Lys is shown in FIG. 2, and the mass spectrum is shown in FIG. 5.
Example 3: solid phase Synthesis of Dh-beta-Ala-His-Asp.
(1) Example 1, step (1) was repeated.
(2) Example 1 step (2) was repeated.
(3) Connecting amino acids: Fmoc-Asp (OtBu) -OH, PyBOP (hexa-BOP)Benzotriazol-1-yl-oxytripyrrolidinophosphorus fluorophosphates), HOBT (1-Hydroxybenzotriazole), NMM (N-methylmorpholine) and Rink-NH2Adding the resin into a reactor in sequence according to a molar ratio of 3:3:3:6:1, adding about 2/3 dimethylformamide in the volume of the container, oscillating at room temperature for 1.5-2h, performing suction filtration, washing the resin for 6 times by using the dimethylformamide, detecting the connection condition of the amino acid by using a color developing agent, and completely reacting when all resin particles are colorless.
(4) Example 1 step (4) was repeated.
(5) Example 1 step (5) was repeated.
(6) Example 1 step (6) was repeated.
(7) Example 1 step (7) was repeated.
(8) Example 1 step (8) was repeated.
(9) And (3) analysis: the obtained crude deuterohemin tripeptide product is detected by reversed-phase preparative liquid chromatography, and the result is shown in figure 3, wherein the main peak of Dh-beta-Ala-His-Asp appears at about 12 min. The main peak was subjected to MS detection, and the molecular weight was determined to be 886.35, which was consistent with the theoretical molecular weight. The HPLC analysis pattern of Dh-beta-Ala-His-Asp is shown in figure 3, and the mass spectrum is shown in figure 6.
Example 4: solid phase synthesis of Dh-beta-Ala-His-Thr-Val-Glu-Lys.
(1) Example 1, step (1) was repeated.
(2) Example 1 step (2) was repeated.
(3) Connecting amino acids: Fmoc-Lys (BOC) -OH, PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HOBT (1-Hydroxybenzotriazol), NMM (N-methylmorpholine) and Rink-NH2Adding the resin into a reactor in sequence according to a molar ratio of 3:3:3:6:1, adding about 2/3 dimethylformamide in the volume of the container, oscillating at room temperature for 1.5-2h, performing suction filtration, washing the resin for 6 times by using the dimethylformamide, detecting the connection condition of the amino acid by using a color developing agent, and completely reacting when all resin particles are colorless.
(4) Repeating steps (2) and (3) and connecting the subsequent Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Thr (tBu) -OH, Fmoc-His (Trt) -OH and Fmoc-beta-Ala-OH.
(5) Example 1 step (5) was repeated.
(6) Example 1 step (6) was repeated.
(7) Example 1 step (7) was repeated.
(8) Example 1 step (8) was repeated.
(9) And (3) analysis: the obtained crude product of deuterohemin hexapeptide is detected by reversed phase preparative liquid chromatography, and the main peak of Dh-beta-Ala-His-Thr-Val-Glu-Lys appears in about 15 min. The main peak was subjected to MS detection, and the molecular weight was determined to be 1228.75, which was consistent with the theoretical molecular weight. The mass spectrum of Dh-beta-Ala-His-Thr-Val-Glu-Lys is shown in FIG. 7.
Example 5: preparation of Dh-beta-Ala-His-Glu-glucose oxidase-copper phosphate co-immobilized hybrid nanoflower
(1) Preparing an enzyme solution: to the reaction vessel, 50mmol/L of phosphate buffer pH7.4 containing 1mg/mL of Dh-. beta. -Ala-His-Glu and 1mg/mL of glucose oxidase was added, and the mixture was allowed to stand at room temperature.
(2) Preparing the nanoflower: slowly dropwise adding 120mmol/L CuSO into the solution obtained in the step (1)4Aqueous solution, so that CuSO is in solution4The final concentration of (A) was 0.8 mmol/L.
(3) Self-assembly: standing at 18-28 deg.C for 80 hr.
(4) And (3) nano-flower treatment: centrifuging at 5000rpm for 80min, and discarding supernatant to obtain precipitate.
(5) Adding ultrapure water to 2/3 volume of the reaction container, performing ultrasonic treatment in an ultrasonic cleaner for 2min, and repeating the step (4) for 3 times; then adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, carrying out ultrasonic treatment in an ultrasonic cleaner for 2min, and repeating the step (4) for 2 times; adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, performing ultrasonic treatment in an ultrasonic cleaner for 6min, and repeating the step (4) for 2 times.
(6) And (3) placing the precipitate obtained by the treatment in the step (5) into a reaction container, heating in water bath at 50-55 ℃ until the absolute ethyl alcohol is completely evaporated, thus obtaining the product.
(7) And (5) detecting the product by a scanning electron microscope after the product is dripped. The scanning electron microscope image of the Dh-beta-Ala-His-Glu-glucose oxidase-copper phosphate co-immobilized hybrid nanoflower is shown in fig. 8.
Example 6: preparation of Dh-beta-Ala-His-Lys-glucose oxidase-copper phosphate co-immobilized hybrid nanoflower
(1) Preparing an enzyme solution: to the reaction vessel, 50mmol/L of phosphate buffer pH7.4 containing 1mg/mL of Dh-. beta. -Ala-His-Glu and 1mg/mL of glucose oxidase was added, and the mixture was allowed to stand at room temperature.
(2) Preparing the nanoflower: slowly dropwise adding 120mmol/L CuSO into the solution obtained in the step (1)4Aqueous solution, so that CuSO is in solution4The final concentration of (A) was 0.5 mmol/L.
(3) Self-assembly: standing at 15-25 deg.C for 108 h.
(4) And (3) nano-flower treatment: the rotation speed is 7000rpm, centrifugation is carried out for 5min, and the supernatant is discarded, leaving the precipitate.
(5) Adding ultrapure water to about 2/3 volume of the reaction container, performing ultrasonic treatment in an ultrasonic cleaner for 3min, and repeating the step (4)4 times; then adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, carrying out ultrasonic treatment in an ultrasonic cleaner for 2min, and repeating the step (4) for 1 time; adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, performing ultrasonic treatment in an ultrasonic cleaner for 8min, and repeating the step (4) for 1 time.
(6) And (3) placing the precipitate obtained by the treatment in the step (5) into a reaction container, heating in water bath at 40-45 ℃ until the absolute ethyl alcohol is completely evaporated, thus obtaining the product.
(7) And (5) detecting the product by a scanning electron microscope after the product is dripped.
Example 7: preparation of Dh-beta-Ala-His-Asp-glucose oxidase-copper phosphate co-immobilized hybrid nanoflower
(1) Preparing an enzyme solution: to the reaction vessel, 50mmol/L of phosphate buffer pH7.4 containing 1mg/mL of Dh-. beta. -Ala-His-Glu and 1mg/mL of glucose oxidase was added, and the mixture was allowed to stand at room temperature.
(2) Preparing the nanoflower: slowly dropwise adding 120mmol/L CuSO into the solution obtained in the step (1)4Aqueous solution, so that CuSO is in solution4The final concentration of (A) was 1 mmol/L.
(3) Self-assembly: standing at 35-45 deg.C for 80 hr.
(4) And (3) nano-flower treatment: centrifuging at 5000rpm for 8min, discarding supernatant, and collecting precipitate.
(5) Adding ultrapure water to 2/3 volume of the reaction container, performing ultrasonic treatment in an ultrasonic cleaner for 1min, and repeating the step (4) for 2 times; then adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, carrying out ultrasonic treatment in an ultrasonic cleaner for 3min, and repeating the step (4) for 2 times; adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, performing ultrasonic treatment in an ultrasonic cleaner for 7min, and repeating the step (4) for 2 times.
(6) And (3) placing the precipitate obtained by the treatment in the step (5) into a reaction container, heating in water bath at 55-60 ℃ until the absolute ethyl alcohol is completely evaporated, thus obtaining the product.
(7) And (5) detecting the product by a scanning electron microscope after the product is dripped.
Example 8: preparation of Dh-beta-Ala-His-Thr-Val-Glu-Lys-glucose oxidase-copper phosphate co-immobilized hybrid nanoflower
(1) Preparing an enzyme solution: to the reaction vessel, 50mmol/L of phosphate buffer pH7.4 containing 1mg/mL of Dh-. beta. -Ala-His-Glu and 1mg/mL of glucose oxidase was added, and the mixture was allowed to stand at room temperature.
(2) Preparing the nanoflower: slowly dropwise adding 120mmol/L CuSO into the solution obtained in the step (1)4Aqueous solution, so that CuSO is in solution4The final concentration of (A) was 0.8 mmol/L.
(3) Self-assembly: standing at 18-28 deg.C for 80 hr.
(4) And (3) nano-flower treatment: centrifuging at 4000rpm for 10min, and discarding the supernatant to obtain precipitate.
(5) Adding ultrapure water to 2/3 volume of the reaction container, performing ultrasonic treatment in an ultrasonic cleaner for 2min, and repeating the step (4) for 3 times; then adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, carrying out ultrasonic treatment in an ultrasonic cleaner for 1min, and repeating the step (4) for 1 time; adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, performing ultrasonic treatment in an ultrasonic cleaner for 5min, and repeating the step (4) for 2 times.
(6) And (3) placing the precipitate obtained by the treatment in the step (5) into a reaction container, heating in water bath at 45-50 ℃ until the absolute ethyl alcohol is completely evaporated, thus obtaining the product.
(7) And (5) detecting the product by a scanning electron microscope after the product is dripped.
Example 9: Dh-beta-Ala-His-Glu-glucose oxidase-copper phosphate co-immobilized hybrid nanoflower for detecting glucose
(1) Preparing a reaction system: adding 800 μ L of 50mmol/L phosphate buffer solution with pH7.0 and 100 μ L of 10mmol/L TMB aqueous solution into multiple EP tubes containing 0.25mg of co-immobilized hybrid nanoflower, and adding 100 μ L of 50mmol/L phosphate buffer solution with pH7.0, 100 μ L of 100 μmol/L glucose aqueous solution, 100 μ L of 250 μmol/L glucose aqueous solution, 100 μ L of 500 μmol/L glucose aqueous solution, 100 μ L of 1000 μmol/L glucose aqueous solution, 100 μ L of 2000 μmol/L glucose aqueous solution, 100 μ L of 5000 μmol/L glucose aqueous solution, 100 μ L of 10000 μmol/L glucose aqueous solution, 100 μ L of 15000 μmol/L glucose aqueous solution into the different EP tubes to make the final concentration of glucose in the EP tubes be 0 μmol/L, 100 μ L, 10. mu. mol/L, 25. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L, 200. mu. mol/L, 500. mu. mol/L, 1000. mu. mol/L, 1500. mu. mol/L; and after the addition is finished, the mixture is vortexed for 10-30s to be fully and uniformly mixed.
(2) And (3) carrying out a nanoflower catalytic cascade reaction: the EP tube was left standing in a 37 ℃ water bath for 15min to allow complete reaction.
(3) Centrifuging the system: placing the EP pipe in a centrifuge to process for 2min at the rotating speed of 6000rpm, so that the nanoflower is separated from the system solution; then 800. mu.L of the supernatant was used for UV detection.
(4) Ultraviolet detection: detecting the absorbance value of the supernatant at the wavelength of 652nm of a spectrophotometer by taking the concentration of glucose as an abscissa, A652nmFor the ordinate, a regression curve was constructed, the regression equation was fitted, the glucose concentration and A652nmA linear relationship; the regression plot of the glucose assay is shown in fig. 9.
(5) Detecting glucose with unknown concentration by using nanoflower: repeating the steps (1) to (4) to obtain the absorbance value A652nmSubstituting into regression equation to obtain ordinate value, and obtaining abscissa value as glucose concentration (μmol/L).
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
1. A preparation method of a double-enzyme co-immobilized copper nanoflower material is characterized by comprising the following steps:
(1) preparing an enzyme solution: adding a phosphate buffer solution in which a deuterohemin short peptide compound and glucose oxidase are dissolved into a reaction container, and placing the reaction container at room temperature;
(2) preparing the nanoflower: slowly dripping CuSO into the solution obtained in the step (1)4Aqueous solution, so that CuSO is in solution4The final concentration of (A) is 0.5-1 mmol/L;
(3) self-assembly: standing at 15-45 deg.C for 36-108 hr;
(4) and (3) nano-flower treatment: centrifuging for 5-10min, discarding supernatant, and collecting precipitate;
(5) sequentially adding ultrapure water and absolute ethyl alcohol to clean the precipitate;
(6) and (5) placing the precipitate obtained by the treatment in the step (5) into a reaction container, heating in a water bath until the absolute ethyl alcohol is completely evaporated, and obtaining the product.
2. The method for preparing a double-enzyme co-immobilized copper nanoflower material according to claim 1, wherein in the step (1), the deuterohemin short peptide compound comprises: dh- β -Ala-His-Glu; dh- β -Ala-His-Lys; dh- β -Ala-His-Asp; dh- β -Ala-His-Thr-Val-Glu-Lys.
3. The method for preparing a double-enzyme co-immobilized copper nanoflower material according to claim 1, wherein in the step (1), the concentration of the deuterohemin short peptide compound is 1 mg/mL; the concentration of the glucose oxidase is 1 mg/mL; the concentration of the phosphate buffer was 50mmol/L, and the pH was 7.4.
4. The method for preparing the double-enzyme co-immobilized copper nanoflower material according to claim 1, wherein in the step (2), CuSO is used4The concentration of the aqueous solution is 120 mmol/L; CuSO in solution4The final concentration of (A) was 0.8 mmol/L.
5. The preparation method of the double-enzyme co-immobilized copper nanoflower material according to claim 1, wherein in the step (5), the specific steps are as follows: adding ultrapure water to 2/3 volume of the reaction container, performing ultrasonic treatment in an ultrasonic cleaner for 1-3min, and repeating the step (4) for 2-4 times; then adding absolute ethyl alcohol to 1/3 volume of the reaction vessel, carrying out ultrasonic treatment in an ultrasonic cleaner for 1-3min, and repeating the step (4) for 1-2 times; adding anhydrous ethanol to 1/3 volume of the reaction vessel, performing ultrasonic treatment in an ultrasonic cleaner for 5-8min, and repeating the step (4) for 1-2 times.
6. The method for preparing the double-enzyme co-immobilized copper nanoflower material according to claim 1, wherein in the step (6), the water bath temperature is 40-60 ℃.
7. The application of the double-enzyme co-immobilized copper nano flower material prepared by the preparation method of the double-enzyme co-immobilized copper nano flower material according to any one of claims 1 to 6 in glucose detection.
8. The use according to claim 7, characterized in that it comprises in particular the following steps:
(1) preparing a reaction system: adding 800 μ L of 50mmol/L pH7.0 phosphate buffer solution and 100 μ L of 10mmol/L TMB aqueous solution into multiple EP tubes containing 0.25mg of co-immobilized hybrid nanoflower, and adding 100 μ L of 50mmol/L pH7.0 phosphate buffer solution and 100 μ L of glucose aqueous solutions with different concentrations into the different EP tubes to make final glucose concentrations in the EP tubes respectively 0 μmol/L, 10 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, 500 μmol/L, 1000 μmol/L and 1500 μmol/L; after the addition is finished, the mixture is swirled for 10 to 30 seconds to be fully and uniformly mixed;
(2) and (3) carrying out a nanoflower catalytic cascade reaction: standing the EP tube in a water bath at 37 ℃ for 15min to fully react;
(3) centrifuging the system: placing the EP pipe in a centrifuge to process for 2min at the rotating speed of 6000rpm, so that the nanoflower is separated from the system solution; then taking 800 mu L of supernatant for ultraviolet detection;
(4) ultraviolet detection: detection at wavelength of 652nm of spectrophotometerAbsorbance value of supernatant, with glucose concentration as abscissa, absorbance value A652nmFor the ordinate, a regression curve was constructed, the regression equation was fitted, the glucose concentration and A652nmA linear relationship;
(5) detecting glucose with unknown concentration by using nanoflower: repeating the steps (1) to (4) to obtain the absorbance value A652nmSubstituting into regression equation to obtain ordinate value, and obtaining abscissa value as glucose concentration.
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