CN116351382A - Copper oxide and ferric oxide nano enzyme and preparation method and application thereof - Google Patents
Copper oxide and ferric oxide nano enzyme and preparation method and application thereof Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/3078—Thermal treatment, e.g. calcining or pyrolizing
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a copper oxide and ferric oxide nano-enzyme, a preparation method and application thereof, and relates to the technical fields of nano-materials and biosensing. The invention provides a preparation method of copper oxide and ferric oxide nano enzyme, which comprises the following steps: cuNO is to 3 ·3H 2 O and Fe 2 (NO 3 ) 3 ·9H 2 Fully mixing O with a precipitator to obtain a mixed solution; placing the mixed solution into a polytetrafluoroethylene high-pressure reaction kettle for reaction, and then separating out a reaction product; calcining the reaction product to obtain CuO/Fe 2 O 3 Nano-enzyme particles. The invention has excellent peroxidase activity. The nano-enzyme has the advantages of simple preparation, high yield, easy storage and excellent stability.In CuO/Fe 2 O 3 As probes, glufosinate and chlortetracycline hydrochloride are used as detection objects, a high-sensitivity portable biological sensing platform is constructed by using a smart phone, and the portable biological sensing platform has even rapid detection capability. The invention belongs to the technical fields of nano materials and biosensing. The method is simple to operate, quick in response and good in practical application prospect in the detection process.
Description
Technical Field
The invention relates to the technical field of nano materials and biosensing, relates to a nano enzyme and a preparation method thereof, in particular to CuO/Fe 2 O 3 A preparation method of nano enzyme.
Background
Nanomaterials having bio-enzyme-like activity are referred to as nanoenzymes. The use of natural enzymes is limited due to their high storage costs, easy inactivation, and stringent storage conditions. In order to solve the problems associated with natural biological enzymes, various nanomaterials having enzyme-like activity have been developed. Nanomaterials have more and more applications such as biosensing and environmental protection due to their enzyme-like activity.
Glufosinate is a broad spectrum herbicide commonly used for annual and perennial dicotyledonous weeds. Its residues can accumulate in the environment and disrupt the natural balance between soil microorganisms. With the increasing use of glufosinate, residue monitoring has attracted considerable attention. Abuse of antibiotics is more likely to cause environmental pollution and increase in bacterial resistance. The aureomycin hydrochloride is added into the feed, so that the emission of animal manure can influence the quantity of microorganisms and the diversity of bacteria in the environment, and if the aureomycin hydrochloride is used as an agricultural fertilizer to directly act on a soil pond, the soil respiration capacity can be reduced, and the soil fertility is weakened.
Various detection methods such as high performance liquid chromatography, gas chromatography, capillary electrophoresis, ion chromatography, etc., which can be expensive instruments, complex sample pretreatment and purification processes, have been developed today, are considered to be disadvantages of these detection methods. Therefore, in order to meet the requirement of rapid detection, it is important to prepare a portable detection sensor.
In the prior art, CN111774057B discloses a high-performance heterojunction material Fe 2 O 3 CuO photoelectric film, the material belongs to the photoelectrochemistry technologyThe technical field can effectively improve the photoelectrochemical property and achieve the purpose of efficiently decomposing water. The above prior art has invented a Fe 2 O 3 The preparation method of the CuO material has the advantages of harsh preparation conditions, complex process and unsuitable detection of glufosinate or chlortetracycline hydrochloride.
In the prior art, CN115267018A discloses a detection technology for glufosinate-ammonium, wherein the glufosinate-ammonium in a sample to be detected is derived by using aromatic acyl chloride, and the derived product can be separated and detected in high performance liquid chromatography, so that the structure and the property of impurities can be conveniently judged. However, the high-efficiency detection of glufosinate still has certain defects, the required detection equipment is high in price, and the operation steps are complicated.
In the prior art, CN109540869B discloses a SERS detection technology for aureomycin hydrochloride, in which surface-enhanced raman detection is performed on aureomycin hydrochloride on a SERS substrate to realize quantitative detection of aureomycin hydrochloride. However, the cost is high, the Au nanoparticle sol needs to be used, the reaction time is long, and the detection result cannot be obtained efficiently and rapidly.
Disclosure of Invention
Aiming at the defects existing in the existing detection methods of glufosinate-ammonium and aureomycin hydrochloride, the invention provides a method for rapidly detecting the contents of glufosinate-ammonium and aureomycin hydrochloride by utilizing a nano enzyme probe and combining a portable detection platform of a smart phone. The nano enzyme is superior to the production and storage cost of the biological enzyme and the portability and the simplicity of the smart phone, so that the characteristics of rapid detection, simple and convenient operation, low detection cost and the like are achieved.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
the preparation process of nanometer copper oxide and ferric oxide enzyme includes the following steps:
s1: cuNO is to 3 ·3H 2 O and Fe 2 (NO 3 ) 3 ·9H 2 Fully mixing O with a precipitator to obtain a mixed solution;
s2: placing the mixed solution into a polytetrafluoroethylene high-pressure reaction kettle for reaction, and then separating out a reaction product;
s3: calcining the reaction product to obtain CuO/Fe 2 O 3 Nano enzyme particles;
further, the precipitant is a mixed solution of 0.56g PVP and 1.2g urea, dissolved in 20mL deionized water and 10mL glycol.
Further, the mixed solution is fully stirred for 30min, and then added into a polytetrafluoroethylene high-pressure reaction kettle for hydrothermal reaction at 90 ℃ for 10h.
Further, after completion of the reaction, a yellow precipitate was obtained by centrifugation, washed alternately with water and ethanol 3 times, and dried under vacuum at 70 ℃.
Further, after the reactant is dried, the reactant is directly calcined for 3 hours at 550 ℃ by using a tube furnace to prepare CuO/Fe 2 O 3 Nano-enzyme particles.
Further, cuO and Fe are prepared by a similar method 2 O 3 Nano enzyme particle and CuO/Fe 2 O 3 The nano-enzyme particles were subjected to comparative analysis.
Further, an enzyme activity test was performed. The catalysts were tested for peroxidase activity using 3,3', 5' -Tetramethylbenzidine (TMB) as chromogenic substrate.
Further, when enzyme activity is tested, cuO/Fe 2 O 3 The suspension is subjected to ultrasonic treatment for 5min.
Further, 50. Mu.L of the liquid was mixed with 100. Mu.LTMB (5 mM), 50. Mu. L H 2 O 2 (10 mM) acetic acid-sodium acetate buffer solution was added, and the total volume after mixing was 2mL.
Further, the mixture was incubated in a 37℃water bath for 20min, and the absorbance was recorded with an ultraviolet-visible spectrophotometer.
Further, cuO/Fe 2 O 3 Detecting CuO and Fe by taking ultraviolet-visible absorption intensity of nano enzyme as reference 2 O 3 The ultraviolet visible absorption intensity of the nano enzyme particles at the same site. It follows that the two are mechanically mixed and do not give rise to enzymatic activity.
It is another object of the present invention to provide a deviceCuO/Fe of the species 2 O 3 The application of the nano-enzyme in the detection of glufosinate-ammonium and chlortetracycline hydrochloride:
(1) The colorimetric analysis of glufosinate comprises the following operation steps:
s1: a series of glufosinate with different concentrations are taken and added into a detection system solution;
s2: placing the system solution into a water bath kettle for incubation;
s3: the absorbance of the system solution was measured.
(2) The chlortetracycline hydrochloride colorimetric analysis comprises the following operation steps:
s1: a series of glufosinate with different concentrations and chlortetracycline hydrochloride are taken and added into a detection system solution;
s2: placing the system solution into a water bath kettle for incubation;
s3: the absorbance of the system solution was measured.
(3) Utilize smart mobile phone to build portable testing platform
S1: adding target molecules with different concentrations into a reaction system to form a composite solution, incubating the composite solution in a water bath, and transferring the composite solution into a glass test tube;
s2: shooting the image by using a smart phone;
s3: segmenting the image by using a Yolo V3 algorithm of a self-development application program, and extracting an average value of RGB or HSV;
s4: a linear Support Vector Machine (SVM) is used to fit the relationship between the average of RGB or HSV values and the target molecule.
The invention has the beneficial effects that:
(1) The invention provides a novel CuO/Fe with excellent enzyme activity 2 O 3 A preparation method of a nano enzyme material. The nano-enzyme has the advantages of simple preparation, high yield, easy storage, excellent stability and the like, and is expected to be used for large-scale industrial production.
(2) The scheme of the invention is to prepare CuO/Fe by a hydrothermal method and a calcination method 2 O 3 The nano enzyme is used for detecting glufosinate-ammonium and aureomycin hydrochloride, and solves the problems of expensive equipment, complex operation and long reaction time in the detection of the substances in the prior artThe effect of rapid quantitative detection is achieved.
(3) The invention constructs a rapid and intelligent analysis platform for glufosinate-ammonium and aureomycin hydrochloride. The platform has the capability of instant and quick detection, is simple to operate, is convenient to carry and can be used quickly and simply.
Drawings
FIG. 1 (A) CuO/Fe 2 O 3 Schematic of the preparation. (B, C, D) CuO, fe 2 O 3 And CuO/Fe 2 O 3 SEM images of (a). (E, F, G) CuO, fe 2 O 3 And CuO/Fe 2 O 3 A TEM image of (a). (H, I) CuO/Fe 2 O 3 HRTEM images of (a). (J) CuO/Fe 2 O 3 Is a EDS image of (C).
FIG. 2 (A) CuO, fe 2 O 3 And CuO/Fe 2 O 3 Is a XRD pattern of (C). (B) CuO, fe 2 O 3 And CuO/Fe 2 O 3 Is a complete XPS spectrum of (C).
FIG. 3 (A) CuO, fe 2 O 3 ,CuO+Fe 2 O 3 And CuO/Fe 2 O 3 Ultraviolet-visible absorption spectrum. (B) CuO/Fe 2 O 3 ，CuO/Fe 2 O 3 +glufosinate, cuO/Fe 2 O 3 Ultraviolet-visible absorption spectrum of + glufosinate + aureomycin hydrochloride. (C) a linear range diagram of glufosinate. (D) linear curve of glufosinate. (E) a linear range diagram of aureomycin hydrochloride. (F) a linear curve of aureomycin hydrochloride.
Fig. 4 (a) is based on the intelligent detection platform principle of deep learning. (B) The smartphone platform detects the color signal and linearity of glufosinate. (C) The smart phone platform detects the aureomycin hydrochloride color signal and linearity.
Detailed Description
In order to more clearly illustrate the technical scheme of the invention, the following detailed description of the embodiments of the invention will be given with reference to the accompanying drawings.
As shown in FIG. 1 (A), a CuO/Fe alloy 2 O 3 The nano enzyme preparation method comprises the steps of preparing by a hydrothermal method and a calcining method, mixing two types of metal salt active agent stable colloidal particles and a precipitator, reacting and washingWashing and calcining to obtain target product CuO/Fe 2 O 3 Nano enzyme.
Wherein the metal salt of copper and iron is CuNO 3 ·3H 2 O and Fe 2 (NO 3 ) 3 ·9H 2 O。
Wherein the precipitant is PVP, urea and the solvent is deionized water and glycol mixed solution.
Wherein the reaction is carried out in a polytetrafluoroethylene high-pressure reaction kettle, and the hydrothermal reaction is carried out at 90 ℃ for 10 hours.
Wherein, deionized water and ethanol are used for washing for three times alternately, and then vacuum drying is carried out.
Wherein, the tube furnace is used for direct calcination during calcination.
The invention is illustrated below with reference to specific examples:
example 1:
in this embodiment, a preparation method of CuO nano-enzyme is disclosed:
s1: 1mmol CuNO 3 ·3H 2 Adding O and 0.56g PVP and 1.2g urea into a mixed solution of 20ml deionized water and 10ml ethylene glycol, fully stirring for 30min, and uniformly mixing;
s2: the mixture is put into a polytetrafluoroethylene high-pressure reaction kettle and subjected to hydrothermal reaction for 10 hours at 90 ℃. After the reaction is finished, obtaining blue precipitate after centrifugal separation, washing for 3 times alternately by using water and ethanol, and drying in vacuum at 70 ℃;
s3: directly calcining the dried product in a tubular furnace at 550 ℃ for 3 hours to obtain CuO/Fe 2 O 3 Nano-enzyme particles.
Example 2:
in this embodiment, a Fe is disclosed 2 O 3 The preparation method of the nano enzyme comprises the following steps:
s1: 0.5mmol Fe 2 (NO 3 ) 3 ·9H 2 Adding O and 0.56g PVP and 1.2g urea into a mixed solution of 20ml deionized water and 10ml ethylene glycol, fully stirring for 30min, and uniformly mixing;
s2: s2 is the same as in example 1. After the reaction is finished, obtaining yellow precipitate after centrifugal separation;
s3: as in S3 of example 1, fe is obtained 2 O 3 Nano-enzyme particles.
Example 3:
in this embodiment, a CuO/Fe is disclosed 2 O 3 The preparation method of the nano enzyme comprises the following steps:
s1: 1mmol CuNO 3 ·3H 2 O，0.5mmol Fe 2 (NO 3 ) 3 ·9H 2 Adding O and 0.56g PVP and 1.2g urea into a mixed solution of 20ml deionized water and 10ml ethylene glycol, fully stirring for 30min, and uniformly mixing;
s2: s2 as in example 2;
s3: as in S3 of example 1, cuO/Fe was obtained 2 O 3 Nano-enzyme particles.
Example 4:
based on examples 1-3, for CuO, fe 2 O 3 、CuO/Fe 2 O 3 Test of nano enzyme activity:
s1: the nanoenzyme suspension was sonicated to obtain 50. Mu.L of liquid with 100. Mu.LTMB (5 mM), 50. Mu. L H 2 O 2 (10 mM) adding acetic acid-sodium acetate buffer solution, and mixing to obtain total volume of 2mL;
s2: the mixture was incubated in a 37℃water bath for 20min, and the absorbance was recorded with an ultraviolet-visible spectrophotometer.
CuO/Fe in a certain reaction time 2 O 3 /TMB/H 2 O 2 The oxidation product generated by the system reaction has characteristic absorption peak at 652nm and is visually dark blue, and the absorbance of oxidized TMB (oxTMB) at 652nm is recorded by an ultraviolet-visible spectrophotometer, thereby proving CuO/Fe 2 O 3 Peroxidase activity of nanoenzymes. CuO/Fe 2 O 3 /TMB/H 2 O 2 The UV-visible absorption intensity of the system at 652nm was set to a reference value (100%). CuO and Fe in a peroxidase-like active system 2 O 3 The system was calculated to be 18.61% and 35.41%. With Fe 2 O 3 In comparison, cuO and Fe 2 O 3 Does not increase the peroxidase-like activity of the mixture of (a),indicating CuO and Fe 2 O 3 Is unable to produce enzyme activity (FIG. 3A).
Example 5:
in this example, colorimetric analysis of glufosinate-ammonium and aureomycin hydrochloride:
in order to realize visual detection, a series of glufosinate-ammonium and aureomycin hydrochloride with different concentrations are added into CuO/Fe 2 O 3 /TMB/H 2 O 2 In the system. Incubate in a 37℃water bath for 20min and record absorbance with an ultraviolet-visible spectrophotometer.
The absorbance value of ox-TMB (652 nm) was proportional to the glufosinate concentration in the range of 30 to 400mM (FIG. 3C). The resulting linear relationship is y= 1.14027-0.000923X, with a detection Limit (LOD) of 16.05 μm based on 3σ/b (where σ is the standard deviation of the blank signal and b is the slope of the regression line) (fig. 3D). Further exploration of CuO/Fe 2 O 3 The effect of detecting aureomycin hydrochloride was analyzed, and its linear relationship was y=0.64184+0.03725x (1-10 μm), and 3σ/b-based LOD was 0.71 μm (fig. 3e, f). Glufosinate can reduce the absorbance of the system, while chlortetracycline hydrochloride alone cannot change the color of the system greatly.
Example 6:
in this embodiment, a portable detection platform is built by using a smart phone:
s1: adding target molecules with different concentrations into a reaction system to form a composite solution, and transferring the composite solution into a glass test tube;
s2: shooting the image by using a smart phone;
s3: segmenting the image by using a Yolo V3 algorithm of a self-development application program, and extracting an average value of RGB or HSV;
s4: a linear Support Vector Machine (SVM) is used to fit the relationship between the average of RGB or HSV values and the target molecule.
The (g+r)/B model was chosen and the fit equation y=0.0008x+1.6253 for glufosinate detection was automatically displayed on the smartphone screen (r2=0.9814) (fig. 4B). Also, as shown in fig. 4C, a portable smart phone analysis biosensor for detecting chlortetracycline hydrochloride was established. Different modalities are intelligently obtained according to the R, G, B, H, S, V value obtained through calculation. The B model was finally chosen to fit the equation y= -6.75886x+154.3191 (r2=0.991).
The embodiments of the invention disclosed above are only useful to help illustrate the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (10)
1. CuO/Fe 2 O 3 The preparation method of the nano enzyme is characterized by comprising the following steps:
s1: cuNO is to 3 ·3H 2 O and Fe 2 (NO 3 ) 3 ·9H 2 Fully mixing O with a precipitator to obtain a mixed solution;
s2: placing the mixed solution into a polytetrafluoroethylene high-pressure reaction kettle for reaction, and then separating out a reaction product;
s3: calcining the reaction product to obtain CuO/Fe 2 O 3 Nano enzyme particles;
s4: the activity of the nanoenzyme was detected.
2. The CuO/Fe according to claim 1 2 O 3 The preparation method of the nano enzyme particles is characterized by comprising the following steps: in S1, 0.56g PVP and 1.2g urea were used as precipitants, dissolved in a mixed solution of 20mL deionized water and 10mL ethylene glycol.
3. The CuO/Fe according to claim 1 2 O 3 The preparation method of the nano enzyme particles is characterized by comprising the following steps: s2, performing hydrothermal reaction at 90 ℃ in a polytetrafluoroethylene high-pressure reaction kettle by utilizing a hydrothermal method, wherein the reaction is performed for 10 hours, and the separation mode adopts centrifugal separationAfter that, deionized water and ethanol were alternately used for rinsing 3 times, and dried at 70 ℃.
4. The CuO/Fe according to claim 1 2 O 3 The preparation method of the nano enzyme particles is characterized by comprising the following steps: s3, directly calcining the dried product in a tubular furnace at 550 ℃ for 3 hours by using a calcination method to obtain CuO/Fe 2 O 3 Nano-enzyme particles.
5. CuO/Fe as prepared in any one of claims 1 to 4 2 O 3 The nano-enzyme is applied to the detection of glufosinate-ammonium and is characterized by comprising the following steps:
s1: a series of glufosinate with different concentrations are taken and added into a detection system solution;
s2: placing the system solution into a water bath kettle for incubation;
s3: the absorbance of the system solution was measured.
6. The use according to claim 5, wherein: the detection solution system in S1 is CuO/Fe 2 O 3 /TMB/H 2 O 2 The system.
7. CuO/Fe as prepared in any one of claims 1 to 4 2 O 3 The nano-enzyme is applied to the detection of chlortetracycline hydrochloride and is characterized by comprising the following steps:
s1: a series of glufosinate with different concentrations and chlortetracycline hydrochloride are taken and added into a detection system solution;
s2: placing the system solution into a water bath kettle for incubation;
s3: the absorbance of the system solution was measured.
8. The use according to claim 7, wherein: the detection solution system in S1 is CuO/Fe 2 O 3 /TMB/H 2 O 2 A glufosinate system.
9. The portable detection platform built by the smart phone is characterized by comprising the following steps:
s1: adding target molecules with different concentrations into a reaction system to form a composite solution, incubating the composite solution in a water bath, and transferring the incubated composite solution into a glass test tube;
s2: shooting the image by using a smart phone;
s3: dividing the image and collecting information;
s4: fitting the relationship between the data.
10. The assay platform of claim 9, wherein: s2, acquiring images by using a portable intelligent mobile phone; s3, segmenting an image and acquiring information of average values of RGB or HSV by using a YoloV3 algorithm in a self-development program; and S4, fitting the relation between the average value of RGB or HSV values and the target molecule concentration by using a linear support vector machine SVM.
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