CN113527701B - Tb-GMP/CeO2Composite material, preparation method thereof and method for detecting ziram - Google Patents

Tb-GMP/CeO2Composite material, preparation method thereof and method for detecting ziram Download PDF

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CN113527701B
CN113527701B CN202110724605.5A CN202110724605A CN113527701B CN 113527701 B CN113527701 B CN 113527701B CN 202110724605 A CN202110724605 A CN 202110724605A CN 113527701 B CN113527701 B CN 113527701B
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solution
ceo
gmp
ziram
concentration
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CN113527701A (en
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王鹏
王冬伟
周志强
刘东晖
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China Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/008—Supramolecular polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
    • C08K3/20—Oxides; Hydroxides
    • C08K3/22—Oxides; Hydroxides of metals
    • G—PHYSICS
    • 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
    • G—PHYSICS
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
    • C08K3/20—Oxides; Hydroxides
    • C08K3/22—Oxides; Hydroxides of metals
    • C08K2003/221—Oxides; Hydroxides of metals of rare earth metal
    • C08K2003/2213—Oxides; Hydroxides of metals of rare earth metal of cerium
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K2201/00—Specific properties of additives
    • C08K2201/011—Nanostructured additives

Abstract

The invention provides Tb-GMP/CeO2The composite material is in a spherical structure, the size of the composite material is 10-50 nm, and physical parameters of an X-ray photoelectron spectrum are as follows: terbium (3 d)3/2)1277.1 eV. Tb-GMP/CeO provided by the invention2The composite material is combined with tyrosinase to form an enzyme biosensor, so that thiram in a sample can be detected.

Description

Tb-GMP/CeO2Composite material, preparation method thereof and method for detecting ziram
Technical Field
The invention belongs to the technical field of pesticide detection, and particularly relates to Tb-GMP/CeO2A composite material and a preparation method thereof, and a method for detecting ziram.
Background
Ziram is a widely used dithiocarbamate fungicide that can be used to prevent fungal diseases in seeds and crops. At present, the standard detection method for the residual thiram in agricultural products in China is a gas chromatography. In the process of measuring the ziram by the gas chromatography, the ziram needs to be heated at high temperature under the strong acid condition to be converted into carbon disulfide, and then the indirect measurement of the ziram content is realized by measuring the carbon disulfide by the gas chromatography. The method for indirectly measuring ziram is tedious in process and time-consuming, needs strong acid, and does not conform to the principle of reducing the use of dangerous goods in green chemistry. Furthermore, the method may be disturbed by sulfur-containing substances in the substrate of the agricultural product, leading to erroneous results, and is not suitable for detecting the agricultural product fumigated with carbon disulfide. And the chromatographic analysis can only be carried out in a laboratory, and the requirement of rapidly detecting the residual ziram on site in real time is difficult to meet. Therefore, it is necessary to develop a suitable rapid detection and analysis method for ziram residue in order to meet the requirement of rapidly detecting ziram residue on site in real time. The ziram inhibits tyrosinase activity, so that a tyrosinase biosensor can be developed for detecting the ziram.
Lanthanide metal coordination polymers are a novel class of lanthanide metal-ligand complexes. Some molecules with at least two lanthanide metal ion binding sites are used as ligands, and through repeated coordination of metal ion-ligand-metal ion-ligand, nano-sized lanthanide metal coordination polymers are assembled. Nucleotides are compounds consisting of bases (purines or pyrimidines), ribose (or deoxyribose) and phosphate groups, contain a plurality of atoms with lone pair electrons, can bind to lanthanide metal ions through coordination bonds, and thus can serve as ligand molecules. Because both a base moiety and a phosphate moiety are present in the nucleotide molecule, and multiple oxygen atoms are present in the phosphate group, lanthanide metal ions can form coordination polymers with nucleotides through coordination. Due to Tb3+、Eu3+And Ce3+The excited state of the nucleotide overlaps with the triplet state of the base molecule, so that the nucleotide can transfer absorbed light energy to lanthanide metal ions to realize ligand-sensitized luminescence. Wherein, Tb3+Tb-GMP, a polymer formed by coordination assembly with disodium 5' -Guanylate (GMP) which transfers absorbed light energy directly to Tb3+Thereby enhancing Tb3+The fluorescence intensity of (2). Tb-GMP and its composite material have been used as fluorescent probe for detecting pesticide residue by fluorescence spectrometry. However, the prior art does not report how to use Tb-GMP composite materials for detecting ziram.
Nano CeO2Ce in the structure of the catalyst participating in redox reaction3+And Ce4+Can be relatively simply converted into each other, so that the nano CeO2Can be used as a catalyst for oxidation-reduction reaction. At present, CeO nanoparticles2It has been reported to have catalytic abilities similar to those of various enzymes catalyzing redox reactions, including oxidase, peroxidase, catalase, superoxide dismutase, and the like. Nano CeO2The catalyst can be used for absorption spectrum analysis and detection by catalyzing certain chromogen reagents to generate oxidation color reaction. Nano CeO2Has been used as a fluorescent probe for detecting pesticide residue by absorption spectrometry. However, there is no prior art on how to use nano CeO2To detect fortuneAnd 5, zinc measurement report.
Disclosure of Invention
In view of the above, the present invention is directed to providing Tb-GMP/CeO2The Tb-GMP/CeO composite material and the preparation method thereof and the method for detecting ziram2The composite material is capable of detecting ziram in a sample.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides Tb-GMP/CeO2Composite material of Tb-GMP/CeO2The structural physical property parameters of the composite material are as follows: a spherical structure with a size of 10-50 nm; the physical parameters of the X-ray photoelectron spectrum are as follows: terbium (3 d)3/2)1277.1eV and terbium (3 d)5/2)1242.5eV, cerium (3 d)3/2)904.4eV, cerium (3 d)5/2) 886.1eV, oxygen 1s 531.3eV, and phosphorus 2p 133.8 eV.
The invention also provides Tb-GMP/CeO in the technical scheme2The preparation method of the composite material comprises the following steps:
1) mixing 5' -disodium guanylate solution with CeO2Mixing the solution, and stirring for the first time to obtain a first stirred material; the time for the first stirring is 20 min;
2) mixing the first stirred material obtained in the step 1) with a terbium chloride solution, and then stirring for the second time to obtain a second stirred material; the time for the second stirring is 30 min;
3) centrifuging the second stirred material obtained in the step 2), wherein the obtained precipitate is Tb-GMP/CeO2A composite material.
The step 1) of mixing 5' -guanylic acid disodium solution with CeO2The volume ratio of the solution is 1: 0.15;
the concentration of the disodium 5' -guanylate solution is 4.07 mg/mL;
the CeO2The concentration of the solution is 2.5 mg/mL;
the CeO2CeO in solution2Is nano-scale CeO2；
The volume ratio of the terbium chloride solution in the step 2) to the disodium 5' -guanylate solution is 1: 1;
the concentration of the terbium chloride solution is 3.74 mg/mL;
the conditions of the centrifugation in the step 3) comprise: the rotation speed of the centrifugation is 12000rpm, and the time of the centrifugation is 10 min.
The invention also provides Tb-GMP/CeO prepared by the technical scheme2The method for detecting ziram by using the composite material comprises the following steps:
a. mixing the tyrosinase solution with a sample, and standing to obtain a standing solution;
b. mixing the standing solution obtained in the step a with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
c. c, mixing the reaction liquid obtained in the step b with Tb-GMP/CeO in the technical scheme2Mixing and oscillating a composite material, a 3,3 ', 5, 5' -tetramethylbenzidine solution and a citric acid-disodium hydrogen phosphate buffer solution to obtain an oscillating liquid, measuring an absorption spectrum of the oscillating liquid to obtain maximum absorbance, and converting the maximum absorbance into an enzyme inhibition rate;
the enzyme inhibition rate is (A)z-A0)/(A-A0)×100，AzRepresents the maximum absorption intensity in the absorption spectrum, A, in the presence of ziram0Represents the maximum absorption intensity in the absorption spectrum in the absence of ziram, and A represents the maximum absorption intensity in the absorption spectrum;
d. substituting the enzyme inhibition rate obtained in the step c into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: the enzyme inhibition rate was 16.32 xc +0.37, and the unit of c was μ g/L.
The invention also provides Tb-GMP/CeO prepared by the technical scheme2The method for detecting ziram by using the composite material comprises the following steps:
(1) mixing the tyrosinase solution with a sample, and standing to obtain a standing solution;
(2) mixing the standing solution obtained in the step (1) with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
(3) reacting the reaction solution obtained in the step (2) with Tb-GMP/CeO in the technical scheme2Mixing and oscillating a composite material, a 3,3 ', 5, 5' -tetramethylbenzidine solution and a citric acid-disodium hydrogen phosphate buffer solution to obtain an oscillating liquid, photographing the oscillating liquid by using a smart phone, reading RGB values in a photo, converting the RGB values into S values, and substituting the S values into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: s is 0.0779 xc +0.0487, with c being μ g/L.
Preferably, the volume ratio of the tyrosinase solution to the sample in the step a or (1) is 1: 1;
the concentration of the tyrosinase solution is 175U/mL;
the standing conditions comprise: the standing time was 5min, and the ambient temperature of standing was 37 ℃.
Preferably, the volume ratio of the tyrosine solution to the Tris-HCl buffer solution and the sample in the step b or (2) is 10:8: 1;
the concentration of the tyrosine solution is 2.5 mM;
the concentration of the Tris-HCl buffer solution is 10mM, and the pH value is 7.4;
the reaction conditions include: the ambient temperature of the reaction was 37 ℃ and the reaction time was 30 min.
Preferably, Tb-GMP/CeO is used in said step c or (3)2The composite material is Tb-GMP/CeO2Mixing the solution with the raw materials, wherein Tb-GMP/CeO2The volume ratio of the solution to the 3,3 ', 5, 5' -tetramethylbenzidine solution and the citric acid-disodium hydrogen phosphate buffer solution is 100:75: 225;
the Tb-GMP/CeO2The concentration of the solution is 2 mg/mL;
the concentration of the 3,3 ', 5, 5' -tetramethyl benzidine solution is 10 mM;
the concentration of the citric acid-disodium hydrogen phosphate buffer solution is 0.2M, and the pH value is 3.0;
the conditions of the oscillation include: the oscillating speed is 500rpm, and the oscillating time is 10 min;
and c, measuring the maximum absorbance of the oscillating liquid at 500-750 nm in the step c.
The invention also provides Tb-GMP/CeO prepared by the technical scheme2The method for detecting ziram by using the composite material comprises the following steps:
A. mixing the tyrosinase solution with a sample, and standing to obtain a standing solution;
B. mixing the standing solution obtained in the step A with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
C. the reaction liquid obtained in the step B and Tb-GMP/CeO in the technical scheme2Mixing and oscillating the composite material and ultrapure water to obtain an oscillating liquid, measuring a fluorescence emission spectrum of the oscillating liquid to obtain maximum fluorescence intensity, and converting the maximum fluorescence intensity into an enzyme inhibition rate;
(FD-F) as an enzyme inhibitor0)/(F-F0) X 100, FD represents the maximum fluorescence intensity in the fluorescence emission spectrum in the presence of ziram, F0Represents the maximum fluorescence intensity in the fluorescence emission spectrum in the absence of ziram, and F represents the maximum fluorescence intensity in the fluorescence emission spectrum;
D. substituting the enzyme inhibition rate obtained in the step C into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: enzyme inhibition rate of 33.21 × log10c +15.96, the unit of c is mug/L.
Preferably, the volume ratio of the tyrosinase solution to the sample in the step A is 1: 1;
the concentration of the tyrosinase solution is 80U/mL;
the standing conditions comprise: standing for 15min at 37 deg.C;
the volume ratio of the tyrosine solution to the Tris-HCl buffer solution to the sample in the step B is 10:8: 1;
the concentration of the tyrosine solution is 2.5 mM;
the concentration of the Tris-HCl buffer solution is 10mM, and the pH value is 7.4;
the reaction conditions include: the reaction environment temperature is 37 ℃, and the reaction time is 30 min;
Tb-GMP/CeO in the step C2The composite material is Tb-GMP/CeO2Mixing the solution with the raw materials, wherein Tb-GMP/CeO2The volume ratio of the solution to the ultrapure water is 1: 79;
the fluorescence excitation wavelength of the fluorescence emission spectrum of the measurement shaking solution was 286 nm.
The invention provides Tb-GMP/CeO2The invention discloses a composite material, a preparation method thereof and a method for detecting ziram by utilizing Tb-GMP/CeO2The mechanism of the composite material for detecting the ziram is as follows: in Tb-GMP/CeO2Tyrosine and tyrosinase mixed system, tyrosine (monophenol) is catalyzed by tyrosinase to generate hydroxylation reaction to generate dopa (bisphenol), and the bisphenol and Tb-GMP/CeO2Has stronger binding capacity than monophenol, therefore, Tb-GMP/CeO2The binding capacity with dopa is stronger, which results in the fluorescence emission performance (the mechanism is dopa to Tb-GMP/CeO2Fluorescence inner filtering effect) and catalytic activity (mechanism is that the material is more difficult to contact with TMB for catalytic reaction after being combined with dopa). The zinc thiram has an inhibiting effect on tyrosinase activity, so that the tyrosinase catalytic activity is weakened and the dopa generation is reduced in the presence of the zinc thiram, and compared with the Tb-GMP/CeO in the absence of the zinc thiram2The fluorescence emission performance and the catalytic activity of the catalyst are changed. And (3) measuring the fluorescence intensity, absorbance or color, and establishing a fluorescence spectrum, an absorption spectrum and a smart phone detection method of the ziram.
Drawings
FIG. 1 shows Tb-GMP/CeO2A Scanning Electron Microscope (SEM) image of (a);
FIG. 2 shows Tb-GMP/CeO2SEM and corresponding element distribution image of (a);
FIG. 3 shows Tb-GMP/CeO2XPS full spectrum analysis of (a);
FIG. 4 shows Tb-GMP/CeO2Fluorescence excitation and emission spectra of (a);
FIG. 5 shows Tb-GMP/CeO2The absorption spectrum of (a);
FIG. 6 shows Tb-GMP/CeO2With nano CeO2Absorption spectra of catalytic TMB oxidation;
FIG. 7 shows Tb-GMP/CeO in the presence of different concentrations of ziram2-absorption spectrum of TMBox under catalysis of tyrosinase-tyrosine system;
FIG. 8 is a linear equation between Fumei zinc concentration and IE (absorption spectroscopy);
FIG. 9 shows Tb-GMP/CeO in the presence of different concentrations of ziram2-fluorescence emission spectrum of the tyrosinase-tyrosine system;
FIG. 10 is a linear equation between the logarithm of the concentration of ziram and IE (fluorescence spectroscopy);
FIG. 11 shows different concentrations of ziram versus tyrosinase-Tb-GMP/CeO2The influence of the system color;
FIG. 12 is a linear equation between the logarithm of the concentration of ziram and IE (smartphone method);
fig. 13 is a condition optimization experiment for detecting ziram by absorption spectroscopy: solution pH (A); tyrosinase concentration (B); reaction time (C) of tyrosinase with tyrosine; reaction temperature (D) and mixing time (E) of tyrosine and ziram
FIG. 14 is a condition-optimized experiment for detecting Fumei zinc by fluorescence spectroscopy: Tb-GMP/CeO2Time (A) of mixing reaction with tyrosinase catalysis system; tyrosinase concentration (B); reaction time (C) of tyrosinase with tyrosine; reaction temperature (D) and mixing time (E) of tyrosine and ziram
FIG. 15 shows the selectivity of detection of ziram by absorption spectroscopy; in the figure, 1-10 are respectively ziram, sodium ion, calcium ion, zinc ion, vitamin C, glucose, glycine, metalaxyl, tebuconazole and azoxystrobin
FIG. 16 shows the selectivity of fluorescence spectroscopy for detection of ziram; in the figure, 1-10 are respectively ziram, sodium ion, calcium ion, zinc ion, vitamin C, glucose, glycine, metalaxyl, tebuconazole and azoxystrobin
Fig. 17 shows the formula for converting RGB values into S values.
Detailed Description
The invention provides Tb-GMP/CeO2Composite material of Tb-GMP/CeO2The structural physical property parameters of the composite material are as follows: spherical structure, size 10 ℃50 nm; the physical parameters of the X-ray photoelectron spectrum are as follows: terbium (3 d)3/2)1277.1eV and terbium (3 d)5/2)1242.5eV, cerium (3 d)3/2)904.4eV, cerium (3 d)5/2) 886.1eV, oxygen 1s 531.3eV, and phosphorus 2p 133.8 eV. In the present invention, the Tb-GMP/CeO2The particle size of the composite material is 10-50 nm, and the composite material is spherical nano particles. In the present invention, the Tb-GMP/CeO2Fluorescence spectrum of composite material: maximum excitation wavelength: 286nm, maximum emission wavelength: 545 nm. Absorption spectrum: the absorption peak is about 245 nm.
The invention also provides Tb-GMP/CeO in the technical scheme2The preparation method of the composite material comprises the following steps:
1) mixing 5' -disodium guanylate solution with CeO2Mixing the solution, and stirring for the first time to obtain a first stirred material; the first stirring time is 20 min;
2) mixing the first stirred material obtained in the step 1) with a terbium chloride solution, and then stirring for the second time to obtain a second stirred material; the second stirring time is 30 min;
3) centrifuging the second stirred material obtained in the step 2), wherein the obtained precipitate is Tb-GMP/CeO2A composite material.
In the invention, 5' -disodium guanylate solution and CeO2Mixing the solution, and then carrying out first stirring to obtain a first stirred material; the time of the first stirring is 20 min.
In the present invention, the disodium 5' -guanylate solution is mixed with CeO2The volume ratio of the solution is preferably 1: 0.15. In the present invention, the concentration of the disodium 5' -guanylate solution is preferably 4.07 mg/mL. In the present invention, the CeO2The concentration of the solution is preferably 2.5 mg/mL. In the present invention, the CeO2CeO in solution2Preferably nano-sized CeO2。
The invention is to the nano-scale CeO2The preparation method of (A) is not particularly limited, and nanoscale CeO is prepared by adopting the routine method of a person skilled in the art2In one embodiment of the present invention, the nano-sized CeO2Preparation method of (1) preferred packageThe method comprises the following steps:
mixing cerium nitrate with a glycol solution to obtain a mixture;
mixing and stirring the mixture and ammonia water to obtain a stirred material;
centrifuging the stirred substance to obtain a precipitate of nano-CeO2。
In the present invention, the cerium nitrate is preferably cerium nitrate hexahydrate. In the present invention, the ratio of the mass of the cerium nitrate to the volume of the ethylene glycol solution is preferably 2.52g:100 mL. In the invention, the solvent of the ethylene glycol solution is water, and the volume ratio of the ethylene glycol to the water is preferably 1: 1.
In the present invention, the mixture is preferably heated to 60 ℃ and mixed with aqueous ammonia, and stirred for preferably 3 hours. In the present invention, the stirring is preferably vigorous stirring.
In the present invention, the conditions of the centrifugation preferably include: the rotating speed of the centrifugation is preferably 12000rpm, and the time of the centrifugation is preferably 10 min.
Obtaining nano-grade CeO2Then preferably mixing with sodium citrate solution, performing ultrasonic treatment, mixing the obtained ultrasonic material with ethanol to obtain precipitate, washing the precipitate with ethanol, and drying to prepare Tb-GMP/CeO2Raw materials of the composite material. In the present invention, the concentration of the sodium citrate solution is preferably 30 g/L. In the present invention, the sodium citrate prevents cerium oxide from agglomerating to grow in size; the ethanol precipitates cerium oxide. In the present invention, the time of the ultrasound is preferably 30min, and the frequency of the ultrasound is not particularly limited in the present invention. The invention preferably collects the sediment by centrifugation, the rotation speed of the centrifugation is 12000rpm, and the time of the centrifugation is 10 min. In the present invention, the number of ethanol washes is preferably 3.
Mixing the obtained first stirring object with a terbium chloride solution, and then carrying out second stirring to obtain a second stirring object; the time of the second stirring is 30 min. In the present invention, the volume ratio of the terbium chloride solution to the disodium 5' -guanylate solution is preferably 1: 1. In the present invention, the concentration of the terbium chloride solution is preferably 3.74 mg/mL.
Centrifuging the obtained second stirring substance to obtain Tb-GMP/CeO precipitate2A composite material. In the present invention, the conditions of the centrifugation preferably include: the rotation speed of the centrifugation is preferably 12000rpm, and the time of the centrifugation is preferably 10 min.
The invention also provides Tb-GMP/CeO prepared by the technical scheme2The method for detecting ziram by using the composite material comprises the following steps:
a. mixing the tyrosinase solution with a sample, and standing to obtain a standing solution;
b. mixing the standing solution obtained in the step a with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
c. c, mixing the reaction liquid obtained in the step b with Tb-GMP/CeO in the technical scheme2Mixing and oscillating a composite material, a 3,3 ', 5, 5' -tetramethylbenzidine solution and a citric acid-disodium hydrogen phosphate buffer solution to obtain an oscillating liquid, measuring an absorption spectrum of the oscillating liquid to obtain maximum absorbance, and converting the maximum absorbance into an enzyme inhibition rate;
the enzyme inhibition rate is (A)z-A0)/(A-A0)×100，AzRepresents the maximum absorption intensity in the absorption spectrum, A, in the presence of ziram0Represents the maximum absorption intensity in the absorption spectrum in the absence of ziram, and A represents the maximum absorption intensity in the absorption spectrum;
d. substituting the enzyme inhibition rate obtained in the step c into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: the enzyme inhibition rate was 16.32 xc +0.37, and the unit of c was μ g/L.
The method comprises the steps of mixing a tyrosinase solution with a sample, and standing to obtain a standing solution.
In the present invention, the sample preferably comprises fruit, preferably comprising apples, and produce, preferably comprising rice. In the present invention, the volume ratio of the tyrosinase solution to the sample is preferably 1: 1. In the present invention, the concentration of the tyrosinase solution is preferably 175U/mL, and the tyrosinase solution is preferably prepared by using Tris-HCl buffer (concentration: 10mM, pH 7.4) as a solvent. In the present invention, the conditions of the standing preferably include: the time for standing is preferably 5min, and the ambient temperature for standing is preferably 37 ℃.
The obtained standing solution is mixed with a tyrosine solution and a Tris-HCl buffer solution for reaction to obtain a reaction solution. In the invention, the volume ratio of the tyrosine solution to the Tris-HCl buffer solution to the sample is preferably 10:8: 1. In the present invention, the concentration of the tyrosine solution is preferably 2.5 mM. In the present invention, the concentration of the Tris-HCl buffer is 10mM, and the pH value is 7.4. In the present invention, the conditions of the reaction preferably include: the ambient temperature of the reaction is preferably 37 ℃ and the reaction time is preferably 30 min.
The invention combines the obtained reaction liquid with Tb-GMP/CeO in the technical scheme2Mixing and oscillating a composite material, a 3,3 ', 5, 5' -tetramethylbenzidine solution and a citric acid-disodium hydrogen phosphate buffer solution to obtain an oscillating liquid, measuring an absorption spectrum of the oscillating liquid to obtain maximum absorbance, and converting the maximum absorbance into an enzyme inhibition rate; the enzyme inhibition rate is (A)z-A0)/(A-A0)×100，AzRepresents the maximum absorption intensity in the absorption spectrum, A, in the presence of ziram0Represents the maximum absorption intensity in the absorption spectrum in the absence of ziram, and A represents the maximum absorption intensity in the absorption spectrum, where A is 0.7 and A is00.095, a and a0Is a fixed value.
In the present invention, the Tb-GMP/CeO2The composite material is preferably Tb-GMP/CeO2Mixing the solution with the raw materials, wherein Tb-GMP/CeO2The volume ratio of the solution to the 3,3 ', 5, 5' -tetramethylbenzidine solution and the citric acid-disodium hydrogen phosphate buffer solution is preferably 100:75: 225. In the present invention, the Tb-GMP/CeO2The concentration of the solution is preferably 2 mg/mL. In the present invention, the concentration of the 3,3 ', 5, 5' -tetramethylbenzidine solution is preferably 10 mM. In the present invention, the concentration of the citric acid-disodium hydrogen phosphate buffer is preferably 0.2M, and the pH is 3.0. In the present inventionIn the present invention, the conditions of the oscillation preferably include: the rotation speed of the oscillation is preferably 500rpm, and the time of the oscillation is preferably 10 min. In the invention, the oscillation liquid preferably detects the maximum absorbance at 500-750 nm.
Substituting the obtained enzyme inhibition rate into a linear equation to obtain the concentration of the ziram in the sample; the linear equation is: the enzyme inhibition rate was 16.32 xc +0.37, and the unit of c was μ g/L.
In the present invention, the obtaining of the linear equation preferably includes: the samples were replaced with ziram solutions of a range of concentrations, and the remaining steps are as above and will not be described further. According to the invention, acetonitrile is preferably used for preparing the ziram solution with different concentrations, wherein the concentrations are as follows: 10. mu.g/L, 50. mu.g/L, 100. mu.g/L, 250. mu.g/L, 400. mu.g/L, 500. mu.g/L. In the present invention, the linear equation detects the linear range of ziram: 0.1-5 mu g/L, and the detection limit is 0.035 mu g/L.
The invention also provides Tb-GMP/CeO prepared by the technical scheme2The method for detecting ziram by using the composite material is characterized by comprising the following steps of:
(1) mixing the tyrosinase solution with a sample, and standing to obtain a standing solution;
(2) mixing the standing solution obtained in the step (1) with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
(3) reacting the reaction solution obtained in the step (2) with Tb-GMP/CeO according to claim 12Mixing and oscillating a composite material, a 3,3 ', 5, 5' -tetramethylbenzidine solution and a citric acid-disodium hydrogen phosphate buffer solution to obtain an oscillating liquid, photographing the oscillating liquid by using a smart phone, reading RGB values in a photo, converting the RGB values into S values, and substituting the S values into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: s is 0.0779 xc +0.0487, with c being μ g/L.
In the present invention, the shaking solution obtained in steps (1) - (3) is the same as the above technical solution, and is not described herein again. After obtaining the oscillation liquid, taking a picture of the oscillation liquid by using a smart phone, reading an RGB value in the picture, converting the RGB value into an S value (a conversion formula is shown in figure 17), and substituting the S value into a linear equation to obtain the concentration of the ziram in the sample; the linear equation is: s is 0.0779 xc +0.0487, with c being μ g/L. The present invention preferably uses software with a read photo (e.g., ImageJ) to obtain RGB values in the photo.
The invention also provides Tb-GMP/CeO prepared by the technical scheme2The method for detecting ziram by using the composite material comprises the following steps:
A. mixing the tyrosinase solution with a sample, and standing to obtain a standing solution;
B. mixing the standing solution obtained in the step A with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
C. mixing the reaction solution obtained in the step B with Tb-GMP/CeO according to claim 12Mixing and oscillating the composite material and ultrapure water to obtain an oscillating liquid, measuring a fluorescence emission spectrum of the oscillating liquid to obtain maximum fluorescence intensity, and converting the maximum fluorescence intensity into an enzyme inhibition rate;
(FD-F) as an enzyme inhibitor0)/(F-F0) X 100, FD represents the maximum fluorescence intensity in the fluorescence emission spectrum in the presence of ziram, F0Represents the maximum fluorescence intensity in the fluorescence emission spectrum in the absence of ziram, and F represents the maximum fluorescence intensity in the fluorescence emission spectrum;
D. substituting the enzyme inhibition rate obtained in the step C into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: enzyme inhibition rate of 33.21 × log10c +15.96, the unit of c is mug/L.
The method comprises the steps of mixing a tyrosinase solution with a sample, and standing to obtain a standing solution. In the present invention, the volume ratio of the tyrosinase solution to the sample is preferably 1: 1. In the present invention, the sample preferably includes fruit, tap water and agricultural products, the fruit preferably includes apple, and the agricultural products preferably include rice and soybean. In the invention, the concentration of the tyrosinase solution is preferably 80U/mL; in the present invention, preferably, a solution of tyrosinase is prepared using Tris-HCl buffer (concentration: 10mM, pH 7.4) as a solvent. In the present invention, the conditions of the standing preferably include: the time for standing is preferably 15min, and the ambient temperature for standing is preferably 37 ℃.
The obtained standing solution is mixed with a tyrosine solution and a Tris-HCl buffer solution for reaction to obtain a reaction solution. In the invention, the volume ratio of the tyrosine solution to the Tris-HCl buffer solution to the sample is preferably 10:8: 1. In the present invention, the concentration of the tyrosine solution is preferably 2.5 mM. In the present invention, the concentration of the Tris-HCl buffer is preferably 10mM, and the pH value is preferably 7.4. In the present invention, the conditions of the reaction preferably include: the ambient temperature of the reaction is preferably 37 ℃ and the reaction time is preferably 30 min.
The invention combines the obtained reaction liquid with Tb-GMP/CeO in the technical scheme2Mixing and oscillating the composite material and ultrapure water to obtain an oscillating liquid, measuring a fluorescence emission spectrum of the oscillating liquid to obtain maximum fluorescence intensity, and converting the maximum fluorescence intensity into an enzyme inhibition rate; (FD-F) as an enzyme inhibitor0)/(F-F0) X 100, FD represents the maximum fluorescence intensity in the fluorescence emission spectrum in the presence of ziram, F0Represents the maximum fluorescence intensity in the fluorescence emission spectrum in the absence of ziram, F represents the maximum fluorescence intensity in the fluorescence emission spectrum, F is 1561.6, F0137.6, F and F0Are all fixed values.
In the present invention, the Tb-GMP/CeO2The composite material is preferably Tb-GMP/CeO2Mixing the solution with the raw materials, wherein Tb-GMP/CeO2The volume ratio of the solution to the ultrapure water is preferably 1: 79. In the present invention, the fluorescence excitation wavelength for measuring the fluorescence emission spectrum of the shaking liquid is preferably 286 nm.
Substituting the obtained enzyme inhibition rate into a linear equation to obtain the concentration of the ziram in the sample; the linear equation is: enzyme inhibition rate of 33.21 × log10c +15.96, the unit of c is mug/L.
In the present invention, the obtaining manner of the linear equation preferably includes: the samples were replaced with ziram solutions of a range of concentrations, and the remaining steps are as above and will not be described further. According to the invention, acetonitrile is preferably used for preparing the ziram solution with different concentrations, wherein the concentrations are as follows: 50. mu.g/L, 100. mu.g/L, 250. mu.g/L, 500. mu.g/L, 1000. mu.g/L, 5000. mu.g/L and 10000. mu.g/L. In the present invention, the linear equation detects the linear range of ziram: 0.5-100 mug/L, and the detection limit is 0.046 mug/L.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Tb-GMP/CeO2Preparing a composite material:
Tb-GMP/CeO synthesis by two-step method2. Firstly, the nanometer CeO is synthesized by a precipitation method2The specific synthesis steps are as follows: 2.52g of cerium nitrate hexahydrate is weighed into a 200mL beaker and dissolved in 100mL of water-ethylene glycol mixed solution (volume ratio is 1: 1). After heating the solution to 60 ℃, 20mL of ammonia was added rapidly and stirred vigorously for 3 h. Cooling the mixed solution, and centrifuging at 12000rpm for 10min to obtain yellow nanometer CeO2And (4) precipitating. Washing nanometer CeO for many times by using ethanol and ultrapure water2Until the washing liquid is neutral. Washing the nanometer CeO2Mixing with 100mL sodium citrate solution (concentration of 30g/L), and after 30min of ultrasonic treatment, adding ethanol continuously until no precipitate is formed. The mixture was centrifuged at 12000rpm for 10min, the supernatant was discarded, and the precipitate was washed three times with ethanol. Freeze drying the washed precipitate to obtain nanometer CeO2。
Tb-GMP/CeO synthesis by stirring method2The method comprises the following specific steps: using ultrapure water as a solvent, nano CeO with a concentration of 2.5mg/mL was prepared2A solution, and a terbium chloride hexahydrate solution at a concentration of 3.74 mg/mL. A GMP solution having a concentration of 4.07mg/mL was prepared using HEPES buffer (concentration: 0.1M, pH 7.4). Taking 1mL of GMP solution, adding 0.15mL of nano CeO into a 10mL plastic centrifuge tube2The solution was mixed and stirred for 20 min. Then 1mL of terbium chloride hexahydrate solution was rapidly added, and stirring was continued for 30 min. After the reaction is finished, centrifuging the mixed solution at 12000rpm for 10min, removing the supernatant, washing the precipitate for three times by using ultrapure water, and then freeze-drying to obtain Tb-GMP/CeO2. To Tb-GMP/CeO2Adding 2mL of ultrapure water, whirling and carrying out ultrasonic treatment until the dispersion is uniform for later use.
Tb-GMP/CeO2The physical parameters of the X-ray photoelectron spectrum of the composite material are as follows: terbium (3d3/2)1277.1eV, terbium (3d5/2)1242.5eV, cerium (3d3/2)904.4eV, cerium (3d5/2)
886.1eV, oxygen 1s 531.3eV, and phosphorus 2p 133.8eV (FIG. 3).
Tb-GMP/CeO2The composite material is spherical nano particles (figure 1), the particle diameter is 25-50nm, the X-ray energy spectrum analysis shows that the composite material contains terbium, cerium and phosphorus elements (figure 2), and the fluorescence spectrum: maximum excitation wavelength: 286nm, maximum emission wavelength: 545nm (FIG. 4). Absorption spectrum: absorption peak about 245nm (FIG. 5); Tb-GMP/CeO2The composite material can catalyze the oxidation of 3,3 ', 5, 5' -tetramethylbenzidine, and the absorption spectrum of the product has an absorption peak at 652nm (figure 6).
Example 2
Tb-GMP/CeO prepared in example 12Composite material for detecting ziram
First, a tyrosinase solution was prepared at a concentration of 175U/mL using Tris-HCl buffer (concentration: 10mM, pH 7.4) as a solvent. Acetonitrile is used as a solvent to prepare a series of thiram solutions with concentration (10 mug/L, 50 mug/L, 100 mug/L, 250 mug/L, 400 mug/L and 500 mug/L) for establishing a standard curve method to quantitatively detect thiram.
And (3) putting 5 mu L of tyrosinase solution into a 2mL plastic centrifuge tube, adding 5 mu L of zinc thiram solution into the centrifuge tube, fully mixing the two, and standing for 5min at 37 ℃. Then, 50. mu.L of 2.5mM tyrosine solution and 40. mu.L of 10mM Tris-HCl buffer (pH 7.4) were added to the centrifuge tube, mixed well, and left to stand at 37 ℃ for reaction for 30 min. After the reaction is finished, 100 mu L of Tb-GMP/CeO is added into a centrifuge tube2Aqueous solution (concentration 2mg/mL), 75 μ L of TMB (3,3 ', 5, 5' -tetramethylbenzidine) solution, and 225 μ L of citric acid-disodium hydrogen phosphate buffer (pH 3). After the mixed solution was shaken at room temperature at 500rpm for 10 minutes, it was put into a cuvette and put into an ultraviolet-visible spectrophotometer, and the absorption spectrum was measured and the maximum absorbance was recorded. The experimental result shows that the greater the concentration of the ziram, the maximum absorption of the solutionThe greater the photometric value (FIG. 7), the absorbance of the solution in the presence of different concentrations of ziram was converted into the enzyme inhibition (IE,%), IE ═ AZ-A0)/(A-A0) X 100 (in the formula, Az and A0 respectively represent Tb-GMP/CeO in the presence or absence of ziram2-maximum absorbance of TMB oxide catalyzed by tyrosinase-tyrosine system. A represents Tb-GMP/CeO2-maximum absorbance of TMB oxide under tyrosine catalysis, wherein a ═ 0.7, a00.095, a and a0Fixed value), a linear equation between IE and the logarithm of the concentration of the ziram is established, and the quantitative detection of the ziram can be realized by a standard curve method (figure 8).
The linear equation is: the enzyme inhibition rate was 16.32 xc +0.37, and the unit of c was μ g/L.
In the linear range: 0.1-5 mu g/L, and the detection limit is 0.035 mu g/L.
Example 3
Tb-GMP/CeO prepared in example 12Composite material for detecting ziram
First, a tyrosinase solution was prepared at a concentration of 80U/mL using Tris-HCl buffer (concentration: 10mM, pH 7.4) as a solvent. Acetonitrile is used as a solvent to prepare a series of zinc thiram solutions with concentrations (50 mug/L, 100 mug/L, 250 mug/L, 500 mug/L, 1000 mug/L, 5000 mug/L and 10000 mug/L) for establishing a standard curve method to quantitatively detect the zinc thiram.
And (3) putting 5 mu L of tyrosinase solution into a 2mL plastic centrifuge tube, adding 5 mu L of zinc thiram solution into the centrifuge tube, fully mixing the two, and standing at 37 ℃ for 15 min. Then, 50. mu.L of 2.5mM tyrosine solution and 40. mu.L of 10mM Tris-HCl buffer (pH 7.4) were added to the centrifuge tube, mixed well, and left to stand at 37 ℃ for reaction for 30 min. After the reaction, 5 mu L of Tb-GMP/CeO was added into the centrifuge tube2An aqueous solution (concentration: 2mg/mL), and 395. mu.L of ultrapure water. After the mixed solution was shaken at room temperature at 500rpm for 15 minutes, it was put into a cuvette and measured with a fluorescence spectrometer (fluorescence excitation wavelength: 286nm), and the maximum fluorescence intensity was recorded. The experimental results show that the higher the concentration of ziram, the higher the maximum fluorescence intensity value of the solution (FIG. 9), and different concentrations of ziram are presentThe maximum fluorescence intensity of the solution was converted into enzyme inhibition (IE,%), IE ═ FZ-F0)/(F-F0) X 100 (in the formula, Fz and F0Tb-GMP/CeO in the presence or absence of ziram2-maximum fluorescence intensity in the fluorescence emission spectrum of the tyrosinase-tyrosine system. F represents Tb-GMP/CeO2-maximum fluorescence intensity in the tyrosine fluorescence emission spectrum. Wherein F is 1561.6, F0137.6, F and F0All are fixed values), a linear equation between IE and the logarithm of the concentration of the ziram is established, and the quantitative detection of the ziram can be realized by a standard curve method (figure 10).
The linear equation is: enzyme inhibition rate of 33.21 × log10c +15.96, the unit of c is mug/L.
Linear range: 0.5-100 mug/L, and the detection limit is 0.046 mug/L.
Example 4
Tb-GMP/CeO prepared in example 12Composite material for detecting ziram
The difference of the detection method from the example 2 is that the cuvette containing the mixed solution is finally photographed by using a smart phone instead of using an ultraviolet-visible spectrophotometer for measurement. The experimental result shows that the larger the concentration of the ziram, the color of the solution gradually changes (fig. 11), the RGB value of the mixed solution area in the photo is read by using ImageJ software, the RGB value is converted into the S value, a linear equation between the S value and the concentration of the ziram is established, and the quantitative detection of the ziram can be realized by a standard curve method (fig. 12).
The linear equation: s is 0.0779 xc +0.0487, with c being μ g/L.
Linear range: 0.1-5 mu g/L.
Example 5
The same method as in example 2 was used to detect ziram in the samples:
an additive recovery experiment was performed to verify the ability of the method to detect ziram residue in real samples (tap water, soy and apple). Tap water is taken from college of university of agriculture in China, and after the tap water passes through a 0.22-micron filter membrane, the standard solution of ziram is diluted, and water samples containing ziram with different concentrations (0.05, 0.25 or 0.5mg/L) are respectively prepared. The detection procedure is the same as that for the detection of ziram in the standard solution. The rice and apple samples are purchased from local supermarkets, and the method for extracting the residual ziram in the agricultural product samples comprises the following steps: accurately weighing 5g of the mashed sample into a 50mL plastic centrifuge tube, adding 25 μ L of zinc thiram solution (with the concentration of 10, 50 or 100mg/L respectively), and standing for 30 min. 10mL of a mixed extract (volume ratio of 1:1) of PBS (pH 8, containing 10mM EDTA) and acetonitrile was added to the tube, and vortexed for 10 min. The extract was then filtered using a 0.22 μm filter. During detection, 10 mu L of extracting solution is taken, and the rest detection steps are the same as the steps for detecting the ziram in the standard solution.
The experimental results are as follows: absorption spectroscopy
Recovery (%). measured concentration/added concentration X100
Relative Standard Deviation (RSD): refers to the relative standard deviation of recovery from triplicate experiments.
TABLE 1 recovery and RSD of residual Fumei zinc in real samples
Figure GDA0003513647110000121
Figure GDA0003513647110000131
Note: the adding concentration and the measured concentration of the ziram in the apple and rice samples are mg/kg, and the adding concentration and the measured concentration of the ziram in the tap water samples are mg/L.
As can be seen from Table 1, the recovery rate and RSD of the ziram detected by the method in the actual sample are both good, and can meet the national relevant standards (GB 2763-2019).
Example 6
The same method as in example 4 was used to detect ziram in the samples:
an additive recovery experiment was performed to verify the ability of the method to detect ziram residue in real samples (tap water, soy and apple). Tap water is taken from college of agriculture university in China, and after the tap water passes through a 0.22 mu m filter membrane, the standard solution of the ziram is diluted, and water samples containing ziram with different concentrations (0.05mg/L, 0.25mg/L or 0.5mg/L) are respectively prepared. The detection procedure is the same as that for the detection of ziram in the standard solution. The rice and apple samples are purchased from local supermarkets, and the method for extracting the residual ziram in the agricultural product samples comprises the following steps: accurately weighing 5g of the mashed sample into a 50mL plastic centrifuge tube, adding 25 μ L of zinc thiram solution (with the concentration of 10mg/L, 50mg/L or 100mg/L respectively), and standing for 30 min. 10mL of a mixed extract (volume ratio of 1:1) of PBS (pH 8, containing 10mM EDTA) and acetonitrile was added to the tube, and vortexed for 10 min. The extract was then filtered using a 0.22 μm filter. During detection, 10 mu L of extracting solution is taken, and the rest detection steps are the same as the steps for detecting the ziram in the standard solution.
The experimental results are as follows: smart phone method:
TABLE 2 recovery of residual Fumei zinc and RSD in the actual samples
Figure GDA0003513647110000132
Figure GDA0003513647110000141
Note: the adding concentration and the measured concentration of the ziram in the apple and rice samples are mg/kg, and the adding concentration and the measured concentration of the ziram in the tap water samples are mg/L.
As can be seen from Table 2, the recovery rate and RSD of the ziram detected by the method in the actual sample are both good, and can meet the national relevant standards (GB 2763-2019).
Example 7
Detection was carried out in the same manner as in example 2, i.e. fluorescence spectroscopy: tap water is taken from college of agriculture university in China, filtered by a 0.22 mu m filter membrane, and then the standard solution of the ziram is diluted to respectively prepare water samples containing ziram with different concentrations (0.1mg/L, 0.5mg/L or 5 mg/L). The detection procedure is the same as that for the detection of ziram in the standard solution. The rice and apple samples are purchased from local supermarkets, and the method for extracting the residual ziram in the agricultural product samples comprises the following steps: accurately weighing 5g of the mashed sample into a 50mL plastic centrifuge tube, adding 25 μ L of zinc thiram solution (with the concentration of 20mg/L, 100mg/L or 1000mg/L respectively), and standing for 30 min. 10mL of a mixed extract (volume ratio of 1:1) of PBS (pH 8, containing 10mM EDTA) and acetonitrile was added to the tube, and vortexed for 10 min. The extract was then filtered using a 0.22 μm filter. During detection, 10 mu L of extracting solution is taken, and the rest detection steps are the same as the steps for detecting the ziram in the standard solution.
The experimental results are as follows:
table 3 recovery and RSD of residual Fumei zinc in practical sample
Figure GDA0003513647110000142
Figure GDA0003513647110000151
Note: the adding concentration and the measured concentration of the ziram in the apple and rice samples are mg/kg, and the adding concentration and the measured concentration of the ziram in the tap water samples are mg/L.
As can be seen from Table 3, the recovery rate and RSD of the ziram detected by the method in the actual sample are both good, and can meet the national relevant standards (GB 2763-2019).
Example 8
The method for detecting ziram is the same as the method in the embodiment 2, namely the absorption spectroscopy is adopted to detect ziram, the influence of the pH value, the concentration of tyrosinase, the time and the temperature of the mixed reaction of tyrosinase and tyrosine and the time of the mixed reaction of ziram and tyrosinase on the detection result is examined, and the results are as follows:
as can be seen from A in FIG. 13 and Table 4, when the pH of the solution was 3, the absorbance value of the oxidation product of TMB was the largest, and thus Tb-GMP/CeO2The pH of the solution at the time of catalytic TMB oxidation was determined to be 3.
As can be seen from B in FIG. 13 and Table 5, when the tyrosinase concentration reached 1.75U/mL, and the tyrosinase concentration continued to increase, the absorbance change of the TMB oxidized product was small, indicating that the tyrosinase concentration may have been excessive. Therefore, considering that the concentration of tyrosinase is reduced as much as possible to improve the sensitivity of detection of ziram, 1.75U/mL is selected as the concentration of tyrosinase. Optimization experiments of tyrosinase and tyrosine mixing reaction time showed that after 30min of reaction, the change in absorbance of the TMB oxidation product was small (C in FIG. 13 and Table 6). Therefore, 30min is selected as the time for the mixed reaction of tyrosinase and tyrosine. The absorbance of the TMB oxidation product reached a maximum at 37 deg.C (D in FIG. 13 and Table 7). Therefore, 37 ℃ was chosen as the reaction temperature.
As can be seen from E in FIG. 13 and Table 8, the absorbance of TMB oxidation product did not change substantially after 5min of the mixing reaction time of ziram and tyrosinase. Therefore, 5min is selected as the time for mixing and reacting the ziram and the tyrosinase.
TABLE 4 pH influence results
Figure GDA0003513647110000161
TABLE 5 results of tyrosinase concentration effects
Figure GDA0003513647110000162
TABLE 6 results of the effect of the mixing reaction time of tyrosinase and tyrosine
Figure GDA0003513647110000163
TABLE 7 influence of reaction temperature
Figure GDA0003513647110000164
Figure GDA0003513647110000171
TABLE 8 results of the time-dependent mixing reaction of ziram with tyrosinase
Figure GDA0003513647110000172
Example 9
The same method as that of example 3, namely fluorescence spectroscopy detection, is adopted to examine the influence of the reaction time, the concentration of tyrosinase, the time and the temperature of the mixed reaction of tyrosinase and tyrosine, and the time of the mixed reaction of ziram and tyrosinase on the detection result, and the results are as follows:
as can be seen from A in FIG. 14 and Table 9, when Tb-GMP/CeO2Tb-GMP/CeO is mixed with tyrosinase catalysis system for reaction for 15min2The fluorescence intensity of the enzyme reaches the lowest level, which shows that the combination effect of the enzyme catalysis product and Tb-GMP/CeO2 is basically complete, so Tb-GMP/CeO2The time for mixing reaction with tyrosinase catalysis system was determined to be 15 min.
As can be seen from B in FIG. 14 and Table 10, when the tyrosinase concentration reached 0.8U/mL, the tyrosinase concentration, Tb-GMP/CeO, continued to be increased2The change in fluorescence intensity of (a) is small, indicating that the tyrosinase concentration may have been excessive. Therefore, considering that the concentration of tyrosinase is reduced as much as possible to improve the sensitivity of detection of ziram, 0.8U/mL is selected as the concentration of tyrosinase. The optimization experiment of the mixed reaction time of tyrosinase and tyrosine shows that Tb-GMP/CeO reacts for 30min2The change in fluorescence intensity of (2) was small (C in fig. 14 and table 11). Therefore, 30min is selected as the time for the mixed reaction of tyrosinase and tyrosine. Tb-GMP/CeO at 37 deg.C2The fluorescence intensity of (2) reached the lowest level (D in FIG. 14 and Table 12). Therefore, 37 ℃ was chosen as the reaction temperature.
As can be seen from E and Table 13 in FIG. 14, Tb-GMP/CeO was observed after the time for the mixed reaction of ziram and tyrosinase reached 15min2Does not substantially change in fluorescence intensity. Therefore, 15min is selected as the time for mixing and reacting the ziram and the tyrosinase.
TABLE 9 Tb-GMP/CeO2Mixing with tyrosinase-catalyzed systemTime-dependent results of the reaction
Figure GDA0003513647110000173
Figure GDA0003513647110000181
TABLE 10 results of tyrosinase concentration effects
Figure GDA0003513647110000182
TABLE 11 results of the effect of the mixing reaction time of tyrosinase and tyrosine
Figure GDA0003513647110000183
TABLE 12 reaction temperatures
Figure GDA0003513647110000184
TABLE 13 results of the time-dependent mixing reaction of ziram with tyrosinase
Figure GDA0003513647110000185
Figure GDA0003513647110000191
Example 10
Selectivity of the detection method:
absorption spectroscopy: selecting several metal ions (sodium ion, calcium ion, zinc ion), biochemical substances (vitamin C, glucose, glycine) and bactericide (metalaxyl, tebuconazole, azoxystrobin) with larger usage amount, and performing the same operationThe procedure was carried out to determine the Tb-GMP/CeO ratio for each substance2-the influence of the absorbance of the TMB-tyrosinase-tyrosine color reaction system. The concentration of all non-targets was 25. mu.g/L. The results are shown in FIG. 15.
As can be seen from FIG. 15, compared with ziram (concentration of 2.5. mu.g/L), other non-target substances still have no obvious inhibition effect on tyrosinase at high concentration (25. mu.g/L), indicating that the specificity of the method for detecting ziram is better.
Example 11
The detection method used in example 3, fluorescence spectroscopy:
selecting several metal ions (sodium ions, calcium ions and zinc ions), biochemical substances (vitamin C, glucose and glycine) and bactericides (metalaxyl, tebuconazole and azoxystrobin) with larger using amount, and determining Tb-GMP/CeO ratio of each substance by the same operation steps2-influence of the maximum fluorescence intensity of the tyrosinase-tyrosine system. The concentration of all non-targets was 50. mu.g/L, and the results are shown in FIG. 16.
The experimental results are as follows: it can be seen that compared with ziram (the concentration is 5 mug/L), other non-target substances still have no obvious inhibition phenomenon on tyrosinase under the condition of high concentration (50 mug/L), and the selectivity of the method for detecting ziram is also better.
As can be seen from the above examples, the Tb-GMP/CeO provided by the present application is used2The composite material can quickly detect the ziram in the sample.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. Tb-GMP/CeO2Composite material, characterized in that the Tb-GMP/CeO2The structural physical property parameters of the composite material are as follows: a spherical structure with a size of 10-50 nm; the physical parameters of the X-ray photoelectron spectrum are as follows: terbium (3 d)3/2):1277.1eV terbium (3 d)5/2)：1242.5eV, cerium (3 d)3/2):904.4eV, cerium (3 d)5/2):886.1eV, oxygen 1s:531.3eV, phosphorus 2p:133.8 eV.
2. Tb-GMP/CeO according to claim 12The preparation method of the composite material is characterized by comprising the following steps:
1) mixing 5' -disodium guanylate solution with CeO2Mixing the solution, and stirring for the first time to obtain a first stirred material; the time for the first stirring is 20 min;
2) mixing the first stirred material obtained in the step 1) with a terbium chloride solution, and then stirring for the second time to obtain a second stirred material; the time for the second stirring is 30 min;
3) centrifuging the second stirred material obtained in the step 2), wherein the obtained precipitate is Tb-GMP/CeO2A composite material.
3. The method according to claim 2, wherein the step 1) of mixing the disodium 5' -guanylate solution with CeO2The volume ratio of the solution is 1: 0.15;
the concentration of the disodium 5' -guanylate solution is 4.07 mg/mL;
the CeO2The concentration of the solution is 2.5 mg/mL;
the CeO2CeO in solution2Is nano-scale CeO2；
The volume ratio of the terbium chloride solution in the step 2) to the disodium 5' -guanylate solution is 1: 1;
the concentration of the terbium chloride solution is 3.74 mg/mL;
the conditions of the centrifugation in the step 3) comprise: the rotation speed of the centrifugation is 12000rpm, and the time of the centrifugation is 10 min.
4. Use of Tb-GMP/CeO according to claim 12The method for detecting ziram by using the composite material is characterized by comprising the following steps of:
a. mixing the tyrosinase solution with a sample, and standing to obtain a standing solution; the sample is apple, rice, river water or tap water;
b. mixing the standing solution obtained in the step a with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
c. reacting the reaction solution obtained in the step b with Tb-GMP/CeO according to claim 12Mixing and oscillating a composite material, a 3,3 ', 5, 5' -tetramethylbenzidine solution and a citric acid-disodium hydrogen phosphate buffer solution to obtain an oscillating liquid, measuring an absorption spectrum of the oscillating liquid to obtain maximum absorbance, and converting the maximum absorbance into an enzyme inhibition rate;
the enzyme inhibition rate is (A)z-A0)/(A-A0)×100，AzRepresents the maximum absorption intensity in the absorption spectrum, A, in the presence of ziram0Represents the maximum absorption intensity in the absorption spectrum in the absence of ziram, and A represents the maximum absorption intensity in the absorption spectrum;
d. substituting the enzyme inhibition rate obtained in the step c into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: the enzyme inhibition rate was 16.32 xc +0.37, and the unit of c was μ g/L.
5. Use of Tb-GMP/CeO according to claim 12The method for detecting ziram by using the composite material is characterized by comprising the following steps of:
(1) mixing the tyrosinase solution with a sample, and standing to obtain a standing solution; the sample is apple, rice, river water or tap water;
(2) mixing the standing solution obtained in the step (1) with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
(3) reacting the reaction solution obtained in the step (2) with Tb-GMP/CeO according to claim 12Mixing and oscillating a composite material, a 3,3 ', 5, 5' -tetramethylbenzidine solution and a citric acid-disodium hydrogen phosphate buffer solution to obtain an oscillating liquid, photographing the oscillating liquid by using a smart phone, reading RGB values in a photo, converting the RGB values into S values, and substituting the S values into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: s is 0.0779 xc +0.0487, with c being μ g/L.
6. The detection method according to claim 4 or 5, wherein the volume ratio of the tyrosinase solution to the sample in the step a or (1) is 1: 1;
the concentration of the tyrosinase solution is 175U/mL;
the standing conditions comprise: the standing time was 5min, and the ambient temperature of standing was 37 ℃.
7. The detection method according to claim 4 or 5, wherein the volume ratio of the tyrosine solution to the Tris-HCl buffer solution and the sample in the step b or (2) is 10:8: 1;
the concentration of the tyrosine solution is 2.5 mM;
the concentration of the Tris-HCl buffer solution is 10mM, and the pH value is 7.4;
the reaction conditions include: the ambient temperature of the reaction was 37 ℃ and the reaction time was 30 min.
8. The detection method according to claim 4 or 5, wherein Tb-GMP/CeO in step c or (3)2The composite material is Tb-GMP/CeO2Mixing the solution with the raw materials, wherein Tb-GMP/CeO2The volume ratio of the solution to the 3,3 ', 5, 5' -tetramethylbenzidine solution and the citric acid-disodium hydrogen phosphate buffer solution is 100:75: 225; the raw materials are 3,3 ', 5, 5' -tetramethyl benzidine solution and citric acid-disodium hydrogen phosphate buffer solution;
the Tb-GMP/CeO2The concentration of the solution is 2 mg/mL;
the concentration of the 3,3 ', 5, 5' -tetramethyl benzidine solution is 10 mM;
the concentration of the citric acid-disodium hydrogen phosphate buffer solution is 0.2M, and the pH value is 3.0;
the conditions of the oscillation include: the oscillating speed is 500rpm, and the oscillating time is 10 min;
and c, measuring the maximum absorbance of the oscillating liquid at 500-750 nm in the step c.
9. Use of Tb-GMP/CeO according to claim 12The method for detecting ziram by using the composite material is characterized by comprising the following steps of:
A. mixing the tyrosinase solution with a sample, and standing to obtain a standing solution; the sample is apple, rice, river water or tap water;
B. mixing the standing solution obtained in the step A with a tyrosine solution and a Tris-HCl buffer solution, and then reacting to obtain a reaction solution;
C. mixing the reaction solution obtained in the step B with Tb-GMP/CeO according to claim 12Mixing and oscillating the composite material and ultrapure water to obtain an oscillating liquid, measuring a fluorescence emission spectrum of the oscillating liquid to obtain maximum fluorescence intensity, and converting the maximum fluorescence intensity into an enzyme inhibition rate;
(FD-F) as an enzyme inhibitor0)/(F-F0) X 100, FD represents the maximum fluorescence intensity in the fluorescence emission spectrum in the presence of ziram, F0Represents the maximum fluorescence intensity in the fluorescence emission spectrum in the absence of ziram, and F represents the maximum fluorescence intensity in the fluorescence emission spectrum;
D. substituting the enzyme inhibition rate obtained in the step C into a linear equation to obtain the concentration of the ziram in the sample;
the linear equation is: enzyme inhibition rate of 33.21 × log10c +15.96, the unit of c is mug/L.
10. The assay of claim 9, wherein the volume ratio of the tyrosinase solution to the sample in step a is 1: 1;
the concentration of the tyrosinase solution is 80U/mL;
the standing conditions comprise: standing for 15min at 37 deg.C;
the volume ratio of the tyrosine solution to the Tris-HCl buffer solution to the sample in the step B is 10:8: 1;
the concentration of the tyrosine solution is 2.5 mM;
the concentration of the Tris-HCl buffer solution is 10mM, and the pH value is 7.4;
the reaction conditions include: the reaction environment temperature is 37 ℃, and the reaction time is 30 min;
Tb-GMP/CeO in the step C2The composite material is Tb-GMP/CeO2Mixing the solution with the raw materials, wherein Tb-GMP/CeO2The volume ratio of the solution to the ultrapure water is 1: 79;
the fluorescence excitation wavelength of the fluorescence emission spectrum of the measurement shaking solution was 286 nm.
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