阅读说明：本技术 一种纳米材料离子液体复合修饰电极及其制备方法与在己烯雌酚测定中的应用 (Nano-material ionic liquid composite modified electrode, preparation method thereof and application of nano-material ionic liquid composite modified electrode in diethylstilbestrol determination ) 是由 赵晓娟 吴俊铨 邹玉婷 刘功良 白卫东 于 2021-07-26 设计创作，主要内容包括：本发明涉及己烯雌酚检测技术领域,具体公开了一种纳米材料离子液体复合修饰电极及其制备方法与在己烯雌酚测定中的应用。所述的纳米材料离子液体复合修饰电极的制备方法,其包括如下步骤：(1)将氧化石墨烯、多壁碳纳米管以及[BMIM]PF-(6)分散在水中,得到GO-MWCNT-(S)-[BMIM]PF-(6)分散液；(2)将GO-MWCNT-(S)-[BMIM]PF-(6)分散液涂覆在玻碳电极表面即得所述的纳米材料离子液体复合修饰电极。以本发明所述的纳米材料离子液体复合修饰电极作为工作电极,在本发明所述方波伏安条件下测定畜禽肉中己烯雌酚的含量,具有线性范围广、检出限低,以及抗干扰能力强的优点。(The invention relates to the technical field of diethylstilbestrol detection, and particularly discloses a nano-material ionic liquid composite modified electrode, a preparation method thereof and application thereof in diethylstilbestrol detection. The preparation method of the nano material ionic liquid composite modified electrode comprises the following steps: (1) mixing graphene oxide, multi-walled carbon nanotubes and [ BMIM ]]PF 6 Dispersing in water to obtain GO-MWCNT S ‑[BMIM]PF 6 A dispersion liquid; (2) mixing GO-MWCNT S ‑[BMIM]PF 6 And coating the dispersion liquid on the surface of the glassy carbon electrode to obtain the nano material ionic liquid composite modified electrode. The nano-material ionic liquid composite modified electrode is used as a working electrode, the content of diethylstilbestrol in livestock and poultry meat is measured under the square wave voltammetry condition, and the nano-material ionic liquid composite modified electrode has the advantages of wide linear range, low detection limit and strong anti-interference capability.)

1. A preparation method of a nano material ionic liquid composite modified electrode is characterized by comprising the following steps:

(1) mixing graphene oxide, multi-walled carbon nanotubes and [ BMIM ]]PF₆Dispersing in water to obtain GO-MWCNT_S-[BMIM]PF₆A dispersion liquid;

(2) mixing GO-MWCNT_S-[BMIM]PF₆And coating the dispersion liquid on the surface of the glassy carbon electrode to obtain the nano material ionic liquid composite modified electrode.

2. The method for preparing the nanomaterial ionic liquid composite modified electrode according to claim 1, wherein in the step (1), graphene oxide, multi-walled carbon nanotubes and [ BMIM ]]PF₆And the dosage ratio of water is 5-7 mg: 3-5 mg: 70-90 muL: 3-5 mL;

most preferably, graphene oxide, multi-walled carbon nanotubes, [ BMIM ] in step (1)]PF₆And the dosage ratio of water is 6mg:4mg:80 muL: 4 mL.

3. The preparation method of the nano-material ionic liquid composite modified electrode according to claim 1,

the glassy carbon electrode in the step (2) is pretreated by the following method: alpha-Al for glassy carbon electrode₂O₃Polishing the powder, cleaning, and then putting the powder into a base solution for cyclic voltammetry scanning;

the base solution is 0.4-0.6 mol/L H₂SO₄A solution; the specific condition of the cyclic voltammetry scanning is that the scanning speed of 50mV/s is within the potential range of-1.0V until the cyclic voltammetry scanning is stable.

4. The preparation method of the nano-material ionic liquid composite modified electrode according to claim 1,

GO-MWCNT in step (2)_S-[BMIM]PF₆The dosage of the dispersion liquid is 4-6 mu L;

most preferably, the GO-MWCNT in step (2)_S-[BMIM]PF₆The amount of the dispersion was 5. mu.L.

5. The nano-material ionic liquid composite modified electrode prepared by the preparation method of any one of claims 1 to 4.

6. The application of the nanomaterial ionic liquid composite modified electrode in diethylstilbestrol determination in claim 5.

7. The application of the nanomaterial ionic liquid composite modified electrode in determining the content of diethylstilbestrol in livestock and poultry meat according to claim 5.

8. The application of claim 7, wherein the nano-material ionic liquid composite modified electrode is used as a working electrode, and the square wave voltammetry is adopted to measure the content of diethylstilbestrol in livestock and poultry meat, and the method specifically comprises the following steps:

(1) preparing a sample solution to be detected;

(2) the nano-material ionic liquid composite modified electrode of claim 5 is used, the response current value of the sample liquid to be detected is measured by adopting a square wave voltammetry method, the concentration of diethylstilbestrol is calculated according to a linear regression equation, and the content of diethylstilbestrol in the livestock and poultry meat is obtained through conversion.

9. The use according to claim 8, wherein the specific test conditions of the square wave voltammetry in step (2) are: and (3) placing the three-electrode electrochemical system in PBS (phosphate buffer solution) containing diethylstilbestrol and having the pH value of 7.0, standing for 300s, and then performing square wave voltammetric scanning under the conditions that the potential is 0.3-0.9V, the potential increment is 12mV, the square wave amplitude is 50mV, and the square wave frequency is 50 Hz.

10. The use according to claim 8,

the linear regression equation is as follows: 3.2951C-0.4889; wherein C is the concentration of diethylstilbestrol, and the unit is mu mol/L; i is the response current value of diethylstilbestrol, and the unit is muA.

Technical Field

The invention relates to the technical field of diethylstilbestrol detection, and in particular relates to a nano-material ionic liquid composite modified electrode, a preparation method thereof and application thereof in diethylstilbestrol detection.

Background

Diethylstilbestrol (DES) is a synthetic non-steroidal estrogen of formula C₁₈H₂₀O₂Once applied to poultry farming as growth promoters; studies have shown that the residue of diethylstilbestrol in livestock and poultry meat can cause serious harm to human health. Therefore, the method has very important significance for measuring the content of the diethylstilbestrol in the livestock and poultry meat.

The existing methods for measuring diethylstilbestrol include liquid chromatography, fluorescence spectrophotometer method, electrophoresis method and sensor method. The sensor method is attracting attention because of its advantages of simplicity, rapidness, low cost, etc. Due to carbon nanomaterials such as Graphene Oxide (GO) and multi-walled carbon nanotubes (MWCNT)_S) Has the advantages of excellent physical properties, good electrocatalytic activity and the like, and is widely used for preparing detection electrodes of electrochemical sensors. However, the stacking phenomenon of the carbon nanomaterial in the electrode made of the single carbon nanomaterial is serious, which is not favorable for the carbon nanomaterial to exert excellent electrochemical performance.

Disclosure of Invention

In order to solve at least one technical problem in the prior art, the invention firstly provides a preparation method of a nano material ionic liquid composite modified electrode.

The technical problem to be solved by the invention is realized by the following technical scheme:

a preparation method of a nano material ionic liquid composite modified electrode comprises the following steps:

(1) mixing graphene oxide, multi-walled carbon nanotubes and [ BMIM ]]PF₆Dispersing in water to obtain GO-MWCNT_S-[BMIM]PF₆A dispersion liquid;

(2) mixing GO-MWCNT_S-[BMIM]PF₆And coating the dispersion liquid on the surface of the glassy carbon electrode to obtain the nano material ionic liquid composite modified electrode.

Research shows that graphene oxide, multi-wall carbon nano-tube and [ BMIM ]]PF₆Co-preparing the raw materials to obtain the nano materialA sub-liquid composite modified electrode; on one hand, stacking conditions between graphene oxide on the electrode and the multi-walled carbon nanotube are obviously reduced, and compatibility and stability of the multi-walled carbon nanotube are improved; meanwhile, the adsorption capacity of the nano-material ionic liquid composite modified electrode on diethylstilbestrol is greatly improved; after the three materials are compounded, a synergistic effect is generated, and the performance superior to that of a single material can be exerted.

Preferably, the preparation method of the nanomaterial ionic liquid composite modified electrode comprises the steps of (1) graphene oxide, multi-walled carbon nanotubes and [ BMIM ]]PF₆And the dosage ratio of the water is 5-7 mg: 3-5 mg: 70-90 mu L: 3-5 mL.

Most preferably, graphene oxide, multi-walled carbon nanotubes, [ BMIM ] in step (1)]PF₆And the dosage ratio of water is 6mg:4mg:80 muL: 4 mL.

Preferably, the glassy carbon electrode described in step (2) is pretreated by the following method: alpha-Al for glassy carbon electrode₂O₃Polishing the powder, cleaning, and then putting the powder into a base solution for cyclic voltammetry scanning;

the base solution is 0.4-0.6 mol/L H₂SO₄A solution; the specific condition of the cyclic voltammetry scanning is that the scanning speed of 50mV/s is within the potential range of-1.0V until the cyclic voltammetry scanning is stable.

Preferably, the GO-MWCNT in step (2)_S-[BMIM]PF₆The amount of the dispersion is 4-6 mu L.

Most preferably, the GO-MWCNT in step (2)_S-[BMIM]PF₆The amount of the dispersion was 5. mu.L.

The invention also provides the nano material ionic liquid composite modified electrode prepared by the preparation method.

The invention also provides an application of the nano-material ionic liquid composite modified electrode in diethylstilbestrol determination.

The invention also provides an application of the nano-material ionic liquid composite modified electrode in determination of the content of diethylstilbestrol in livestock and poultry meat.

Preferably, the method comprises the following steps of taking a nano-material ionic liquid composite modified electrode as a working electrode, and measuring the content of diethylstilbestrol in livestock and poultry meat by adopting a square wave voltammetry method:

(1) preparing a sample solution to be detected;

(2) the nano-material ionic liquid composite modified electrode of claim 5 is used, the response current value of the sample liquid to be detected is measured by adopting a square wave voltammetry method, the concentration of diethylstilbestrol is calculated according to a linear regression equation, and the content of diethylstilbestrol in the livestock and poultry meat is obtained through conversion.

Preferably, the specific test conditions of the square wave voltammetry in step (2) are: placing a three-electrode electrochemical system in a PBS (phosphate buffer solution) solution containing diethylstilbestrol and having a pH value of 7.0, standing for 300s, and then performing square wave voltammetric scanning under the conditions that the potential is 0.3-0.9V, the potential increment is 12mV, the square wave amplitude is 50mV, and the square wave frequency is 50 Hz;

the linear regression equation is as follows: 3.2951C-0.4889; wherein C is the concentration of diethylstilbestrol, and the unit is mu mol/L; i is the response current value of diethylstilbestrol, and the unit is muA.

Further research by the inventor shows that the content of diethylstilbestrol in livestock meat is measured under the square wave voltammetry condition by using the nano-material ionic liquid composite modified electrode as a working electrode, and is 2.0 multiplied by 10^-7～1.0×10^{- 5}Has a good linear relation in the mol/L range (r is 0.991) and a detection limit (S/N is 3) of 6.7 multiplied by 10^-8mol/L。

Meanwhile, the nano material ionic liquid composite modified electrode is used as a working electrode, and K with the concentration of 100 times is used under the square wave volt-ampere condition⁺、Na⁺、Ca²⁺、Mg²⁺、Fe³⁺、Cl^-、NO₃ ^-、SO₄ ^2-Does not interfere with the determination of diethylstilbestrol; glucose at a 50-fold concentration did not interfere with the assay of diethylstilbestrol; in the 10-fold concentration of the structural analogs, estradiol and estriol did not interfere with the measurement of diethylstilbestrol.

And the dienestrol and the hexestrol can generate an oxidation peak near 0.7V, and the peak has certain interference on the electrochemical software to automatically read the oxidation peak current of the diethylstilbestrol at 0.6V. If the sample contains the dienestrol and the hexestrol, an electrochemical software can be used for manually taking a peak function to read the oxidation peak current value of the diethylstilbestrol and calculate the content of the diethylstilbestrol, or the interference of the dienestrol and the hexestrol needs to be eliminated during the pretreatment of the sample so as to obtain the accurate content of the diethylstilbestrol.

Preferably, the preparation method of the sample solution to be tested comprises the following steps:

mincing livestock and poultry meat, adding acetonitrile, and extracting to obtain an extract supernatant; adding n-hexane into the extracted supernatant, standing, taking the lower layer liquid, removing the solvent, and adding an absolute ethyl alcohol solvent to obtain a sample liquid to be detected;

wherein the dosage ratio of the livestock meat to the acetonitrile is 1g: 8-15 mL; the volume ratio of the supernatant to the n-hexane is 1-3: 1.

The inventor further researches show that the preparation method of the sample liquid to be detected can effectively extract and enrich diethylstilbestrol in the livestock and poultry meat, and simultaneously can reduce the interference of dienestrol and hexanestrol on the determination method.

Preferably, the livestock meat is pork, chicken or beef.

Has the advantages that: the invention provides a brand-new nano material ionic liquid composite modified electrode; the electrode is made of graphene oxide, multi-walled carbon nanotubes and [ BMIM ]]PF₆Is prepared by raw materials together; the three materials generate a synergistic effect after being compounded, and can exert better performance than a single material; the diethylstilbestrol adsorbent has stronger adsorption capacity on diethylstilbestrol; the nano-material ionic liquid composite modified electrode is used as a working electrode, the content of diethylstilbestrol in livestock and poultry meat is measured under the square wave voltammetry condition, and the nano-material ionic liquid composite modified electrode has the advantages of wide linear range, low detection limit and strong anti-interference capability.

Drawings

FIG. 1 is an SEM image of various modified electrodes; wherein, FIG. 1A is SEM picture of GO/GCE, FIG. 1B is SEM picture of MWCNTs/GCE, FIG. 1C is SEM picture of GO-MWCNTs/GCE, FIG. 1D is GO-MWCNTs- [ BMIM]PF₆SEM image of/GCE.

FIG. 2 shows a 1X 10 modified electrode pair^-6A graph of electrochemical response of mol/L DES; wherein a, b, c and d represent significant differences.

FIG. 3 is GO-MWCNT_S-[BMIM]PF₆SWV profile of/GCE in DES solutions of different concentrations; wherein the inset is a plot of current response versus concentration for DES, and C is_{Diethylstilbestrol}(a→g)：0，2×10^-7，3×10^-7，5×10^-7，1×10^-6，5×10^-6，1×10^-5mol/L。

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to these examples in any way.

Example 1 preparation of nanomaterial ionic liquid composite modified electrode

(1) Mixing 6mg of graphene oxide, 4mg of multi-walled carbon nanotube and 80 μ L of [ BMIM ]]PF₆Adding into a centrifuge tube filled with 4mL of ultrapure water, vortexing on a vortex mixer for 5min, and ultrasonically dispersing for 2h to obtain GO-MWCNT_S-[BMIM]PF₆A dispersion liquid;

(2) taking 5 mu L of prepared GO-MWCNT_S-[BMIM]PF₆Dropping the dispersion liquid on the surface of a glassy carbon electrode, drying the glassy carbon electrode at room temperature, and leaching the electrode with ultrapure water to obtain the nano-material ionic liquid composite modified electrode (GO-MWCNT)_S-[BMIM]PF₆/GCE)；

The glassy carbon electrode is pretreated by the following method before dripping: taking glassy carbon electrode, using 1.0, 0.3, 0.05 μm alpha-Al in sequence₂O₃Polishing the powder on a special polishing fluff pad, rinsing the powder with ultrapure water, and then respectively adding nitric acid (V)_{Nitric acid}：V_{Water (W)}1:1), absolute ethanol and ultrapure water, each for 1 minute, then at 0.5mol/L H₂SO₄And (3) taking the solution as a base solution, and carrying out cyclic voltammetry scanning on the glassy carbon electrode at a scanning speed of 50mV/s within a potential range of-1.0V until the glassy carbon electrode is stable.

Comparative example 1

Respectively using 6mg of graphene oxide and 4mg of multi-wall carbon nano-particlesGO-MWCNT prepared by taking tube as raw material according to method_S/GCE。

Comparative example 2

MWCNTs were prepared using 4mg of multiwall carbon nanotubes as starting materials by the method described above_S/GCE。

Comparative example 3

GO/GCE is prepared by taking 6mg of graphene oxide as a raw material according to the method.

GO/GCE, MWCNTs/GCE, GO-MWCNTs/GCE and GO-MWCNT prepared in the above examples and comparative examples by SEM_S-[BMIM]PF₆The results of the characterization of the/GCE are shown in FIG. 1. The GO/GCE surface is covered with a number of corrugated structures as a result of pi-pi interactions between graphene oxide sheets; a network structure aggregated by strong van der waals force between adjacent carbon nanotubes can be observed on the MWCNTs/GCE; when GO and MWCNTs are mixed, ultrasonically dispersed and modified on the surface of a glassy carbon electrode, the stacking condition of carbon nanotubes dispersed by graphene oxide sheets on the surface of GO-MWCNTs/GCE is obviously reduced; and GO-MWCNT_S-[BMIM]PF₆The surface of the/GCE is distributed with a uniform reticular structure, and the stacking phenomenon of the mixture system is further reduced.

Example 2 electrochemical Performance testing of nanomaterial Ionic liquid composite modified electrode

The specific conditions of square wave voltammetry are: and (3) placing the three-electrode electrochemical system in a PBS (phosphate buffer solution) solution with the pH value of 7.0 containing DES (data encryption standard), standing for 300s, and then performing square wave voltammetry scanning under the conditions that the potential is 0.3-0.9V, the potential increment is 12mV, the square wave amplitude is 50mV, and the square wave frequency is 50 Hz.

(1) Electrochemical response test

GO-MWCNT prepared by the above examples and comparative examples_S-[BMIM]PF₆/GCE、GO-MWCNT_S/GCE、MWCNT_SThe total of four modified electrodes of/GCE and GO/GCE are examined for different electrode pairs of 1 × 10^-6Electrochemical response of mol/L DES, and significance analysis is carried out. The results are shown in FIG. 2, GO-MWCNT_SThe response current of/GCE to DES is obviously larger than that of GO/GCE and MWCNT_S/GCE(P<0.05), this may be becauseGO can be used as a surfactant to disperse MWCNTs, so that the specific surface area, the conductivity and the DES action sites are increased, and on the other hand, the oxygen-containing functional group contained in GO can form a hydrogen bond with phenolic hydroxyl in DES molecules, so that the adsorption capacity to DES is enhanced. When the composite material contains high-conductivity ionic liquid BMIM]PF₆Prepared GO-MWCNT_S-[BMIM]PF₆The response sensitivity of the/GCE to DES is further improved, and the response current value is obviously larger than that of other three modified electrodes (P)<0.05), further explaining that the composite nano material has synergistic effect, increases the specific surface area of the modified electrode and the active action site on DES, and is beneficial to the DES in the solution to be adsorbed on the surface of the modified electrode to generate oxidation reaction, so GO-MWCNT is selected and utilized_S-[BMIM]PF₆The composite material is used for modifying the glassy carbon electrode.

(2) Reproducibility test

Using the same GO-MWCNT_S-[BMIM]PF₆The concentration of the/GCE pair is 1X 10^-6The DES solution of mol/L is tested, 10 times of continuous scanning is carried out, and the RSD of the response current value is 4.8% (n is 10), which indicates GO-MWCNT_S-[BMIM]PF₆the/GCE has better test repeatability.

6-branched GO-MWCNT were prepared according to the same method as in example 1_S-[BMIM]PF₆(ii)/GCE and concentration of 1X 10 under optimum conditions^-6When the DES solution of mol/L is measured, the RSD of the response current value is 4.4% (n is 6), which indicates GO-MWCNT_S-[BMIM]PF₆the/GCE has good manufacturing reproducibility.

(3) Stability test

3-branched GO-MWCNT were prepared in the same manner as in example 1_S-[BMIM]PF₆(GCE) concentration 1X 10 on day 1^-6The DES solution of mol/L and the peak current value thereof are recorded, and then the DES solution is placed in a refrigerator at 4 ℃ to be protected from light for storage, and the modified electrode is measured once every other day by 1 multiplied by 10^-6mol/L DES solution. GO-MWCNT at day 8_S-[BMIM]PF₆The response current value of the/GCE to DES can reach 96.7% of the initial current, which shows that the modified current can maintain good stability in one week.

(4) Interference testing

Some common ion, compound and structural analogue pairs were examined for 1X 10^-6Interference of mol/L DES assay. The results show that K is 100 times the concentration⁺、Na⁺、Ca²⁺、Mg²⁺、Fe³⁺、Cl^-、NO₃ ^-、SO₄ ^2-Do not interfere with the determination of DES; glucose at 50-fold concentration did not interfere with DES determination; in the 10-fold concentration of structural analogs, estradiol and estriol did not interfere with DES determination, while dienestrol and estriol produced oxidation peaks near 0.7V, which interfered with DES measurement of oxidation peak current at 0.6V. When a sample is measured, whether the two structural analogs are contained in the sample can be judged according to the peak appearance condition of square wave volt-ampere of a sample solution. When the sample does not contain the two substances, the electrochemical method can accurately determine the DES content in the sample; if the sample contains structural analogues, the oxidation peak current value of the DES can be read by utilizing the manual peak-taking function of electrochemical software, and the content of the DES can be calculated, or interfering substances are removed during the pretreatment of the sample to obtain the accurate content of the DES.

(5) Linear range and detection limit

Square Wave Voltammetry (SWV) is adopted to investigate DES solutions with different concentrations in GO-MWCNT_S-[BMIM]PF₆Current response on/GCE. As a result, as shown in FIG. 3, the DES had a response current value (I, μ A) and a concentration (C, μmol/L) of 2.0X 10^-7～1.0×10^-5Has good linear relation in the mol/L range, and the linear equation is that I is 3.2951C-0.4889 (r is 0.991), and the detection limit is 6.7 multiplied by 10^-8mol/L。

Example 3 sample determination

(1) Preparing a sample solution to be detected;

(2) the nano-material ionic liquid composite modified electrode prepared in the embodiment 1 is used, the response current value of the sample liquid to be detected is measured by adopting a square wave voltammetry method, the concentration of diethylstilbestrol is calculated according to a linear regression equation, and the content of diethylstilbestrol in livestock and poultry meat can be obtained through further conversion;

the specific test conditions of the square wave voltammetry in the step (2) are as follows: placing a three-electrode electrochemical system in a PBS (phosphate buffer solution) solution containing diethylstilbestrol and having a pH value of 7.0, standing for 300s, and then performing square wave voltammetric scanning under the conditions that the potential is 0.3-0.9V, the potential increment is 12mV, the square wave amplitude is 50mV, and the square wave frequency is 50 Hz;

the linear regression equation is as follows: 3.2951C-0.4889; wherein C is the concentration of diethylstilbestrol, and the unit is mu mol/L; i is the response current value of diethylstilbestrol, and the unit is muA.

The preparation method of the sample liquid to be detected comprises the following steps: weighing 1.0g of minced chicken, pork or beef sample in a 50mL centrifuge tube, adding 10mL of acetonitrile, vortex, mixing uniformly, extracting for 15min, performing ultrasonic extraction for 10min, centrifuging at 7000r/min for 10min, transferring the supernatant to a second centrifuge tube, adding 10mL of acetonitrile into the residue, repeating the extraction twice, and combining the supernatants. And adding 10mL of normal hexane into the second centrifugal tube, standing for 15min, taking the lower layer liquid, carrying out rotary evaporation at 50 ℃ to dryness, and adding 5mL of absolute ethyl alcohol for dissolving to obtain the sample liquid to be detected.

The results show that the livestock and poultry meat samples do not have the characteristic oxidation peak of DES, which indicates that the tested samples do not contain DES.

Further, 3 DES standard solutions with different concentrations, i.e., low, medium and high, were added to the above samples, and the samples were processed in the same manner and then measured in parallel for 3 times, with the results shown in table 1, where the standard recovery rates of the pork, chicken and beef samples were 85.0% to 90.3%, 84.5% to 90.2%, 85.8% to 88.6%, and RSD were 2.1% to 2.9%, 0.8% to 3.6%, and 0.2% to 3.4%, respectively. In addition, the three livestock meat samples are verified and determined by an HPLC method according to the national standard GB/T5009.108-2003, and the three livestock meat samples are determined to contain no DES and GO-MWCNT_S-[BMIM]PF₆The results of the/GCE assay remained consistent, indicating that GO-MWCNT was used_S-[BMIM]PF₆The method has good accuracy and reliability in the determination of the diethylstilbestrol in the chicken, the pork and the beef by the GCE.

TABLE 1 results of recovery test of meat samples of livestock and poultry