A molecularly imprinted sensor for detecting alpha-linolenic acid and a preparation method and application thereof

Abstract

The application relates to the technical field of analytical detection, in particular to a molecular imprinting sensor for detecting alpha-linolenic acid and a preparation method and application thereof. The molecular imprinting sensor is prepared by in-situ growth of ZIF-8 on the surface of MXene material, addition of thionine as a probe to obtain MXene@ZIF-8-Thi composite material, coating of the composite material on the surface of a SPE electrode, polymerization of a molecular imprinting polymer film on the electrode surface by using o-phenylenediamine and 3,4-ethylenedioxythiophene as functional monomers and alpha-linolenic acid as a template molecule, and preparation of the molecular imprinting sensor after elution of the template molecule. The molecular imprinting sensor can rapidly and accurately detect the content of alpha-linolenic acid in pork and other samples, realizes high-sensitivity and high-selectivity detection of alpha-linolenic acid, and has a wide application prospect in the field of food safety detection due to the simple preparation method and low manufacturing cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analytical detection, and particularly relates to a molecular imprinting sensor for detecting alpha-linolenic acid as well as a preparation method and application thereof. BACKGROUND

[0002] Alpha-linolenic acid (ALA) is an unsaturated fatty acid essential for performing key functions in the human body, which can improve blood lipid conditions, promote brain development, reduce the risk of cardiovascular diseases and alleviate inflammatory reactions. However, excessive intake of unsaturated fatty acids can also cause the development of diseases such as cardiovascular diseases, cancer, inflammation and autoimmune diseases. Therefore, it is crucial to establish a reliable method for detecting ALA.

[0003] So far, the detection of ALA in pork samples is mainly determined by gas chromatography (GC), and in addition, gas chromatography-flame ionization (GC-FID) and near-infrared spectroscopy (NIRS) can also accurately detect ALA. However, the complex pretreatment steps and expensive instrument equipment hinder their practical application in the modern supply chain. In contrast, electrochemical sensors are of great concern due to their rapid response, simple operation and low cost.

[0004] Therefore, it is an urgent technical problem in the art to develop an electrochemical sensor for detecting alpha-linolenic acid. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a preparation method of a molecular imprinting sensor for detecting alpha-linolenic acid, comprising: growing ZIF-8 on the surface of MXene material in situ to obtain MXene@ZIF-8 material; dissolving thi (Thi) and MXene@ZIF-8 material in a chitosan solution to obtain MXene@ZIF-8-Thi composite material; coating the MXene@ZIF-8-Thi composite material on the surface of a screen-printed electrode (SPE) to obtain a MXene@ZIF-8-Thi / SPE electrode; using o-phenylenediamine (O-PD) and 3,4-ethylenedioxythiophene (EDOT) as functional monomers and alpha-linolenic acid as a template molecule, a molecular imprinting polymer film (MIP) is polymerized on the surface of the MXene@ZIF-8-Thi / SPE electrode, and after eluting the template molecule, the molecular imprinting sensor (MIP / MXene@ZIF-8-Thi / SPE) is obtained.

[0006] The MXene@ZIF-8 material with a heterostructure is a composite nanomaterial, which has high conductivity, good dispersibility and large surface area, and the modified SPE electrode can provide stronger electron transfer capacity and higher stability, thereby providing a good foundation for subsequent molecular imprinting.

[0007] O-PD and EDOT are used as bifunctional monomers, in which the amino group (-NH2) of O-PD and the carboxylic group (-COOH) of ALA can be combined by hydrogen bond, and the thiophene ring and alkyl chain of EDOT interact with the hydrophobic long chain of ALA by van der Waals force. The two functional monomers can form multiple non-covalent interactions with the template molecule, thereby improving the selectivity and affinity of the combination.

[0008] In addition, the bifunctional monomers participate in the formation of the cross-linked network, thereby improving the recognition ability and stability of the material, and further improving the selectivity and reliability of the detection.

[0009] Preferably, the MXene material is Ti3C2T x two-dimensional material.

[0010] In some embodiments, the step of growing ZIF-8 in situ on the surface of the MXene material comprises: dissolving the MXene material and zinc acetate in a methanol solvent to form a mixed solution, then ultrasonicating, and then adding 2-methylimidazole, stirring, and standing to obtain the MXene@ZIF-8 material.

[0011] Preferably, in the mixed solution, the concentration of the MXene material is 1-20 mg / mL (preferably 5-15 mg / mL); and / or, the concentration of zinc acetate is 10-80 mg / mL (preferably 20-30 mg / mL); and / or, the mass ratio of 2-methylimidazole to zinc acetate is 1: (1-2.5).

[0012] Preferably, after adding 2-methylimidazole, magnetic stirring is performed for 12-24 h, and then standing for 4 h to obtain the MXene@ZIF-8 material.

[0013] In some embodiments, in the mixed solution of thionine, MXene@ZIF-8 material and chitosan, the concentration of thionine is 5-10 mg / mL; and / or, the concentration of MXene@ZIF-8 material is 0.5-2 mg / mL (preferably 0.8-1.2 mg / mL); and / or, the mass concentration of chitosan is 0.2%-1%.

[0014] In some embodiments, the step of polymerizing a molecularly imprinted polymer film on the surface of the MXene@ZIF-8-Thi / SPE electrode comprises: dissolving o-phenylenediamine, 3,4-ethylenedioxythiophene and alpha-linolenic acid in a PBS solution, and performing electro-polymerization by cyclic voltammetry.

[0015] Preferably, in the mixture of o-phenylenediamine, 3,4-ethylenedioxythiophene, α-linolenic acid and PBS solution, the concentration of o-phenylenediamine is 2-8 mM (preferably 3-5 mM); and / or, the concentration of 3,4-ethylenedioxythiophene is 4-12 mM (preferably 5-7 mM); and / or, the concentration of α-linolenic acid is 1-4 mM (preferably 1-3 mM).

[0016] Preferably, the conditions of the electro-polymerization include: a voltage of 0.4-1.2 V, 15-30 cycles of electro-polymerization, and a scanning speed of 40-60 mV / S.

[0017] In some embodiments, the electrode is removed of surface impurities before use.

[0018] Preferably, the method of removing surface impurities includes: placing the electrode in a 0.01-0.1 M phosphate buffer solution (pH = 7.2-7.4) and activating by constant potential method (preferably 1.7 V).

[0019] Preferably, the activation time is 180 s to 300 s (more preferably 180 s).

[0020] In some embodiments, the elution reagent for the template molecule is a mixture of methanol and acetic acid; preferably in a volume ratio of 1:1.

[0021] Further, the present application provides a molecularly imprinted sensor for detecting α-linolenic acid, which is prepared by the preparation method of any one of the above embodiments.

[0022] Further, the present application provides the use of the molecularly imprinted sensor in detecting the content of α-linolenic acid in a sample.

[0023] In some embodiments, the sample includes but is not limited to plant samples, animal samples (such as pork, etc.), processed products or biomedical samples.

[0024] In some embodiments, the form of the sample includes but is not limited to solid, liquid or powder.

[0025] Further, the present application provides a method for detecting the content of α-linolenic acid, which includes: connecting the molecularly imprinted sensor to an electrochemical workstation and detecting the content of α-linolenic acid in a sample by differential pulse voltammetry.

[0026] Preferably, the detection conditions of the differential pulse voltammetry include: a potential of -0.6-0.1 V, a potential increment of 0.004 V, an amplitude of 0.05 V, a pulse width of 0.05 s, a pulse period of 0.5 s, and a sampling width of 0.02 s.

[0027] In the specific implementation process, the pretreatment method of the meat sample includes: stirring the meat sample and placing it in an ethanol solution for vigorous shaking, and then centrifuging to obtain a supernatant, and diluting the supernatant with a PBS buffer solution (preferably 0.01M) and then (preferably 10 times) for detection.

[0028] Compared with the prior art, the present application has the beneficial effects that:

[0029] The present application provides a molecular imprinting sensor for detecting alpha-linolenic acid, which can quickly and accurately detect the content of alpha-linolenic acid in pork and other samples, realizing high sensitivity and high selectivity detection of alpha-linolenic acid. The molecular imprinting sensor of the present application has simple preparation method and low manufacturing cost, and has broad application prospect in the field of food safety detection. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a preparation flowchart of the molecular imprinting sensor of Example 1.

[0031] Figure 2 is a detection performance test curve of different molecular imprinting sensors. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application. In the embodiments provided in the present specification, the specific technology or conditions are not specified, which are according to the technology or conditions described in the literature in the art, or according to the product manual. The reagents or instruments used are not specified by the manufacturer, which are conventional products that can be purchased through regular channels. The MXene in the following examples is Ti3C2T x , which is purchased from Jiangsu Xianfeng Nanometer Material Technology Co., Ltd.

[0033] Example 1

[0034] The present embodiment provides a molecular imprinting sensor for detecting alpha-linolenic acid, and the preparation flowchart is as shown in Figure 1 , and the preparation method is as follows:

[0035] (1) The SPE electrode is placed in 0.01M phosphate buffer (pH=7.2~7.4), and activated by constant potential method (1.7V) for 180s to remove impurities on the surface of the electrode.

[0036] (2) 0.7g Ti3C2T x(10 mg / mL) and 2.2 g zinc acetate dihydrate (C4H 10 O6Zn·2H2O) were added to 70 mL of methanol solvent and ultrasonically treated for 4 h.

[0037] (3) 3.3 g of 2-methylimidazole (2-MI) was added to the above solution mixture, magnetically stirred for 24 h, and left to stand for 4 h to obtain a MXene@ZIF-8 material, which was centrifuged and dried with methanol.

[0038] (4) 10 mg of Thi (10 mg / mL) and 1 mg of MXene@ZIF-8 (1 mg / mL) were dissolved in 1 mL of a 0.2% chitosan solution to mix, and after ultrasonic treatment to uniformly disperse, a MXene@ZIF-8-Thi composite material was obtained. 5 μL was dropped on the surface of the SPE working electrode and dried to obtain a MXene@ZIF-8-Thi / SPE electrode.

[0039] (5) 0.433 mg of O-PD (4 mM), 0.853 mg of EDOT (6 mM), and 0.557 mg of ALA (2 mM) were dissolved in 1 mL of a PBS solution, and a molecularly imprinted polymer film was polymerized on the surface of the MXene@ZIF-8-Thi / SPE electrode by cyclic voltammetry (CV) at a scan rate of 50 mV / s in a potential range of 0.4-1.2 V for 15 cycles, and then eluted with a methanol / acetic acid mixture (1:1, v / v) for 5 min to obtain a molecularly imprinted sensor, which was named MIP / MXene@ZIF-8-Thi / SPE or MIP (O-PD&EDOT) / MXene@ZIF-8-Thi / SPE.

[0040] Example 2

[0041] The detection performance of the molecularly imprinted sensor prepared in Example 1 was tested, and the steps were as follows:

[0042] A series of ALA-phosphate buffer solutions (pH = 7.2-7.4) with concentrations of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM were prepared, respectively. The molecularly imprinted sensor of Example 1 was connected to an electrochemical workstation (BIOSYS P20), and the differential pulse voltammetry detection was performed (potential-0.6~0.1V, potential increment 0.004V, amplitude 0.05V, pulse width 0.05s, pulse period 0.5s, sampling width 0.02s). As the concentration of the ALA solution increased, the oxidation peak of Thi gradually decreased. The peak current of the oxidation peak obtained in the blank solution was taken as I0, and the peak currents obtained from the standard ALA solutions with different concentrations were taken as I x(x = 1, 2, 3…), the formula ΔI = I0- I x , respectively, ΔI x is calculated, and a set of ALA concentration logarithm and ΔI relationship curve is obtained, as shown in Figure 2 , the linear detection range is 1 nM-10 mM.

[0043] Example 3

[0044] This embodiment detects the accuracy of the molecularly imprinted sensor prepared in Example 1. Specifically, the ALA content in a pork sample is detected, and the steps are as follows:

[0045] (1) Take 2.5 g of pork sample and stir it into 10 mL of ethanol solution and shake vigorously;

[0046] (2) Place the above solution in a centrifuge and centrifuge at 6000 r / min for 2 min;

[0047] (3) Take the supernatant, dilute it 10 times with 0.01 M PBS buffer solution, and then connect it to the electrochemical workstation using the molecularly imprinted sensor prepared in Example 1, and test it by differential pulse voltammetry.

[0048] At the same time, the same pork sample is tested by gas chromatography (GC) as a control, and the conditions of gas chromatography are as follows:

[0049] (1) Capillary chromatographic column: polydicyclopentylsiloxane strong polar stationary phase, column length 100 m, inner diameter 0.25 mm, film thickness 0.2 μm;

[0050] (2) Injector temperature: 270℃;

[0051] (3) Detector temperature: 280℃;

[0052] (4) Programmed temperature: initial temperature 100℃, duration 13 min; 100℃-180℃, temperature rise rate 10℃ / min, hold for 6 min; 180℃-200℃, temperature rise rate 1℃ / min, hold for 20 min; 200℃-230℃, temperature rise rate 4℃ / min, hold for 10.5 min.

[0053] (5) Carrier gas: nitrogen.

[0054] (6) Split ratio: 100:1.

[0055] (7) Injection volume: 1.0 μL.

[0056] (8) The detection conditions should meet the requirements of theoretical plate number (n) at least 2000 / m, and separation degree (R) at least 1.25.

[0057] The same pork sample was tested 6 times, and the results are shown in Table 1. The results show that the RSD of the test results in Example 1 is 10.94%, the results are consistent and close to the concentration detected by gas chromatography, indicating that the detection results of the molecular imprinted sensor in Example 1 are accurate and reliable.

[0058] Table 1. Test results of ALA in pork samples

[0059]

[0060] Comparative Example 1

[0061] This comparative example provides a molecularly imprinted sensor for detecting α-linolenic acid. The preparation method differs from Example 1 only in that EDOT is replaced with an equal amount of pyrrole (PY). The resulting molecularly imprinted sensor is named MIP(O-PD&PY) / MXene@ZIF-8-Thi / SPE.

[0062] The detection performance was tested according to the method in Example 2, and the results are as follows: Figure 2 As shown, the detection range of this molecular imprinting sensor is 10nM-100μM, and its detection performance is not as good as that of Example 1.

[0063] Comparative Example 2

[0064] This comparative example provides a molecularly imprinted sensor for detecting α-linolenic acid. The preparation method differs from Example 1 only in that MXene is replaced with an equal amount of graphene oxide (GO). The resulting molecularly imprinted sensor is named MIP(O-PD&EDOT) / GO@ZIF-8-Thi / SPE.

[0065] The detection performance was tested according to the method in Example 2, and the results are as follows: Figure 2 As shown, the detection range of this molecular imprinting sensor is 1nM-10μM, and its detection performance is not as good as that of Example 1.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a molecularly imprinted sensor for detecting α-linolenic acid, characterized by, Comprising: In-situ growth of ZIF-8 on the surface of MXene material to obtain MXene@ZIF-8 material; The thiophene is dissolved in a chitosan solution with the MXene@ZIF-8 material to obtain a MXene@ZIF-8-Thi composite material; the MXene@ZIF-8-Thi composite material is coated on the surface of an SPE electrode to obtain a MXene@ZIF-8-Thi / SPE electrode; o-phenylenediamine and 3,4-ethylenedioxythiophene are used as functional monomers, and alpha-linolenic acid is used as a template molecule; a molecularly imprinted polymer film is polymerized on the surface of the MXene@ZIF-8-Thi / SPE electrode; and after elution of the template molecule, the molecularly imprinted sensor is obtained; and the MXene material is Ti3C2T x two-dimensional material.

2. The production method according to claim 1, characterized by, The step of in-situ growth of ZIF-8 on the surface of MXene material comprises: dissolving MXene material and zinc acetate in methanol solvent to obtain a mixed solution, then ultrasonicating, then adding 2-methylimidazole, stirring and standing to obtain MXene@ZIF-8 material.

3. The production method according to claim 2, characterized by, In the mixed solution, the concentration of MXene material is 1-20 mg / mL; and / or, the concentration of zinc acetate is 10-80 mg / mL; and / or, the mass ratio of 2-methylimidazole to zinc acetate is 1: (1-2.5).

4. The production method according to claim 2, characterized by, After adding 2-methylimidazole, magnetic stirring for 12-24 h, then standing for 4 h to obtain MXene@ZIF-8 material.

5. The preparation method according to claim 1, characterized in that, In the mixed solution of thionine, MXene@ZIF-8 material and chitosan, the concentration of thionine is 5-10 mg / mL; and / or, the concentration of MXene@ZIF-8 material is 0.5-2 mg / mL; and / or, the mass concentration of chitosan is 0.2%-1%.

6. The method of claim 1, wherein, The step of polymerizing molecularly imprinted polymer film on the surface of MXene@ZIF-8-Thi / SPE electrode comprises: dissolving o-phenylenediamine, 3,4-ethylenedioxythiophene and alpha-linolenic acid in PBS solution, and then performing electro-polymerization by cyclic voltammetry.

7. A molecularly imprinted sensor for detecting alpha-linolenic acid, characterized by, It is prepared by the preparation method of any one of claims 1-6.

8. Use of the molecularly imprinted sensor of claim 7 in detecting the content of alpha-linolenic acid in a sample.

9. A method for detecting the content of α-linolenic acid, characterized by, Comprising: After connecting the molecularly imprinted sensor of claim 7 to an electrochemical workstation, the content of alpha-linolenic acid in a sample is detected by differential pulse voltammetry.

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