Molecular imprinting sensor for detecting alpha-linolenic acid as well as preparation method and application of molecular imprinting sensor

Abstract

The invention relates to the technical field of analysis and detection, in particular to a molecular imprinting sensor for detecting alpha-linolenic acid as well as a preparation method and application of the molecular imprinting sensor. The molecularly imprinted sensor is prepared by the following steps: growing ZIF-8 on the surface of an MXene material in situ, and adding thionine as a probe to prepare an MXene (at) ZIF-8-Thi composite material; coating the surface of an SPE (Solid Phase Extraction) electrode with the coating; then, o-phenylenediamine and 3, 4-ethylenedioxythiophene are adopted as functional monomers, alpha-linolenic acid is adopted as a template molecule, a molecularly imprinted polymer film is polymerized on the surface of the electrode, and after the template molecule is eluted, the molecularly imprinted sensor is prepared. The molecularly imprinted sensor disclosed by the invention can be used for rapidly and accurately detecting the content of alpha-linolenic acid in samples such as pork and the like, and high-sensitivity and high-selectivity detection of alpha-linolenic acid is realized. The preparation method of the molecularly imprinted sensor is simple, the manufacturing cost is low, and the molecularly imprinted sensor has a wide application prospect in the field of food safety detection.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a molecular imprinting sensor for detecting alpha-linolenic acid, and a preparation method and application thereof. Background Art

[0002] Alpha-linolenic acid (ALA), an unsaturated fatty acid, is essential for key human functions, including improving blood lipid profiles, promoting brain development, reducing the risk of cardiovascular disease, and alleviating inflammation. However, excessive intake of unsaturated fatty acids can also contribute to the development of diseases such as cardiovascular disease, cancer, inflammation, and autoimmune disorders. Therefore, establishing reliable ALA detection methods is crucial.

[0003] To date, the detection of ALA in pork samples has primarily been performed using gas chromatography (GC). Gas chromatography-flame ionization (GC-FID) and near-infrared spectroscopy (NIRS) can also accurately detect ALA. However, complex pretreatment steps and expensive instrumentation hinder their practical application in modern supply chains. In contrast, electrochemical sensors have attracted considerable attention due to their rapid response, ease of operation, and low cost.

[0004] Therefore, developing an electrochemical sensor for detecting α-linolenic acid has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] In order to solve the above technical difficulties, the present invention provides a method for preparing a molecular imprinting sensor for detecting α-linolenic acid, comprising: in situ growing ZIF-8 on the surface of a MXene material to prepare a MXene@ZIF-8 material; dissolving thionine (Thi) and the MXene@ZIF-8 material in a chitosan solution to prepare a MXene@ZIF-8-Thi composite material; coating the MXene@ZIF-8-Thi composite material on the surface of a screen-printed electrode (SPE) to prepare a MXene@ZIF-8-Thi / SPE electrode; using o-phenylenediamine (O-PD) and 3,4-ethylenedioxythiophene (EDOT) as functional monomers and α-linolenic acid as a template molecule to polymerize a molecular imprinting polymer (MIP) on the surface of the MXene@ZIF-8-Thi / SPE electrode, and eluting the template molecule to prepare the molecular imprinting sensor (MIP / MXene@ZIF-8-Thi / SPE).

[0006] Among them, the MXene@ZIF-8 material with a heterogeneous structure is a composite nanomaterial with high conductivity, good dispersibility and large surface area. The SPE electrode modified with it can provide stronger electron transfer ability and higher stability, 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 acid group (-COOH) of ALA can be combined through hydrogen bonds, while the thiophene ring and alkyl chain of EDOT interact with the hydrophobic long chain of ALA through van der Waals forces. These two functional monomers can form multiple non-covalent interactions with the template molecule, thereby improving the selectivity and affinity of the binding.

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

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

[0010] In some embodiments, the step of in situ growing ZIF-8 on the surface of a MXene material includes: dissolving the MXene material and zinc acetate in a methanol solvent to prepare a mixed solution, ultrasonicating the solution, and then adding 2-methylimidazole, stirring the solution, and allowing the solution to stand to obtain a 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 to 24 hours, and then the mixture is allowed to stand for 4 hours 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 a MXene@ZIF-8-Thi / SPE electrode includes dissolving o-phenylenediamine, 3,4-ethylenedioxythiophene, and α-linolenic acid in a PBS solution and performing electropolymerization using 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 electropolymerization conditions include: a voltage of 0.4-1.2 V, a number of electropolymerization cycles of 15-30 cycles, and a scanning speed of 40-60 mV / S.

[0017] In some embodiments, the electrode is freed of surface impurities prior to use.

[0018] Preferably, the method for removing surface impurities comprises: placing the electrode in a 0.01-0.1 M phosphate buffer solution (pH=7.2-7.4) and activating the electrode using a 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 reagent for eluting the template molecule is a mixture of methanol and acetic acid; preferably, the volume ratio is 1:1.

[0021] Furthermore, the present invention provides a molecular imprinting sensor for detecting α-linolenic acid prepared by the preparation method of any of the above embodiments.

[0022] Furthermore, the present invention provides the use of the molecular imprinting 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 sample is in a form including, but not limited to, solid, liquid, or powder.

[0025] Furthermore, the present invention provides a method for detecting the content of α-linolenic acid, comprising: connecting the molecular imprinting 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: potential -0.6~0.1V, potential increment 0.004V, amplitude 0.05V, pulse width 0.05s, pulse period 0.5s, and sampling width 0.02s.

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

[0028] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a molecular imprinting sensor for detecting α-linolenic acid. This sensor can quickly and accurately detect the α-linolenic acid content in samples such as pork, achieving highly sensitive and selective detection of α-linolenic acid. The molecular imprinting sensor of the present invention has a simple preparation method and low production cost, and has broad application prospects in the field of food safety testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the preparation process of the molecular imprinting sensor in Example 1.

[0030] Figure 2 It is the detection performance test curve of different molecular imprinting sensors. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, if the specific technology or conditions are not specified, they shall be carried out in accordance with the technology or conditions described in the literature in this field, or in accordance with the product instructions. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels. The MXene in the following embodiments is Ti3C2T x MXene was purchased from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd.

[0032] Example 1 This embodiment provides a molecular imprinting sensor for detecting α-linolenic acid. The schematic diagram of its preparation process is shown in FIG. Figure 1 As shown, the preparation method is as follows: (1) The SPE electrode was placed in 0.01 M phosphate buffer (pH = 7.2-7.4) and activated using a constant potential method (1.7 V) for 180 s to remove impurities on the electrode surface.

[0033] (2) 0.7g Ti3C2T x (final concentration 10 mg / mL) and 2.2 g zinc acetate dihydrate (C4H 10O6Zn·2H2O) was added into 70 mL of methanol solvent and ultrasonicated for 4 h.

[0034] (3) 3.3 g of 2-methylimidazole (2-MI) was added to the above solution and mixed, then magnetically stirred for 24 h and allowed to stand for 4 h to obtain MXene@ZIF-8 material, which was then centrifuged and washed and dried with methanol.

[0035] (4) Dissolve 10 mg of Thi (10 mg / mL) and 1 mg of MXene@ZIF-8 (1 mg / mL) in 1 mL of 0.2% chitosan solution and mix them. Ultrasonicate until uniformly dispersed to obtain a MXene@ZIF-8-Thi composite material. 5 μL of the mixture was dropped onto the surface of the SPE working electrode and dried to obtain a MXene@ZIF-8-Thi / SPE electrode.

[0036] (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 PBS solution. Cyclic voltammetry (CV) was performed at a scan rate of 50 mV / s in the potential range of 0.4-1.2 V for 15 cycles to polymerize the molecular imprinted polymer film on the surface of the MXene@ZIF-8-Thi / SPE electrode. The molecular imprinted polymer film was then eluted with a methanol / acetic acid mixture (1:1, v / v) for 5 min to obtain a molecular imprinted sensor, which was named MIP / MXene@ZIF-8-Thi / SPE or MIP (O-PD&EDOT) / MXene@ZIF-8-Thi / SPE.

[0037] Example 2 In this example, the detection performance of the molecular imprinting sensor prepared in Example 1 was tested, and the steps were as follows: ALA-phosphate buffer (pH = 7.2-7.4) with a concentration of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM was prepared, and the molecular imprinting sensor of Example 1 was connected to an electrochemical workstation (BIOSYS P20). Differential pulse voltammetry was used for detection (potential -0.6-0.1 V, potential increment 0.004 V, amplitude 0.05 V, pulse width 0.05 s, pulse period 0.5 s, sampling width 0.02 s). 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 of different concentrations were recorded as I. x (x=1,2,3…), by the formula ΔI=I0-I x , respectively calculate ΔI x, thus obtaining a set of relationship curves between the logarithm of ALA concentration and ΔI, such as Figure 2 As shown, the linear detection range is 1 nM-10 mM.

[0038] Example 3 This example tests the accuracy of the molecular imprinting sensor prepared in Example 1. Specifically, this example tests the ALA content in pork samples using the following steps: (1) Take 2.5 g of pork sample, mince it, and place it in 10 mL of ethanol solution and shake it vigorously; (2) Place the above solution in a centrifuge and centrifuge at 6000 rpm for 2 minutes; (3) The supernatant was diluted 10 times with 0.01 M PBS buffer solution and connected to an electrochemical workstation using the molecular imprinting sensor prepared in Example 1, and tested by differential pulse voltammetry.

[0039] At the same time, the same pork sample was tested by gas chromatography (GC) as a control. The GC conditions were as follows: (1) Capillary column: polydicyanopropylsiloxane strong polar stationary phase, column length 100m, inner diameter 0.25mm, film thickness 0.2μm; (2) Injector temperature: 270°C; (3) Detector temperature: 280°C; (4) Programmed heating: initial temperature 100 °C, hold for 13 min; 100 °C~180 °C, heating rate 10 °C / min, hold for 6 min; 180 °C~200 °C, heating rate 1 °C / min, hold for 20 min; 200 °C~230 °C, heating rate 4 °C / min, hold for 10.5 min.

[0040] (5) Carrier gas: nitrogen.

[0041] (6) Split ratio: 100:1.

[0042] (7) Injection volume: 1.0 μL.

[0043] (8) The detection conditions should meet the requirements of theoretical plate number (n) of at least 2000 / m and resolution (R) of at least 1.25.

[0044] The same pork sample was tested six times. The results are shown in Table 1. The results show that the RSD of the test result of Example 1 was 10.94%, and the results were consistent and close to the concentration detected by gas chromatography, indicating that the detection results of the molecular imprinting sensor of Example 1 are accurate and reliable.

[0045] Table 1 Test results of ALA in pork samples

[0046] Comparative Example 1 This comparative example provides a molecular imprinting sensor for detecting α-linolenic acid. The preparation method is different from that of Example 1 only in that EDOT is replaced by an equal amount of pyrrole (PY). The prepared molecular imprinting sensor is named MIP (O-PD&PY) / MXene@ZIF-8-Thi / SPE.

[0047] The detection performance was tested according to the method of Example 2. The results are as follows Figure 2 As shown, the detection range of the molecular imprinting sensor is 10 nM-100 μM, and the detection effect is not as good as that of Example 1.

[0048] Comparative Example 2 This comparative example provides a molecular imprinting sensor for detecting α-linolenic acid. The preparation method is different from that of Example 1 only in that MXene is replaced with an equal amount of graphene oxide (GO). The prepared molecular imprinting sensor is named MIP (O-PD & EDOT) / GO@ZIF-8-Thi / SPE.

[0049] The detection performance was tested according to the method of Example 2. The results are as follows Figure 2 As shown, the detection range of the molecular imprinting sensor is 1 nM-10 μM, and the detection effect is not as good as that of Example 1.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a molecular imprinting sensor for detecting α-linolenic acid, characterized in that: include: In situ growth of ZIF-8 on the surface of MXene material to prepare MXene@ZIF-8 material; Thionine and MXene@ZIF-8 materials were dissolved in chitosan solution to prepare a MXene@ZIF-8-Thi composite material; the MXene@ZIF-8-Thi composite material was coated on the surface of a SPE electrode to prepare a MXene@ZIF-8-Thi / SPE electrode; o-phenylenediamine and 3,4-ethylenedioxythiophene were used as functional monomers, and α-linolenic acid was used as a template molecule to polymerize a molecular imprinting polymer film on the surface of the MXene@ZIF-8-Thi / SPE electrode, and the molecular imprinting sensor was prepared after eluting the template molecules.

2. The preparation method according to claim 1, characterized in that The MXene material is Ti3C2T x Two-dimensional materials.

3. The preparation method according to claim 1, characterized in that The steps of in situ growing ZIF-8 on the surface of MXene material include: dissolving MXene material and zinc acetate in methanol solvent, ultrasonicating the mixture, adding 2-methylimidazole, stirring and letting it stand to obtain MXene@ZIF-8 material.

4. The preparation method according to claim 3, characterized in that In the mixed solution, the concentration of the 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).

5. The preparation method according to claim 3, characterized in that After adding 2-methylimidazole, the mixture was magnetically stirred for 12 to 24 hours and then allowed to stand for 4 hours to obtain the MXene@ZIF-8 material.

6. The preparation method according to claim 1, characterized in that In a 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%.

7. The preparation method according to claim 1, characterized in that The steps of polymerizing a molecular imprinting polymer film on the surface of the MXene@ZIF-8-Thi / SPE electrode include: dissolving o-phenylenediamine, 3,4-ethylenedioxythiophene and α-linolenic acid in a PBS solution, and performing electropolymerization using cyclic voltammetry.

8. A molecular imprinting sensor for detecting α-linolenic acid, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the molecular imprinting sensor according to claim 8 in detecting the content of α-linolenic acid in a sample.

10. A method for detecting the content of α-linolenic acid, characterized in that: include: After the molecular imprinting sensor according to claim 8 is connected to an electrochemical workstation, the α-linolenic acid content in the sample is detected by differential pulse voltammetry.

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