Histamine molecularly imprinted electrochemical sensor based on carbon-based nano composite material as well as preparation method and application of histamine molecularly imprinted electrochemical sensor

Abstract

The invention belongs to the technical field of food safety detection, and particularly relates to a histamine molecular imprinting electrochemical sensor based on a carbon-based nano composite material and a preparation method and application of the histamine molecular imprinting electrochemical sensor. Aiming at the problems of poor selectivity, high detection limit or complicated operation and the like of the existing histamine detection technology, a Co3O4 / N-HCS (cobaltosic oxide / nitrogen-doped hollow carbon sphere) composite material is prepared by adopting a solvothermal method, o-phenylenediamine (o-PD) is taken as a functional monomer, a molecularly imprinted polymer membrane (MIP) is constructed on the surface of a glassy carbon electrode (GCE) by adopting an electropolymerization method, and the histamine detection electrode is prepared. The MIP / Co3O4 / N-HCS / GCE sensor with specific recognition sites on histamine is formed. The sensor has high selectivity, high sensitivity and good stability, is simple and convenient to operate and rapid in detection, can efficiently realize accurate detection of histamine in food, and is suitable for safety monitoring scenes of various foods such as fermented food, aquatic products and dairy products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety detection, and particularly relates to a histamine molecular imprinting electrochemical sensor based on a carbon-based nanocomposite material and a preparation method and application thereof. BACKGROUND

[0002] Histamine (HIS) is a typical biological amine, which is widely present in aquatic products, fermented foods and dairy products and is generated by the decarboxylation of histidine by microorganisms. Excessive intake of histamine can cause capillary dilation, hypotension, skin flushing and other acute poisoning symptoms, and long-term intake can also cause chronic diseases such as Alzheimer's disease and asthma, which poses a serious threat to human health. According to the reports of the European Food Safety Authority (EFSA) and the Centers for Disease Control and Prevention (CDC) of the United States, histamine poisoning is one of the common foodborne disease inducers worldwide, so it is of great importance to develop efficient and sensitive histamine detection technology to ensure food safety. At present, the detection methods of histamine mainly include chromatography, spectroscopy, immunoassay and traditional electrochemical sensing technology. Among them, high performance liquid chromatography (HPLC) and gas chromatography (GC) are the "gold standard" for detection, but they require derivatization pretreatment, are complicated to operate and depend on expensive equipment and professional personnel, and are difficult to achieve on-site rapid detection; fluorescence and spectrophotometry are easily interfered by food matrix and have poor selectivity; enzyme-linked immunosorbent assay (ELISA) can be used for rapid screening, but the reagent kit is costly, the enzyme storage conditions are harsh, and the results are semi-quantitative; traditional electrochemical sensors have the advantages of miniaturization and fast response, but their selectivity depends on the specificity of enzymes and are easily interfered by reducing substances, which is difficult to meet the precise detection requirements of histamine in complex food matrix. Therefore, the technical scheme of the present application is proposed. SUMMARY

[0003] In view of the problems of poor selectivity, high detection limit, complicated operation or high cost of the existing detection technology, the present application provides a histamine molecular imprinting electrochemical sensor based on a carbon-based nanocomposite material and a preparation method and application thereof, which can realize high selectivity, high sensitivity and rapid detection of histamine in food, and has simple preparation process, controllable cost and is suitable for on-site detection.

[0004] The present application provides a preparation method of a histamine molecular imprinting electrochemical sensor based on a carbon-based nanocomposite material, comprising the following steps: (I) Preparation of nitrogen-doped hollow carbon spheres: (I-1) Mix deionized water, isopropanol and ammonia water, and dropwise add tetraethyl orthosilicate for stirring to generate silica seeds; continue to dropwise add tetraethyl orthosilicate for reaction, and then centrifuge, wash and dry in sequence to obtain silica powder; (I-2) dispersing the silica powder in deionized water, and then adding hexadecyl trimethyl ammonium bromide, resorcinol, ethanol, ammonia, formaldehyde in sequence, centrifuging after reaction to obtain silica-coated resorcinol-formaldehyde resin; (I-3) calcining the silica-coated resorcinol-formaldehyde resin in an inert atmosphere, and etching with hydrofluoric acid to obtain nitrogen-doped hollow carbon spheres; (II) preparation of three cobalt tetraoxide / nitrogen-doped hollow carbon spheres: (II-1) dispersing the nitrogen-doped hollow carbon spheres in ethanol, and then adding cobalt acetate and ammonia in sequence for reaction; (II-2) centrifuging, washing and drying in sequence after reaction to obtain three cobalt tetraoxide / nitrogen-doped hollow carbon spheres; (III) preparation of a histamine molecularly imprinted electrochemical sensor (III-1) polishing, cleaning, activating and testing a glassy carbon electrode in sequence to obtain a treated glassy carbon electrode; (III-2) dispersing the three cobalt tetraoxide / nitrogen-doped hollow carbon spheres in deionized water, and coating on the surface of the treated glassy carbon electrode to obtain a coated electrode; (III-3) immersing the coated electrode in a buffer solution containing histamine and o-phenylenediamine for electro-polymerization, and finally washing to obtain a histamine molecularly imprinted electrochemical sensor.

[0005] Preferably, in step (I-1): dropping tetraethyl orthosilicate and stirring at 35-40℃ for 30-40min to generate silica seeds; and / or, continuously dropping tetraethyl orthosilicate and reacting at 35-40℃ for 2-3h; and / or, the drying temperature is 60-70℃.

[0006] Preferably, in step (I-3): the inert atmosphere is nitrogen atmosphere; and / or, the calcination is performed by heating at 2-3℃ / min to 800-850℃, and constant temperature calcination for 2-3h.

[0007] Preferably, in step (II-1), the reaction temperature is 150-160℃, and the reaction time is 3-4h.

[0008] Preferably, in step (III-1), the polishing is performed by polishing in sequence with 1.0μm, 0.3μm and 0.05μm alumina polishing powder.

[0009] Preferably, in step (III-3), the electro-polymerization is performed at a voltage range of -0.1-0.9V and a scan rate of 50mV / s for 10 cycles.

[0010] Based on the same technical concept, the application further provides a histamine molecular imprinting electrochemical sensor based on carbon-based nanocomposite prepared by the above preparation method. The sensor is of MIP / Co3O4 / N-HCS / GCE structure, which comprises a glassy carbon electrode (GCE), a cobalt trioxide / nitrogen-doped hollow carbon sphere (Co3O4 / N-HCS) composite layer coated on the surface of the glassy carbon electrode, and a molecular imprinting polymer (MIP) film electro-polymerized on the surface of the composite layer.

[0011] Based on the same technical concept, the application further provides an application of the histamine molecular imprinting electrochemical sensor based on carbon-based nanocomposite in food detection. The food is aquatic products, fermented foods, dairy products, etc. The detection adopts a three-electrode system, the histamine molecular imprinting electrochemical sensor based on carbon-based nanocomposite is used as a working electrode, Ag / AgCl is used as a reference electrode, and platinum wire is used as an auxiliary electrode, and the detection method is differential pulse voltammetry.

[0012] Preferably, the detection condition of the differential pulse voltammetry is that 5.0mM of [Fe(CN)6] 3- / 4- is used as a redox probe, the potential range is -0.1-0.6V, the pulse amplitude is 0.05V, the pulse width is 0.05s, the sampling width is 0.0167s, the pulse interval is 0.2s, and the standing time is 2s.

[0013] Preferably, before the food sample is detected, the food sample needs to be pretreated: liquid food is filtered through a 0.22μm microporous filter membrane, mixed with a phosphate buffer solution with pH=6.5 at a volume ratio of 1:1, solid food is first homogenized and broken, extracted with a phosphate buffer solution with pH=6.5, centrifuged to obtain supernatant, filtered through a 0.22μm microporous filter membrane, and then detected. And / or, the linear range of detection is 0.1-100μM, the detection limit is 41.0nM (the calculation method is 3σ / S, wherein σ is the signal standard deviation of 10 blank experiments, and S is the slope of the calibration curve), the sample detection recovery rate is 107.0%-116.6%, and the relative standard deviation is less than 5%.

[0014] The application has the following beneficial effects: 1. The Co3O4 / N-HCS composite material is prepared by a solvothermal method, the high specific surface area and high conductivity of N-HCS can inhibit the agglomeration of Co3O4, improve the electron transmission efficiency, the catalytic activity of Co3O4 further enhances the response signal of the sensor, and the synergistic effect of the two significantly improves the sensitivity.

[0015] 2. The molecularly imprinted polymer film is constructed by electropolymerization with o-phenylenediamine as a functional monomer, the film thickness and uniformity can be accurately controlled, the specific recognition sites formed have high selectivity for histamine, and the influence of coexisting interferents can be effectively excluded.

[0016] 3. The sensor has simple preparation process, does not need complex equipment, is convenient to operate, rapid in detection (single detection time < 30 min), and has lower cost than ELISA kit and chromatographic detection equipment, and is suitable for on-site rapid detection.

[0017] 4. The sensor has wide linear range (0.1-100 μM) and low detection limit (41.0 nM), can cover the key concentration range of possible excessive histamine in food, meets the detection requirements of samples with different pollution degrees, and has excellent recovery rate and stability of actual samples and strong practicability. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0019] Figure 1 is the SEM diagram of (A) Co3O4, (B) Co3O4 / N-HCS, and (C) MIP / Co3O4 / N-HCS.

[0020] Figure 2 is the EDS spectrum diagram of Co3O4 / N-HCS.

[0021] Figure 3 is the TEM diagram of (A) and (B) Co3O4 / N-HCS.

[0022] Figure 4 is the XRD diagram of N-HCS, Co3O4, and Co3O4 / N-HCS.

[0023] Figure 5 is the DPV current difference Delta is the quantitative relationship diagram of I and HIS concentration. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0025] Embodiments

[0026] The present embodiment provides a preparation method of a histamine molecule imprinting electrochemical sensor based on carbon-based nanocomposite material, comprising the following steps: (I) Preparation of Co3O4 / N-HCS composite material (I-1) Synthesis of N-HCS: 23.5 mL of deionized water, 63.3 mL of isopropyl alcohol and 13 mL of 25% ammonia water are mixed, 0.6 mL of tetraethyl orthosilicate (TEOS) is added dropwise under 35°C water bath, and stirring is carried out for 30 min to generate SiO2 seed crystal; 5 mL of TEOS is continuously added dropwise, and reaction is carried out at 35°C for 2 h; centrifugation, washing and drying at 60°C are carried out to obtain SiO2 powder; 0.8 g of SiO2 powder is ultrasonically dispersed in 70 mL of deionized water, 2.3 g of cetyltrimethylammonium bromide (CTAB), 0.35 g of resorcinol, 28 mL of ethanol and 0.1 mL of ammonia water are added, 0.5 mL of formaldehyde is added dropwise after stirring at 35°C for 30 min, stirring is carried out for 6 h, and centrifugation is carried out after standing overnight to obtain SiO2@RF (SiO2 wrapped resorcinol-formaldehyde resin), and drying is carried out at 60°C; SiO2@RF is heated to 800°C at a rate of 2°C / min in a nitrogen atmosphere, and constant temperature calcination is carried out for 2 h to obtain SiO2@C; SiO2 template is removed by etching with 10% hydrofluoric acid (HF), and washing and drying are carried out to obtain N-HCS.

[0027] (I-2) Synthesis of Co3O4 / N-HCS: 40 mg of N-HCS is ultrasonically dispersed in 25 mL of ethanol, 0.5 g of cobalt acetate (Co(Ac)2·4H2O) is added, and ultrasonic is carried out for 20 min; 2.5 mL of ammonia water is added dropwise, and stirring is carried out for 1 h; the mixed solution is transferred to a 40 mL polytetrafluoroethylene high-pressure reaction kettle, reaction is carried out at 150°C for 3 h; centrifugation is carried out after natural cooling, ethanol and deionized water are alternately washed for 3 times, and drying is carried out at 60°C for 6 h to obtain Co3O4 / N-HCS composite material.

[0028] (II) Preparation of MIP / Co3O4 / N-HCS / GCE sensor (II-1) Glassy carbon electrode (GCE) pretreatment: GCE was polished with 1.0 μm, 0.3 μm, 0.05 μm alumina polishing powder in turn, and ultrasonic cleaned with distilled water and anhydrous ethanol alternately; the electrode was placed in 0.5 M H2SO4, and activated by cyclic voltammetry (CV); then the electrode was placed in 5.0 mM [Fe(CN)6] 3- / 4- solution (containing 0.1 M KCl) for CV test, and when the redox peak potential difference was ≤ 80 mV, the electrode pretreatment was completed.

[0029] (II-2) Preparation of modified electrode: Co3O4 / N-HCS was ultrasonically dispersed in deionized water to prepare a 2 mg / mL dispersion; 6 μL of the dispersion was dropped on the surface of the pretreated GCE and naturally dried to obtain Co3O4 / N-HCS / GCE; the electrode was immersed in a pH 6.5 phosphate buffer (PBS) containing 3 mM histamine (HIS) and 9 mM o-phenylenediamine (o-PD), and 10 cycles of CV electropolymerization were performed at a voltage range of -0.1-0.9 V and a scan rate of 50 mV / s; after polymerization, the electrode surface was washed with deionized water, and then placed in methanol for 15 min to remove the template molecule HIS to obtain the MIP / Co3O4 / N-HCS / GCE sensor.

[0030] Detection example (I) Test conditions: A three-electrode system (MIP / Co3O4 / N-HCS / GCE as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the auxiliary electrode) was used for testing on a CHI660E electrochemical workstation. Cyclic voltammetry (CV): potential range -0.1-0.6 V, scan rate 50 mV / s, and static time 2 s; electrochemical impedance spectroscopy (EIS): frequency 0.1 Hz-100 kHz, amplitude 0.005 V, and direct current potential as open circuit voltage. Differential pulse voltammetry (DPV): 5.0 mM [Fe(CN)6] 3- / 4- (containing 0.1 M KCl) as the probe, potential range -0.1-0.6 V, pulse amplitude 0.05 V, pulse width 0.05 s, sampling width 0.0167 s, pulse interval 0.2 s, and static time 2 s.

[0031] (II) Performance results: 2.1 Linear range and detection limit: Under the optimized conditions, the linear range of the sensor for detecting histamine was 0.1-100 μM, and the linear regression equation was ΔI (μA) = 3.719 log[C] + 30.915 (R 2=0.991); Based on 10 blank experiments, the detection limit was as low as 41.0 nM (3σ / S, where σ is the standard deviation of the blank signal and S is the slope of the calibration curve).

[0032] 2.2 Selectivity and anti-interference: Anti-interference experiments showed that the sensor's recognition signal for histamine was significantly higher than that for coexisting interfering substances such as histidine, phenylalanine, dopamine, tyramine, and uric acid, with a current response difference of ≥3.5 times, indicating its high selectivity.

[0033] 2.3 Stability and reproducibility: The relative standard deviation (RSD) of 2.78% (<5%) for the detection of 0.5 μM histamine by 6 parallel-prepared sensors was 2.78% (<5%). After 6 consecutive days of testing, the response signal retention rate reached 96.2% and the daily average decay rate was only 0.63%, demonstrating excellent stability.

[0034] 2.4 Actual Sample Testing: Spiking recovery experiments were conducted on commercially available beer samples. At spiked concentrations of 1 μM and 5 μM, the recoveries were 116.6% and 107.0%, respectively, with RSDs of 3.17% and 2.22% (both <5%), verifying its reliability in practical applications.

[0035] Sample pretreatment: Take commercially available beer samples, filter them through a 0.22μm microporous membrane, and mix them with PBS buffer at pH 6.5 at a 1:1 volume ratio to obtain the sample to be tested.

[0036] (III) Characterization Results Depend on Figure 1 It can be seen that the scanning electron microscope image of pure Co3O4 shows a uniform spherical nanoparticle structure with a narrow particle size distribution. Figure 1 A), the Co3O4 / N-HCS composite material exhibits good dispersibility, with Co3O4 nanoparticles uniformly distributed on the N-HCS surface (A). Figure 1 B). This structure effectively inhibits the aggregation of Co3O4, improves the stability of the material, and increases the contact area between the material and the target molecule (HIS), providing more active sites for HIS adsorption. After electropolymerization modification, the surface roughness of MIP / Co3O4 / N-HCS is significantly increased ( Figure 1 (C) This morphological change indicates that molecularly imprinted polymers (MIPs) have been successfully introduced into the surface of the composite material.

[0037] Depend on Figure 2The presence of C, N, Co, and O peaks in the energy-dispersive X-ray spectrum of Co3O4 / N-HCS confirms the presence of N-HCS and Co3O4 in the sample. Key characteristics are as follows: the C peak (0 keV) indicates the presence of the N-HCS support; the O peak (0.5 keV) and Co peak (6-8 keV) together verify the formation of Co3O4; and the N peak (0.4 keV) confirms nitrogen doping. The absence of other impurity peaks in the spectrum indicates material purity and uniform elemental distribution, confirming the successful synthesis of Co3O4 / N-HCS.

[0038] Depend on Figure 3 It can be seen that the transmission electron microscope images of the Co3O4 / N-HCS nanomaterials show that N-HCS has a hollow structure, and the Co3O4 is about 16 nm thick and uniformly distributed on the surface of N-HCS. Figure 3 A) This is beneficial for increasing the specific surface area of ​​the material, increasing the number of active sites, and promoting the adsorption of HIS on the material surface. The high-resolution image shows that the lattice spacing of Co3O4 is 0.202 nm, corresponding to the (400) crystal plane of Co3O4, further proving that the composite material contains Co3O4.

[0039] Depend on Figure 4 X-ray diffraction analysis of N-HCS showed a broad diffraction peak at 2θ = 23.41°, indicating that N-HCS contained an amorphous carbon phase after high-temperature heat treatment. The diffraction peaks of Co3O4 at 2θ = 19.00°, 32.27°, 36.85°, 44.81°, 55.66°, 59.36°, 59.36°, 65.24°, and 77.38° correspond to the (111), (220), (311), (400), (422), (511), (440), and (533) crystal planes of the spinel structure Co3O4, which is consistent with the Co3O4 standard card (JCPDS: 42-1467). The simultaneous detection of typical characteristic peaks of Co3O4 and N-HCS in the spectrum of the Co3O4 / N-HCS composite material further confirms the successful composite of Co3O4 and N-HCS nanomaterials.

[0040] Depend on Figure 5 It can be seen that the current difference Δ I It showed a good linear correlation with HIS concentration in the range of 0.1–100 μM, and its linear regression equation was Δ. I (μA) = 3.719 log[ C ] + 30.915 (R 2 =0.991).

[0041] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of the changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a carbon-based nanocomposite-based histamine molecularly imprinted electrochemical sensor, characterized by, The method comprises the following steps: (I) preparation of nitrogen-doped hollow carbon spheres: (I-1) mixing deionized water, isopropyl alcohol and ammonia water, and adding tetraethyl orthosilicate dropwise for stirring to generate silica seeds by reaction; continuing to add tetraethyl orthosilicate dropwise for reaction, and then sequentially centrifuging, washing and drying to obtain silica powder; (I-2) dispersing the silica powder in deionized water, and then sequentially adding cetyltrimethylammonium bromide, resorcinol, ethanol, ammonia water and formaldehyde, and centrifuging after reaction to obtain silica-coated resorcinol-formaldehyde resin; (I-3) calcining the silica-coated resorcinol-formaldehyde resin in an inert atmosphere, and etching with hydrofluoric acid to obtain nitrogen-doped hollow carbon spheres; (II) preparation of cobalt tetraoxide / nitrogen-doped hollow carbon spheres: (II-1) dispersing the nitrogen-doped hollow carbon spheres in ethanol, and then sequentially adding cobalt acetate and ammonia water for reaction; (II-2) sequentially centrifuging, washing and drying after reaction to obtain cobalt tetraoxide / nitrogen-doped hollow carbon spheres; (III) preparation of a histamine molecularly imprinted electrochemical sensor (III-1) polishing, cleaning, activating and testing a glassy carbon electrode in sequence to obtain a treated glassy carbon electrode; (III-2) dispersing the cobalt tetraoxide / nitrogen-doped hollow carbon spheres in deionized water, and coating on the surface of the treated glassy carbon electrode to obtain a coated electrode; (III-3) immersing the coated electrode in a buffer solution containing histamine and o-phenylenediamine for electropolymerization, and finally cleaning to obtain a histamine molecularly imprinted electrochemical sensor.

2. The method for preparing a histamine molecularly imprinted electrochemical sensor based on carbon-based nanocomposite according to claim 1, characterized in that, In step (I-1): the tetraethyl orthosilicate is added dropwise for stirring at 35-40°C for 30-40 min to generate silica seeds by reaction; and / or, the tetraethyl orthosilicate is continuously added dropwise for reaction at 35-40°C for 2-3 h; and / or, the drying temperature is 60-70°C.

3. The method for preparing a histamine molecularly imprinted electrochemical sensor based on carbon-based nanocomposite according to claim 1, characterized in that, In step (I-3): the inert atmosphere is a nitrogen atmosphere; and / or, the calcination is performed by heating at 2-3°C / min to 800-850°C, and then constant temperature calcination for 2-3 h.

4. The method for preparing a histamine molecularly imprinted electrochemical sensor based on carbon-based nanocomposite according to claim 1, characterized in that, In step (II-1), the reaction temperature is 150-160°C, and the reaction time is 3-4 h. 5.The method for preparing a histamine molecularly imprinted electrochemical sensor based on carbon-based nanocomposites according to claim 1, characterized in that, In step (III-1), the polishing is performed by sequentially polishing with 1.0 μm, 0.3 μm and 0.05 μm alumina polishing powder.

6. The method for preparing a histamine molecularly imprinted electrochemical sensor based on carbon-based nanocomposite according to claim 1, wherein, In step (III-3), the electropolymerization is performed by scanning at a rate of 50 mV / s for 10 cycles within a voltage range of -0.1-0.9 V.

7. The histamine molecularly imprinted electrochemical sensor based on carbon-based nanocomposites obtained by the preparation method of any one of claims 1-6.

8. Use of the carbon-based nanocomposite-based histamine molecularly imprinted electrochemical sensor according to claim 7 for food detection, characterized in that, The detection is performed by using a three-electrode system, taking the histamine molecularly imprinted electrochemical sensor based on carbon-based nanocomposites as a working electrode, Ag / AgCl as a reference electrode, and a platinum wire as an auxiliary electrode, and the detection method is differential pulse voltammetry.

9. Use of the carbon-based nanocomposite-based histamine molecularly imprinted electrochemical sensor according to claim 8 in food detection, characterized by, The detection condition of the differential pulse voltammetry is: 5.0 mM of [Fe(CN)6] 3- 4- as a redox probe, the potential range is -0.1-0.6 V, the pulse amplitude is 0.05 V, the pulse width is 0.05 s, the sampling width is 0.0167 s, the pulse interval is 0.2 s, and the resting time is 2 s.​ 10. Use of the carbon-based nanocomposite-based histamine molecularly imprinted electrochemical sensor according to claim 8 in food detection, characterized in that, Before detecting the food sample, it needs to be pretreated: liquid food is filtered through 0.22 μm microporous filter membrane, mixed with phosphate buffer solution with pH = 6.5 at a volume ratio of 1:1; solid food is first homogenized and broken, extracted with phosphate buffer solution with pH = 6.5, centrifuged to take supernatant, filtered through 0.22 μm microporous filter membrane and then detected; And / or, the linear range of detection is 0.1-100 μM, the detection limit is 41.0 nM, the recovery rate of sample detection is 107.0%-116.6%, and the relative standard deviation is less than 5%.

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