Method for detecting content of crystal violet and / or malachite green in fish product
By combining low-temperature cleaning and magnetic solid-phase extraction technology, chitosan-coated magnetic nanoparticles are used to efficiently enrich crystal violet and malachite green in aquatic products, solving the problems of insufficient detection sensitivity and sample interference in the prior art, achieving a fast and accurate detection effect.
Patent Information
- Application Number
- CN202510180820.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art lacks sensitivity when detecting low concentrations of crystal violet and malachite green in aquatic products, and complex sample matrix interference leads to inaccurate detection results. The traditional method has a long detection time and high cost.
Low-temperature cleaning technology was used to remove fat interference in the samples, and magnetic solid-phase extraction was carried out in combination with chitosan-coated magnetic nanoparticles (CS@MNPs). It was separated by an external magnetic field and used acidified acetonitrile as a desorption solvent to achieve efficient enrichment and quantitative analysis of the target pollutants.
It significantly improves the sensitivity and accuracy of detection, shortens the detection time, reduces the dependence on high-end instruments, has high repeatability and stability, and meets the needs of modern aquatic product inspection for rapid, sensitive and efficient.
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Figure CN119985768A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for detecting the content of crystal violet and / or malachite green in fish products. Background Art
[0002] Crystal violet (CV) and malachite green (MG) are triphenylmethane compounds with strong antibacterial and antifungal effects, and are therefore commonly used as synthetic chemical antimicrobial agents in aquaculture. However, these compounds are strictly regulated in many countries and regions due to their potential carcinogenicity and reproductive toxicity.
[0003] Studies in recent years have shown that malachite green and crystal violet pose a risk of persistent residues in fish. As the concentrations of these pollutants in aquatic products are low, existing technologies have low sensitivity in detecting low-concentration pollutants, often leading to inaccurate test results. In addition, the complex sample matrix in aquatic products can interfere with the test process, affecting the accuracy and reliability of the test, especially for samples with high fat content, which can increase the error in the test results.
[0004] Although magnetic solid phase extraction (MSPE) and colorimetric analysis techniques are widely used in food safety testing, they still face the problem of improving sensitivity and shortening detection time. Therefore, it is of great significance to develop a rapid, sensitive and economical method for the detection of CV and MG in aquatic food. Summary of the invention
[0005] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a method for detecting the content of crystal violet and / or malachite green in fish products.
[0006] According to one aspect of the present invention, a method for detecting the content of crystal violet and / or malachite green in fish products is provided, comprising: adding a magnetic adsorbent to the fish product after low-temperature treatment, the magnetic adsorbent consisting of Fe3O4 nanoparticles and functional shells coated on the surface of the Fe3O4 nanoparticles, so that the crystal violet and / or malachite green are adsorbed to the surface of the magnetic adsorbent through hydrogen bonding; separating the magnetic adsorbent from the fish product by an external magnetic field; using acidified acetonitrile as a desorption solvent, desorbing the crystal violet and / or malachite green from the magnetic adsorbent to obtain a desorption solution; analyzing the desorption solution to obtain the relative or absolute content of the crystal violet and / or malachite green.
[0007] Preferably, the magnetic adsorbent has an average diameter of 128.6 nm, a magnetization value of 29.534 emu g-1, and an average potential peak value of +28.5 mV in aqueous solution.
[0008] Preferably, the method for low-temperature treatment of the fish product comprises: treating the fish product with acetonitrile and then placing the fish product at -40°C for 48 hours.
[0009] Preferably, the preparation method of the magnetic adsorbent includes: storing Fe3O4 nanoparticles in an ethanol-water solution to form a suspension; ultrasonically treating the suspension in anhydrous ethanol, adding APTES (3-aminopropyltriethoxysilane) and adjusting the pH to 11; collecting the Fe3O4 nanoparticles after heating in a water bath, washing them with ethanol and storing them in water to form a magnetic fluid; mixing the magnetic fluid with glutaraldehyde, and reacting it with a chitosan solution after ultrasonic treatment to obtain the magnetic adsorbent.
[0010] Preferably, the water bath heating condition is reflux in a 60° C. water bath for 5 hours.
[0011] Preferably, the pH is adjusted to 11 using aqueous ammonia.
[0012] Preferably, analyzing the desorption solution to obtain the relative or absolute content of the crystal violet and / or malachite green comprises: performing chromatographic analysis using a UHPLC system, using a ZORBAX Eclipse XDB-C18 column for separation; using acetonitrile as phase A and 0.125 mol / L ammonium acetate, pH=4.5 as phase B, and performing gradient elution: maintaining phase A at 80% for the first 1.5 minutes, then increasing to 95% at 2 minutes, reducing phase A to 65% at 3 minutes, and maintaining it for 2 minutes before returning to 80%; the flow rate is set to 0.85 mL / min, the injection volume is 5 μL, and the detection wavelengths are set to 588 nm and 617 nm, respectively.
[0013] In current food safety testing technologies, the detection of banned substances in aquatic products (such as crystal violet and malachite green) faces many challenges. These challenges mainly come from the insufficient sensitivity of existing technologies in detecting low-concentration pollutants, which makes it difficult to ensure the accuracy and reliability of the test results. Especially in complex food samples, such as eels and other scaleless fish, due to the particularity of their physiological structure, they are prone to adsorb foreign pollutants, which makes the existing traditional detection methods face sample interference problems during the extraction and detection process, and often require a long processing time, increasing the detection cost.
[0014] In response to the above problems, the present invention proposes an innovative method combining low-temperature cleaning (LTC) and magnetic solid phase extraction (MSPE) technology for rapid and highly sensitive detection of pollutants such as crystal violet and malachite green in fish products. This method utilizes low-temperature cleaning to effectively remove interfering substances in the sample, and combined with the high sensitivity of magnetic materials, it can efficiently extract pollutants in aquatic products, significantly improving the accuracy and sensitivity of detection. Compared with traditional methods, the present invention can shorten the detection time, reduce dependence on high-end instruments, and has high repeatability and stability, meeting the requirements of modern aquatic product detection for rapid, sensitive and efficient detection. Through the application of this method, the level of aquatic product safety supervision can be effectively improved to ensure the food safety of consumers. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:
[0016] Figure 1 Characterization of magnetic nanoparticles provided according to an embodiment of the present invention: (a) FT-IR spectra of CS@MNPs and Fe3O4 nanoparticles; (b) hysteresis curve; (c) Zeta potential; (d) particle size.
[0017] Figure 2 Optimized conditions of the MSPE process provided according to an embodiment of the present invention: (a) pH; (b) adsorbent dosage; (c) extraction time; (d) elution solvent; (e) elution volume; (f) elution time; (g) reuse of CS@MNPs.
[0018] Figure 3 1 is a UHPLC chromatogram of the chemical structural formulas, calibration curves, standard samples and spiked sample extracts of MG and CV provided according to the embodiments of the present invention. DETAILED DESCRIPTION
[0019] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods.
[0020] To date, a variety of methods have been applied to detect malachite green and crystal violet, including ultra-high performance liquid chromatography (UPLC), surface-enhanced Raman scattering (SERS), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), high performance liquid chromatography-tandem ultraviolet spectroscopy (HPLC-UV), high performance liquid chromatography-diode array detector (HPLC-DAD), and ultraviolet-visible spectrophotometry (UV-Vis). However, most chromatography-based analytical methods, such as HPLC-MS / MS, HPLC-DA, etc., are not only time-consuming due to the separation procedures, but also can only be used in laboratories due to their large instrument size. However, no matter which analytical method is used, it is of great significance to extract and enrich the test substances from complex food matrices efficiently and specifically. In order to minimize the potential influence of the matrix on the sample, appropriate sample pretreatment techniques must be implemented. There are many sample pretreatment methods, including micro-solid phase extraction (μ-SPE), solid phase microextraction (SPME), magnetic solid phase extraction (MSPE), and liquid phase extraction (LPE). Compared with other techniques, MSPE has the advantages of simple operation, fast phase separation, and high enrichment efficiency. Therefore, it can be considered a more suitable method for various complex sample types, such as aquatic products. However, the adsorption capacity of magnetic particles with only a magnetic core may not be sufficient. How to enhance the adsorption capacity of target analytes and overcome matrix interference and achieve reuse is an urgent technical problem to be solved in this field.
[0021] The present invention provides a method for detecting the content of crystal violet and / or malachite green in fish products, comprising: adding a magnetic adsorbent into the fish product after low-temperature treatment, wherein the magnetic adsorbent is composed of Fe3O4 nanoparticles and functional shells coated on the surface of the Fe3O4 nanoparticles, so that the crystal violet and / or malachite green are adsorbed to the surface of the magnetic adsorbent through hydrogen bonding; separating the magnetic adsorbent from the fish product through an external magnetic field; using acidified acetonitrile as a desorption solvent, desorbing the crystal violet and / or malachite green from the magnetic adsorbent to obtain a desorption solution; and analyzing the desorption solution to obtain the relative or absolute content of the crystal violet and / or malachite green.
[0022] (1) Preparation of magnetic adsorbent (preparation of CS@MNPs)
[0023] Fe3O4 nanoparticles were synthesized by chemical coprecipitation and stored in a mixture of ethanol and water (100 mL, volume ratio 3:1). The prepared suspension (12 mL) was added to 288 mL of anhydrous ethanol and sonicated. APTES (18 mL) was added and the pH was adjusted to 11 with aqueous ammonia to introduce amide groups on the Fe3O4 surface. The solution was refluxed in a 60°C water bath for 5 h. The functionalized Fe3O4 nanoparticles were collected by a magnet, thoroughly washed 5 times with ethanol, and stored in 20 mL of water. This process was repeated 5 times to obtain 100 mL of magnetic fluid.
[0024] In above-mentioned suspension (100mL), add glutaraldehyde (10mL, 25%), this mixture is handled 3 hours under 20kHz ultrasonic wave (500 watts, pulse 9 seconds open 1 second close) condition, is divided into 6 stages, and each stage is separated by 30 minutes.Gained carbonyl-magnetic nanoparticle is adding chitosan solution (100mL) and reacting overnight to form chitosan magnetic composite material under stirring.Each step is all separated nanoparticle from solution to remove unreacted glutaraldehyde and chitosan by external magnetic field.The chitosan magnetic nano composite material that finally obtains is dried overnight at 40 ℃.
[0025] (2) Characterization of magnetic adsorbents (preparation of CS@MNPs)
[0026] The functional groups of CS@MNPs nanoparticles were detected using a Thermo Scientific iN10 Fourier transform infrared spectrometer (FT-IR, USA). The magnetic properties of CS@MNPs were measured at room temperature using a vibrating sample magnetometer 7404 (VSM, USA) produced by LakeShore Cryotronic. The shape and size of CS@MNPs were characterized using a nanoparticle size and Zeta potential analyzer (Nano ZS90, MalvernInstruments, UK).
[0027] (3) Adsorption experiment
[0028] In the adsorption experiment, CS@MNPs were used as adsorbents, while CV and MG were used as adsorbed solutes. To determine the required adsorbent dosage, different doses of adsorbent (0.05 g to 0.25 g) were added to a 25 mg / L standard solution. Liquid samples were taken every 5 to 160 minutes to determine the concentration trend of the standard solution. CV and MG were detected at wavelengths of 584 nm and 617 nm, respectively, using a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan). All data were analyzed and fitted using Origin2021 (USA).
[0029] (4) Molecular docking analysis
[0030] Molecular docking was used to explore the interactions between surface functional groups and target compounds (CV and MG) during the extraction process. The chemical structures of surface functional groups, CV and MG were drawn using Chemdraw 20.0. Before starting docking, a The docking box was used to wrap the chitosan molecule. Molecular docking analysis was performed using AutoDock Vina 1.2.3. The comprehensiveness of the global search was set to 32, and the docking conformation with the highest binding energy was considered as the bound conformation. Finally, the docking results were visualized and analyzed using PyMol 2.5.5.
[0031] (5) Low temperature processed fish products
[0032] Eel samples were purchased from a supermarket and thawed at room temperature. Eel samples were thoroughly cleaned and cut into pieces of equal size (10 g each). Standard solutions at different concentration levels were added to prepare spiked samples. The spiked samples were ground into a paste, and 10 g of the sample was taken and added to 30 mL of acetonitrile for crude extraction. The mixture was then placed at -40 °C for 48 hours to low-temperature purify to remove fat interference. A specific eel sample was proven to be free of CV and MG by the FDA traditional test method, and this sample was used for all preliminary, optimization, and method evaluation experiments.
[0033] (6) Enrichment of crystal violet and malachite green in eel samples
[0034] The pH of the sample supernatant was adjusted to 11 with 0.1 M NaOH and diluted to 100 mL. CS@MNPs were added and extracted for 50 minutes. Then eluted with 5 mL of acidified acetonitrile (acetonitrile and 3% glacial acetic acid, volume ratio 93:3), and the elution effect was enhanced by ultrasound and vortex oscillation for 90 seconds respectively. A permanent magnet was used for solid-liquid separation, and the supernatant was collected and blown dry with nitrogen. After re-adding 500 μL of acetonitrile and vortex mixing for 120 seconds, the sample was passed through a 0.22 μm filter membrane for UHPLC analysis.
[0035] (7) HPLC analysis
[0036] Chromatographic analysis was performed using a UHPLC system (Agilent 1290 Infinity II, USA), which was equipped with a manual injector, degasser, quaternary pump and variable wavelength detector (VWD). A ZORBAX Eclipse XDB-C18 column (150 mm length, 4.6 mm inner diameter, 5 μm pore size, Agilent, USA) was used for separation. Acetonitrile was used as phase A and ammonium acetate (0.125 mol / L, pH = 4.5) was used as phase B for gradient elution: Phase A was maintained at 80% for the first 1.5 minutes, and then increased to 95% at 2 minutes. Phase A was reduced to 65% at 3 minutes and restored to 80% after 2 minutes. The flow rate was set to 0.85 mL / min, the injection volume was 5 μL, and the detection wavelengths were set to 588 nm and 617 nm, respectively. SPSS (USA) software was used for one-way analysis of variance to determine whether there were significant differences between the groups.
[0037] (8) Regeneration of CS@MNPs
[0038] The used CS@MNPs were washed with methanol and deionized water and dried overnight at 60 °C. The recovered adsorbents were stored in sealed centrifuge tubes at room temperature for reuse.
[0039] (9) Optimization of magnetic solid phase extraction (MSPE) conditions
[0040] 1) Sample pH: Figure 2 In a, pH values of 5, 6, 7, 8, 9 and 11 were optimized. There was a significant increase at pH=11.
[0041] 2) Adsorbent dosage: Figure 2 b shows the adsorption capacity of CV and MG at different adsorbent dosages (30, 40, 50, 60, 70, 80, and 90 mg). 50 mg was considered to be the ideal adsorbent dosage.
[0042] 3) Extraction time: according to Figure 2 The extraction results of CS@MNPs at different times in c show that when the time reaches 50 minutes, the adsorption process reaches the highest extraction efficiency.
[0043] 4) Elution solvent: Figure 2 d shows that under the same conditions, acidified acetonitrile has the strongest elution ability.
[0044] 5) Elution volume: Figure 2 As shown in e, the peak area reaches the best when the elution volume is 5 mL.
[0045] 6) Elution time: Figure 2 f shows that the best result was achieved at 180 seconds.
[0046] 7) Recycling of CS@MNPs: Figure 2 g It can be seen that the CS@MNPs synthesized in the present invention exhibit relatively stable reusability during 25 cycles of use.
[0047] (10) Detection method performance evaluation
[0048] Figure 3The chemical structural formulas, calibration curves, and chromatograms of standard spiked samples and extracts of MG and CV are shown. The method adopted in the present invention successfully extracted CV and MG from eel samples. According to the chromatographic results, CV and MG showed good linear relationships in the concentration ranges of 1-2000 ng / mL and 0.5-2000 ng / mL. The limits of detection (LOD) and limits of quantification (LOQ) were determined by the peak concentrations of signal-to-noise ratio of 3 (S / N=3) and signal-to-noise ratio of 10 (S / N=10). The HPLC method developed in the present invention covered the LOD ranges of CV and MG, which were 0.18 and 0.54 ng / g, respectively, both of which were lower than the maximum residue limits set by the European Union (MRL and MRPL were 2 ng / g). The LOQs of CV and MG were 0.63 and 1.8 ng / g, respectively. In order to achieve optimal performance over a wider range of analyte concentrations, spiked samples of 10 ng / g and 100 ng / g were used for recovery testing. To determine the intra-day and inter-day reproducibility and recovery, the samples were tested within one day and within three consecutive days. To ensure data accuracy, each experiment was repeated three times. The recoveries of MG and CV were 83.19% to 108.73% within the day and 83.73% to 112.88% between the days. The relative standard deviation (RSD) of the intra-day reproducibility of MG and CV was between 1.45% and 4.81%, and the RSD of the inter-day reproducibility was between 2.58% and 7.60%. Although the inter-day RSD value was higher than the intra-day, the overall RSD range was still within an acceptable range. Therefore, the method developed by the present invention performed well in terms of reproducibility and recovery, indicating that the method has high reliability and accuracy.
[0049] (11) Actual sample analysis
[0050] Three different brands of eels were purchased from the local market and randomly numbered. All actual samples were processed according to the method of the present invention. 100 ng / mL of mixed standard was added to prepare spiked samples. Compared with the chromatographic peaks of the mixed standard, there was no significant difference between the spiked samples and the mixed samples. Therefore, it can be considered that the selected samples do not contain CV and MG.
[0051] (12) Characterization results of CS@MNPs
[0052] 1) Fourier transform infrared spectroscopy (FT-IR)
[0053] FT-IR spectra of Fe3O4 and CS@MNPs are shown in Figure 1 a. Bare Fe3O4 particles at 582cm -1 The stretching vibration band of Fe—O groups is shown at 586 cm -1The CH stretching vibration bands of the methyl and methylene groups on the chitosan chain appear at 2880 cm -1 and 2950cm -1 The C-N stretching vibration absorption peak and the C-O bending vibration absorption peak appear at 1020 cm -1 and 1412cm -1 The former is formed by the carbon and nitrogen atoms in the amino group, and the latter represents the hydroxyl groups on the polysaccharide chain. The N—H bending vibration absorption peak appears at 1631 cm -1 The infrared spectrum of CS@MNPs showed vibration peaks consistent with the functional groups on the chitosan polysaccharide chain, indicating that chitosan was successfully coated on the Fe3O4 nanoparticles.
[0054] 2) Hysteresis curve (VSM) analysis method
[0055] like Figure 1 The hysteresis curve at room temperature shows that the magnetization intensity measurement range is 5×10 -7 Up to 10 3 The results show that it has superparamagnetism, with a magnetization value of 29.534emu g-1.
[0056] 3) Zeta potential and laser particle size analysis
[0057] At neutral pH, chitosan has a positive charge. Figure 1 c, the average potential peak of CS@MNPs in aqueous solution is +28.5mV. The absolute value of the Zeta potential is about 30mV, which indicates that the nanoparticles have strong electrostatic repulsion under this condition and are not easy to aggregate, confirming the good stability of CS@MNPs. Dynamic light scattering (DLS) measurement results show that the average diameter of CS@MNPs is 128.6nm, ensuring that it can fully contact the sample.
[0058] In the prior art, magnetic solid phase extraction technology is a sample pretreatment method based on magnetic particles, which can efficiently enrich and extract target pollutants from liquid samples. The specific steps are: (1) Add magnetic nanoparticles to the sample to bind to the target pollutants. (2) Use a magnetic field to separate the particles from the liquid sample and extract the enriched pollutants. (3) Perform subsequent colorimetric analysis or other detection methods. Although this method is widely used in liquid samples and can effectively separate target substances, it still has certain limitations in dealing with fat interference and improving detection sensitivity and accuracy.
[0059] The present invention proposes a method based on the combination of low temperature cleaning (LTC) and magnetic solid phase extraction (MSPE) for rapid and highly sensitive detection of crystal violet and malachite green in aquatic products. The magnetic solid phase extraction (MSPE) method uses magnetic nanoparticles to bind to the target substance to be tested, and then separates the solid and liquid through a magnetic field, thereby improving the analysis efficiency. It includes: adding functionalized magnetic nanoparticles to the sample to be tested to bind to CV and MG; then, separating the particles from the solution through an external magnetic field to achieve pre-concentration of the target substance. Low temperature cleaning (LTC) technology helps to remove fat interference in the sample, so that it shows good results in the treatment of complex matrix samples. The method first uses low temperature cleaning to effectively remove fat interference in the sample, and then combines magnetic nanoparticles (MNPs) to enrich the target pollutants. This process can improve the detection sensitivity and reduce the interference of the sample matrix, and finally perform efficient quantitative analysis through colorimetry.
[0060] Compared with the traditional detection method, the specific steps of the detection method of low temperature cleaning (LTC) combined with magnetic solid phase extraction (MSPE) of the present invention are as follows: first, the fat interference in the aquatic product sample is removed by low temperature cleaning, and then the chitosan-coated magnetic nanoparticles (CS@MNPs) are combined for efficient CV and MG enrichment. CS@MNPs have good stability and adsorption capacity, and can increase the enrichment concentration of the target substance after efficient solid-liquid separation. Compared with the traditional detection method, this method performs well in sensitivity, ease of operation and cost control. Finally, HPLC is used to quantitatively analyze the extracted samples to achieve rapid and sensitive CV and MG detection. The greatest advantage of the present invention is its high sensitivity, simplicity and low cost, and it can effectively identify trace pollutants in aquatic products in rapid detection. The combination of low temperature cleaning and MSPE technology can effectively reduce matrix interference in aquatic products at higher sensitivity. The detection limits of CV and MG of the present invention are reduced to 0.18ng / g and 0.54ng / g, respectively, meeting the needs of high sensitivity and rapid detection of food safety.
[0061] Comparative Example:
[0062] Synthesis of β-CDP: Referring to the literature (Raeisi et al., 2017), 1.135 g of β-CD and 25 mL of dry DMF were added to a 250 mL round-bottom flask, and 0.18 g of oil-suspended NaH was slowly added under nitrogen protection, and stirred at room temperature for 24 hours to generate β-CD oxygen anions. Subsequently, 0.644 g of BTDA was added and stirred at 60 ° C for 24 hours. After cooling, the reaction mixture was added to 80 mL of acetone for precipitation, filtered, washed with acetone in turn, and vacuum dried for 12 hours.
[0063] Preparation of MNPs-CDP: Fe3O4@SiO2NPs were prepared according to the literature (Raeisi et al., 2017; Li et al., 2015). 0.5g Fe3O4@SiO2 was dispersed in 50mL dry toluene, and 1.0mL EPO was added after ultrasonic homogenization. The mixture was stirred at 80℃ under nitrogen protection for 24 hours, and Fe3O4@SiO2-GP was obtained by magnetic separation. Subsequently, 0.5g Fe3O4@SiO2-GP was added to 7mL dry DMF containing 2.0g β-CDP, refluxed at 80℃ for 24 hours, washed with DMF and ethanol in turn after magnetic separation, and dried in vacuum at 80℃ for 6 hours.
[0064] Take 50mL containing 100μg L -1 The target substance and 10% NaCl (w / v) sample solution were adjusted to pH 5.0, 11.0 mg MNPs-CDP was added, and stirred for 10 minutes until adsorption equilibrium. After magnetic separation of the adsorbent, 200 μL methanol / acetic acid (95:5, v / v) was used for desorption for 2 minutes, and the eluate was collected and 20 μL was injected into HPLC for analysis.
[0065] The Fe-O characteristic peaks of MNPs-EPO and MNPs-CDP are located at 633 cm -1 and 625cm -1 1000–1100cm -1 The peaks at 2852 and 2934 cm-1 in the MNPs-EPO spectrum are attributed to the Si-O-Si stretching vibration of Fe3O4 coated with SiO2. -1 The peaks at 1656 and 1718 cm are the CH bonds of EPO. -1 The peak at is the carbonyl group (ester group and ketone group) of β-CDP, indicating that β-CDP is successfully grafted onto the surface of MNPs. MNPs-CDP is uniformly spherical with an average particle size of 40-50nm.
[0066] The extraction time (2–15 min) and desorption time (1–5 min) were optimized by the single factor method, and the optimal extraction time was determined to be 10 min and the desorption time was 2 min. The eluent type experiment showed that methanol / acetic acid (95:5, v / v) had the best effect, and the recovery rate was the highest when the volume was 200 μL. The sample volume experiment showed that 50 mL was the optimal volume. Electrostatic interaction, hydrogen bonding and inclusion complex formation are the main adsorption mechanisms. At pH 5.0, the carboxylic acid group of MNPs-CDP is ionized and interacts electrostatically with the positively charged dye molecules; at low pH, it interacts through hydrogen bonding.
[0067] The detection limits (LOD) in the comparative example are 0.03 μg / L for crystal violet (CV) and 0.03 μg / L for malachite green (MG) (equivalent to 30 ng / g). The detection limits of CV in the present invention are 166 times lower than those in the comparative example, and the detection limits of MG are 55.5 times lower than those in the prior art.
[0068] In the embodiment of the present invention, chitosan-coated magnetic nanoparticles improve the stability and adsorption capacity of the material. Chitosan provides a protective layer for MNPs to prevent oxidation aggregation and makes them superparamagnetic. Surface amino modification is used to achieve ion exchange and hydrogen bonding of CV and MG. This design not only improves the adsorption capacity of the material, but also has good regeneration performance, so that CS@MNPs can maintain a stable adsorption effect after 25 cycles of use, and has the characteristics of economy and environmental friendliness.
[0069] Molecular docking analysis reveals the binding mechanism between the target and chitosan. Molecular docking analysis was used to study the adsorption mechanism of CV and MG during the extraction process, and it was found that CV and MG were mainly bound to chitosan through hydrogen bonding interactions. The analysis results showed that the difference in the number of hydrogen bonds was the main reason for the difference in the adsorption amount of CV and MG, providing a theoretical basis for the design of functional groups on the surface of materials.
[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for detecting the content of crystal violet and / or malachite green in fish products, characterized in that: include: Adding a magnetic adsorbent to the fish product after low temperature treatment, wherein the magnetic adsorbent is composed of Fe3O4 nanoparticles and functional group shells coated on the surface of the Fe3O4 nanoparticles, so that the crystal violet and / or malachite green are adsorbed to the surface of the magnetic adsorbent through hydrogen bonding; separating the magnetic adsorbent from the fish product by an external magnetic field; Using acidified acetonitrile as a desorption solvent, desorbing the crystal violet and / or malachite green from the magnetic adsorbent to obtain a desorption solution; The desorption solution is analyzed to obtain the relative or absolute content of the crystal violet and / or malachite green.
2. The method according to claim 1, characterized in that The magnetic adsorbent has an average diameter of 128.6 nm, a magnetization value of 29.534 emu g-1, and an average potential peak value of +28.5 mV in aqueous solution.
3. The method according to claim 1, characterized in that The method for low-temperature treatment of the fish product comprises: treating the fish product with acetonitrile and then placing the fish product at -40°C for 48 hours.
4. The method according to claim 1, characterized in that The preparation method of the magnetic adsorbent comprises: The Fe3O4 nanoparticles are stored in an ethanol-water solution to form a suspension; The suspension was sonicated in anhydrous ethanol, and the pH was adjusted to 11 after adding APTES (3-aminopropyltriethoxysilane); After heating in a water bath, the Fe3O4 nanoparticles are collected, washed with ethanol and stored in water to form a magnetic fluid; The magnetic fluid is mixed with glutaraldehyde, and reacted with chitosan solution after ultrasonic treatment to obtain the magnetic adsorbent.
5. The method according to claim 4, characterized in that The water bath heating condition was reflux in a 60°C water bath for 5 hours.
6. The method according to claim 4, characterized in that The pH was adjusted to 11 using aqueous ammonia.
7. The method according to claim 1, characterized in that Analyzing the desorption solution to obtain the relative or absolute content of the crystal violet and / or malachite green comprises: Chromatographic analysis was performed using a UHPLC system, and a ZORBAX Eclipse XDB-C18 column was used for separation; Acetonitrile was used as phase A and 0.125 mol / L ammonium acetate, pH = 4.5, was used as phase B for gradient elution: Phase A was maintained at 80% for the first 1.5 minutes, then increased to 95% at 2 minutes, and phase A was reduced to 65% at 3 minutes, maintained for 2 minutes, and then restored to 80%; The flow rate was set to 0.85 mL / min, the injection volume was 5 μL, and the detection wavelengths were set to 588 nm and 617 nm, respectively.