Preparation method of electrochemical sensor based on Ag NPs / Au NFs / copper wire structure and application of electrochemical sensor in detection of EPA and DHA in fish oil

Abstract

The invention discloses a preparation method of an electrochemical sensor based on an AgNPs / Au NFs / copper wire structure and application of the electrochemical sensor to detection of EPA and DHA in fish oil, and is characterized by comprising the following steps: synthesizing Au NFs on a copper wire by an electrochemical deposition method, and directly sputtering Ag NPs on the surface of the Au NFs by magnetron sputtering to obtain the electrochemical sensor based on the Ag NPs / AuNFs / copper wire structure; the fish oil sample detection method comprises the following steps: carrying out catalytic methyl esterification on a fish oil sample through a potassium hydroxide-methanol solution and BF3, and converting glyceride type EPA / DHA into methyl ester type EPA / DHA; a to-be-detected solution subjected to pretreatment and an electrochemical sensor are stirred to react and then subjected to Raman detection, the content of DHA and EPA in a fish oil sample is calculated according to the relation between the concentration of DHA-Me / EPA-Me and the characteristic peak signal intensity of DHA-Me / EPA-Me, and the method has the advantages of being simple, rapid and high in sensitivity and accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting the content of EPA and DHA in fish oil, and in particular to a preparation method of an electrochemical sensor based on AgNPs / AuNFs / copper wire structure and application of the electrochemical sensor to detection of EPA and DHA in fish oil. BACKGROUND

[0002] Surface-Enhanced Raman Scattering (SERS) technology, as a highly sensitive molecular detection method, has been widely used in the fields of biomedicine, environmental monitoring and chemical analysis in recent years. SERS realizes the detection of low-concentration or even single-molecule level target substances by enhancing the Raman signal on the surface of metal nanostructures. However, traditional SERS substrates usually rely on noble metal (such as gold and silver) nanostructures, which have high preparation cost, poor stability, and are easily disturbed in practical application, limiting their large-scale application. Therefore, developing new types of low-cost and high-stability SERS substrates has become a research hotspot.

[0003] Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two functional long-chain polyunsaturated fatty acids, mainly existing in the fat of fish that feed on phytoplankton. Studies have shown that DHA and EPA in fish oil can treat and relieve conditions including hyperlipidemia, diabetes, cancer, inflammation and neurodegenerative diseases. Health products containing fish oil are popular among consumers, especially deep-sea fish oil products that claim to have high DHA and EPA content, which are of great commercial value. Therefore, it is very important to monitor the true content of DHA and EPA in fish oil products.

[0004] Gas chromatography (GC) is an internationally recognized method for detecting the composition and content of fatty acids in oil and fat products. However, GC analysis has many processing steps, such as a large amount of sample preparation, a lengthy measurement process, complex large equipment and laboratory testing environmental conditions. Therefore, in market regulation applications, GC cannot fully meet the rapid monitoring needs of the qualitative and quantitative detection of EPA and DHA in fish oil products. Therefore, it is urgent to develop a convenient, rapid and intuitive detection technology for the qualitative and quantitative detection of DHA and EPA in fish oil products. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a preparation method of an electrochemical sensor based on AgNPs / AuNFs / copper wire structure capable of enhancing SERS intensity and a method for quantitatively detecting the content of DHA and EPA in fish oil by combining with methyl esterification treatment, which has the advantages of simplicity, rapidness, high sensitivity and accuracy.

[0006] The application solves the above technical problems by adopting the technical scheme of a preparation method of an electrochemical sensor based on an Ag NPs / Au NFs / copper wire structure, characterized by comprising the following steps:

[0007] Step 1, synthesizing Au NFs on a copper wire through an electrochemical deposition method to obtain an Au NFs / copper wire structure;

[0008] Step 2, directly sputtering Ag NPs on the surface of the Au NFs / copper wire structure through a magnetron sputtering method to obtain the electrochemical sensor based on the Ag NPs / Au NFs / copper wire structure.

[0009] Further, step 1 is specifically as follows: through the electrochemical deposition method, taking the copper wire as a working electrode, an Ag / AgCl electrode as a reference electrode, and a platinum wire as a counter electrode, adopting a chronoamperometry method in an ethanol electrolyte containing 8-12 mM HAuCl4 and 0.2-0.3 M H2SO4, applying a deposition voltage of-1.5 V, and depositing for 450-650 s to grow Au nanoflowers (Au NFs) on the surface of the copper wire; after the reaction is completed, the copper wire is washed with deionized water and dried to obtain the Au NFs / copper wire structure.

[0010] Preferably, the deposition time is 450-650 s.

[0011] Further, step 2 is specifically as follows: depositing Ag NPs on the surface of the Au NFs / copper wire structure through the magnetron sputtering method, controlling the sputtering time to be 30-120 s, growing Ag nanoparticles (Ag NPs) on the surface of the Au NFs to obtain the electrochemical sensor based on the Ag-NPs / Au-NF / copper wire structure.

[0012] Preferably, the sputtering time is 60 s.

[0013] The application further provides a method for detecting EPA and DHA in fish oil by using the electrochemical sensor, comprising the following steps:

[0014] Step 1, pretreating a fish oil sample before methyl esterification

[0015] Take 0.20 g of fish oil sample in a reaction bottle, add 4 mL of 0.5 mol / L KOH-methanol solution, after nitrogen purging, connect the reflux device, and react in boiling water bath for 15-25 min; then add 8 mL of 14 vt% BF3-methanol solution, continue to boil for 15-25 min; after the reaction is completed, cool to room temperature, add 5 mL of isooctane and 25 mL of saturated NaCl solution, shake for 0.5-2 min, then extract in the refrigerator; transfer the upper organic phase to a glass tube, add anhydrous MgSO4 to remove water, filter to obtain the test solution containing DHA-methyl ester (DHA-Me) and EPA-methyl ester (EPA-Me); wherein the mixing ratio of fish oil sample, KOH-methanol solution, BF3-methanol solution, isooctane and saturated NaCl solution is 0.2 g:4 mL:8 mL:5 mL:25 mL; the fish oil sample is methyl esterified by potassium hydroxide-methanol solution and BF3 catalyst, and the glyceride type EPA / DHA is converted into methyl ester type (EPA-Me / DHA-Me), so as to eliminate the influence of molecular structure difference on SERS response;

[0016] Step 2, standard curve establishment

[0017] Prepare DHA-Me and EPA-Me standard solutions respectively, the concentration range is 10 -3 mg / mL to 10 -6 mg / mL, dilute to the target concentration with isooctane or ethanol, collect the SERS spectra of all standard solutions, extract the peak height, half-peak width and peak area parameters of the characteristic peaks, and establish the standard curve between the DHA-Me concentration and the characteristic peak signal intensity, and the standard curve between the EPA-Me concentration and the characteristic peak signal intensity through the PLS calibration model;

[0018] Step 3, detection of EPA and DHA content in fish oil

[0019] Take 5 mL of the test solution obtained in step 1 and the electrochemical sensor, stir for 1-10 minutes, dry after standing, and use the Raman detector to detect, obtain the SERS signal intensity of DHA-Me at 869 cm -1 and the SERS signal intensity of EPA-Me at 955 cm -1 , according to the relationship between DHA-Me and the characteristic peak signal intensity and the relationship between EPA-Me concentration and the characteristic peak signal intensity, calculate the content of DHA and EPA in the fish oil sample.

[0020] Further, the Raman detector detection conditions are: using 532 nm Raman laser excitation, power is 30 mW, collection time is 10 s.

[0021] Compared with the prior art, the preparation method of the electrochemical sensor based on the Ag NPs / Au NFs / copper wire structure and the application of the electrochemical sensor to the detection of EPA and DHA in fish oil have the following advantages: firstly, the rose-shaped structure of the Au NFs provides abundant Raman "hot spots", and the addition of the Ag NPs further enhances the electromagnetic field effect, so that the SERS signal is significantly enhanced. In addition, the methylesterification treatment converts the DHA and EPA in the fish oil into DHA-Me and EPA-Me, which facilitates the capture and adsorption of the SERS substrate and avoids the problem of inconsistent response intensity when the triglyceride type and diglyceride type DHA and EPA are directly detected, thereby improving the sensitivity and accuracy of the detection.

[0022] In summary, the preparation method of the electrochemical sensor based on the Ag NPs / Au NFs / copper wire structure and the application of the electrochemical sensor to the detection of EPA and DHA in fish oil have the following advantages: firstly, the rose-shaped structure of the Au NFs provides abundant Raman "hot spots", and the addition of the Ag NPs further enhances the electromagnetic field effect, so that the SERS signal is significantly enhanced. In addition, the methylesterification treatment converts the DHA and EPA in the fish oil into DHA-Me and EPA-Me, which facilitates the capture and adsorption of the SERS substrate and avoids the problem of inconsistent response intensity when the triglyceride type and diglyceride type DHA and EPA are directly detected, thereby improving the sensitivity and accuracy of the detection. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For scanning electron microscope (SEM) observation of the morphology change of Au NFs under different deposition times, wherein a is the deposition time 450s, b is the deposition time 500s, c is the deposition time 550s, d is the deposition time 600s, and e is the deposition time 650s;

[0024] Figure 2 For scanning electron microscope (SEM) observation of the morphology change of Au NFs under different sputtering times, wherein a is the sputtering time 30s, b is the sputtering time 60s, c is the sputtering time 90s, and d is the sputtering time 120s;

[0025] Figure 3 For EDS determination of the horizontal distribution of Au, Ag, Cu, C and O elements of the electrochemical sensor based on the Ag-NPs / Au-NF / copper wire structure;

[0026] Figure 4 (a) is the SERS response change graph of Au NFs prepared under different deposition times under different Raman shifts, (b) is the SERS response change graph of Ag NPs / Au NFs / copper wire structure prepared under different sputtering times under different Raman shifts, (c) is the SERS response change of Ag NPs / copper wire, Au NFs / copper wire and the sensor under different Raman shifts, and (d) is the SERS signal intensity comparison column chart of Ag NPs / copper wire and Au NFs / copper wire and the sensor.

[0027] Figure 5 (a) is the SERS intensity curve of Au NPs / Au NFs / copper wire structure-based electrochemical sensor at different Raman shifts and CV standard solution concentrations, (b) is the linear relationship diagram of the characteristic peak intensity at 1620 cm -1 and the logarithm of CV molecular concentration;

[0028] Figure 6 is the repeatability and uniformity analysis of Ag NPs / Au NFs / copper wire structure-based electrochemical sensor, wherein (a) is the SERS intensity curve of 50 CVs with a concentration of 10 -5 M at different Raman shifts; (b) is the SERS intensity histogram of 50 CVs with a concentration of 10 -5 M; (c) is the SERS intensity mapping in a randomly selected 600×600 μm 2 region, and (d) is the signal uniformity analysis of the SERS sensor;

[0029] Figure 7 is the SERS spectrum of two substances of DHA and EPA detected by three SERS substrates, wherein (a) is DHA, and (b) is EPA;

[0030] Figure 8 (a) is the SERS spectrum of free DHA, triglyceride DHA, diglyceride DHA and methyl ester DHA at different Raman shifts, (b) is the SERS response intensity histogram of free DHA, triglyceride DHA, diglyceride DHA and methyl ester DHA at characteristic peaks, (c) is the SERS spectrum of free EPA, triglyceride EPA, diglyceride EPA and methyl ester EPA at different Raman shifts, and (d) is the SERS response intensity histogram of free EPA, triglyceride EPA, diglyceride EPA and methyl ester EPA at characteristic peaks;

[0031] Figure 9 (a) is the SERS peak shape of triglyceride DHA before and after methyl esterification treatment, (b) is the SERS peak shape of diglyceride DHA before and after methyl esterification treatment, (c) is the SERS peak shape of triglyceride EPA before and after methyl esterification treatment, and (d) is the SERS peak shape of diglyceride EPA before and after methyl esterification treatment;

[0032] Figure 10 (a) is the SERS intensity of the methyl ester DHA standard solution with a concentration of 10.0-10 -6 mg / mL, (b) is the calibration curve of the logarithm of the concentration of methyl ester DHA and the SERS intensity at its characteristic peak, (c) is the SERS intensity of the methyl ester EPA standard solution with a concentration of 10.0-10 -6SERS intensity of the methyl ester type EPA standard solution of 0.1 mg / mL, (d) is the calibration curve of the logarithm of the concentration of methyl ester type EPA and the SERS intensity at its characteristic peak;

[0033] Figure 11 Linear relationship between the true value and the predicted value of DHA-Me concentration in the standard curve of the PLS calibration model construction, (b) linear relationship between the true value and the predicted value of EPA-Me concentration in the standard curve of the PLS calibration model construction. DETAILED DESCRIPTION

[0034] The application will be further described in detail below with reference to the embodiments combined with the accompanying drawings.

[0035] Reagents: Hydrated tetrachloroauric acid (III) (HAuCl4 x H2O, purity 99.9%) and silver nitrate (AgNO3, purity 99.9%) were purchased from Shanghai Aldrin Biochemical Technology Co., Ltd. (Shandong, China). The model molecule crystal violet (CV, purity 99%) was provided by Sinopharm Chemical Reagent Co., Ltd. (Shenyang, China). Docosahexaenoic acid (DHA, purity 99.3%), eicosapentaenoic acid (EPA, purity 99.3%), docosahexaenoic acid methyl ester (DHA-Me, purity 99.3%), eicosapentaenoic acid methyl ester (EPA-Me, purity 99.3%), triglyceride type docosahexaenoic acid (DHA-TG, purity 99.3%), triglyceride type eicosapentaenoic acid (EPA-TG, purity 99.3%), diglyceride type docosahexaenoic acid (DHA-DG, purity 99.3%), and diglyceride type eicosapentaenoic acid (EPA-DG, purity 99.3%) were purchased from Beijing North-Plus Biotech Co., Ltd. (Beijing, China). Potassium hydroxide (KOH, purity 99.9%), methanol (CH3OH, purity 99.8%), boron trifluoride methanol solution (BF3-CH3OH, content 14% in methanol), 2,2,4-trimethylpentane (C8H 18 , purity 99.9%), sodium chloride (NaCl, purity 99.0%), and magnesium sulfate (MgSO4, purity 99.5%) were purchased from Sigma-Aldrich Biotech Co., Ltd. (Shanghai, China). Fish oil (#1, #2, #3, and #4) was provided by Ningbo Today Food Co., Ltd. (Ningbo, China). Copper wire was purchased from Ningbo Yufeng Steel Co., Ltd. (Ningbo, China). Most of the above reagents were of analytical purity, and the solutions were prepared using Merck ultrapure water or ethanol.

[0036] Instrument: Current-time (i-t) curves were measured on an electrochemical workstation (CHI-660A15246). The morphology of the V-chip was characterized by field emission scanning electron microscopy (SEM, SU-70, Hitachi, secondary electron mode). Energy dispersive X-ray spectroscopy (EDS, EDAX TEAM Apollo XL) and elemental mapping were used to determine the elemental composition and distribution. Surface-enhanced Raman spectroscopy (SERS) was collected using a compact Raman spectrometer (BWS 415, B&W Tek) equipped with a 532 nm semiconductor laser. The integration time was 10 seconds, and the laser power was 20 mW or 30 mW.

[0037] Specific embodiment one, the preparation method of the electrochemical sensor based on AgNPs / AuNFs / copper wire structure, comprising the following steps,

[0038] Step 1, by electrochemical deposition method, taking copper wire (0.8 cm x 1.5 cm) as working electrode, Ag / AgCl electrode as reference electrode, platinum wire as counter electrode, using chronoamperometry in the ethanol electrolyte containing 10 mM HAuCl4 and 0.25 M H2SO4, applying deposition voltage of-1.5 V, deposition time is 450-650 s, growing Au nanoflower (AuNFs) on the surface of copper wire, after the reaction, washing with deionized water and drying;

[0039] Step 2, depositing Ag NPs on the surface of Au NFs by magnetron sputtering, controlling the sputtering time for 30-120 s, growing Ag nanoparticles (Ag NPs) on the surface of Au NFs, forming an electrochemical sensor based on Ag-NPs / Au-NF / copper wire structure.

[0040] The above Ag NPs preparation method is as follows: first, weigh 17 mg of silver nitrate and 10 mL of deionized water to synthesize 10 mmol / L silver nitrate solution; then take 3.3 mL of 4 g / L sodium hydroxide solution and 11.6 mg of hydroxylamine hydrochloride, respectively, add water to 100 mL to obtain a reducing agent mixture for synthesizing Ag NPs; quickly add 2 mL of silver nitrate solution to 18 mL of reducing agent mixture to obtain yellow-green Ag NPs, filter through a 0.22 μm Millipore membrane, and store in a 4°C refrigerator for standby.

[0041] Specific embodiment two, parameter optimization of the electrochemical sensor prepared by the method of specific embodiment one.

[0042] 1. Deposition time optimization

[0043] The morphology of Au NFs under different deposition times was observed by scanning electron microscopy (SEM), and the results are as followsFigure 1 As shown. Figure 1 As shown in (a), when the deposition time is 450s, the clusters do not show obvious flower-like shapes, but rather small protrusions similar to those formed by the aggregation of gold nanoparticles. Figure 1 (b) and Figure 1 As shown in (c), as the deposition time gradually increases, the hierarchical structure of gold nanoparticles becomes more obvious, forming a rose petal-like shape. Figure 1 As shown in (d), when the deposition time is 600s, the AuNFs are more graded, the petals are thin and sharp, the area of ​​the gold nanostructures is correspondingly larger, and the sharp edges of the Au NFs are extended. Figure 1 As shown in (e), at a deposition time of 650 s, the Au NFs petals overgrow, and the spaces between the petals are filled with a large number of small lumps of material, causing the flower-like structure to gradually disappear, resulting in a spiny spherical structure. The results indicate that a deposition time of 600 s is the optimal condition, forming a uniform rosette-like structure with abundant 3D tips and nanogaps, providing a high density of "hotspots" for SERS.

[0044] 2. Sputtering time optimization

[0045] The morphology changes of Au NFs under different sputtering times were observed by scanning electron microscopy (SEM). Figure 2 As shown. Figure 2 As shown in (a), when the sputtering time is 30s, the AgNPs are sparsely distributed and the morphology of AuNFs is not significantly changed. Figure 2 As shown in (b), when the sputtering time is 60s, Ag NPs evenly cover the surface of Au NFs, leaving gaps between petals and forming high-density “hot spots”. Figure 2 As shown in (c), when the sputtering time is 90s, the gaps on the surface of Au NFs are gradually covered by the Ag layer, and the sharp crystal 3D edges of the petals are gradually passivated. Figure 2 As shown in (d), at a sputtering time of 120 seconds, the silver nanoparticles completely encapsulate the AuNFs, and the rosette-like structure disappears. The results indicate that a sputtering time of 60 seconds is optimal, allowing the Ag NPs to evenly cover the Au NFs surface, preserving the petal structure while significantly enhancing the electromagnetic field. Ultimately, the optimal preparation conditions were determined to be: AuNFs deposition time of 600 seconds and AgNPs sputtering time of 60 seconds, for further experiments.

[0046] The electrochemical sensor based on Ag-NPs / Au-NF / copper wire structure prepared under the optimal conditions was analyzed by dispersive X-ray spectroscopy (EDS). Figure 3 Energy dispersive X-ray spectroscopy (EDS) and elemental mapping analysis confirmed that Ag, Au, Cu, C, and O elements were uniformly distributed on the sensor surface, confirming that the AgNPs / AuNFs / copper wire structure had been successfully constructed.

[0047] Specific embodiment three, performance analysis of the electrochemical sensor prepared by the method of specific embodiment one.

[0048] 1. Ag / Au synergistic electromagnetic enhancement effect

[0049] Experimental group 1-4: the preparation method of the electrochemical sensor based on Ag NPs / Au NFs / copper wire structure is the same as that in the above specific embodiment one, the difference is that the deposition time in step 1 is 600s, and the sputtering time in step 2 is set to 30s, 60s, 90s and 120s respectively.

[0050] Control group 1: preparation method of Ag NPs / copper wire structure: an electrochemical sensor of Ag NPs / copper wire structure is obtained by directly sputtering Ag NPs on the copper wire through magnetron sputtering technology, and the sputtering time is 60s.

[0051] Control group 2: preparation method of Au NFs / copper wire structure: Au NFs are prepared on the pre-cleaned copper wire by electrochemical deposition method. In an ethanol electrolyte containing 10mM HAuCl4 and 0.25M H2SO4, the electrochemical deposition time is 450s, 500s, 550s, 600s and 650s respectively under the condition of deposition voltage-1.5V by using chronoamperometry (i-t) technology. Au NFs grown under these conditions are uniformly distributed on the copper wire substrate, and an electrochemical sensor of Au NFs / copper wire structure is obtained.

[0052] Experimental method: experimental groups 1-4 and control groups 1 and 2 are collected by a compact Raman spectrometer (BWS 415, B&W Tek company) equipped with a 532nm semiconductor laser for surface enhanced Raman spectroscopy (SERS), the integration time is 10 seconds, and the laser power is 20 milliwatts or 30 milliwatts, and the results are shown in Figure 4 .

[0053] As can be seen from (a) in Figure 4 , the Raman intensity of the Au NFs / copper wire structure prepared in the control group 2 is the highest when the deposition time is 600s; as can be seen from (b) in Figure 4 , among experimental groups 1-4, the Raman intensity of the electrochemical sensor of Ag NPs / Au NFs / copper wire structure with sputtering time of 60s is the highest.

[0054] The Ag NPs / Au NFs / copper wire sensor prepared under the optimal conditions is compared with the sensors prepared in the control group 1 and the control group 2 with a deposition time of 600s, Figure 4 (c) and Figure 4As shown in (d), the SERS signal intensity of the composite substrate is increased by more than 4 times, which is attributed to the synergistic electromagnetic enhancement effect of Ag / Au. The physical combination of AgNPs and Au NFs has a significant synergistic effect in stability and SERS signal enhancement.

[0055] 2. Detection limit analysis of electrochemical sensors

[0056] Experimental method: The initial concentration was 10 -3 M crystal violet (CV) solution (solvent is ultrapure water), the concentration gradient is prepared by stepwise dilution method. -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 A series of standard solutions of M were prepared. CV solutions of different concentrations were drop-coated on the surface of an electrochemical sensor based on an AgNPs / AuNFs / copper wire structure prepared under optimal conditions, left to stand at room temperature to complete molecular adsorption, and then dried with nitrogen. The SERS detection parameters were set as follows: laser wavelength: 532 nm, power: 30 mW; integration time: 10 seconds; focusing area: 5 points were randomly selected on the sensor surface for detection and the average value was taken. CV at 1620 cm -1 The characteristic peak (C-C ring stretching vibration) at was selected as the target peak.

[0057] Depend on Figure 5 (a) It can be seen that in 10 -9 The peak is still clearly discernible at low concentrations, indicating the sensor's high capture ability for trace molecules. -1 The peak intensity (y) is the ordinate, and the calibration curve is drawn. Figure 5 From (b), we can see that the linear regression equation is y = 58468.78 + 6480.88 × lgC (CV), R 2 =0.995. The limit of detection (LOD) was 8.35×10 -10 M, which indicates that the electrochemical sensor based on AgNPs / AuNFs / copper wire structure has excellent low concentration detection capability, R 2 The value = 0.995 indicates a strong correlation between concentration and signal intensity. This result demonstrates that the sensor has the ability to efficiently capture and accurately quantify trace amounts of target compounds, providing a methodological basis for the ultrasensitive detection of EPA / DHA in fish oil.

[0058] 3. Repeatability and uniformity

[0059] (1) Repeatability test

[0060] According to the method of embodiment one, AgNPs / AuNFs / copper wire electrochemical sensors were prepared in five independent batches, and the same deposition time (Au NFs: 600 s; Ag NPs sputtering: 60 s), voltage (-1.5 V) and cleaning process were ensured for each batch. Ten sensors were taken from each batch, and each sensor was detected for 10 times, for a total of 50 groups of data. -5 A crystal violet (CV) standard solution of M was prepared, and 10 times of detection was performed for each batch of sensor, for a total of 50 groups of data. The detection conditions were 532 nm laser, 30 mW power, and integration time of 10 s. The sensor surface was cleaned with ethanol before each detection to eliminate the interference of residual molecules. The intensity of the 1620 cm -1 peak was recorded, and the relative standard deviation (RSD) between different batches and within the same batch was calculated. The results are shown in FIG. 16. Figure 6 As shown in FIG. 16(a), the RSD of the intensity of the 1620 cm -1 peak of the sensors in the five batches was 9.07%, indicating that the preparation process was stable and the performance was consistent between different batches. As shown in FIG. 16(b), the RSD of the 10 times of detection within the same batch was less than 10%, indicating that the repeatability of the sensors within the same batch was excellent. Figure 6

[0061] (2) Uniformity test

[0062] A 600x600 μm 2 region on the surface of the sensors in the same batch was randomly selected, and SERS spectra were collected at each point. The intensity of the 1620 cm -1 peak was extracted, and a two-dimensional intensity distribution map was generated. The RSD of the peak intensity of all points was calculated to evaluate the uniformity of the signal distribution on the surface of the sensors. The results are shown in FIG. 16. Figure 6 As shown in FIG. 16(c), the SERS intensity mapping showed that the signal intensity was uniformly distributed with weak changes and no obvious abnormal fluctuations. The results are shown in FIG. 16(d). Figure 6 As shown in FIG. 16(d), the RSD of the peak intensity of all detection points was 12.42%, indicating that the nanostructures were uniformly distributed on the surface of the sensors, and the density of the Raman "hot spots" was high and the spatial distribution was consistent.

[0063] In summary, through the repeated detection of CV by the sensors in five independent batches (RSD = 9.07%) and the intensity mapping analysis of the 600x600 μm 2 region on the surface, it was verified that the AgNPs / AuNFs / copper wire electrochemical sensors had excellent preparation repeatability and signal uniformity. This performance guaranteed the reliability and stability of the sensors in the detection of complex actual samples (such as fish oil), and provided high-confidence technical support for food safety supervision.

[0064] Embodiment four, influence of the physical conformation of the surface of the substrate material on the Raman enhancement effect of the electrochemical sensor.

[0065] ​The physical conformation of the surface of the substrate material (such as nanostructure shape, gap density) and the molecular structure of the detected substance jointly determine the distribution and intensity of the Raman "hot spot". Noble metals (gold, silver) are widely used due to the local surface plasmon resonance (LSPR) effect, among which gold has high stability and silver has stronger signal enhancement capability.

[0066] The substrate material AgNCs is synthesized by a typical hydrothermal method: prepare 75 mM CTAB solution (60°C, 10 min), 10 mM silver amine (AgOH·2NH3) solution and 1.5 mM glucose solution in a ratio of 1:1:2 by volume, and add them into a beaker in turn; then continuously stir the obtained mixture to obtain a uniform solution, place it in the inner liner of a reaction kettle, and put it into a 120°C oven for 8 hours, take it out after cooling, centrifuge and wash, and store it at 4°C for standby.

[0067] The preparation method of the substrate material Au@Ag NPs includes the following steps:

[0068] Step 1, the preparation method of Au NPs is as follows: gold nanometer solution is synthesized by citric acid reduction method, and silver-coated gold nanometer solution is prepared by seed-induced growth method. First, the synthesis of gold nanometer solution is as follows: 515 μL of 2wt% chloroauric acid solution is mixed in 100 mL of ultrapure water, heated to boiling, and 1 mL of 1wt% sodium citrate solution is added under continuous stirring. About 1 min later, the mixed solution suddenly turns purple red, indicating that gold nanoparticles have been synthesized. The solution is boiled for 30 min under continuous stirring, and then filtered through a 0.22 μm Millipore membrane when the solution temperature decreases to room temperature. AuNPs solution containing nanoparticles with a particle size of about 38 nm is obtained and stored at 4°C.

[0069] Step 2, 10 mL of the above Au NPs solution is taken in a conical flask, 1.5 mL of 0.1 mol / L ascorbic acid is added under magnetic stirring, and 3.5 mL of 10 -3 mol / L AgNO3 solution is continuously added at a rate of 1 drop / min under stirring. When the volume of silver nitrate increases, the color of Au@Ag NPs changes from wine red to bright orange, i.e. Au@Ag NPs with a silver shell thickness of 7 nm are synthesized.

[0070] The physical form of the combination of the three substrate materials prepared above and the detected molecules determines the Raman enhancement effect, such as Figure 7 (a) and Figure 7As shown in (b), the response intensity values of the characteristic peaks of DHA and EPA under the same detection conditions were AgNPs / AuNFs>Ag NCs>Au@Ag NPs, and the angular Ag NCs with sharp tips and the flower-like AuNFs with sharp 3D edges could both generate more SERS "hot spots" than the spherical Au@Ag NPs. Therefore, the physical conformation of the substrate material and the molecular structure of the detected substance combined significantly affect the density and intensity of the Raman "hot spots".

[0071] Specific embodiment five, a method for detecting the content of EPA and DHA in fish oil based on an Ag NPs / AuNFs / copper wire structure electrochemical sensor, comprising the following steps:

[0072] Step 1, sample methyl esterification pretreatment

[0073] Weigh 0.20 g of fish oil sample into a 50 mL reaction bottle, add 4 mL of 0.5 mol / L KOH-methanol solution, nitrogen purge for 3 min, then connect the reflux device, and boil in water bath for 20 min; then add 8 mL of 14% BF3-methanol solution, continue to boil for 20 min; after the reaction is completed, cool to room temperature, add 5 mL of isooctane and 25 mL of saturated NaCl solution, shake for 1 min, then extract in the refrigerator for 20 min; transfer the upper organic phase to a 5 mL glass tube, add anhydrous MgSO4 to remove water, filter to obtain the test solution containing methyl ester type DHA (DHA-Me) and methyl ester type EPA (EPA-Me) (the fish oil sample is methyl esterified by potassium hydroxide-methanol solution and BF3 catalyst, and the glyceride type EPA / DHA is converted into methyl ester type (EPA-Me / DHA-Me), eliminating the influence of molecular structure difference on SERS response);

[0074] Step 2, preparation of standard solution

[0075] Prepare single standard solutions of free DHA, EPA, triglyceride type (DHA-TG, EPA-TG), diglyceride type (DHA-DG, EPA-DG), and methyl ester type (DHA-Me, EPA-Me) respectively, with a concentration range of 10 -3 mg / mL to 10 -6 mg / mL, diluted with isooctane or ethanol to the target concentration;

[0076] Step 3, sample detection standard curve establishment

[0077] Collect the SERS spectra (800-1800 cm -1 ) of all standard solutions, extract the characteristic peaks (DHA-Me: 869 cm -1 , EPA-Me: 955 cm -1) peak height, half-peak width and peak area, and the standard curve between the concentration of DHA-Me and the signal intensity of the characteristic peak, and the standard curve between the concentration of EPA-Me and the signal intensity of the characteristic peak were constructed by PLS calibration model;

[0078] Step 4, detection of EPA and DHA content in fish oil

[0079] 5 mL of the sample obtained in step 1 was taken and stirred with the electrochemical sensor for 5 minutes, and after standing and drying, the SERS signal intensity of DHA-Me at 869 cm -1 and the SERS signal intensity of EPA-Me at 955 cm -1 were obtained by using 532 nm Raman laser excitation with a power of 30 mW and a collection time of 10 s. According to the relationship between the concentration of DHA-Me and the signal intensity of the characteristic peak, and the relationship between the concentration of EPA-Me and the signal intensity of the characteristic peak, the content of DHA and EPA in fish oil was quantitatively detected.

[0080] Specific embodiment six, the pretreatment optimization of the EPA and DHA content detection method in specific embodiment five.

[0081] 1. The sample to be tested: free DHA / EPA, triglyceride DHA / EPA, diglyceride DHA / EPA and methyl ester DHA / EPA were all diluted to 10 -3 mg / mL with isooctane. The methyl esterization treatment method is as follows: the fish oil sample is subjected to methyl esterization treatment (0.5 mol / L KOH-CH3OH solution and 14% BF3-CH3OH solution, boiling water bath reaction for 40 minutes), and different ester types (triglyceride type, diglyceride type) of DHA and EPA are all converted into methyl ester type, and methyl ester type DHA and EPA are obtained after extraction.

[0082] 2. Experimental method

[0083] 5 mL of the sample to be tested was taken and stirred with the electrochemical sensor for 5 minutes, and after standing and drying, the SERS signal intensity of DHA-Me at 869 cm -1 and the SERS signal intensity of EPA-Me at 955 cm -1 were obtained by using 532 nm Raman laser excitation with a power of 30 mW and a collection time of 10 s. Each sample was detected 5 times, and the average value was taken. According to the relationship between the concentration of DHA-Me and the signal intensity of the characteristic peak, and the relationship between the concentration of EPA-Me and the signal intensity of the characteristic peak, the content of DHA and EPA in fish oil was quantitatively detected.

[0084] 3. Analysis of experimental results

[0085] Firstly, by comparison, it was found that free DHA and methyl ester DHA Figure 8 (a) and free EPA and methyl ester EPA (Figure 8 c) The detected SERS spectra are consistent, and the peaks are clearly visible, especially at 869cm -1 955cm at EPA -1 By taking these two characteristic peaks and drawing a SERS intensity comparison bar graph, we can intuitively see the difference between the two molecular structures of DHA ( Figure 8 b) and EPA( Figure 8 d) There is almost no difference in the SERS response intensity at the characteristic peak, which indicates that the difference of one methyl group between free DHA and methyl ester DHA, and between free EPA and methyl ester EPA does not affect the change of SERS vibration spectrum.

[0086] Secondly, after SERS detection of triglyceride DHA, diglyceride DHA and methyl ester DHA, as well as triglyceride EPA, diglyceride EPA and methyl ester EPA, it was found that the peak shape characteristics of triglyceride DHA and diglyceride DHA were consistent with those of methyl ester DHA ( Figure 8 a), the peak shape characteristics of triglyceride EPA and diester EPA are also consistent with those of methyl ester EPA ( Figure 8 c). And through the 869cm of DHA -1 955cm at EPA -1 The response intensity histogram of the characteristic peak at ( Figure 8 As can be seen in Figures b and d, the response intensity is ranked from highest to lowest in the order of methyl ester > triglyceride > diglyceride. This indicates that direct detection of triglyceride and diglyceride DHA and EPA cannot accurately determine their true content. Therefore, further investigation is needed to explore the relationship between the SERS response intensities of the characteristic peaks of methyl ester DHA and EPA obtained after methyl esterification of these esters.

[0087] The results are as follows Figure 9 As shown in (a)-(d), the SERS response intensity of triglyceride / diglyceride DHA / EPA is enhanced after methylation. It is speculated that because the molecular volume and stereostructure of triglyceride and diglyceride types are large, it is very difficult for all three hydrocarbon ends to bind tightly to the SERS substrate. Therefore, the SERS response intensity of DHA and EPA obtained cannot truly reflect the total content of DHA and EPA in the triglyceride or diglyceride types. After the methylation reaction, the molecular stereostructure of the triglyceride or diglyceride type is broken and completely decomposed into DHA methyl ester or EPA methyl ester. The single-chain DHA methyl ester or EPA methyl ester is more likely to bind stably to the SERS substrate, thereby calibrating the true content of DHA and EPA in different ester types of oils. From the above, it can be seen that methylation treatment can eliminate the interference of molecular stereostructure on SERS binding and achieve uniform detection of target molecules.

[0088] Specific embodiment seven, sensitivity analysis of the EPA and DHA content detection method in specific embodiment five.

[0089] 1. Gradient dilution of methyl ester standard solution:

[0090] Methyl ester type DHA (DHA-Me) and EPA (EPA-Me) standard were dissolved in isooctane, and the initial concentration was set to 10.0 mg / mL. Using step-by-step dilution method, standard solutions with concentration gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 mg / mL were prepared in turn, a total of 6 gradients. Each dilution was completed by accurate pipetting (error <0.1%), ensuring the accuracy of the concentration.

[0091] 2. SERS detection and data acquisition:

[0092] Take 5 mL standard solution and AgNPs / AuNFs / copper wire electrochemical sensor stirring for 5 minutes, dry after standing, using 532 nm Raman laser excitation, power is 30 mW, collection time is 10 s, get DHA-Me in 869 cm -1 SERS signal intensity and EPA-Me in 955 cm -1 SERS signal intensity, each sample was repeated 5 times, take the average value. The results are shown in Figure 10 , Figure 10 (a) (DHA-Me) and Figure 10 (c) (EPA-Me) show the change of SERS signal intensity of characteristic peak under different concentrations, the signal intensity decreases monotonously with the decrease of concentration.

[0093] 3. Standard curve fitting:

[0094] With concentration logarithm (lgC) as abscissa and characteristic peak intensity (I) as ordinate, draw the calibration curve, as Figure 10 (b), Figure 10 (d). Further indicates the calibration curve of the concentration logarithm of methyl ester type DHA and methyl ester type EPA and the SERS intensity at their respective characteristic peaks, R 2 all up to 0.998. Therefore, according to the three times signal-to-noise ratio, the detection limit (LOD) of methyl ester type DHA and methyl ester type EPA is finally determined as 7.24 x 10 -7 mg / mL and 4.57 x 10 -7mg / mL, which indicated that the electrochemical sensor had excellent detection ability for DHA and EPA methyl esters and could meet the needs of quantitative analysis of the real content of DHA and EPA after the fish oil sample was subjected to methyl esterification treatment.

[0095] Specific embodiment eight, accuracy analysis of the standard curve established in specific embodiment seven.

[0096] 1. Sample preparation and data collection: 40 groups of DHA-Me and EPA-Me mixed standard solutions were prepared, and the concentration gradient was 1-12 mg / mL. The SERS spectra in the range of 800-1800 cm -1 were collected by using the electrochemical sensor based on Ag NPs / Au NFs / copper wire structure. The peak height, half-peak width and peak area parameters of the characteristic peaks of DHA-Me (869 cm -1 ) and EPA-Me (955 cm -1 ) were extracted as independent variables for quantitative analysis.

[0097] 2. Standard curve verification: 40 groups of data were randomly divided into a training set (30 groups) and a test set (10 groups). Based on the training set data, the standard curves of DHA-Me and EPA-Me were constructed by PLS calibration model, and the prediction ability of the standard curve was further verified by using the test set, and the correlation coefficient (R 2 ) and root mean square error (RMSE) of the predicted concentration and the actual concentration were calculated. As shown in Figure 11 (a), the determination coefficient (R 2 ) of DHA methyl ester (DHA-Me) was 0.985, and the root mean square error (RMSE) was 1.53; as shown in Figure 11 (b), the R 2 of EPA methyl ester (EPA-Me) was 0.988, and the RMSE was 1.48. The predicted concentration of DHA-Me and EPA-Me in the test set was highly consistent with the actual concentration. The prediction error was stable, and the relative standard deviation (RSD) was less than 5%. The high correlation coefficient (R 2 >0.98) and low RMSE (<1.55) indicated that the standard curve could accurately reflect the concentration change of DHA-Me and EPA-Me.

[0098] Specific embodiment nine, accuracy analysis of the EPA and DHA content detection method in specific embodiment five.

[0099] After 4 fish oil samples (Fish oil #1-#4) were subjected to methyl esterification treatment, the contents of DHA-Me and EPA-Me were detected by SERS and GC-MS, respectively, and the relative error of SERS results and GC-MS was <10% (Table 1, 1), which could verify that the sensor also had excellent accuracy.

[0100] Table 1 compares the performance of SERS method and GC-MS in DHA detection

[0101]

[0102] Table 2 compares the performance of SERS method and GC-MS in EPA detection

[0103]

[0104] The above GC-MS detection method is as follows: the specific experimental process of internal standard method is as follows: 0.20 g of fish oil sample is weighed in a 50 mL reaction bottle and 50 μL of internal standard solution (C11:0 triglyceride) is added, 4 mL of 0.5 mol / L potassium hydroxide-methanol solution is added, a small amount of zeolite is added, nitrogen is blown for 3 min (to prevent oxidation), the reflux device is immediately connected, and the reaction is carried out in boiling water for 20 min, and the bottle is shaken several times during the reaction; after 20 min of reaction, 8 mL of 14% boron trifluoride-methanol solution (to further make the methyl esterification reaction complete) is added, and the boiling reaction is continued for 20 min. After the reaction is completed, the sample is taken out and cooled to room temperature, 5 mL of isooctane and 25 mL of saturated sodium chloride solution are added, and the mixture is shaken for 1 min, and then saturated sodium chloride solution is added to the neck of the bottle, and the mixture is extracted in the refrigerator for 20 min. The upper organic phase is transferred to a 5 mL glass tube, anhydrous magnesium sulfate is added to remove water, and then the mixture is filtered through an organic filter membrane and loaded into a new test tube. 100 μL of the above methyl esterified organic phase is taken, 900 μL of isooctane is added for dilution, the mixture is mixed, and then the mixture is detected by GC-MS.

[0105] The chromatographic conditions are as follows: DB-23 capillary column (60 m x 0.25 mm i.d. x 0.25 μm film thickness) is used. The carrier gas is helium, and the flow rate is 1.0 mL / min. The injection mode is split mode, and the split ratio is 30:1. The initial temperature of the column oven is 60°C, which is maintained for 3 min, then increased to 160°C at a rate of 15°C / min, maintained for 0 min, then increased to 210°C at a rate of 8°C / min, maintained for 0 min, and finally increased to 230°C at a rate of 3.15°C / min, maintained for 10 min.

[0106] The mass spectrometry conditions are as follows: an electron impact source of 70 eV is used. The interface temperature is 270°C, the ion source temperature is 230°C, and the quadrupole rod temperature is 150°C. The scanning range is m / z 35-500.

[0107] The detection limit of traditional GC-MS method for DHA and EPA is usually 1.5-2.0 ng / mL, while the LOD of the present method is reduced to sub-nanogram level, with about 3-4 times higher sensitivity. GC-MS requires complex pretreatment (derivatization, chromatographic separation) and long analysis time, while the present method shortens the total time consumption through methyl esterification combined with SERS detection. SERS method has lower detection limit, shorter sample processing time (methyl esterification + detection only needs 40 minutes), and does not require complex instruments, suitable for on-site rapid screening.

[0108] In summary, based on the AgNPs / AuNFs / copper wire structure electrochemical sensor, the standard curve (R 2 = 0.998) was constructed by gradient dilution of methyl esterification standard solution, realizing high sensitivity detection of DHA-Me and EPA-Me (LOD is 0.724 ng / mL and 0.457 ng / mL, respectively). The detection limit is significantly lower than that of traditional GC-MS method, and does not require complex instruments and lengthy process, providing an efficient solution for rapid and accurate quantification of EPA and DHA in fish oil.

[0109] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present application shall also fall within the protection scope of the present application.

Claims

1. A method for preparing an electrochemical sensor based on an Ag NPs / Au NFs / copper wire structure, characterized in that The following steps are involved: Step 1: synthesize Au NFs on copper wire by electrochemical deposition to obtain an AuNFs / copper wire structure; Step 2: Ag NPs were directly sputtered on the surface of the Au NFs / copper wire structure by magnetron sputtering to obtain an electrochemical sensor based on the Ag NPs / AuNFs / copper wire structure.

2. The method for preparing an electrochemical sensor based on an Ag NPs / Au NFs / copper wire structure according to claim 1, characterized in that Step 1 is as follows: using electrochemical deposition, copper wire is used as the working electrode, Ag / AgCl electrode is used as the reference electrode, and platinum wire is used as the counter electrode. The chronoamperometric method is used in an ethanol electrolyte containing 8-12mM HAuCl4 and 0.2-0.3M H2SO4, and a deposition voltage of -1.5V is applied for a deposition time of 450 to 650s to grow Au nanoflowers on the surface of the copper wire. After the reaction is completed, rinse with deionized water and dry to obtain an AuNFs / copper wire structure.

3. The method for preparing an electrochemical sensor based on an Ag NPs / Au NFs / copper wire structure according to claim 2, wherein: The deposition time is 450 to 650 seconds.

4. The method for preparing an electrochemical sensor based on an Ag NPs / Au NFs / copper wire structure according to claim 1, characterized in that Step 2 is as follows: Ag NPs are deposited on the surface of the Au NFs / copper wire structure by magnetron sputtering, and the sputtering time is controlled to be 30 to 120 s to obtain an electrochemical sensor based on the Ag-NPs / Au-NF / copper wire structure.

5. The method for preparing an electrochemical sensor based on an Ag NPs / Au NFs / copper wire structure according to claim 4, wherein: The sputtering time is 60s.

6. A method for detecting EPA and DHA in fish oil using the electrochemical sensor according to any one of claims 1 to 5, characterized in that The following steps are involved: Step 1: Pre-treatment of fish oil sample for methyl esterification Weigh 0.20g of fish oil sample into a reaction flask, add 4mL of 0.5mol / LKOH-methanol solution, purge with nitrogen, connect a reflux device, and react in a boiling water bath for 15-25min; then add 8mL of 14vt% BF3-methanol solution and continue boiling for 15-25min; after the reaction, cool to room temperature, add 5mL of isooctane and 25mL of saturated NaCl solution, shake for 0.5-2min, and place in a refrigerator for extraction; transfer the upper organic phase to a glass tube, add anhydrous MgSO4 to remove water, and filter to obtain a test solution containing methyl ester DHA and methyl ester EPA; wherein the mixing ratio of fish oil sample, KOH-methanol solution, BF3-methanol solution, isooctane and saturated NaCl solution is 0.2g:4mL:8mL:5mL:25mL; Step 2: Establish a standard curve Prepare standard solutions of DHA-Me and EPA-Me respectively, with concentrations ranging from 10 -3 mg / mL to 10 -6 mg / mL, diluted with isooctane or ethanol to the target concentration, collected SERS spectra of all standard solutions, extracted parameters such as peak height, half-peak width and peak area of ​​the characteristic peaks, and constructed standard curves between methyl ester DHA concentration and characteristic peak signal intensity and between methyl ester EPA concentration and characteristic peak signal intensity using the PLS calibration model; Step 3: Detection of EPA and DHA content in fish oil Take 5 mL of the test solution obtained in step 1 and stir it with the electrochemical sensor for 1-10 minutes. After standing and drying, use a Raman detector to detect DHA-Me at 869 cm -1 The SERS signal intensity of EPA-Me at 955 cm -1 The SERS signal intensity under the condition of δ=0.04 was used, and the content of DHA and EPA in the fish oil sample was calculated according to the relationship between DHA-Me and its characteristic peak signal intensity and the relationship between EPA-Me concentration and its characteristic peak signal intensity.

7. The method for detecting EPA and DHA in fish oil according to claim 6, wherein: The detection conditions of the Raman detector are as follows: using 532 nm Raman laser excitation, a power of 30 mW, and an acquisition time of 10 s.

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