Method for detecting phthalic acid ester substances in milk based on Au-Ag-IP6 SERS (Surface Enhanced Raman Scattering) substrate

Abstract

The invention belongs to the technical field of food safety detection, and particularly discloses a method for detecting PAEs in milk based on an Au-Ag-IP6SERS substrate, and the method comprises the following steps: preparing gold nanoparticles; au (at) Ag (at) IP6 nanoparticles are prepared; preparing an Au (at) Ag (at) IP6NPs SERS substrate; preparing a phthalic hydrazide standard substance and a phthalic acid ester standard substance; preparing the phthalylhydrazine generated by the reaction of the phthalic acid ester; preparing a milk standard sample and performing pretreatment; preparing an SERS sample to be detected; and performing Raman detection. According to the method, more than thirty PAEs can be uniformly converted into the phthalylhydrazine molecules, so that not only is the non-water-soluble problem of the PAEs solved, but also the total amount of all PAEs can be detected at the same time; compared with a detection method taking other gold and silver nanoparticles as an SERS substrate, the scheme has the advantages that the phthalylhydrazine molecules can be specifically captured, the sensitivity is higher, and the detection limit is lower. After the SERS substrate is prepared, PAEs can be detected only through simple pretreatment, and PAEs in milk can be rapidly detected within 15 min.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and specifically relates to a method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate. Background Art

[0002] Phthalates (PAEs), as a type of plasticizer, are widely used in the medical, industrial and human life fields [1]. They are often added to products such as food packaging, toys, cosmetics and medical devices to make plastics elastic and improve their strength and durability. The global annual consumption of PAEs is close to 100,000 tons. China is the largest plasticizer market, producing more than 45,000 tons of PAEs each year, accounting for 45% of the global total. However, PAEs are extremely harmful to the human body. They enter the human body through breathing, oral cavity, skin contact, etc., are adsorbed by solid particles, and eventually accumulate in the body. It is known that PAEs have been detected in human tissues and secretions (such as lungs, colon, breast milk and placenta). PAEs are environmental estrogens with reproductive and developmental toxicity. They have the potential to cause carcinogenesis, teratogenesis, and mutagenesis. They can cause systemic dysfunction, affect the endocrine system, affect the development of the nervous system of newborns, and cause precocious puberty in females. They may also affect embryonic development and uterine mucosal tissue hyperplasia through placental lipid and zinc metabolism, and have a great impact on the developing male reproductive system[5]. There are many types of PAEs, more than dozens of them. In China, only four PAEs, di(2-ethylhexyl) phthalate (DEHP), diallyl phthalate (DAP), diisononyl phthalate (DINP), and dibutyl phthalate (DBP), are allowed to be used in food packaging. PAEs should be banned in oily foods and infant foods[6]. The maximum residue levels of DEHP, DINP, and DBP are 1.5, 9.0, and 0.3 mg kg-1, respectively. However, due to the fat-soluble characteristics of PAEs, PAEs will still migrate from plastics into food, especially packaged milk. Among them, DBP and DEHP are the two most prominent species in packaged milk, accounting for about 47% of the total PAEs content in milk. Therefore, it is crucial to develop a simple and rapid method to detect PAEs molecules in packaged milk.

[0003] Surface enhanced Raman spectroscopy (SERS) is a spectroscopic technique that can provide rich chemical fingerprint information of the object to be tested at the single-molecule scale. Since Fleischmann et al. discovered in 1974 that the Raman signal of pyridine molecules adsorbed on the rough silver electrode surface was greatly improved, SERS technology has developed rapidly. When molecules are adsorbed on rough metal surfaces (such as silver, copper, gold, etc.), due to the effect of light, the plasma energy on the metal surface can be excited to a high energy level, and coupled with the electric field of the light wave, and resonate, so that the local electromagnetic field on the metal surface is significantly enhanced. This molecular signal on the rough silver surface is as high as 106 times that in the solution, making the originally weak Raman signal detectable. SERS technology not only has simple pre-treatment, but also has the advantages of high sensitivity, high selectivity, non-destructiveness and rapid analysis. It has a wide range of applications in many fields, including biomedicine, environmental monitoring, food safety, etc. At present, the detection methods for phthalates (PAEs) include traditional analytical methods, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and high performance liquid chromatography (HPLC), which have high detection sensitivity and accuracy, but at the same time, its pretreatment process is very complicated. The quantitative analysis of PAEs by selective identification using biosensor technology requires regular replacement and maintenance of sensors, which is time-consuming and labor-intensive.

[0004] Therefore, it is very necessary to develop a low-cost, simple, rapid and highly sensitive method for detecting PAEs in packaged milk. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a method for detecting phthalates (PAEs) in packaged milk based on Au@Ag@IP6 SERS substrate. Au@Ag@IP6 is used as the SERS substrate. Due to the strong metal complexation between IP6 and metal nanoparticles and the hydrogen bonding between IP6 and phthalohydrazide (PHAH2), phthalohydrazide molecules are tightly adsorbed in the "hot spot" area of ​​metal nanoparticles, thereby achieving quantitative detection of PHAH2 and indirectly detecting PAEs. This study provides a new diagnostic approach for whether the plasticizer in milk exceeds the standard. More importantly, it is expected to become a general technology for accurately detecting PAEs in other systems such as edible oil and urine.

[0006] In order to achieve the above objectives, one of the technical solutions of the present invention is: a method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate, comprising the following steps:

[0007] S1: Preparation of gold nanoparticles;

[0008] S2: Preparation of Au@Ag@IP6 nanoparticles: The gold nanoparticles prepared in step S1 were used as seeds to prepare Au@Ag nanoparticles capped with inositol hexaphosphate (IP6) (Au@Ag@IP6 NPs);

[0009] S3: Preparation of Au@Ag@IP6 NPs SERS substrate: The Au@Ag@IP6 NPs prepared in step S2 were evenly spread on the gold sheet and dried;

[0010] S4: Preparation of phthalic acid hydrazide standard and phthalic acid ester (PAEs) standard;

[0011] S5: preparing phthalic acid hydrazide generated by the reaction of phthalic acid esters (PAEs): diluting the phthalic acid esters (PAEs) standard obtained in step S4, adding hydrazine hydrate, and reacting in a water bath to obtain phthalic acid hydrazide;

[0012] S6: preparing a milk spiked sample and pre-treating it: adding the phthalate ester (PAEs) standard prepared in step S4 to the milk to prepare a milk spiked sample, adding ethanol to the milk spiked sample, ultrasonically centrifuging, taking the supernatant through a membrane and drying, adding deionized water to redissolve, extracting the phthalate ester with n-hexane, adding ethanol to redissolve the phthalate ester, and then adding hydrazine hydrate to react under water bath conditions;

[0013] S7: Preparation of SERS samples: Different concentrations of phthalic hydrazide standards, different concentrations of phthalic hydrazide generated by the reaction of phthalic esters (PAEs), and spiked simulation detection of phthalic esters (PAEs) in milk system were added to the Au@Ag@IP6 NPs SERS substrate prepared in S3;

[0014] S8: Raman detection: Perform Raman detection on the SERS sample prepared in step S7, and compare and analyze the measured SERS spectra.

[0015] The analysis results showed that they all had characteristic spectral peaks at 712, 1026 and 1376 cm-1, which can be attributed to CN vibration, aromatic ring vibration and ring stretching vibration peaks of 2,3-naphthylidene compounds respectively. The results showed that the quantitative detection of phthalates (PAEs) in milk can be achieved by converting (PAEs) in milk into phthaloyl hydrazide using hydrazine hydrate.

[0016] In a preferred embodiment of the present invention, the method for preparing gold nanoparticles in step S1 is as follows: 150-250 mL of 0.05-0.015 wt% chloroauric acid aqueous solution is boiled for 5-15 min, 1-3 mL of 0.5-0.15 wt% sodium citrate is added, stirring is continued for 10-20 min, and the mixture is cooled to room temperature to obtain gold nanoparticles having a particle size of 35-55 nm and an ultraviolet absorption wavelength mainly in the range of 500-550 nm.

[0017] In a preferred embodiment of the present invention, the preparation method of Au@Ag@IP6 NPs in step S2 comprises taking 4-7 mL of 15-25 mM silver nitrate aqueous solution, adding 7-10 mL of 0.5-1.5 mM IP6 aqueous solution thereto; taking 50-60 mL of deionized water, adding 5-15 mL of the gold nanoparticles prepared in step S1, adding 2-3 mL of 0.5-1.5 wt % sodium citrate and 0.5-1.5 wt % ascorbic acid while stirring, and 8-12 mL of AgNO3 is added at a speed of 160-220 μL / min with stirring to obtain Au@Ag@IP6 NPs with a particle size of 100-200 nm.

[0018] The role of IP6 is to complex with strong metals to form an ultra-thin coating on the Ag shell to adsorb phthaloyl hydrazide molecules and obtain super strong SERS enhancement.

[0019] In a preferred embodiment of the present invention, the gold sheet in step S3 needs to be soaked in acetone for 20-40 minutes, then soaked in piranha solution for 20-40 minutes to remove organic matter, and finally rinsed with ultrapure water for several times until clean.

[0020] In a preferred embodiment of the present invention, the preparation method of the phthalic acid hydrazide standard in step S4 is to dilute the phthalic acid hydrazide to a concentration of 0.1-1000 mg L using ethanol and water in a volume ratio of 1:1. -1 The preparation method of the phthalic acid hydrazide standard and the phthalic acid ester (PAEs) standard is to dilute the stock solution of the phthalic acid ester (PAEs) standard with ethanol to a concentration of 0.1-1000 mg L -1 .

[0021] In a preferred embodiment of the present invention, in step S5, the volume ratio of the phthalate ester (PAEs) standard to hydrazine hydrate is (3-5):1, the water bath temperature is 75-85°C, and the reaction time is 5-15 min.

[0022] PAEs are reacted into phthaloyl hydrazide in ethanol, and hydrazine hydrate is used to convert water-insoluble phthalate compounds into water-soluble phthaloyl hydrazide. The converted phthaloyl hydrazide is adsorbed on the surface of Au@Ag@IP6NPs and enters the "SERS hotspot".

[0023] In a preferred embodiment of the present invention, step S6 simulates the environment in which plastic migrates into milk by adding spikes to milk.

[0024] In a preferred embodiment of the present invention, the signal molecule detected by Raman in step S7 is a phthalic acid hydrazide molecule.

[0025] In a preferred embodiment of the present invention, the step S7 includes a portable Raman spectrometer or other types of Raman spectrometers.

[0026] In a preferred embodiment of the present invention, the excitation wavelength of Raman detection in step S7 is 785 nm, the power is 500 mw, and the time is 150 ms.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention constructs a platform for detecting phthalates in milk based on SERS, wherein the Au@Ag@IP6NPs SERS substrate provides SERS "hot spots" for closer adsorption of phthaloyl hydrazide molecules, so that the present invention is fast and convenient, highly sensitive and highly specific, and can achieve fingerprint recognition. This method can achieve rapid detection of phthalate molecules in milk within 15 minutes;

[0029] 2. The present invention can uniformly convert more than 30 types of phthalates into phthaloyl hydrazide, which not only solves the problem of non-water solubility of phthalates, but also can realize the simultaneous detection of multiple types of phthalates and the detection of the total amount of all types of phthalates;

[0030] 3. Compared with the detection method using gold sol as SERS substrate, the present invention has higher detection sensitivity and lower detection limit, and can better capture phthalic acid hydrazide molecules. After the SERS substrate is prepared, only simple pretreatment is required to detect phthalate, thereby realizing rapid measurement of the Raman signal of phthalate in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the principle of the present invention;

[0032] Figure 2 This is a comparison of the surface enhanced Raman spectra of phthalic hydrazide standard and phthalic hydrazide reacted with PAEs standard;

[0033] Figure 3 The concentration is 10 μg L -1 , 50 μg L -1 , 100 μg L -1 , 150 μg L -1 , 200 μg L -1 Surface enhanced Raman spectrum of phthalic acid hydrazide standard;

[0034] Figure 4 The concentration is 10 μg L -1 , 50 μg L -1 , 100 μg L -1 , 150 μg L -1 , 200 μg L -1 Surface enhanced Raman spectrum of phthalic acid hydrazide generated by reaction of a mixed sample of six phthalate standards with hydrazine hydrate;

[0035] Figure 5 The concentration of the mixed sample of six phthalates before reaction and after reaction at 1376 cm -1 Relationship diagram of peak area at ;

[0036] Figure 6 The actual milk system was spiked with a concentration of 100 μg L -1 Six different PAEs and 100 μg L -1 Surface enhanced Raman spectra of mixed samples of six PAEs and a blank sample of milk;

[0037] Figure 7 This is the surface enhanced Raman spectrum of a mixed sample of six PAEs spiked with different concentrations in an actual milk system. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0039] Example 1

[0040] Preparation of gold nanoparticles (AuNPs): 200 mL of chloroauric acid aqueous solution (0.01 wt%) was added to a 250 mL round-bottom flask and stirred until boiling. After boiling for 10 min, 2 mL of sodium citrate (1 wt%) was added and stirred for 15 min.

[0041] Preparation of Au@Ag@IP6 nanoparticles (Au@Ag@IP6NPs): Prepare IP6-capped Au@Ag nanoparticles. Prepare silver nitrate aqueous solution in advance, take 5.6mL of 20mM silver nitrate aqueous solution to a 15mL centrifuge tube, and add 8.4mL of 1mMIP6 aqueous solution to it. First, take 55mL of Milli-Q water, add 10mL of 45nm gold seeds prepared by S1, add 2.4mL of sodium citrate (1wt%) and ascorbic acid (1wt%) while stirring, and use a stepper motor to add 9.6mL of AgNO3 to the round-bottom flask at a speed of 200μL / min. Stir until the nitrate is added to obtain Au@Ag@IP6NPs with a particle size of 150nm;

[0042] Preparation of Au@Ag@IP6NPs SERS substrate: Take 5mL of prepared 150nm Au@Ag@IP6NPs, centrifuge at 3500rpm for 5minutes, remove the supernatant, wash it again with 1mM IP6, and finally adjust the volume to 100μL. Take 3μL on the gold sheet and dry it naturally to obtain a uniform SERS substrate;

[0043] Preparation of phthalic acid hydrazide standard and six different PAEs standards: Wash the glass bottle with ethanol and dry it. Weigh 20 mg of phthalic acid hydrazide powder into a glass bottle, add 20 mL of ethanol and water (ethanol: water = 1:1) to prepare a 1000 ppm standard. Dilute this standard step by step to 100 mg L -1 , 10mg L -1 , 1mg L -1 , 100 μg L -1 , 10 μg L -1 Six PAEs standards were prepared, including di(2-ethylhexyl) phthalate (DEHP), diallyl phthalate (DAP), diisononyl phthalate (DINP), dibutyl phthalate (DBP), di-n-octyl phthalate (DNOP), dicyclohexyl phthalate (DCHP) and dihexyl phthalate (DHP). 20 mg of each stock solution was weighed into six glass bottles, and 20 ml of ethanol was added to prepare 1000 mg L -1 The standard was then diluted to 100 mg L -1 , 10mg L -1 , 1mg L -1 , 100 μg L -1 , 10 μg L -1 Weigh 3 mg of each of the six PAEs standard solutions and mix them in a glass bottle. Add 18 ml of ethanol to prepare 1000 mg L-1 The standard solution was then diluted to 100 mg L -1 , 10mg L -1 , 1mgL -1 , 100 μg L -1 , 10 μg L -1 .

[0044] Reaction of PAEs to phthaloyl hydrazide: Absorb 10 μg L -1 , 50 μg L -1 , 100 μg L -1 , 150μgL -1 , 200 μg L -1 1 mL of each of the six PAEs standard solution mixtures were placed in five 3 mL small glass bottles, and 250 μL of hydrazine hydrate was added to each of them. The mixture was reacted in a water bath at 60°C for 10 minutes to obtain phthaloyl hydrazide.

[0045] Preparation and pretreatment of milk spiked samples: Preparation of 50 μg L -1 , 100 μg L -1 , 150 μg L -1 Milk spiked with standard sample (weigh 0.2g milk, add 10μL, 20μL, 30μL 1mg L -1 The milk spiked sample was pre-treated by adding 4 mL of ethanol, ultrasonicating for 10 min, centrifuging at 5000 rpm / min for 2 min, removing the supernatant, filtering through a membrane, drying, adding 1 mL of deionized water for reconstitution, extracting with 400 μL of n-hexane, drying, adding 1 mL of ethanol for reconstitution, adding 250 μL of hydrazine hydrate, and water bathing at 60°C for 10 minutes.

[0046] Preparation of SERS samples: 3 μL of phthalic acid hydrazide standards with different concentrations and phthalic acid hydrazide with different concentrations reacted with PAEs were added to the Au@Ag@IP6NPs SERS substrate prepared above, and the spiked simulation detection of phthalates in the milk system was performed;

[0047] The prepared SERS samples were subjected to Raman detection using a portable Raman detector, with an excitation light wavelength of 785 nm, a power of 500 mw, and a time of 150 ms; and the measured SERS spectra were compared and analyzed.

[0048] 100 μg L -1 SERS spectra of phthalic acid hydrazide reacted with PAEs and phthalic acid hydrazide standard at this concentration ( Figure 2 ) Comparative analysis found that they are all at 712cm-1 、1026cm -1 and 1376cm -1 Characteristic spectral peaks appear at , which can be attributed to CN vibration, aromatic ring vibration and ring stretching vibration peaks of 2,3-naphthyridine compounds.

[0049] The results showed that the quantitative detection of PAEs in milk could be achieved by converting PAEs in milk into phthaloyl hydrazide using hydrazine hydrate.

[0050] The schematic diagram of surface Raman spectroscopy test of phthaloyl hydrazide molecules is shown in the figure. Figure 1 As shown; for 100 μg L -1 Surface enhanced Raman spectroscopy was performed on phthalic hydrazide generated by the reaction of six different phthalate standard solutions with hydrazine hydrate and phthalic hydrazide standards of the same concentration. The results are as follows: Figure 2 As shown; for a concentration of 10 μg L -1 , 50 μg L -1 , 100μgL -1 , 150 μg L -1 , 200 μg L -1 The surface enhanced Raman spectroscopy test of phthalic acid hydrazide standard was carried out, and the results were as follows Figure 3 As shown; for a concentration of 10 μg L -1 , 50 μg L -1 , 100 μg L -1 , 150 μg L -1 , 200 μg L -1 The surface enhanced Raman spectroscopy test of the phthalic acid hydrazide generated by the reaction of the six phthalate standard solutions with hydrazine hydrate was performed. The results are as follows Figure 4 As shown in the figure, the concentration of the mixed sample of six phthalates before reaction and after reaction at 1376cm -1 The relationship between the peak area Figure 5 As shown; the actual milk system was spiked with a concentration of 100 μg L -1 Six different phthalates and their mixed samples and milk blank samples were tested by surface enhanced Raman spectroscopy. The results are as follows Figure 6 As shown; for the spiked concentration in milk of 50 μg L -1 , 100 μg L -1 and 150 μg L -1 The surface enhanced Raman spectroscopy of the six phthalate mixed samples was tested. Figure 7 shown.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate, characterized in that: The following steps are involved: S1: Preparation of gold nanoparticles; S2: Preparation of Au@Ag@IP6 nanoparticles: The gold nanoparticles prepared in step S1 were used as seeds to prepare inositol hexaphosphate-capped Au@Ag nanoparticles Au@Ag@IP6 NPs; S3: Preparation of Au@Ag@IP6 NPs SERS substrate: The Au@Ag@IP6 NPs prepared in step S2 were evenly spread on the gold sheet and dried; S4: Preparation of phthalic acid hydrazide standards and phthalate standards; S5: preparing phthalic acid hydrazide generated by the reaction of phthalic acid esters: diluting the phthalic acid ester standard obtained in step S4, adding hydrazine hydrate, and reacting in a water bath to obtain phthalic acid hydrazide; S6: preparing a milk spiked sample and pre-treating it: adding the phthalate standard prepared in step S4 to the milk to prepare a milk spiked sample, adding ethanol to the milk spiked sample, performing ultrasonic centrifugation, taking the supernatant, passing it through a membrane and drying it, adding deionized water to redissolve it, extracting the phthalate with n-hexane, adding ethanol to redissolve the phthalate, and then adding hydrazine hydrate to react under water bath conditions; S7: Preparation of SERS samples: Different concentrations of phthalic acid hydrazide standard, different concentrations of phthalic acid hydrazide generated by the reaction of phthalic acid esters, and spiked simulation detection of phthalic acid esters in milk system were added to the Au@Ag@IP6 NPs SERS substrate prepared in S3; S8: Raman detection: Perform Raman detection on the SERS sample prepared in step S7, and compare and analyze the measured SERS spectra.

2. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: The method for preparing gold nanoparticles in step S1 is as follows: 150-250 mL of 0.05-0.015 wt% chloroauric acid aqueous solution is boiled for 5-15 min, 1-3 mL of 0.5-0.15 wt% sodium citrate is added, stirring is continued for 10-20 min, and the mixture is cooled to room temperature to obtain gold nanoparticles with a particle size of 50-60 nm and an ultraviolet absorption wavelength of 500 nm-550 nm.

3. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: The preparation method of Au@Ag@IP6 NPs in step S2 includes taking 4-7mL of 15-25mM silver nitrate aqueous solution, adding 7-10mL of 0.5-1.5mM IP6 aqueous solution thereto; taking 50-60mL of deionized water, adding 5-15mL of the gold nanoparticles prepared in step S1, adding 2-3mL of 0.5-1.5wt% sodium citrate and 0.5-1.5wt% ascorbic acid while stirring, and 8-12mL of AgNO3 is added at a speed of 160-220μL / min to obtain Au@Ag@IP6 NPs with a particle size of 100-200nm.

4. Detection of phthalates in milk based on Au@Ag@IP6 SERS substrate as claimed in claim 1 A qualitative method characterized in that The gold sheet in step S3 needs to be soaked in acetone for 20-40 minutes, then soaked in piranha solution for 20-40 minutes to remove organic matter, and finally rinsed with ultrapure water several times until clean.

5. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: The preparation method of the phthalic acid hydrazide standard in step S4 is to dilute the phthalic acid hydrazide to a concentration of 0.1-1000 mg L using ethanol and water in a volume ratio of 1:

1. -1 The preparation method of the phthalic acid ester standard is to dilute the stock solution of the phthalic acid ester standard with ethanol to a concentration of 0.1-1000 mg L -1 .

6. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: In step S5, the volume ratio of the phthalate standard to the hydrazine hydrate is (3-5):

1.

7. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: The water bath temperature in steps S5 and S6 is 75-85° C., and the reaction time is 5-15 min.

8. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: The signal molecule detected by Raman in step S7 is a phthaloyl hydrazide molecule.

9. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: In step S7, the Raman detection is performed using a portable Raman spectrometer or other types of Raman spectrometers.

10. The method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate according to claim 1, characterized in that: The excitation wavelength of Raman detection in step S7 is 785 nm, the power is 500 mw, and the time is 150 ms.

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