CN112903657B

CN112903657B - Surface enhanced Raman spectroscopy detection method for melamine and formaldehyde

Info

Publication number: CN112903657B
Application number: CN202110120617.7A
Authority: CN
Inventors: 胡玉玲; 葛琨; 李攻科
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-01-28
Filing date: 2021-01-28
Publication date: 2022-06-24
Anticipated expiration: 2041-01-28
Also published as: CN112903657A

Abstract

The invention discloses a surface enhanced Raman spectroscopy detection method of melamine and formaldehyde. The formaldehyde SERS substrate provided by the invention has the advantages of specific selectivity, high sensitivity, signal stability and reproducibility. The preparation method of the formaldehyde SERS substrate is simple, the cost is low, and the prepared detection kit is convenient to carry and easy to store. In the SERS detection method of melamine and formaldehyde, two substrates are adopted, and the detection of melamine and formaldehyde can be realized by preparing the liquid to be detected once.

Description

Surface enhanced Raman spectroscopy detection method for melamine and formaldehyde

Technical Field

The invention belongs to the technical field of chemical detection, and particularly relates to a surface enhanced Raman spectroscopy detection method of melamine and formaldehyde.

Background

Food safety issues arising from the migration of hazards within food contact materials into food products are of increasing concern. Melamine tableware is formed by polymerizing melamine monomer and formaldehyde monomer according to a certain proportion, and is widely used due to the advantages of low price, simple manufacture, firmness, durability, easy cleaning and the like. However, melamine and formaldehyde in melamine tableware have the risk of migrating into food, and it is very important to determine the migration risk of melamine and formaldehyde in melamine tableware simultaneously. In addition, some merchants have replaced melamine resins with urea formaldehyde resins in order to reduce production costs, which greatly increases consumer food risk. Therefore, the establishment of an accurate, efficient and economic method for analyzing melamine and formaldehyde in melamine tableware is of great significance.

At present, the detection method of the hazardous substances in domestic food contact materials is mainly high performance liquid chromatography, and the method has the inconvenience of respective detection, time consumption, high cost and the like, so that the requirement of on-site rapid detection cannot be met. Surface-enhanced Raman spectroscopy (SERS, abbreviated in english) has many advantages such as high sensitivity, strong specificity, and rapid detection, and is very easy to realize on-site rapid detection. However, in the actual detection process, the reproducibility and selectivity of SERS analysis and detection are all deficient, and the detection of actual complex matrix samples cannot be met.

Disclosure of Invention

The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides the formaldehyde SERS substrate which can realize quantitative detection of formaldehyde and has better selectivity and reproducibility.

The invention also provides a preparation method of the formaldehyde SERS substrate.

The invention also provides a method for SERS detection of formaldehyde.

The invention also provides a SERS detection method of melamine and formaldehyde.

The invention also provides a detection kit containing the formaldehyde SERS substrate.

According to one aspect of the invention, the formaldehyde SERS substrate comprises a substrate and a composite material loaded on the substrate, wherein the composite material comprises reduced graphene oxide, silver nanoparticles and 3-methyl-2-benzothiazolinone hydrazone hydrochloride.

According to the invention, a pi-pi bond can be formed between the 3-methyl-2-benzothiazolinone hydrazone hydrochloride and the reduced graphene oxide, so that the 3-methyl-2-benzothiazolinone hydrazone hydrochloride is modified on the surfaces of the reduced graphene oxide and silver nanoparticles, and the selective detection of formaldehyde is realized. On the other hand, the reduced graphene oxide is negatively charged, the tail end of the 3-methyl-2-benzothiazolinone hydrazone hydrochloride molecule is positively charged due to the amino group, the two molecules have an electrostatic effect, the bonding force of the two molecules is further enhanced, and more 3-methyl-2-benzothiazolinone hydrazone hydrochloride is modified on the surface of the reduced graphene oxide, so that the selectivity and the detection sensitivity of the formaldehyde SERS substrate to formaldehyde are improved; the reduced graphene oxide also has natural chemical inertness, so that the stability of the silver nanoparticles and the stability of the 3-methyl-2-benzothiazolinone hydrazone hydrochloride can be protected, and the detection specificity of the formaldehyde SERS substrate can be stably maintained for a long time.

In some embodiments of the present invention, the mass ratio of the reduced graphene oxide to the silver nanoparticles to the 3-methyl-2-benzothiazolinone hydrazone hydrochloride is (0.1 to 0.15) mg/mL: (1.0-1.5) nmol/L: 0.5 mg/mL.

In some preferred embodiments of the present invention, the substrate is chromatography paper.

According to another aspect of the invention, the preparation method of the formaldehyde SERS substrate is provided, and the composite material is loaded on the surface of the substrate.

In some embodiments of the present invention, the preparation method of the formaldehyde SERS substrate is more specifically performed by: and loading the reduced graphene oxide and the silver nanoparticles on the surface of the substrate, and then loading the 3-methyl-2-benzothiazolinone hydrazone hydrochloride.

According to another aspect of the present invention, a method for SERS detection of formaldehyde is provided, which includes the following steps:

s1: the relation between the peak value and the concentration of the characteristic Raman displacement position of the formaldehyde is obtained by adopting the formaldehyde SERS substrate;

s2: and adding the to-be-detected product on the formaldehyde SERS substrate, detecting to obtain the peak value of the characteristic Raman displacement position of the formaldehyde, and calculating by combining the relation between the peak value of the characteristic Raman displacement position of the formaldehyde and the concentration to obtain the concentration of the formaldehyde in the to-be-detected product.

In some embodiments of the present invention, the sample is obtained by immersing melamine tableware in a migration solution.

In some preferred embodiments of the present invention, the migration solution is at least one selected from the group consisting of water, ethanol, and acetic acid. The migration liquid is used for migrating formaldehyde in melamine tableware, is simple to operate and is suitable for SERS detection of formaldehyde.

In some more preferred embodiments of the present invention, the ethanol has a volume concentration of 10% to 12%; more preferably, the mass concentration of acetic acid is 3% to 5%.

According to another aspect of the present invention, a method for SERS detection of melamine and formaldehyde is provided, which includes the following steps:

s1: the relation between the peak value and the concentration of the characteristic Raman displacement position of the formaldehyde is obtained by adopting the formaldehyde SERS substrate, and the relation between the peak value and the concentration of the characteristic Raman displacement position of the melamine is obtained by adopting the melamine SERS substrate;

s2: adding a to-be-detected article to the formaldehyde SERS substrate, detecting to obtain a characteristic Raman displacement peak value of formaldehyde, adding the to-be-detected article and a melamine adapter to the melamine SERS substrate, detecting to obtain a characteristic Raman displacement peak value of melamine, and calculating to obtain the concentrations of the to-be-detected variety formaldehyde and the melamine by combining the relation between the characteristic Raman displacement peak value and the concentration of the formaldehyde and the relation between the characteristic Raman displacement peak value and the concentration of the melamine S1.

In some embodiments of the present invention, the melamine SERS substrate includes a substrate, and a composite material supported on the substrate, the composite material including silver nanoparticle-coated silicon spheres. The silver nanoparticles are uniformly coated on the surface of the silicon ball, so that a better SERS enhancement effect is ensured, and the silver nanoparticles are loaded on the substrate, so that adsorption points are provided for the melamine aptamer loaded on the substrate, and the selection specificity and sensitivity of the melamine SERS substrate to melamine are improved.

In some preferred embodiments of the present invention, the mass concentration-to-mass ratio of the silver nanoparticles to the silicon spheres is (1.0 to 1.5) nmol/L: 200 mg.

In some more preferred embodiments of the present invention, the substrate is chromatography paper.

In some more preferred embodiments of the present invention, the preparation method of the melamine SERS substrate is prepared by loading the composite material on the surface of the substrate.

In some more preferred embodiments of the present invention, the preparation method of the melamine SERS substrate is more specifically performed by: and coating the silver nano particles on the silicon spheres, and loading the silicon spheres coated with the silver nano particles on the surface of the substrate.

In some more preferred embodiments of the present invention, the above-mentioned specific operation of coating the silver nanoparticles onto the silicon spheres is: mixing Ag with water⁺And dispersing the mixed solution of the silicon balls into the polyvinylpyrrolidone solution.

In some more preferred embodiments of the present invention, the mass concentration ratio of melamine to melamine aptamer is 0.75 μmol/L: (0.4-5) mg/L.

In some more preferred embodiments of the present invention, the melamine aptamer has the sequence 5 '-thiol-TTTTTTTTTTTTTTTTTTTT-3'.

In some more preferred embodiments of the present invention, the sample is obtained by immersing melamine tableware in a migration solution.

In some more preferred embodiments of the present invention, the number of the wetting is 3 to 4.

In some more preferred embodiments of the present invention, the temperature of the impregnation is 50 to 120 ℃, the time of the impregnation is 2 to 2.5 hours, and the temperature of the impregnation is more preferably 60 to 80 ℃.

In some more preferred embodiments of the present invention, the migration solution is preferably acetic acid. The transfer liquid is used for transferring melamine and formaldehyde in melamine tableware, is simple to operate and is suitable for SERS detection of the melamine and the formaldehyde.

According to another aspect of the invention, a detection kit is provided, which includes the formaldehyde SERS substrate and the melamine SERS substrate.

In some preferred embodiments of the present invention, the detection kit further comprises ethanol for storing the melamine SERS substrate. When the melamine SERS substrate is stored in the ethanol solution, the storage time of the melamine SERS substrate can be obviously prolonged.

The technical scheme of the invention has the beneficial effects that:

(1) according to the formaldehyde SERS substrate and the melamine SERS substrate, the composite material is distributed on the substrate, so that hot spots of the whole substrate are uniformly distributed, and the sensitivity of the substrate is enhanced; the 3-methyl-2-benzothiazolinone hydrazone hydrochloride is loaded on a formaldehyde SERS substrate to carry out formaldehyde detection, and the melamine aptamer is adopted to carry out melamine detection, so that the specific selectivity, the sensitivity and the repeatability of the detection can be obviously improved. The silicon spheres coated by the reduced graphene oxide and silver nanoparticles can play a good role in protecting the stability of the substrate, so that the stability of the substrate is improved, and the stability of SERS signals is improved.

(2) The preparation method of the formaldehyde SERS substrate and the melamine SERS substrate is simple, the cost is low, the prepared detection kit is convenient to carry and easy to store, and the storage stability and the storage time of the melamine SERS substrate can be obviously improved by storing the melamine SERS substrate in an ethanol solution.

(3) The melamine tableware migration method is simple and effective, can realize the detection of melamine and formaldehyde by one-time migration, and is suitable for SERS field detection.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a TEM image of a formaldehyde SERS substrate prepared in example 1 of the present invention.

FIG. 2 is a TEM image of a melamine SERS substrate prepared according to example 3 of the present invention.

Fig. 3 is an ultraviolet-visible extinction spectrum of reduced graphene oxide (a), silver nanoparticles (b), and a reduced graphene oxide-silver nanoparticle mixture (c) in the preparation process of example 2.

Fig. 4 shows uv-vis extinction spectra of the silicon spheres (a) and the silver nanoparticle-coated silicon spheres (b) in the preparation process of example 3.

FIG. 5 is an XPS spectrum of the 3-methyl-2-benzothiazolinone hydrazone hydrochloride supported pre-formaldehyde SERS substrate C1s in example 1.

FIG. 6 is an XPS spectrum of a formaldehyde SERS substrate C1s after loading 3-methyl-2-benzothiazolinone hydrazone hydrochloride in example 1.

FIG. 7 is an XPS spectrum of the 3-methyl-2-benzothiazolinone hydrazone hydrochloride supported pre-formaldehyde SERS substrate N1s in example 1.

FIG. 8 is an XPS spectrum of the formaldehyde SERS substrate N1s after loading 3-methyl-2-benzothiazolinone hydrazone hydrochloride in example 1.

FIG. 9 shows that the concentration of formaldehyde is 1275cm^-1Peak to response concentration standard curve.

FIG. 10 shows melamine concentrations at 686cm^-1Peak to response concentration standard curve.

Fig. 11 is a graph showing response of the substrate (a) of comparative example 1, the substrate (b) of comparative example 2, the substrate (c) of comparative example 3, and the formaldehyde SERS substrate (d) of example 1 to formaldehyde SERS signals.

Fig. 12 is a graph showing response of chromatography paper (a), a substrate (b) of comparative example 4, a substrate (c) of comparative example 5, and a melamine SERS substrate (d) of example 3 to melamine SERS signals.

Fig. 13 is a bar graph of SERS response of different melamine analogs and melamine.

Fig. 14 is a bar graph of SERS response of different formaldehyde analogs and formaldehyde.

FIG. 15 is a histogram of SERS response to formaldehyde using the formaldehyde SERS substrate of example 1 for 14 consecutive days.

FIG. 16 is a histogram of the SERS response to melamine using the melamine SERS substrate of example 3 for 14 consecutive days.

Detailed Description

The idea of the invention and the resulting technical effects will be clearly and completely described below in connection with the embodiments, so that the objects, features and effects of the invention can be fully understood. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

The embodiment prepares a formaldehyde SERS substrate, and the specific process is as follows:

s1: preparing silver nano particles: 100mL of 10mmol/L silver nitrate solution is measured and added into a 250mL three-neck flask, the three-neck flask is heated in an oil bath, magnetic stirring and condensation reflux are kept during the heating process, after the three-neck flask is heated to a boiling state, magnetic stirring is continued, 1mL of 2% (w/w) sodium citrate solution is rapidly added, the three-neck flask is continuously refluxed for 1h under the heating and stirring, the mixture is stirred and cooled to the room temperature, and the concentration of the mixture is doubled through a centrifugal mode (6000rpm,10min) to obtain the required silver nanoparticles.

S2: preparation of formaldehyde SERS substrate: and (3) uniformly mixing the silver nanoparticles prepared in the S1 and 0.2mg/mL reduced graphene oxide according to a ratio of 1:1(v: v), and then incubating for 12h in the dark. Then, 5mL of the mixed solution is loaded on 13mm chromatographic paper through a syringe, and the chromatographic paper loaded with the silver nanoparticles and the reduced graphene oxide and 1mL of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with the concentration of 500 microgram/mL are incubated in the dark for 12 hours to obtain the formaldehyde SERS substrate.

Example 2

s1: preparing silver nanoparticles: 100mL of 10mmol/L silver nitrate solution is measured and added into a 250mL three-neck flask, the three-neck flask is heated in an oil bath, magnetic stirring and condensation reflux are kept during the heating process, after the three-neck flask is heated to a boiling state, magnetic stirring is continued, 1mL of 2% (w/w) sodium citrate solution is rapidly added, the three-neck flask is continuously refluxed for 1h under the heating and stirring, the mixture is stirred and cooled to the room temperature, and the concentration of the mixture is doubled through a centrifugal mode (6000rpm,10min) to obtain the required silver nanoparticles.

S2: preparation of formaldehyde SERS substrate: and uniformly mixing the silver nanoparticles (Ag) prepared in the S1 with 0.2mg/mL reduced graphene oxide (rGO) according to the ratio of 1.2:1(v: v), and incubating for 12h in a dark place. Then, 5mL of the mixed solution is loaded on 13mm chromatographic paper through a syringe, and the chromatographic paper loaded with the silver nanoparticles and the reduced graphene oxide and 1.5mL of 3-methyl-2-benzothiazolinone hydrazone hydrochloride with the concentration of 500 microgram/mL are incubated in the dark for 12 hours to obtain the formaldehyde SERS substrate.

Comparative example 1

The comparative example prepares a substrate, and is different from the example 1 in that reduced graphene oxide and silver nanoparticles are not added, and the specific process is as follows: and (3) incubating chromatographic paper and 1mL of 3-methyl-2-benzothiazolinone hydrazone hydrochloride with 500 mu g/mL in the dark for 12 hours to obtain a substrate.

Comparative example 2

This comparative example prepared a substrate, which was different from example 1 in that no silver nanoparticles were added, and the specific procedure was: loading 5mL of reduced graphene oxide with the mass concentration of 0.2mg/mL on 13mm chromatographic paper through a syringe, and then incubating the chromatographic paper loaded with the reduced graphene oxide and 1mL of 500 mug/mL 3-methyl-2-benzothiazolinone hydrazone hydrochloride in a dark place for 12 hours to obtain the substrate.

Comparative example 3

The comparative example prepares a substrate, and is different from the substrate prepared in example 1 in that reduced graphene oxide is not added, and the specific process is as follows: 5mL of silver nanoparticles (prepared by the same method as S1 in example 1) are loaded on a 13mm chromatographic paper by a syringe, and the chromatographic paper loaded with the silver nanoparticles and 1mL of 3-methyl-2-benzothiazolinone hydrazone hydrochloride of 500 mug/mL are incubated in the dark for 12h to obtain the substrate.

Example 3

This embodiment has prepared a melamine SERS basement, and the concrete process is:

s1: synthesis of silicon spheres: respectively adding 90mL of absolute ethyl alcohol and 13.5mL of ammonia water into a 250mL round-bottom flask, then dropwise adding 9.0mL of tetraethyl orthosilicate, stirring for 24 hours, finally respectively cleaning 3 times by using the absolute ethyl alcohol and ultrapure water, and drying to obtain silicon spheres with the diameter of about 430 nm.

S2 synthesis of silicon spheres coated by silver nanoparticles: and dispersing the silicon spheres prepared by 200mgS1 into 20mL of absolute ethyl alcohol, and ultrasonically dispersing for 30min, and meanwhile, preparing 0.2mol/L silver ammonium solution by using 2mol/L sodium hydroxide, 0.2mol/L silver nitrate and 5% ammonia water. 10mL of 0.2mol/L silver ammonium solution was added to 20mL of anhydrous ethanol-dispersed silica sphere solution, and the mixture was stirred for 1 hour. 1g of polyvinylpyrrolidone with the molecular weight of 58000 is taken and dispersed into 50mL of absolute ethyl alcohol, and then the mixture of the silicon spheres and the silver ammonium solution is added into the polyvinylpyrrolidone solution and reacts for 7h at 70 ℃. And finally, washing the silicon spheres with absolute ethyl alcohol for 3 times, and drying the silicon spheres in vacuum for 24 hours to obtain the silicon spheres coated with the silver nanoparticles.

S3: synthesizing a melamine SERS substrate: preparing 1.5mg/mL of silicon spheres coated by the silver nanoparticles, taking 5mL of the solution by using an injector, and loading the silicon spheres coated by the silver nanoparticles on 13mm chromatographic paper in a filtering manner to obtain the melamine SERS substrate.

Comparative example 4

This comparative example prepared a substrate, which was different from example 3 in that no silver nanoparticles were added, and the specific procedure was: the substrate was obtained by loading 1.5mg/mL silica spheres (prepared as S1 in example 3) in a syringe to 5mL of the above solution and filtering the solution to obtain a silica sphere supported on 13mm chromatography paper.

Comparative example 5

This comparative example prepared a substrate, which differs from example 3 in that no silica spheres were added, by the specific procedure: taking 5mL of the above solution of 1.5mg/mL of silver nanoparticles (the preparation method of the silver nanoparticles is S1 in example 1) by using a syringe, and loading the silver nanoparticles on 13mm chromatographic paper by a filtration mode to obtain the substrate.

Example 4

The embodiment is a method for SERS detection of formaldehyde, which comprises the following specific processes:

s1: preparing formaldehyde standard solutions with mass concentrations of 0.60mg/L, 0.75mg/L, 1.00mg/L, 2.00mg/L, 3.00mg/L, 5.00mg/L, 7.50mg/L and 10.00mg/L respectively, incubating 1.00mL of the formaldehyde standard solutions with the formaldehyde SERS substrate prepared in example 1 for 10min, taking out, incubating with 1mL of 10.0mg/mL ferric ammonium sulfate dodecahydrate solution for 10min, directly detecting by using a Delta Nu Raman instrument, wherein the excitation intensity is 48mV, the integration time is 5s, continuously detecting each solution with concentration for 3 times, taking the average value and the relative deviation, and drawing 1275cm^-1The peak-formaldehyde concentration standard curve at the raman shift is shown in fig. 9.

S2: taking 7 melamine dishes in 7 beakers, adopting 250mL of acetic acid (4%, w/v) as migration liquid, migrating at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃ for 2h respectively, repeating the migration for three times, discarding the migration liquid after the previous two times of migration, drying, then performing the next time, keeping the supernatant migration liquid for the last time, drying the supernatant migration liquid with acetic acid by nitrogen, and re-dissolving the supernatant liquid with water to 250mL as a liquid to be detected. Passing the seven groups of solutions to be detected through a 0.22 mu m microporous filter membrane, and collecting the filtrate for later use.

S3: respectively taking 1mL of solution to be tested and the formaldehyde SERS substrate prepared in the embodiment 1, incubating for 10min, taking out, incubating for 10min with 1mL of 10.0mg/mL ferric ammonium sulfate dodecahydrate solution, performing SERS test, continuously detecting each solution to be tested for 3 times, and calculating 1275cm of 3 data^-1Substituting the average value and relative standard deviation of the peak value into a formaldehyde concentration standard curve to obtain the melamine dish sample at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, and 100 deg.CThe formaldehyde concentrations in the migrated liquid after migration with acetic acid (4%, w/v) were 0, 0.60 (+ -0.05) mg/L, 0.95 (+ -0.06) mg/L, 2.30 (+ -0.09) mg/L, 6.46 (+ -0.28) mg/L, 10.61 (+ -0.64) mg/L, 14.85 (+ -0.89) mg/L, respectively. It can be seen that the concentration of formaldehyde in the melamine tableware transfer liquid increases with the increase of the transfer temperature.

Example 5

The embodiment is a method for SERS detection of melamine and formaldehyde, which comprises the following steps:

s1: melamine standard solutions with mass concentrations of 0.40mg/L, 0.50mg/L, 0.60mg/L, 0.70mg/L, 1.00mg/L, 1.50mg/L, 3.00mg/L and 5.00mg/L are prepared respectively, 1.00mL of the melamine standard solution with each mass concentration and activated melamine aptamer are taken respectively (0.5 mL of 5mmol/L tris (2-carbonylethyl) phosphate is added into 0.5mL of 1.5 mu mol/L melamine aptamer, and the mixture is incubated for 1h in dark place for activation) according to the following ratio of 1:1(v: v), incubating the mixture with the melamine SERS substrate prepared in example 3 in the dark for 12h, washing the mixture for 3 times with 10mmol/L phosphate buffer solution (PBSpH7.4), directly detecting the mixture by using a Delta Nu Raman spectrometer, wherein the excitation intensity is 48mV, the integration time is 5s, continuously detecting the solution with each concentration for 3 times, taking an average value and a relative deviation, and drawing a 686cm^-1Peak at raman shift-standard curve for melamine concentration. The results are shown in FIG. 10.

S2: the standard curve of formaldehyde concentration was plotted as in S1 of example 4.

S3: taking 3 melamine dishes in 3 beakers, dividing the melamine dishes into a group A, a group B and a group C, wherein the three groups respectively and correspondingly adopt 250mL of water, ethanol (20%, v/v) and acetic acid (4%, w/v) as migration liquid, migrating the melamine dishes at 70 ℃ for 2 hours, repeating the migration for three times, discarding the migration liquid after the previous two times of migration, drying the migration liquid, then performing the next time, preserving and taking supernatant migration liquid for the last time, performing nitrogen blow-drying on the ethanol in the group B and the acetic acid in the group C, re-dissolving the melamine dishes with water to 250mL as a liquid to be detected, and directly taking the group A as the liquid to be detected. Passing the three groups of solutions to be detected through a 0.22 mu m microporous filter membrane, and collecting the filtrate for later use.

S4: respectively mixing the solution to be detected and the activated aptamer (the activation method is the same as S1) according to the proportion of 1:1(v: v) ratio mixing allAfter the mixing, the substrate was incubated with the melamine SERS substrate prepared in example 3 in the dark for 12 hours, washed with 10mmol/L phosphate buffer solution (PBS pH7.4) for 3 times, SERS detection was performed, each solution to be detected was continuously detected for 3 times, and 686cm of 3 data was calculated^-1And substituting the average value and the relative standard deviation of the peak values into a melamine concentration standard curve to obtain melamine concentration standard curves of the melamine dish sample, wherein the melamine concentration in migration liquid after the melamine dish sample is migrated by water, ethanol (20 percent, v/v) and acetic acid (4 percent, w/v) is 0, 0 and 0.59 (+/-0.03) mg/L respectively. It can be seen that the use of acetic acid effectively migrates the melamine in melamine dishware.

S5: respectively taking 1mL of solution to be tested and the formaldehyde SERS substrate prepared in the embodiment 1, incubating for 10min, taking out, incubating for 10min with 1mL of 10.0mg/mL ferric ammonium sulfate dodecahydrate solution, performing SERS test, continuously detecting each solution to be tested for 3 times, and calculating 1275cm of 3 data^-1The mean values and relative standard deviations of the peak values are substituted into a formaldehyde concentration standard curve, and the formaldehyde concentrations in migration liquid of the melamine dish sample after migration through water, ethanol (20%, v/v) and acetic acid (4%, w/v) are respectively 0.49 (+ -0.02) mg/L, 0.41 (+ -0.02) mg/L and 2.30 (+ -0.09) mg/L. It can be seen that the use of acetic acid effectively migrates formaldehyde from melamine dishware.

Test examples

1. TEM images of the formaldehyde SERS substrate prepared in example 1 and the melamine SERS substrate prepared in example 3 correspond to those shown in fig. 1 and 2, respectively. It can be seen from fig. 1 that silver nanoparticles are uniformly loaded on the surface and inside of the reduced graphene oxide, and are very densely distributed, so that a better SERS enhancement effect can be ensured. As can be seen from fig. 2, the silver nanoparticles uniformly grow on the surface of the silicon sphere, so that a good SERS enhancement effect can be ensured.

2. Fig. 3 shows the ultraviolet-visible extinction spectra of the reduced graphene oxide (a), the silver nanoparticles (b), and the reduced graphene oxide-silver nanoparticle mixture (c) in the preparation process of example 2. As can be seen from fig. 3, compared with the reduced graphene oxide (a), the silver nanoparticles (b) and the reduced graphene oxide and silver nanoparticle mixture (c) both have a significant absorption peak at 410nm, indicating that the reduced graphene oxide and silver nanoparticle mixture has an excellent SERS enhancement effect.

3. Fig. 4 shows uv-visible extinction spectra of the silicon spheres (a) and the silver nanoparticle-coated silicon spheres (b) in the preparation process of example 3. As can be seen from fig. 4, the silver nanoparticle-coated silicon spheres (b) exhibited a significant absorption peak at 410nm, compared to the silicon spheres (a), indicating the successful synthesis of silver nanoparticle-coated silicon spheres.

4. To further verify that the 3-methyl-2-benzothiazolinone hydrazone hydrochloride in example 1 is successfully loaded on the formaldehyde SERS substrate, X-ray photon energy spectrum (XPS) analysis is performed on the formaldehyde SERS substrate before and after the 3-methyl-2-benzothiazolinone hydrazone hydrochloride is loaded, and the results are shown in fig. 5 to 8, where fig. 5 is an XPS spectrum of the formaldehyde SERS substrate C1s before loading, fig. 6 is an XPS spectrum of the formaldehyde SERS substrate C1s after loading, fig. 7 is an XPS spectrum of the formaldehyde SERS substrate N1s before loading, and fig. 8 is an XPS spectrum of the formaldehyde SERS substrate N1s after loading.

As can be seen from the comparison of FIG. 5 and FIG. 6, FIG. 7 and FIG. 8, the C-N bond in C1s shifts from 285.9eV to 286.9eV after the 3-methyl-2-benzothiazolinone hydrazone hydrochloride is loaded on the formaldehyde SERS substrate; the C-N bond in N1s shifted from 403.2eV to 402.6eV, indicating that 3-methyl-2-benzothiazolinone hydrazone hydrochloride has been successfully supported on formaldehyde SERS substrates.

5. In order to verify the signal response capability of the formaldehyde SERS substrate of the technical scheme of the invention to formaldehyde, a formaldehyde SERS signal response test is carried out on the substrates of the embodiment 1 and the comparative examples 1-3, and the method for acquiring the SERS signal response comprises the following steps: 1mL of a solution to be tested (the preparation method is S2 in example 4, the migration liquid is water) is dripped on a substrate, the solution is taken out after incubation for 10min, and SERS test is carried out after the incubation for 10min with 1mL of 10.0mg/mL ferric ammonium sulfate dodecahydrate solution, so as to obtain SERS signal response. The SERS signal response of the substrates of example 1(d) and comparative examples 1(a), 2(b), 3(c) to formaldehyde is shown in fig. 11.

As can be seen from the SERS signal response conditions in fig. 11d and fig. 11a, b, and c, the formaldehyde SERS substrate of embodiment 1 has the highest SERS enhancement effect on formaldehyde, and can meet the detection requirement.

6. In order to verify the signal response capability of the melamine SERS substrate prepared in example 3 to melamine, the chromatography paper (a), the substrate (b) in comparative example 4, the substrate (c) in comparative example 5 and the substrate (d) in example 3 were subjected to a melamine SERS signal response test, and the SERS signal response was obtained by the following method: the test solution (prepared as S3 in example 5, migration liquid is water) and the activated aptamer (activated as S1 in example 5) were mixed according to the ratio of 1: after being uniformly mixed according to the proportion of 1(v: v), the mixture is incubated with chromatographic paper and the substrates of comparative example 4 and example 3 for 12h in dark, washed with 10mmol/L phosphate buffer solution (PBSpH7.4) for 3 times respectively, and then SERS detection is carried out respectively to obtain SERS signal response. The SERS signal response of the substrates of chromatography paper (a), comparative example 4(b) and example 3(c) to melamine is shown in fig. 12.

As can be seen from the SERS signal response conditions in fig. 12d and fig. 12a, b, and c, the melamine SERS substrate of embodiment 3 has the highest SERS enhancement effect on melamine, and can meet the detection requirement.

7. In order to verify the accuracy of the SERS detection method of melamine and formaldehyde in the embodiment 5 for detecting melamine, the to-be-detected liquid in the embodiment 5 is subjected to the standard adding treatment, wherein the standard adding sample is obtained by adding melamine standard solutions with the amounts of 0.5mg/L, 1.0mg/L and 5.0mg/L to the to-be-detected sample, then SERS detection is carried out, continuous testing is carried out for 3 times, and 686cm of 3 data is calculated^-1And substituting the average value and the relative deviation of the peak values into a melamine concentration standard curve to obtain the melamine concentration in the standard sample, and calculating to obtain the standard sample recovery rate of 91.2-110.0% and the relative standard deviation of 1.8-8.3%.

The detection accuracy of the established SERS analysis method is verified by High Performance Liquid Chromatography (HPLC). The solution to be tested (prepared as S3 in example 5) was subjected to HPLC using a high performance liquid chromatograph equipped with a diode array detector (Singapore Watts Corp.) with a detection wavelength of 230nm, and the selected column was an ES amino column (250 mm. times.4.6 mm, 5 μm). The mobile phase is 5mmol/L sodium dihydrogen phosphate-acetonitrile (volume ratio is 75:25), the flow rate is 1.0mL/min, and the sample amount is 10 μ L. After HPLC detection, the melamine dish sample is obtained, the concentrations of melamine in migration liquid after migration of water, ethanol (20%, v/v) and acetic acid (4%, v/v) are respectively 0, 0 and 0.60 (+/-0.01) mg/L, and the relative deviation with the SERS analysis method is 1.7%, so that the reliability of the analysis method is proved.

8. In order to verify the accuracy of formaldehyde detection by the SERS detection method of melamine and formaldehyde in the embodiment 5, the solution to be detected in the embodiment 5 is subjected to the standard adding treatment, wherein the standard adding sample is obtained by adding 0.5mg/L, 1.0mg/L and 5.0mg/L formaldehyde standard solutions to the sample to be detected, then SERS detection is carried out, continuous testing is carried out for 3 times, and 1275cm of 3 data is calculated^-1And substituting the average value and the relative deviation of the peak values into a standard formaldehyde concentration curve to obtain the formaldehyde concentration in the standard sample, and calculating to obtain the standard sample recovery rate of 94.0-106.0% and the relative standard deviation of 4.7-9.1%.

The detection accuracy of the established SERS analysis method was verified by High Performance Liquid Chromatography (HPLC). The solution to be tested (prepared as S3 in example 5) was subjected to HPLC using a high performance liquid chromatograph equipped with a diode array detector (Singapore Watts Corp.) with a detection wavelength of 360nm, and the chromatographic column selected was a Diamonsil C18 column (250 mm. times.4.6 mm, 5 μm). The mobile phase adopts 0.1 percent trifluoroacetic acid (A) -acetonitrile (B) and adopts gradient elution, and the elution conditions are as follows: 0-5 min: mobile phase B rose from 30% to 70%; 5-10 min: mobile phase B rose from 70% to 80%; 10-12 min: mobile phase B decreased from 80% to 30%; 12-17 min: mobile phase B was maintained at 30%; the flow rate was 1.0mL/min and the amount of sample was 10. mu.L. HPLC detection shows that the concentrations of formaldehyde in migration liquid of the melamine dish sample after migration of water, ethanol (20%, v/v) and acetic acid (4%, v/v) are respectively 0.48 (+ -0.01), 0.40 (+ -0.01) and 2.31 (+ -0.04) mg/L, and relative deviations from the SERS analysis method are respectively 2.1%, 2.5% and 0.4%, thus proving the reliability of the analysis method.

9. In order to verify that the melamine SERS substrate has specific selection on melamine, the melamine SERS substrate is adopted to detect melamine analogues such as ammonia, cyromazine, urea, uric acid, trioxymethylene, cyanuric acid, dicyandiamide and melamine, the detection method refers to the SERS detection method for melamine in example 5, and the result is shown in FIG. 13. As can be seen from fig. 13, when the melamine SERS substrate is used to detect the above eight substances, SERS responses of ammonia, cyromazine, urea, uric acid, trioxymethylene, cyanuric acid, and dicyandiamide are weak, so that the method of the present invention can accurately determine the melamine content.

10. In order to verify the specificity of the formaldehyde SERS substrate to formaldehyde, the formaldehyde SERS substrate is used for detecting formaldehyde analogues such as acetone, dichloromethane, methanol, formic acid, ethanol, acetonitrile, acetaldehyde and formaldehyde, and the SERS spectrogram is shown in FIG. 14. As can be seen from fig. 14, when the formaldehyde SERS substrate of the present invention is used to detect the above eight substances, SERS responses of acetone, dichloromethane, methanol, formic acid, ethanol, acetonitrile, and acetaldehyde are weak, so that the method of the present invention can accurately determine the content of formaldehyde.

11. In order to detect the stability of the formaldehyde SERS substrate and the melamine SERS substrate, the melamine SERS substrate is stored by adopting ethanol. The method for detecting the stability comprises the following steps: formaldehyde was detected using the formaldehyde SERS substrate of example 1 using the method of example 5 for 14 consecutive days, and the results are shown in fig. 15; the melamine was measured using the melamine SERS substrate of example 3 using the method of example 5 for 14 consecutive days, and the results are shown in FIG. 16.

As can be seen from fig. 15, the relative standard deviation value of formaldehyde detection for 14 consecutive days is 4.2%, which indicates that the formaldehyde SRES substrate has good stability, wherein the reduced graphene oxide has good chemical inertness, and can protect the stability of the silver nanoparticles and the 3-methyl-2-benzothiazolinone hydrazone hydrochloride, so that the prepared formaldehyde SERS substrate has good stability. As can be seen from FIG. 16, the relative standard deviation of the melamine detection for 14 consecutive days is 4.3%, which indicates that the storage time of the melamine SERS substrate can be significantly improved when the melamine SERS substrate is stored in an ethanol solution, so that the melamine SERS substrate can maintain good stability within 14 days,

the embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. A formaldehyde SERS substrate, comprising: the formaldehyde SERS substrate comprises a substrate and a composite material loaded on the substrate, wherein the composite material comprises reduced graphene oxide, silver nanoparticles and 3-methyl-2-benzothiazolinone hydrazone hydrochloride; the mass ratio of the reduced graphene oxide to the silver nanoparticles to the 3-methyl-2-benzothiazolinone hydrazone hydrochloride is (0.1-0.15) mg/mL: (1.0-1.5) nmol/L: 0.5 mg/mL.

2. A method of preparing the formaldehyde SERS substrate of claim 1, wherein: and loading the composite material on the surface of the substrate to obtain the formaldehyde SERS substrate.

3. A formaldehyde SERS detection method is characterized in that: the method comprises the following steps:

s1: the formaldehyde SERS substrate of claim 1, wherein the relationship between the peak value and the concentration of formaldehyde at the characteristic Raman shift is obtained;

s2: adding a sample to be detected on the formaldehyde SERS substrate as claimed in claim 1, detecting to obtain a peak value at the characteristic Raman shift of formaldehyde, and calculating to obtain the concentration of formaldehyde in the sample to be detected by combining the relation between the peak value at the characteristic Raman shift of formaldehyde and the concentration of formaldehyde S1.

4. A SERS detection method of melamine and formaldehyde is characterized in that: the method comprises the following steps:

s1: the formaldehyde SERS substrate of claim 1 is adopted to obtain the relation between the peak value and the concentration of the characteristic Raman shift position of formaldehyde, and the melamine SERS substrate is adopted to obtain the relation between the peak value and the concentration of the characteristic Raman shift position of melamine;

s2: adding a to-be-detected article to the formaldehyde SERS substrate of claim 1, detecting to obtain a peak value at the characteristic Raman shift of formaldehyde, adding the to-be-detected article and a melamine adapter to the melamine SERS substrate, detecting to obtain a peak value at the characteristic Raman shift of melamine, and calculating to obtain the concentrations of the formaldehyde and the melamine of the to-be-detected variety by combining the relation between the peak value at the characteristic Raman shift of formaldehyde and the concentration and the relation between the peak value at the characteristic Raman shift of melamine and the concentration in S1.

5. The method for SERS detection of melamine and formaldehyde as recited in claim 4, wherein: the melamine SERS substrate comprises a substrate and a composite material loaded on the substrate, wherein the composite material comprises silicon spheres coated by silver nanoparticles.

6. The method for SERS detection of melamine and formaldehyde as recited in claim 4, wherein: the sequence of the melamine aptamer is 5 '-thiol-TTTTTTTTTTTTTTTTTTTT-3'.

7. The method for SERS detection of melamine and formaldehyde as recited in claim 4, wherein: the article to be detected is obtained by soaking melamine tableware in the migration solution.

8. The SERS detection method of melamine and formaldehyde according to claim 7, wherein: the migration liquid is selected from at least one of water, ethanol and acetic acid.

9. A detection kit, characterized in that: comprising the formaldehyde SERS substrate of claim 1 and the melamine SERS substrate of claim 5.

10. The test kit according to claim 9, characterized in that: further comprising ethanol to store the melamine SERS substrate of claim 5.