Method for rapidly detecting androgen steroid based on surface enhanced Raman scattering technology and application thereof

Abstract

The invention relates to a method for rapidly detecting androgen steroid based on a surface enhanced Raman scattering technology and application. The method comprises the following steps: 1) synthesizing a bowl-shaped polydopamine-precious metal substrate material; 2) adding a solution containing a substrate material into the androgen steroid solution; and (3) carrying out surface enhanced Raman scattering (SERS) detection, recording a spectrogram and carrying out data analysis. According to the invention, high-sensitivity detection of steroid hormones is realized by synthesizing a specific nano-structure material and utilizing an SERS technology, and the method is especially suitable for qualitative and quantitative analysis of synthetic steroid hormones in environmental water bodies, biological samples and foods. According to the method, the SERS detection method is utilized for the first time, the endocrine disrupter in the complex biological sample can be rapidly and accurately detected, a chromatography-mass spectrometry detection method which is complex in process and high in cost is avoided, and effective technical support is provided for environmental monitoring and biological detection.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry and biosensing, and in particular relates to a rapid detection method for androgen steroids based on surface enhanced Raman scattering (SERS) and a spectrum construction system thereof. Background Art

[0002] Steroids are a general term for cyclopentane-perhydrophenanthrene compounds, usually including cholesterol, ergosterol, bile acid, vitamin D, sex hormones, adrenal cortex hormones, etc. They are widely distributed in nature, and their specific physiological functions vary. As an endocrine disruptor, androgenic steroids interfere with androgen receptors in organisms and may cause various health problems. In addition, these steroids can enter the human body through diet and environmental exposure and accumulate in foods such as meat and dairy products, increasing the risk to human health. Although existing analytical methods such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) have high sensitivity in detecting steroids, these methods are usually complex and expensive, cumbersome to operate, have a long detection cycle, and require high-cost equipment, and cannot achieve simple portable detection.

[0003] In recent years, SERS technology has been widely used in environmental and biological sample analysis due to its high sensitivity, specificity and label-free detection capabilities. In order to optimize the performance of SERS substrates, many studies have focused on the development of complex metal nanostructures, such as the configuration of metal nanoparticles in pores or cavity structures, which enhance the Raman signal by generating highly concentrated electromagnetic fields. However, the manufacturing process of these complex structures usually involves multiple steps, including template selection and preparation, template surface modification, and coating of metal nanoparticles, which are complicated and expensive, limiting their practical application. Moreover, especially for androgenic steroids with similar structures, how to use SERS to simply and accurately detect androgenic steroids is a technical problem that needs to be solved urgently. Therefore, it is very important to develop a method that can not only optimize the SERS performance to ensure high uniformity and repeatability of the substrate, but also achieve high sensitivity and real-time detection of anabolic androgenic steroids with similar structures. Summary of the invention

[0004] In order to make up for the shortcomings of the existing technology in the field of drug detection, the present invention provides a method for rapid detection of androgen steroids based on surface enhanced Raman scattering (SERS). By synthesizing specific nanostructured materials and using SERS technology to achieve high-sensitivity detection of steroid hormones, it is particularly suitable for qualitative and quantitative analysis of synthetic steroid hormones in environmental water, biological samples and food.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a method for rapid detection of androgenic steroids based on surface enhanced Raman scattering technology, the method comprising the following steps:

[0007] 1) Synthesize bowl-shaped polydopamine@precious metal substrate material;

[0008] 2) adding a solution containing a base material to the androgenic steroid solution;

[0009] 3) Surface enhanced Raman scattering (SERS) detection, recording spectra and performing data analysis.

[0010] The androgenic steroids include androstenedione (C 19 H 26 O2), Adrenosterone (C 19 H 24 O3), 4-Androsten-4-ol-3,17-dione (C 19 H 26 O3) and 5-Androstene-3β,17β-diol (C 19 H 30 O2).

[0011] Further, in step 2), the concentration of the androgen steroid solution is 0.0001-1000 μg / mL, for example, 0.0001 μg / mL, 0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL and 1000 μg / mL;

[0012] Furthermore, in step 2), the volume ratio of the androgen steroid solution to the solution containing the base material is (1-2):1.

[0013] Furthermore, the preparation method of the bowl-shaped polydopamine@precious metal substrate material in step 1) is as follows:

[0014] 1.1 Preparation of bowl-shaped polydopamine (PDA): bowl-shaped polydopamine is obtained by polymerization of bowl-shaped dopamine monomers;

[0015] 1.2 Preparation of bowl-shaped polydopamine@precious metal nanoparticles: Precious metals are in situ reduced and loaded on the surface of bowl-shaped polydopamine.

[0016] Furthermore, step 1.1 includes the following steps:

[0017] 1.1.1 Preparation of dopamine monomer solution: dissolve dopamine hydrochloride, template agent tetramethylbenzidine TMB, and triblock polymer F127 in water to obtain a dopamine monomer solution;

[0018] Furthermore, the concentration of dopamine hydrochloride is 10-15 g / L; the concentration of F127 is 6-10 g / L.

[0019] Furthermore, the mass volume (g / mL) ratio of dopamine hydrochloride to TMB is 1:(1.3-2).

[0020] 1.1.2 Add ammonia water to the dopamine monomer solution and stir to obtain dopamine monomer;

[0021] Furthermore, the mass ratio of the ammonia water to the dopamine monomer solution is (2.5-4):1.

[0022] Furthermore, the stirring temperature is 20-30° C., and the stirring time is 1-3 h.

[0023] 1.1.3 Polymerization of dopamine monomers to obtain bowl-shaped polydopamine PDA;

[0024] Furthermore, the polymerization reaction refers to adding ethanol-water solution to dopamine monomer, the reaction temperature is 100-130° C., and the reaction time is 12-36 hours.

[0025] Furthermore, in the ethanol-water solution, the ethanol concentration is 40-60wt%.

[0026] Furthermore, the step 1.2 comprises the following steps:

[0027] 1.2.1 Dissolve the bowl-shaped polydopamine PDA in water to obtain a bowl-shaped polydopamine dispersion;

[0028] The concentration of the bowl-shaped polydopamine dispersion is 10-20 g / L, preferably 10-15 g / L.

[0029] 1.2.2 Mix the bowl-shaped polydopamine dispersion with the salt solution of the precious metal, stir and react to obtain bowl-shaped polydopamine@precious metal nanoparticles.

[0030] Furthermore, the noble metal is selected from Au or Pt.

[0031] Furthermore, the noble metal salt is selected from chloroauric acid or chloroplatinic acid.

[0032] Furthermore, the solvent of the noble metal salt solution is water.

[0033] Furthermore, the concentration of the noble metal salt solution is 15-100 μM.

[0034] Furthermore, the stirring reaction time is 12-36 hours, and the temperature is 20-30°C.

[0035] Furthermore, the volume ratio of the polydopamine dispersion to the noble metal salt solution is (100-200):1; preferably (100-120):1.

[0036] In some embodiments of the present invention, the volume of the noble metal salt solution is 50-150 μL.

[0037] In the present invention, the PDA@precious metal substrate material is a bowl-shaped mesoporous PDA particle prepared by an emulsion-induced interface anisotropic assembly strategy, which is used for SERS substrate material and has good response performance. By adjusting the amount of TMB, the morphology of PDA nanoparticles can be adjusted from symmetrical nanospheres to bowl-shaped particles, and finally to an asymmetric bowl-shaped structure. At the same time, the use of precious metal precursors such as HAuCl4 optimizes SERS performance, with good uniformity and reproducibility.

[0038] Furthermore, in step 1.2.2, the bowl-shaped polydopamine dispersion is mixed with the salt solution of the noble metal, and the low eutectic mixture is added and stirred for reaction.

[0039] Furthermore, the volume ratio of the eutectic mixture to the polydopamine dispersion is (0.05-0.2):1.

[0040] Further, the eutectic mixture is selected from the system of choline chloride-ethylene glycol or choline chloride-glycerol, wherein the molar ratio of choline chloride:ethylene glycol is 1:(2-4); or the molar ratio of choline chloride:glycerol is 1:(2-4).

[0041] The inventors found that the choline chloride-ethylene glycol or choline chloride-propylene glycol system contains hydrogen bond acceptors and hydrogen bond donors, and the low eutectic mixture formed at room temperature has lower viscosity and higher solubility. Therefore, during the reaction of the mixed solution, the dispersion uniformity of polydopamine can be improved, so that the precious metal can be effectively combined with PDA, the active reaction / loading sites are increased, the loading efficiency of the precious metal is improved, and the agglomeration of the precious metal ions can be prevented, and the dispersion ability of the precious metal is improved. Therefore, the bowl-shaped PDA surface can effectively load more uniform precious metal particles, enriching the surface properties of the substrate material, increasing the specific surface area of ​​the bowl-shaped PDA@precious metal nanoparticles, improving the sensitivity and response ability of the substrate in the SERS detection process, and generating a highly concentrated electromagnetic field enhanced Raman signal, which is more conducive to the detection and differentiation of androgenic steroids with similar structures.

[0042] Furthermore, the substrate material is bowl-shaped PDA@Au nanoparticles.

[0043] Furthermore, in the solution containing the base material in step 2), the solvent is water.

[0044] Furthermore, in step 3), a Raman spectrometer is used to perform SERS detection.

[0045] Furthermore, in step 3), the laser wavelength of the Raman spectrometer is 633 nm.

[0046] Furthermore, step 3) data analysis includes qualitative analysis to detect or identify the type of androgenic steroid.

[0047] The second aspect of the present invention provides a method for rapidly detecting androgenic steroids based on surface enhanced Raman scattering technology, which is used to quantitatively detect the concentration of androgenic steroids, and the method comprises the following steps:

[0048] i) Synthesizing bowl-shaped polydopamine@precious metal substrate materials;

[0049] ii) preparing standard androgenic steroid solutions of different concentrations and adding the solution containing the base material;

[0050] iii) performing SERS detection on standard androgenic steroid solutions of different concentrations, analyzing the concentration-spectrum intensity relationship of androgenic steroids, and establishing a linear regression model between androgenic steroid concentration and SERS spectrum characteristic peak intensity;

[0051] iv) Perform quantitative analysis based on the linear regression model.

[0052] The androgenic steroids include androstenedione (C 19 H 26 O2), Adrenosterone (C 19 H 24 O3), 4-Androsten-4-ol-3,17-dione (C 19 H 26 O3) and 5-Androstene-3β,17β-diol (C 19 H 30 O2).

[0053] Furthermore, the bowl-shaped polydopamine@precious metal substrate material in step i) is the same as the substrate material in step 1) of the first aspect.

[0054] The concentrations of the solutions in step ii) are 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 and 1000 μg / mL, respectively.

[0055] Furthermore, the volume ratio of the standard androgen steroid solution to the base material solution is (1-2):1.

[0056] Furthermore, the concentration of the solution containing the substrate material is 50-200 mM.

[0057] Furthermore, the detection limit (LOD) and linear range were calculated to evaluate the sensitivity and reliability of the method.

[0058] Furthermore, the linear regression model of androstenedione is: Y = 1695.91x + 7262, and the linear determination coefficient R 2 =0.974;

[0059] Furthermore, the linear regression model of adrenal androsterone is: Y = 1804.21x + 7519.38, and the linear determination coefficient R 2 =0.982;

[0060] Furthermore, the linear regression model of 4-androsten-4-ol-3,17-dione is: Y = 2786.2x + 11769.14, and the linear determination coefficient R 2 =0.990;

[0061] Furthermore, the linear regression model of androstenediol is: Y = 1685.01x + 678.23, and the linear determination coefficient R 2 =0.972.

[0062] Furthermore, according to the above linear regression model, the content and concentration of various androgenic steroids in the sample to be tested can be quantitatively detected.

[0063] The third aspect of the present invention provides a verification method for the method for detecting androgenic steroids described in the first aspect or the second aspect, by adding standard androgenic steroid solutions of different concentrations to actual samples, examining the detection value, recovery rate, relative standard deviation RSD, etc., to verify the repeatability and accuracy of the method. Specifically comprising the following steps:

[0064] 3.1 Collect actual samples, filter and wash;

[0065] 3.2 Adding standard androgenic steroid solutions of different concentrations to the actual sample to obtain a mixed solution;

[0066] 3.3 Adding the base material solution to the obtained mixed solution;

[0067] 3.4 Use Raman spectrometer to perform SERS detection and analyze the concentration, recovery rate and relative standard deviation (RSD) of androgenic steroids in actual samples.

[0068] Furthermore, the actual sample in step 3.1 includes but is not limited to tap water and milk, which are filtered, washed, and boiled as needed.

[0069] Furthermore, the filtration uses a 0.2-0.4 μm filter membrane.

[0070] Furthermore, the concentration of the standard androgenic steroid solution in step 3.2 is 0.1-100 μg / mL.

[0071] Furthermore, the laser wavelength of the Raman spectrometer is 633 nm.

[0072] Furthermore, the androgenic steroid includes Androstenedione (C 19 H 26 O2), Adrenosterone (C 19 H 24 O3), 4-Androsten-4-ol-3,17-dione (C 19 H 26 O3) and 5-Androstene-3β,17β-diol (C 19 H 30 O2).

[0073] Furthermore, the base material in step 3.3 is the bowl-shaped polydopamine@precious metal nanoparticles prepared according to step 1) of the first aspect.

[0074] Furthermore, in the detection process of step 3.4, the linear regression model of step iii) in the second aspect is adopted, and the obtained Raman spectrum is baseline corrected and normalized using LabSpec software (version 6.2), so as to obtain the content of androgenic steroids in the actual sample, the recovery rate and the relative standard deviation RSD, and evaluate the accuracy and effectiveness of the method.

[0075] The fourth aspect of the present invention provides a bowl-shaped polydopamine@noble metal nanoparticle and a preparation method thereof, which is prepared by step 1) of the first aspect.

[0076] The fifth aspect of the present invention uses the method for rapid detection of androgenic steroids based on surface enhanced Raman scattering technology described in the first aspect to detect the type and content of androgenic steroids in a substance to be tested. The method comprises the following steps:

[0077] 5.1 Collect the samples to be tested, filter and wash;

[0078] 5.2 The sample to be tested is dispersed in a solvent to obtain a sample solution to be tested;

[0079] 5.3 Add the substrate material solution to the sample solution to be tested;

[0080] 5.4 Use Raman spectrometer to perform SERS detection and analyze the type and content of androgenic steroids in the sample to be tested.

[0081] Furthermore, the filtration uses a 0.2-0.4 μm filter membrane.

[0082] Furthermore, the laser wavelength of the Raman spectrometer is 633 nm.

[0083] Furthermore, the androgenic steroid includes Androstenedione (C 19 H 26 O2), Adrenosterone (C 19 H 24 O3), 4-Androsten-4-ol-3,17-dione (C 19 H 26 O3) and 5-Androstene-3β,17β-diol (C 19 H 30 O2).

[0084] Furthermore, the substrate material is the bowl-shaped polydopamine@noble metal nanoparticles prepared in step 1) of the first aspect, or the bowl-shaped polydopamine@noble metal nanoparticles described in the fourth aspect.

[0085] Furthermore, in step 5.4, the linear regression model of step iii) in the second aspect is used, and the obtained Raman spectrum is baseline corrected and normalized using LabSpec software to obtain the content of androgenic steroids in the sample to be tested.

[0086] The sixth aspect of the present invention is to use the method described in the first aspect, the second aspect or the fifth aspect in the rapid detection of androgen steroids by surface enhanced Raman scattering (SERS). BRIEF DESCRIPTION OF THE DRAWINGS

[0087] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0088] Figure 1 Schematic diagram of the formation process of bowl-shaped PDA mesoporous particles.

[0089] Figure 2 is a SEM image of the PDA particles prepared in Example 1.

[0090] Figure 3 is the SEM image of PDA@Au nanoparticles prepared in Example 1.

[0091] Figure 4 is a SEM image of the PDA particles prepared in Example 2.

[0092] Figure 5 is a SEM image of the PDA particles prepared in Example 3.

[0093] Figure 6 This is a SEM image of the PDA particles prepared in Example 4.

[0094] Figure 7 It is the electromagnetic field distribution curve of the PDA@Au nanoparticles prepared in Examples 1 and 4-6 simulated by FDTD; wherein A is the electromagnetic field distribution curve of the nanoparticles in Example 4 simulated by FDTD, B is the electromagnetic field distribution curve of the nanoparticles in Example 1 simulated by FDTD, C is the electromagnetic field distribution curve of the nanoparticles in Example 5 simulated by FDTD, and D is the electromagnetic field distribution curve of the nanoparticles in Example 6 simulated by FDTD.

[0095] Figure 8 It is the corresponding SERS spectrum of the PDA@Au nanoparticles loaded with 1 μM toluidine blue prepared in Examples 1, 4-6; wherein E is the SERS spectrum of the nanoparticles in Example 4, F is the SERS spectrum of the nanoparticles in Example 1, G is the SERS spectrum of the nanoparticles in Example 5, and H is the SERS spectrum of the nanoparticles in Example 6.

[0096] Fig. 9 is the SEM image of PDA@Au nanoparticles prepared in Example 7.

[0097] Fig.10 The SERS spectra (excitation wavelength is 633 nm) and molecular structures of four anabolic androgenic steroids at 1 mM concentration detected in Example 9.

[0098] Fig.11 These are the SERS spectra and corresponding concentration-response curves of the four androgenic steroids at different concentrations in Example 10, wherein A is Androstenedione, B is Adrenosterone, C is 4-Androsten-4-ol-3,17-dione, and D is the SERS spectrum and corresponding concentration-response curve of 5-Androstene-3β,17β-diol. DETAILED DESCRIPTION

[0099] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0100] Example 1

[0101] A method for preparing a bowl-shaped PDA@Au nanoparticle substrate, the process is as follows Figure 1 As shown, the following steps are included:

[0102] 1.1 Preparation of bowl-shaped porous polydopamine PDA:

[0103] 1.1.1 Preparation of dopamine monomer solution: 0.15 g dopamine hydrochloride, 0.3 mL template TMB, and 0.1 g F127 were dissolved in 10 mL 50% ethanol-water solution to obtain a dopamine monomer solution;

[0104] 1.1.2 Add 0.4 mL of ammonia water to the dopamine monomer solution, stir at 20 °C for 1 h, add ethanol, separate the precipitate, and obtain a bowl-shaped dopamine monomer. The SEM electron microscope image (the scale line is 200 nm) is as follows: Figure 2 As shown, it can be seen that PDA presents an asymmetric bowl-shaped morphology with a large specific surface area;

[0105] 1.1.3 Polymerization of dopamine monomer to obtain bowl-shaped polydopamine PDA: 10 mL of 50% ethanol-water solution was added to the above-obtained precipitate, and the mixture was reacted at 100° C. for 24 h to obtain bowl-shaped PDA.

[0106] 1.2 Preparation of PDA@Au NPs:

[0107] 1.2.1 Dissolve the prepared bowl-shaped PDA in 10 mL of ultrapure water to obtain a PDA dispersion;

[0108] 1.2.2 Mix 10 mL of PDA dispersion with 90 μL of HAuCl4 solution (concentration of 20 μM), stir and react at 25°C for 24 hours, centrifuge at 13000 rpm / min, and wash with ultrapure water 2-3 times to obtain bowl-shaped PDA@Au nanoparticles.

[0109] Figure 3 This is a scanning electron microscope image of the bowl-shaped PDA@Au nanoparticles prepared in Example 1. It can be seen that the precious metal Au can be loaded on the surface of the bowl-shaped PDA, and the bowl-shaped particles have more and richer surface protrusions and a larger specific surface area, which will improve the SERS response ability and is more conducive to improving the sensitivity and accuracy of SERS detection, so that androgen steroid substances with similar structures can be identified and distinguished.

[0110] Example 2

[0111] The other steps are the same as those in Example 1, except that the amount of TMB added in 1.1.1 is different:

[0112] 1.1.1 Preparation of dopamine monomer solution: Dissolve 0.15 g dopamine hydrochloride, 0.25 mL template TMB, and 0.1 g F127 in 10 mL 50% ethanol / water solution to obtain a dopamine monomer solution;

[0113] 1.1.2 Add 0.4 mL of aqueous ammonia to the dopamine monomer solution, stir at 20° C. for 1 h, add ethanol, separate the precipitate, and obtain a bowl-shaped dopamine monomer; finally, bowl-shaped PDA@Au nanoparticles are prepared.

[0114] The SEM electron microscope (the scale line is 200nm) of the bowl-shaped PDA monomer is as follows Figure 4 As shown, it can be seen that some PDA particles have a bowl-shaped structure, a large specific surface area, and better SERS detection response capability.

[0115] Example 3

[0116] The other steps are the same as those in Example 1, except that the amount of TMB added in 1.1.1 is different:

[0117] 1.1.1 Preparation of dopamine monomer solution: 0.15 g of dopamine hydrochloride, 0.2 mL of template TMB, and 0.1 g of F127 were dissolved in 10 mL of 50% ethanol / water solution to obtain a dopamine monomer solution; finally, bowl-shaped PDA@Au nanoparticles were prepared.

[0118] The SEM electron microscope (the scale line is 200nm) of the prepared PDA particles is as follows: Figure 5 As shown, it can be seen that some PDA particles also have a bowl-shaped structure with a large specific surface area.

[0119] Example 4

[0120] The other steps are the same as those in Example 1, except that the amount of TMB added in 1.1.1 is different:

[0121] 1.1.1 Preparation of dopamine monomer solution: 0.15 g dopamine hydrochloride, 0.15 mL template TMB, and 0.1 g F127 were dissolved in 10 mL 50% ethanol / water solution to obtain dopamine monomer solution; finally, PDA@Au nanoparticles were prepared. The SEM electron microscope (scale line is 200 nm) of the prepared PDA particles is as follows: Figure 6 As shown, almost all PDA particles present a spherical structure.

[0122] It can be seen from Examples 1-4 that when the amount of TMB is between 0.2 and 0.3 mL (the mass volume ratio of dopamine hydrochloride to TMB is in the range of 1:1.3-2), bowl-shaped PDA and PDA@Au porous materials with a larger specific surface area can be prepared. However, when the amount of TMB is further reduced to 0.15 mL, the prepared PDA particles are spherical and have a smaller specific surface area. The SERS detection response performance of the PDA@Au particles after loading with precious metals will be reduced.

[0123] Example 5

[0124] The other steps are the same as those in Example 1, except that the concentration of chloroauric acid in 1.2.2 is different:

[0125] 1.2.2 Mix 10 mL of PDA dispersion with 90 μL of HAuCl4 solution (concentration of 50 μM), stir and react at 25°C for 24 hours, centrifuge at 13000 rpm / min, and wash with ultrapure water 2-3 times to obtain bowl-shaped PDA@Au nanoparticles.

[0126] Example 6

[0127] The other steps are the same as those in Example 1, except that the concentration of chloroauric acid in 1.2.2 is different:

[0128] 1.2.2 Mix 10 mL of PDA dispersion with 90 μL of HAuCl4 solution (concentration of 100 μM), stir and react at 25°C for 24 hours, centrifuge at 13000 rpm / min, and wash with ultrapure water 2-3 times to obtain bowl-shaped PDA@Au nanoparticles.

[0129] Further investigation and verification of the influence of substrate materials with different morphologies on SERS response ability:

[0130] The FDTD simulated electromagnetic field distribution curves and the response SERS spectra of toluidine blue of the PDA@Au nanoparticles with different morphologies prepared in Test Examples 1 and 4-6 and the bowl-shaped PDA@Au nanoparticles with different gold loadings were tested. Figure 7 The FDTD (finite difference time domain) simulated electromagnetic field distribution curves of PDA@Au nanoparticles with different morphologies in Examples 1, 4-6 are shown. Among them, AD corresponds to the simulated electromagnetic field distribution curves of the nanoparticles in Examples 4, 1, 5 and 6, respectively.

[0131] It can be seen that the electromagnetic field of the bowl-shaped PDA@Au NPs (B) of Example 1 is significantly stronger than that of the spherical PDA@Au NPs (A) of Example 4, which is attributed to the enhanced absorption of gold nanoparticles on both sides of the bowl-shaped structure. Figure 7 It can be seen from BD that with the increase of the concentration of chloroauric acid, the size of gold nanoparticles in the substrate material increases, so the electromagnetic field of the bowl-shaped PDA@Au NPs is also enhanced.

[0132] The surface enhanced Raman scattering (SERS) activities of the four prepared PDA@Au substrates were evaluated using 1 μM toluidine blue as a Raman reporter. Figure 8 The SERS spectra of the PDA@Au nanoparticles with different morphologies in Examples 1, 4-6 in response to toluidine blue are shown (E is the SERS spectrum of the nanoparticles in Example 4, F is the SERS spectrum of the nanoparticles in Example 1, G is the SERS spectrum of the nanoparticles in Example 5, and H is the SERS spectrum of the nanoparticles in Example 6). It can be seen that all SERS spectra have a peak at 1629 cm –1 A significant peak appears at each position, which can be attributed to the coupling of the C–C stretching vibration of the benzene ring with the C–N–C stretching vibration of the benzene ring and the bending deformation vibration mode of the –NH2 group.

[0133] In summary, PDA@Au nanoparticles have SRES responsive activity. Relatively speaking, the bowl-shaped PDA@Au has a larger specific surface area and the absorption of precious metal particles by the two side structures is enhanced, so the electromagnetic field is enhanced; and as the size of the precious metal increases, the electromagnetic field of PDA@Au NPs will also increase.

[0134] Example 7

[0135] The other steps are the same as those in Example 1, except that a low eutectic mixture is added in step 1.2.2:

[0136] 1.2.2 Mix 10 mL of bowl-shaped PDA dispersion with 90 μL of HAuCl4 solution (concentration of 20 μM), add 1 mL of low eutectic mixture choline chloride-ethylene glycol (the molar ratio of the two is 1:3), stir and react at 25°C for 24 h, centrifuge at 13000 rpm / min, and wash with ultrapure water 2-3 times to finally obtain bowl-shaped PDA@Au nanoparticles.

[0137] Fig. 9This is a scanning electron microscope image of the bowl-shaped PDA@Au nanoparticles prepared in Example 7. It can be seen that compared with Example 1, when the amount of precious metal is the same, the surface of the bowl-shaped PDA prepared in Example 7 can load more Au. This may be due to the addition of the low eutectic mixture, which improves the binding force between PDA and Au, reduces Au agglomeration, and further improves stability, so that more precious metals can be effectively loaded on the PDA surface. On the one hand, the loading amount and effective utilization rate of precious metals are increased. On the other hand, due to more precious metals on the surface of the bowl-shaped PDA, the precious metal particles form more protrusions, enriching the specific surface structure of the substrate material and increasing the specific surface area of ​​the substrate material, thereby further improving the SERS response performance.

[0138] Example 8

[0139] The other steps are the same as those in Example 1, except that chloroplatinic acid is used instead of chloroauric acid in 1.2.2. Finally, PDA@Pt nanoparticles are obtained.

[0140] Example 9

[0141] A method for rapid detection of steroid hormones based on surface enhanced Raman scattering technology for qualitative detection of androgenic steroids comprises the following steps:

[0142] 1) bowl-shaped PDA@Au nanoparticles prepared in Example 1;

[0143] 2) Dissolve four androgenic steroids (Androstenedione, Adrenosterone, 4-Androsten-4-ol-3,17-dione and 5-Androstene-3β,17β-diol) in ultrapure water to prepare standard solutions with a concentration of 1 mM. Take 10 μL of each androgenic steroid standard solution, add 10 μL of bowl-shaped PDA@Au nanoparticle dispersion (concentration of 100 mM), and shake to mix;

[0144] 3) Use Raman spectrometer to perform SERS detection, record spectra and perform data analysis: The mixed solution is dropped on a clean aluminum foil, and the SERS properties are measured using a confocal microscopic Raman spectrometer. The laser wavelength is 633 nm, and the Raman spectrum results are obtained by accumulating for 10 seconds and repeating 3 times; LabSpec software (version 6.2) is used to perform baseline correction and normalization on the obtained Raman spectrum.

[0145] like Fig.10As shown, the bowl-shaped PDA@Au nanoparticles of the present invention can effectively identify and distinguish the corresponding intervals of four androgenic steroids, and can accurately and quickly screen out four androgenic steroids with similar structures according to their molecular vibration peaks and intensities. It has a broad-spectrum effect on the detection and rapid identification of androgenic steroids using the SERS method, solves the problem of only being able to use complex detection methods such as nuclear magnetic resonance or high costs, and proposes new ideas and methods for the analysis and detection of similar substances.

[0146] Example 10

[0147] A method for rapid detection of steroid hormones based on surface enhanced Raman scattering technology for quantitative detection of androgenic steroids comprises the following steps:

[0148] i) bowl-shaped PDA@Au nanoparticles prepared in Example 1;

[0149] ii) Four androgenic steroids (Androstenedione, Adrenosterone, 4-Androsten-4-ol-3,17-dione and 5-Androstene-3β,17β-diol) were dissolved in ultrapure water to prepare standard androgenic steroid solutions with concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 and 1000 μg / mL, respectively; 10 μL of each androgenic steroid standard solution was taken, and 10 μL of bowl-shaped PDA@Au nanoparticle dispersion (concentration of 100 mM) was added to each solution, and the mixture was shaken and mixed;

[0150] iii) Perform SERS detection using a Raman spectrometer, record spectra and perform data analysis: The mixed solution was dropped onto a clean aluminum foil, and the SERS properties were measured using a confocal micro-Raman spectrometer. The laser wavelength was 633 nm, and the Raman spectrum results were obtained by accumulating and repeating for 3 times for 10 seconds. The obtained Raman spectra were baseline corrected and normalized using LabSpec software (version 6.2) to analyze the concentration-spectrum intensity relationship of various androgenic steroids, and a linear regression model (such as Fig.11 shown);

[0151] The linear regression model of androstenedione is: Y = 1695.91x + 7262, and the linear determination coefficient R 2 =0.974; the linear regression model of adrenal androsterone is: Y = 1804.21x + 7519.38, the linear determination coefficient R 2 =0.982; the linear regression model of 4-androsten-4-ol-3,17-dione is: Y=2786.2x+11769.14, the linear determination coefficient R 2=0.990; the linear regression model of androstenediol is: Y = 1685.01x + 678.23, the linear determination coefficient R 2 =0.972.

[0152] iv) Based on this linear regression model, it can be used for the quantitative detection of four androgenic steroids.

[0153] Embodiment 11

[0154] 3.1 Collect actual samples, filter and wash:

[0155] Actual sample 1: A tap water sample was collected 15 minutes after flushing the household drinking water supply system. The collected water sample was boiled for 10 minutes to remove chlorine;

[0156] Actual Sample 2: Pure milk samples purchased from the store were taken and all samples were filtered using a 0.22 μm filter membrane to remove suspended particles.

[0157] 3.2 Each standard androgenic steroid solution was added to the actual samples 1 and 2 to obtain a mixed solution: the concentrations of the four standard androgenic steroid solutions were 0.1, 1 and 100 μg / mL; the volume ratio of the standard androgenic steroid solution to the actual sample was 1:1;

[0158] 3.3 Add the obtained mixed solution into the bowl-shaped PDA@Au nanoparticle substrate material solution obtained in Example 1;

[0159] 3.4 SERS detection was performed using a Raman spectrometer to analyze the concentrations of various androgenic steroids in the actual samples to be tested: the laser wavelength was 633 nm, and the Raman spectrum results were obtained by accumulating and repeating 3 times for 10 seconds (the SERS detection process was the same as in Example 4); and the obtained Raman spectra were baseline corrected and normalized using LabSpec software (version 6.2).

[0160] The test results are shown in Table 1. It can be seen that the SERS detection method of the present invention can detect the concentrations of four standard androgenic steroids with similar structures in actual samples. The SERS method provides good recovery rates ranging from 94.0% to 106.0%, and the relative standard deviation (RSD) is 1.9% to 4.0%. The acceptable recovery rates and RSDs shown in Table 1 verify the potential practicality of the SERS method proposed by the present invention for detecting anabolic androgenic steroids in complex samples.

[0161] Table 1: Detection of synthetic androgenic steroids added to tap water and milk samples

[0162]

[0163]

[0164] *Average of three measurements (±SD)

[0165] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for rapid detection of androgen steroids based on surface enhanced Raman scattering technology, characterized in that: The method comprises the following steps: 1) Synthesize bowl-shaped polydopamine@precious metal substrate material; 2) adding a solution containing a base material to the androgenic steroid solution; 3) Surface enhanced Raman scattering (SERS) detection, recording spectra and performing data analysis; The androgenic steroids include androstenedione (C 19 H 26 O2), Adrenosterone (C 19 H 24 O3), 4-Androsten-4-ol-3,17-dione (C 19 H 26 O3) and 5-Androstene-3β,17β-diol (C 19 H 30 O2).

2. The method according to claim 1, characterized in that The method for preparing the bowl-shaped polydopamine@precious metal substrate material in step 1) comprises the following steps: 1.1 Preparation of bowl-shaped polydopamine PDA: bowl-shaped polydopamine is obtained by polymerization of bowl-shaped dopamine monomers; 1.2 Preparation of bowl-shaped polydopamine@precious metal nanoparticles: Precious metals are in situ reduced and loaded on the surface of bowl-shaped polydopamine particles; Preferably, the step 1.1 comprises the following steps: 1.1.1 Preparation of dopamine monomer solution: dissolve dopamine hydrochloride, template agent tetramethylbenzidine TMB, and triblock polymer F127 in water to obtain a dopamine monomer solution; 1.1.2 Add ammonia water to the dopamine monomer solution and stir to obtain dopamine monomer; 1.1.3 The dopamine monomer is polymerized to obtain bowl-shaped polydopamine.

3. The method according to claim 2, characterized in that The step 1.2 comprises the following steps: 1.2.1 Dissolve the bowl-shaped polydopamine in water to obtain a bowl-shaped polydopamine dispersion: 1.2.2 Mix the bowl-shaped polydopamine dispersion with the salt solution of the precious metal to obtain bowl-shaped polydopamine@precious metal nanoparticles.

4. The method according to claim 2, characterized in that: In step 1.1.1, in the dopamine monomer solution, the concentration of dopamine hydrochloride is 10-15 g / L; the concentration of F127 is 6-10 g / L, and the mass volume (g / mL) ratio of dopamine hydrochloride to TMB is 1:(1.3-2).

5. The method according to claim 4, characterized in that In step 1.1.2, the mass ratio of ammonia water to dopamine monomer solution is (2.5-4):1; the stirring temperature is 20-30°C, and the stirring time is 1-3h; The polymerization reaction temperature in step 1.1.3 is 100-130° C., the reaction time is 12-36 hours, and the polymerization reaction system is ethanol-water.

6. The method according to claim 3, characterized in that In step 1.2.1, the concentration of the bowl-shaped polydopamine dispersion is 10-20 g / L; In step 1.2.2, the noble metal is selected from Au or Pt; Preferably, the noble metal salt is selected from chloroauric acid or chloroplatinic acid; Preferably, the concentration of the noble metal salt solution is 15-100 μM; Preferably, the volume ratio of the polydopamine dispersion to the noble metal salt solution is 100:1 to 200:1; Preferably, the reaction time is 12-36 hours and the temperature is 20-30°C.

7. The method according to claim 1, characterized in that In the solution containing the base material in step 2), the solvent is water; the volume ratio of the androgen steroid solution to the solution containing the base material is (1-2):1; the concentration of the solution containing the base material is 50-200 mM; In step 3), the laser wavelength of Raman spectroscopy detection is 633 nm; the type of the androgenic steroid is detected or identified.

8. A method for rapid detection of androgen steroids based on surface enhanced Raman scattering technology, characterized in that: The method comprises the following steps: i) Synthesizing bowl-shaped polydopamine@precious metal substrate materials; ii) preparing standard androgenic steroid solutions of different concentrations and adding the solution containing the base material; iii) analyzing the concentration-spectrum intensity relationship of the androgenic steroid by performing surface enhanced Raman scattering (SERS) detection on standard androgenic steroid solutions of different concentrations, and establishing a linear regression model between the androgenic steroid concentration and the characteristic peak intensity of the SERS spectrum; iv) Conduct quantitative analysis based on linear regression model; Preferably, the androgenic steroid includes Androstenedione (C 19 H 26 O2), Adrenosterone (C 19 H 24 O3), 4-Androsten-4-ol-3,17-dione (C 19 H 26 O3) and 5-Androstene-3β,17β-diol (C 19 H 30 O2); Preferably, the standard androgenic steroid concentrations are 0.0001 μg / mL, 0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL and 1000 μg / mL, respectively.

9. The method according to claim 8, characterized in that The method for preparing the bowl-shaped polydopamine@precious metal substrate material in step i) comprises the following steps: 1.1 Preparation of bowl-shaped polydopamine PDA: bowl-shaped polydopamine is obtained by polymerization of bowl-shaped dopamine monomers; 1.2 Preparation of bowl-shaped polydopamine@precious metal nanoparticles: Precious metals are in situ reduced and loaded on the surface of bowl-shaped polydopamine particles; Preferably, the step 1.1 comprises the following steps: 1.1.1 Preparation of dopamine monomer solution: dissolve dopamine hydrochloride, template agent tetramethylbenzidine TMB, and triblock polymer F127 in water to obtain a dopamine monomer solution; 1.1.2 Add ammonia water to the dopamine monomer solution and stir to obtain dopamine monomer; 1.1.3 Polymerization of dopamine monomers to obtain bowl-shaped polydopamine; Preferably, the step 1.2 comprises the following steps: 1.2.1 Dissolve the bowl-shaped polydopamine in water to obtain a bowl-shaped polydopamine dispersion: 1.2.2 Mix the bowl-shaped polydopamine dispersion with the salt solution of the precious metal to obtain bowl-shaped polydopamine@precious metal nanoparticles.

10. Use of the method according to any one of claims 1 to 9 in the rapid detection of androgen steroids by surface enhanced Raman scattering.

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