Method and device for raman spectroscopy of small molecule compounds

Abstract

The application provides a Raman spectrum analysis method and device for small molecule compounds, and specifically, the method shields background signals by an aluminum sheet and performs Raman spectrum detection in combination with an enhanced substrate Au@Ag. The method has the advantages of simple operation, rapid reaction, high sensitivity, low cost and obvious characteristic signal peaks. In addition, the small molecule substances in the serum sample can be obtained by removing the protein in the serum sample by using ammonium sulfate, and rich and detailed information of the small molecule substances in the serum sample can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of compound detection technology, and more specifically, this invention relates to a Raman spectroscopy analysis method and apparatus for small molecule compounds. Background Technology

[0002] The detection of small molecule compounds (MW < 1000) is a crucial step in fields such as metabolomics, food safety, and drug analysis. In metabolomics, small molecule metabolites serve as substrates and products of metabolic pathways, and the samples used for detection are typically serum, tissue extracts, etc. In the quality assessment of traditional Chinese medicine or the detection of pesticide residues in fruits and vegetables, the target small molecules may be active ingredients or exogenous contaminants. Current mainstream detection technologies rely on nuclear magnetic resonance (NMR), mass spectrometry (MS), and chromatography-coupled techniques (such as HPLC-MS and GC-MS). While these methods can provide high-precision results, they have limitations such as high instrument costs and complex sample preparation (such as derivatization and chromatographic separation), making it difficult to meet the needs of rapid on-site detection or large-scale screening.

[0003] Raman spectroscopy, as a label-free and non-destructive molecular fingerprinting technique, has become an ideal alternative for the detection of small molecule compounds. Existing research shows that this technique can directly obtain Raman spectra of complex matrices such as serum, extracts of traditional Chinese medicine, and the surfaces of fruits and vegetables, which contain rich information about small molecules. However, its practical application faces three major bottlenecks: (1) Conventional Raman systems are limited by insufficient sensitivity, making it difficult to detect low concentrations of small molecules; (2) Although enhancing the substrate (such as SERS) can improve the signal intensity, large molecules such as proteins in serum or pigments in fruits and vegetables will competitively occupy the "hot spot" region, obscuring the signal of the target small molecule; (3) The differences in physical properties of different matrices (such as viscous serum and porous traditional Chinese medicine) result in a lack of universality in pretreatment methods. These problems seriously restrict the standardized application of Raman technology in cross-disciplinary small molecule detection.

[0004] Therefore, there is an urgent need to develop a Raman spectroscopy detection method with high universality and simplified pretreatment, which can overcome interference from complex matrices and achieve rapid and sensitive detection of small molecule compounds in various sample types such as serum, Chinese medicinal materials, fruits and vegetables, providing a general technical platform for metabolomics, quality monitoring and safety detection. Summary of the Invention

[0005] The purpose of this invention is to provide a Raman detection method for small molecule compounds that is highly universal, has simplified pretreatment, is resistant to background interference, and is rapid and sensitive.

[0006] In a first aspect of the present invention, a Raman spectroscopic analysis method for small molecule compounds is provided, the method comprising the following steps:

[0007] a. Add Au@Ag to the sample to be tested and mix well to form a mixture; and

[0008] b. The mixture is placed in a container containing an aluminum substrate for Raman detection.

[0009] In another alternative, the small molecule compound is an organic or inorganic small molecule, including carbohydrates, amino acids, peptides, fatty acids, nucleotides, vitamins, minerals, alkaloids, aglycones, flavonoids, acids, bases, alcohols, esters, oxides, and / or amines.

[0010] In another example, the sample to be tested is a biological sample or a plant sample.

[0011] In another example, the sample to be tested is a serum sample, chicory seeds, garlic, or scallions.

[0012] In another alternative, the Au@Ag is in a sol state during the mixing reaction.

[0013] In another example, the Au@Ag is in the form of spherical granules.

[0014] In another alternative, the diameter of the spherical Au@Ag is 10-100 nm, preferably 10-50 nm, and more preferably 10-30 nm.

[0015] In another alternative, the Au@Ag has a characteristic absorption peak at 407±10 nm.

[0016] In another preferred embodiment, the Au@Ag is prepared by adding silver nitrate solution and L-ascorbic acid solution to a colloidal gold solution.

[0017] In another preferred embodiment, the Au@Ag is prepared on-the-spot.

[0018] In another alternative, the container containing an aluminum substrate includes: a container made of aluminum or a container formed by placing aluminum sheets or films in the container.

[0019] In another preferred embodiment, the container containing the aluminum substrate is an enzyme-labeled well with an aluminum sheet placed at the bottom.

[0020] In another alternative, the test sample and Au@Ag are mixed directly in a container containing an aluminum substrate.

[0021] In another example, the sample to be tested and Au@Ag were mixed and then transferred to a container containing an aluminum substrate.

[0022] In another alternative example, when the method is used on a serum sample, it includes the following steps:

[0023] a. Add ammonium sulfate to the serum sample to precipitate proteins and separate the supernatant;

[0024] b. Add Au@Ag to the supernatant to form a mixture; and

[0025] c. The mixture is placed in a sample container containing an aluminum substrate for Raman detection.

[0026] In another preferred embodiment, the serum sample is derived from the serum of a patient or a healthy person.

[0027] In another preferred embodiment, the ammonium sulfate is a saturated ammonium sulfate solution.

[0028] In another preferred embodiment, the ammonium sulfate, as a quality control material for the measurement system, has a characteristic Raman signal at 970 cm⁻¹ ± 5 cm⁻¹.

[0029] In another preferred embodiment, the method is used to detect small molecule metabolites in a serum sample, including plasma proteins, growth factors, hormones, inorganic ions, amino acids, glucose, nucleosides, lipids, and sterols.

[0030] In another preferred embodiment, the detection is performed using a Raman spectrometer.

[0031] In a second aspect of the invention, a Raman spectroscopic analysis apparatus for small molecule compounds is provided, the apparatus comprising:

[0032] A. Input module, the input module includes: (1) pre-processing the sample to be tested, and (2) mixing the processed sample with Au@Ag to form a mixture;

[0033] B. A Raman detection module, wherein the Raman detection module is configured to: detect a mixture placed in a container containing an aluminum substrate using a Raman spectrometer; and

[0034] C. Output module, wherein the output module is configured to output Raman characteristic spectral signals.

[0035] In another preferred embodiment, the pretreatment includes: precipitating proteins in the serum sample using ammonium sulfate reagent and separating the supernatant.

[0036] In another preferred embodiment, the pretreatment includes: water-soluble or alcohol-soluble treatment of the plant sample.

[0037] In a third aspect of the invention, a kit for Raman spectroscopy analysis of small molecule compounds is provided, the kit comprising:

[0038] (i) Colloidal gold reagent, silver nitrate reagent, and reducing agent used in the preparation of Au@Ag; and

[0039] (ii) Containers containing an aluminum substrate.

[0040] In another preferred embodiment, the reducing agent is selected from the group consisting of L-ascorbic acid, hydroquinone, sodium borohydride, lithium aluminum hydride, or sodium hydride.

[0041] In another preferred embodiment, the kit further includes (iii) ammonium sulfate reagent.

[0042] In another preferred embodiment, the Au@Ag is prepared on the spot using colloidal gold reagent, silver nitrate reagent, and L-ascorbic acid reagent.

[0043] In another preferred embodiment, the Au@Ag is stable within 0-12 hours, preferably 0-8 hours, after preparation.

[0044] In another preferred embodiment, the kit further includes instructions and a container for holding the reagents.

[0045] In another preferred embodiment, the kit further includes a diluent for diluting the above-described reagents.

[0046] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0047] Figure 1 The measuring device of the detection method of the present invention is shown.

[0048] Figure 2 The Raman spectra of the enzyme-labeled wells with and without aluminum foil are shown.

[0049] Figure 3 A schematic diagram of Raman detection is shown.

[0050] Figure 4 The Raman spectrum of direct detection of serum a is shown.

[0051] Figure 5 The UV-Vis absorption spectrum of Au@Ag is shown.

[0052] Figure 6 The scanning electron microscope image of Au@Ag is shown.

[0053] Figure 7 Transmission electron microscopy (TEM) images of Au@Ag are shown.

[0054] Figure 8 The Raman spectrum of serum a+Au@Ag is shown.

[0055] Figure 9 The Raman spectrum of the supernatant + Au@Ag after protein removal from serum a is shown.

[0056] Figure 10 The Raman spectrum of direct detection of serum b is shown.

[0057] Figure 11 The Raman spectrum of serum b+Au@Ag is shown.

[0058] Figure 12 The Raman spectrum of the supernatant + Au@Ag after protein removal from serum b is shown.

[0059] Figure 13 The Raman spectrum of acetonitrile is shown.

[0060] Figure 14 The Raman spectrum of the supernatant + Au@Ag after removing serum protein a with acetonitrile is shown.

[0061] Figure 15 The Raman spectrum of a saturated ammonium sulfate solution is shown.

[0062] Figure 16 The Raman spectrum of the supernatant + Au@Ag after removing serum protein a with ammonium sulfate is shown.

[0063] Figure 17 The Raman spectrum of a saturated sodium chloride solution is shown.

[0064] Figure 18 The Raman spectrum of the supernatant + Au@Ag after removing serum α protein with sodium chloride is shown.

[0065] Figure 19 The diagram shows the supernatant after removing serum protein a being loaded into the sample well of the silver nanoparticles.

[0066] Figure 20 The Raman spectrum of the silver nanoparticles is shown.

[0067] Figure 21 The Raman spectrum of the supernatant after protein removal from serum a + silver nanoparticles is shown.

[0068] Figure 22 The Raman spectrum of colloidal silver at 50 nm is shown.

[0069] Figure 23 The Raman spectrum of the supernatant after protein removal from serum a + colloidal silver is shown.

[0070] Figure 24 The Raman spectrum of Au@Ag sol is shown.

[0071] Figure 25 The Raman spectrum of the supernatant + Au@Ag after protein removal from serum a is shown.

[0072] Figure 26 The Raman spectrum of colloidal gold at 70 nm is shown.

[0073] Figure 27 The Raman spectrum of chicory seed + colloidal gold is shown.

[0074] Figure 28 The Raman spectrum of chicory seed + colloidal silver is shown.

[0075] Figure 29 The Raman spectrum of chicory seed + Au@Ag is shown.

[0076] Figure 30 The Raman spectrum of garlic + colloidal gold is shown.

[0077] Figure 31 The Raman spectrum for detecting garlic + colloidal silver is shown.

[0078] Figure 32 The Raman spectrum of garlic + Au@Ag is shown.

[0079] Figure 33 The Raman spectrum of the detection of scallion + colloidal gold is shown.

[0080] Figure 34 The Raman spectrum of the detection of scallion + colloidal silver is shown.

[0081] Figure 35 The Raman spectrum of scallion + Au@Ag is shown.

[0082] Figure 36 The Raman spectra of the supernatant after protein removal from serum c and the immediately prepared Au@Ag are shown.

[0083] Figure 37 The Raman spectra of the supernatant after protein removal from serum c and Au@Ag stored for 8 hours are shown.

[0084] Figure 38 The Raman spectra of the supernatant after protein removal from serum c and Au@Ag stored for 24 hours are shown. Detailed Implementation

[0085] Through extensive and in-depth research, the inventors have developed a Raman detection method for small molecule compounds. This method uses an aluminum sheet to shield the background signal and combines it with a surface-enhanced substrate Au@Ag to rapidly detect multiple small molecule compounds in a sample and obtain Raman spectra with distinct characteristic signals. Furthermore, by using ammonium sulfate to remove proteins from serum, more abundant and detailed information on small molecule metabolites in serum samples can be obtained.

[0086] the term

[0087] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0088] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0089] In this invention, the terms "reinforced substrate", "Raman magnetic beads" and "Raman microspheres" are used interchangeably.

[0090] Surface-enhanced Raman spectroscopy

[0091] Raman spectroscopy, a type of molecular vibrational spectroscopy, can reflect the characteristic structure of molecules. However, Raman scattering is a very weak process. Surface-enhanced Raman spectroscopy (SERS) involves adsorbing the analyte onto rough surfaces such as silver, gold, or copper, significantly increasing the intensity of its Raman signal. The corresponding spectrum is called surface-enhanced Raman spectroscopy. SERS has demonstrated significant application potential over existing technologies in molecular imaging and rapid qualitative and quantitative detection of multiple targets with high precision, and has been widely used in the analysis of proteins, bacteria, pathogens, and small molecules.

[0092] ammonium sulfate

[0093] White crystalline powder; aqueous solution is acidic. Insoluble in alcohol, acetone, and ammonia. Hygroscopic; it clumps together after absorbing moisture. Completely decomposes into ammonia, nitrogen, sulfur dioxide, and water when heated above 513℃. Releases ammonia upon reaction with alkalis. Reacts with barium chloride solution to form barium sulfate precipitate. Can also cause protein salting out.

[0094] It is an excellent nitrogen fertilizer (commonly known as fertilizer powder), suitable for general soils and crops. It promotes vigorous growth of branches and leaves, improves fruit quality and yield, and enhances crops' resistance to disasters. It can undergo a double decomposition reaction with salt to produce ammonium chloride, react with aluminum sulfate to produce ammonium alum, and be used with boric acid and other substances to manufacture refractory materials. Adding it to electroplating solutions increases conductivity. It is also a catalyst for food coloring, a nitrogen source for cultivating yeast in fresh yeast production, a dyeing auxiliary for acid dyes, and a deliming agent for leather. Furthermore, it is used in beer brewing, chemical reagents, and battery production. Another important application is in rare earth mining. Mining uses ammonium sulfate as raw material, employing ion exchange to extract rare earth elements from the ore. The leachate is then collected, purified, precipitated, pressed, and calcined to obtain the raw rare earth ore.

[0095] In biology, ammonium sulfate can be used in protein purification processes because it is an inert substance that does not readily react with other bioactive substances. This allows it to protect protein activity to the greatest extent possible during purification. In addition, ammonium sulfate has excellent solubility and can create a high-salt environment, which prepares the protein for precipitation and subsequent high-salt purification.

[0096] Detection methods

[0097] The detection method of this invention is based on surface-enhanced Raman spectroscopy, uses an aluminum sheet for background removal, and combines it with an enhanced substrate Au@Ag, which enables simple and rapid detection of small molecule compounds in various samples.

[0098] In serum samples, ammonium sulfate was used to remove proteins, ultimately obtaining Raman spectral signals with distinct characteristic peaks. The method was validated in different serum samples, showing that it could detect clear characteristic signals in all cases. Furthermore, the method was compared with different protein removal reagents (e.g., acetonitrile, sodium chloride, ammonium sulfate), and ammonium sulfate was ultimately selected as having excellent protein removal performance.

[0099] The detection of serum samples according to the present invention specifically includes the following steps:

[0100] 1. Preparation of Au@Ag sol (for substrate reinforcement);

[0101] 2. Add saturated ammonium sulfate solution to the serum sample to precipitate proteins, and separate the supernatant;

[0102] 3. Mix the supernatant with the Au@Ag sol in the wells containing the aluminum foil;

[0103] 4. Use a Raman spectrometer to detect characteristic signals.

[0104] The Raman spectroscopy analysis method of this invention can also be used to detect small molecule compounds in plant samples such as traditional Chinese medicine materials. By simply mixing the sample with an enhancing substrate Au@Ag, the Raman characteristic signal spectrum of the sample can be obtained rapidly. This method can be used to identify the authenticity and quality of medicinal materials, as well as information on their small molecule components.

[0105] Reagent test kit

[0106] The present invention also provides a kit for the detection method of the present invention, the kit comprising:

[0107] (i) Colloidal gold reagents, silver nitrate reagents and reducing agents used to prepare Au@Ag;

[0108] (ii) Containers containing an aluminum substrate; and

[0109] (iii) Ammonium sulfate reagent.

[0110] The enhanced substrate Au@Ag in the kit can be prepared instantaneously using colloidal gold reagent, silver nitrate reagent, and a reducing agent, and Au@Ag exhibits stability within 8 hours. In a specific embodiment of the invention, Au@Ag sol is prepared using colloidal gold reagent, silver nitrate reagent, and L-ascorbic acid, wherein L-ascorbic acid can be replaced with other reducing agents, such as hydroquinone, sodium borohydride, lithium aluminum hydride, sodium hydride, etc. The kit of the present invention has universal applicability and is simple to operate, and can be used for the detection of biological or plant samples.

[0111] Furthermore, when the kit of the present invention is used to detect serum samples, ammonium sulfate reagent is used to precipitate proteins in the serum, enabling the acquisition of information on various small molecule metabolites with Raman characteristic signals. In a specific embodiment of the present invention, a saturated ammonium sulfate solution is used for protein precipitation, and the saturated ammonium sulfate solution can serve as a quality control for the detection system, exhibiting a characteristic Raman signal at 970 cm⁻¹ ± 5 cm⁻¹.

[0112] Compared with the prior art, the advantages of the present invention are as follows:

[0113] 1. The Raman detection method for small molecule compounds of the present invention is simple to operate, fast to react, highly sensitive and low in cost, and can be used for the detection of various biological or plant samples.

[0114] 2. The present invention uses an aluminum sheet placed at the bottom of the sample container, which can shield the background signal brought by the sample container and improve accuracy.

[0115] 3. This invention utilizes ammonium sulfate to precipitate proteins in serum. Compared with other protein precipitation reagents (acetonitrile, sodium chloride, etc.), the spectral signal is significantly enhanced and the characteristic peaks are obvious, which can present richer and more detailed sample information. Moreover, the serum sample can be detected by Raman spectroscopy after protein precipitation, without the need for complicated sample pretreatment and expensive instruments (such as micro Raman spectroscopy).

[0116] 4. The detection method of the present invention is stable in different serum samples and is expected to be used to construct Raman characteristic standard spectra of small molecule metabolites in serum.

[0117] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0118] reagents

[0119] 1. 20nm colloidal gold (batch number: D241125001, Shanghai Xinpu Biotechnology Co., Ltd.);

[0120] 2. 70nm colloidal gold (batch number: D241205002, Shanghai Xinpu Biotechnology Co., Ltd.);

[0121] 3. 50nm colloidal silver (batch number: D241219001, Shanghai Xinpu Biotechnology Co., Ltd.);

[0122] 4. Silver nanoparticles (batch number: AS120241212, Advanced Polaris Technology Co., Ltd.);

[0123] 5. Silver nitrate (analytical grade, batch number: 20200923, Sinopharm Chemical Reagent Co., Ltd.);

[0124] 6. L-Ascorbic acid (analytical grade, batch number: 20170109, Sinopharm Chemical Reagent Co., Ltd.);

[0125] 7. Acetonitrile (analytical grade, batch number: 20240125, Sinopharm Chemical Reagent Co., Ltd.);

[0126] 8. Ammonium sulfate (analytical grade, batch number: 20140829, Sinopharm Chemical Reagent Co., Ltd.)

[0127] 9. Sodium chloride (analytical grade, batch number: C2019014, Sinopharm Chemical Reagent Co., Ltd.);

[0128] 10. Hydrochloric acid (analytical grade, batch number: 20150413, Sinopharm Chemical Reagent Co., Ltd.).

[0129] All water used in the experiments was deionized water.

[0130] equipment

[0131] 1. CT14RD type benchtop high-speed refrigerated centrifuge (Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.);

[0132] 2. FA1004 electronic balance (Shanghai Sunny Hengping Scientific Instruments Co., Ltd.);

[0133] 3. Leici PHS-25 Digital Display pH Meter (Leici (Shanghai) Technology Co., Ltd.);

[0134] 4. L6S UV-Vis spectrophotometer (Shanghai Yidian Analytical Instrument Co., Ltd.);

[0135] 5. Raman spectrometer (Liqiong (Shanghai) Optoelectronic Technology Co., Ltd.).

[0136] Experimental setup and procedures

[0137] This invention designs and develops a suitable measuring device, see [link / reference]. Figure 1 , Figure 1 It consists of enzyme-labeled wells and an aluminum plate. The enzyme-labeled wells themselves are made of polystyrene (PS), which has a strong Raman signal and can interfere with detection. Therefore, an aluminum plate is placed at the bottom of the well. Aluminum is one of the few materials that almost does not have a Raman signal, thus effectively avoiding interference from the sample cup material. The Raman spectra of the enzyme-labeled wells with and without the aluminum plate are shown below. Figure 2 As shown, it can be seen that almost no impurity background peaks were detected in the Raman spectrum after adding aluminum sheets.

[0138] The sample and the enhanced substrate (Au@Ag sol) were mixed and added to the above measuring device, and Raman spectroscopy was used for detection. Small molecule metabolites in the sample were identified by the presented Raman characteristic spectra. Figure 3 As shown.

[0139] Example 1. Detection of Raman spectra of serum a using three methods

[0140] 1. Direct detection of the Raman spectrum of serum a

[0141] Take 100 μl of serum a and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample cup to obtain the Raman spectrum of serum a. Figure 4 As shown.

[0142] 2. Detection of Raman spectra of serum a+Au@Ag

[0143] 2.1 Preparation of Au@Ag (reinforced substrate)

[0144] Take 1 ml of 20 nm colloidal gold, add 100 μl of 20 mM silver nitrate solution and mix well, then add 100 μl of 20 mM L-ascorbic acid solution, slowly mix with a pipette, let stand, and wait until the solution color turns yellowish-brown to obtain Au@Ag sol (reinforcing substrate). Transfer the solution to a light-protected centrifuge tube / reagent bottle for storage.

[0145] 2.3 Characterization of Au@Ag (enhanced substrate)

[0146] Spectral scanning was performed using a UV-Vis spectrophotometer in the wavelength range of 350–650 nm, and the results are as follows: Figure 5 As shown in the figure, the maximum absorption peak of Au@Ag is located at 407 nm. This position corresponds only to the characteristic absorption peak of silver, and no characteristic absorption peak of gold is observed. This phenomenon initially suggests that the gold core may be completely encapsulated by the silver shell.

[0147] The Au@Ag sol was further characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the results are as follows: Figure 6 and Figure 7 As shown. Figure 6 Scanning electron microscopy images of Au@Ag sol are shown, revealing that the nanoparticles are relatively uniform in size and spherical in shape. Figure 7 The results provide transmission electron microscopy (TEM) images of the Au@Ag sol, clearly showing the internal structure of the nanoparticles: the dark central region is the gold core, while the outer light-colored region is the silver shell, with a thickness of approximately 10 nm. The overall diameter of the Au@Ag nanoparticles is approximately 30 nm. These results not only confirm the core-shell structure of the Au@Ag nanoparticles but also provide important structural information for understanding their optical properties and potential applications.

[0148] 2.4 Detection of Raman spectra of serum a+Au@Ag

[0149] Add 50 μl of Au@Ag sol to the measuring device, then add 50 μl of serum a and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of serum a + Au@Ag. Figure 8 As shown.

[0150] 3. Detection of Raman spectra of serum a supernatant after protein removal + Au@Ag

[0151] 3.1 Serum a protein removal

[0152] Take 500 μl of serum a and slowly add 500 μl of saturated ammonium sulfate solution. Protein will be observed to precipitate rapidly. Mix and shake for 1 min, then centrifuge (12000 rpm, 4℃, 15 min). Take out the supernatant for testing.

[0153] 3.2 Detection of Raman spectra of serum a supernatant after protein removal + Au@Ag

[0154] Add 50 μl of Au@Ag sol to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant + Au@Ag after protein removal from serum a. Figure 9 As shown.

[0155] In this embodiment, by... Figure 4 , Figure 8 and Figure 9 Comparative analysis revealed the following findings: direct detection of serum a samples failed to produce characteristic spectra; the addition of Au@Ag sol also failed to produce significant characteristic signals; however, after removing protein components using a saturated ammonium sulfate solution, Raman spectral signals were successfully observed upon binding to an enhancing substrate. This result indicates that protein removal treatment of serum samples significantly improves their Raman spectral detection capability.

[0156] Example 2. Detection of Raman spectra of serum b using three methods.

[0157] 1. Direct detection of the Raman spectrum of serum b

[0158] 100 μl of serum b was added to the measuring device, and the central region of the sample cup was detected using a Raman spectrometer to obtain the Raman spectrum of serum b. Figure 10 As shown.

[0159] 2. Detection of Raman spectra of serum b+Au@Ag

[0160] The preparation of Au@Ag sol was the same as in Example 1. 50 μl of Au@Ag sol was added to the measuring device, followed by 50 μl of serum b, which was then mixed using a pipette. The central region of the sample cup was detected using a Raman spectrometer to obtain the Raman spectrum of serum b + Au@Ag. Figure 11 As shown.

[0161] 3. Detection of Raman spectra of serum b supernatant after protein removal + Au@Ag

[0162] 3.1 Serum beta protein removal

[0163] Take 500 μl of serum b and slowly add 500 μl of saturated ammonium sulfate solution. Protein will be observed to precipitate rapidly. Mix and shake for 1 min, then centrifuge (12000 rpm, 4℃, 15 min). Take out the supernatant for testing.

[0164] 3.2 Detection of Raman spectra of serum b supernatant after protein removal + Au@Ag

[0165] Add 50 μl of Au@Ag sol to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant + Au@Ag after protein removal from serum b. Figure 12 As shown.

[0166] In this embodiment, by... Figure 10 , Figure 11 and Figure 12 Comparative analysis revealed the following findings: Direct detection of serum b samples failed to produce a characteristic spectrum; however, the addition of Au@Ag sol revealed a weak Raman spectral signal. Nevertheless, after removing the protein components and then binding to Au@Ag sol, the Raman spectral signal was significantly enhanced, exhibiting richer and more detailed characteristic spectral information.

[0167] Example 3. Comparison of Raman spectra of supernatants after precipitating proteins with acetonitrile, ammonium sulfate, and sodium chloride.

[0168] 1. Detection of Raman spectra of the supernatant + Au@Ag after removing serum α protein with acetonitrile.

[0169] 1.1 Acetonitrile precipitates proteins

[0170] Take 500 μl of serum a and slowly add 500 μl of acetonitrile solution. Protein will precipitate rapidly. Sonicate on ice for 30 min, centrifuge (12000 rpm, 4℃, 15 min), and take out the supernatant for testing.

[0171] 1.2 Detection of Raman spectra of the supernatant + Au@Ag after removing serum α protein with acetonitrile

[0172] Add 100 μl of acetonitrile solution to the measuring device, and use a Raman spectrometer to detect the central region of the sample cup to obtain the Raman spectrum of acetonitrile. This spectrum can be used as a control for observation. Figure 13 As shown.

[0173] Add 50 μl of Au@Ag sol to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant + Au@Ag after protein removal from serum a. Figure 14 As shown.

[0174] 2. Detection of Raman spectra of the supernatant + Au@Ag after removal of serum alpha protein using ammonium sulfate.

[0175] 2.1 Ammonium sulfate precipitates proteins

[0176] Take 500 μl of serum a and slowly add 500 μl of saturated ammonium sulfate solution. Protein will be observed to precipitate rapidly. Centrifuge (12000 rpm, 4℃, 15 min), and take out the supernatant for testing.

[0177] 2.2 Detection of Raman spectra of the supernatant + Au@Ag after ammonium sulfate removal of serum α protein

[0178] Add 100 μl of saturated ammonium sulfate solution to the measuring apparatus, and use a Raman spectrometer to detect the central region of the sample cup to obtain the Raman spectrum of the saturated ammonium sulfate solution. This spectrum can be used as a control for observation. Figure 15 As shown.

[0179] Add 50 μl of Au@Ag sol to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant + Au@Ag after protein removal from serum a. Figure 16 As shown.

[0180] 3. Detection of Raman spectra of the supernatant + Au@Ag after removal of serum α protein with sodium chloride.

[0181] 3.1 Sodium chloride precipitates proteins

[0182] Take 500 μl of serum a, slowly add 500 μl of saturated sodium chloride solution, and then adjust the pH to 5.0 with 2M hydrochloric acid. Protein will be observed to precipitate rapidly. Centrifuge (12000 rpm, 4℃, 15 min), and take out the supernatant for testing.

[0183] 3.2 Detection of Raman spectra of the supernatant + Au@Ag after removal of serum α protein with sodium chloride

[0184] Add 100 μl of saturated sodium chloride solution to the measuring apparatus, and use a Raman spectrometer to detect the central region of the sample cup to obtain the Raman spectrum of the saturated sodium chloride solution. This spectrum can be used as a control for observation. Figure 17 As shown.

[0185] Add 50 μl of Au@Ag sol to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant + Au@Ag after protein removal from serum a. Figure 18 As shown.

[0186] In this embodiment, by... Figure 14 , Figure 16 and Figure 18 Comparative analysis leads to the conclusion that serum Raman characteristic spectra can only be successfully observed when saturated ammonium sulfate solution is used as the protein precipitant. This result indicates that saturated ammonium sulfate solution plays a unique and crucial role in enhancing the Raman spectral signal during protein precipitation.

[0187] Example 4: Comparison of reinforcement effects of different reinforcing substrates

[0188] 1. Using silver nanoparticles as a reinforcing substrate

[0189] The Raman spectrum of the silver nanoparticles was obtained by detecting the central region of the sample well using a Raman spectroscopy instrument. This spectrum served as a control for further observation. Figure 20 As shown.

[0190] Take 5 μl of the supernatant after removing serum protein α from ammonium sulfate and place it in the sample well of the silver nanoparticles. Figure 19 As shown, the Raman spectrum of the supernatant of serum a after protein removal and silver nanoparticles was obtained by detecting the central region of the sample well using a Raman spectrometer. Figure 21 As shown.

[0191] 2. Using colloidal silver as a reinforcing substrate

[0192] Add 100 μl of 50 nm colloidal silver solution to the measuring device, and use a Raman spectrometer to detect the central region of the sample cup to obtain the Raman spectrum of the colloidal silver. This spectrum can be used as a control for observation. Figure 22 As shown.

[0193] Add 50 μl of 50 nm colloidal silver solution to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant + colloidal silver after protein removal from serum a. Figure 23 As shown.

[0194] 3. Using Au@Ag as the reinforcing substrate

[0195] Add 10 μl of Au@Ag sol to the measuring device, and use a Raman spectrometer to detect the central region of the sample cup to obtain the Raman spectrum of Au@Ag. This spectrum can be used as a control for observation. Figure 24 As shown.

[0196] Add 50 μl of Au@Ag sol to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant + Au@Ag after protein removal from serum a. Figure 25 As shown.

[0197] In this embodiment, by... Figure 21 , Figure 23 and Figure 25 Comparative analysis clearly shows that serum Raman characteristic spectra can only be successfully visualized when Au@Ag is used as the enhancing substrate. This result highlights the unique advantage of the Au@Ag substrate in enhancing Raman spectral signals, indicating its significant improvement in Raman detection of serum samples.

[0198] In summary, the results demonstrate that the analytical method and apparatus for detecting blood metabolites using surface-enhanced Raman spectroscopy proposed in this invention significantly improve the sensitivity of serum metabolite detection. This method provides an efficient and reliable technical means for metabolomics research, possessing significant research value and application potential.

[0199] Example 5: Testing chicory seeds using three different enhancement substrates and comparing the enhancement effects.

[0200] 1. Using 70nm colloidal gold as the reinforcing substrate

[0201] 100 μl of 70 nm colloidal gold solution was added to the measuring device, and the central region of the sample well was detected using a Raman spectrometer to obtain the Raman spectrum of the 70 nm colloidal gold. This spectrum was used as a control for observation. Figure 26 As shown.

[0202] Weigh 0.1 g of chicory seeds and place them in a test tube. Add 500 μl of 70 nm colloidal gold solution, shake for 30 s to ensure full contact, and let stand at room temperature for 10 min. Extract 100 μl of the sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well to obtain the Raman spectrum of chicory seeds + 70 nm colloidal gold. Figure 27 As shown.

[0203] 2. Using colloidal silver as a reinforcing substrate

[0204] Colloidal silver reference spectrum Figure 22 .

[0205] Weigh 0.1 g of chicory seeds and place them in a test tube. Add 500 μl of 50 nm colloidal silver solution, shake for 30 s to ensure full contact, and let stand at room temperature for 10 min. Extract 100 μl of the sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well to obtain the Raman spectrum of chicory seeds + colloidal silver. Figure 28As shown.

[0206] 3. Using Au@Ag as the reinforcing substrate

[0207] Au@Ag reference spectrum Figure 24 .

[0208] Weigh 0.1g of chicory seeds and place them in a test tube. Add 500μl of Au@Ag sol, shake for 30s to ensure full contact, let stand at room temperature for 10min, extract 100μl of sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well to obtain the Raman spectrum of chicory seeds + Au@Ag. Figure 29 As shown.

[0209] In this embodiment, by... Figure 27 , Figure 28 and Figure 29 Comparative analysis yields the following observations: When colloidal silver is used as the reinforcing substrate, the characteristic spectrum cannot be effectively detected; while when colloidal gold is used as the reinforcing substrate, although a weak Raman spectral signal can be observed, its intensity and information content are both limited, and further observation reveals… Figure 27 It is evident that 70nm colloidal gold itself possesses numerous weak characteristic signals, which may affect the detection of target analytes; therefore, 70nm colloidal gold is unsuitable as an enhancement substrate. However, when Au@Ag is used as the enhancement substrate, the Raman spectral signal is significantly enhanced, exhibiting richer, more detailed, and clearer characteristic spectral information. This discovery not only highlights the superior performance of Au@Ag in Raman spectral enhancement but also provides new ideas and directions for research in related fields.

[0210] Example 6: Detecting garlic using three different enhancement substrates and comparing the enhancement effects.

[0211] 1. Using 70nm colloidal gold as the reinforcing substrate

[0212] Colloidal gold reference spectrum Figure 26 .

[0213] Garlic was roughly chopped into pieces the size of rice grains. 0.1g of garlic was placed in a test tube, and 500μl of 70nm colloidal gold solution was added. The mixture was shaken for 30 seconds to ensure full contact. After standing at room temperature for 10 minutes, 100μl of the sample solution was extracted and added to the measuring device. The central region of the sample well was detected using a Raman spectrometer to obtain the Raman spectrum of garlic + 70nm colloidal gold. Figure 30 As shown.

[0214] 2. Using colloidal silver as a reinforcing substrate

[0215] Colloidal silver reference spectrum Figure 22 .

[0216] Weigh 0.1g of garlic and place it in a test tube. Add 500μl of 50nm colloidal silver solution, shake for 30s to ensure full contact, let stand at room temperature for 10min, extract 100μl of sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well to obtain the Raman spectrum of garlic + colloidal silver. Figure 31 As shown.

[0217] 3. Using Au@Ag as the reinforcing substrate

[0218] Au@Ag reference spectrum Figure 24 .

[0219] Weigh 0.1g of garlic and place it in a test tube. Add 500μl of Au@Ag sol, shake for 30s to ensure full contact, let stand at room temperature for 10min, extract 100μl of sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well to obtain the Raman spectrum of garlic + Au@Ag. Figure 32 As shown.

[0220] In this embodiment, by... Figure 30 , Figure 31 and Figure 32 Comparative analysis revealed the following observations: when colloidal silver was used as the enhancing substrate, the characteristic spectrum could not be effectively detected; while when colloidal gold was used as the substrate, although the characteristic spectrum could be detected, the signal intensity was weak; however, when Au@Ag was used as the enhancing substrate, the originally weak Raman signal was significantly enhanced, with its intensity amplified by 10 times compared to the colloidal gold substrate. This experimental result fully demonstrates the superior performance of Au@Ag in the field of Raman spectroscopy enhancement, providing strong support for its potential in related applications.

[0221] Example 7: Detecting and comparing the enhancement effects of scallions using three different enhanced substrates.

[0222] 1. Using 70nm colloidal gold as the reinforcing substrate

[0223] Colloidal gold reference spectrum Figure 26 .

[0224] Cut scallions into 0.5cm segments, weigh 0.1g of scallions and place them in a test tube. Add 500μl of 70nm colloidal gold solution, shake for 30s to ensure full contact, let stand at room temperature for 10min, extract 100μl of sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well, obtaining the Raman spectrum of chicory seed + 70nm colloidal gold. Figure 33 As shown.

[0225] 2. Using colloidal silver as a reinforcing substrate

[0226] Colloidal silver reference spectrum Figure 22 .

[0227] Weigh 0.1g of scallion and place it in a test tube. Add 500μl of 50nm colloidal silver solution, shake for 30s to ensure full contact, let stand at room temperature for 10min, extract 100μl of sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well to obtain the Raman spectrum of scallion + colloidal silver. Figure 34 As shown.

[0228] 3. Using Au@Ag as the reinforcing substrate

[0229] Au@Ag reference spectrum Figure 24 .

[0230] Weigh 0.1g of scallion and place it in a test tube. Add 500μl of Au@Ag sol, shake for 30s to ensure full contact, let stand at room temperature for 10min, extract 100μl of sample solution and add it to the measuring device. Use a Raman spectrometer to detect the central region of the sample well to obtain the Raman spectrum of scallion + Au@Ag. Figure 35 As shown.

[0231] In this embodiment, by... Figure 33 , Figure 34 and Figure 35 Comparative analysis reveals the following conclusions: when colloidal silver was used as the reinforcing substrate, the characteristic spectra could not be effectively detected; while when colloidal gold was used as the substrate, although the characteristic spectra could be detected, the spectral information provided was relatively limited; however, when Au@Ag was used as the reinforcing substrate, not only did the number of characteristic peaks increase significantly, but the richness of the spectral information was also significantly improved. This finding not only confirms the effectiveness of Au@Ag in reinforcing substrates but also provides a new perspective for further research and applications.

[0232] Example 8: Stability observation of Au@Ag sol

[0233] 1. Experimental preparation

[0234] Prepare 1 ml Au@Ag sol, place it in a brown glass bottle, and store at room temperature for 24 hours; prepare 1 ml Au@Ag sol, place it in a brown glass bottle, and store at room temperature for 8 hours; prepare 1 ml Au@Ag sol immediately for analysis.

[0235] Take 500 μl of serum c and slowly add 500 μl of saturated ammonium sulfate solution. Protein will be observed to precipitate rapidly. Mix and shake for 1 min, then centrifuge (12000 rpm, 4℃, 15 min). Take out the supernatant for testing.

[0236] 2. Using freshly prepared Au@Ag sol as a reinforcing substrate

[0237] Add 50 μl of freshly prepared Au@Ag sol to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant after protein removal from serum c + freshly prepared Au@Ag. Figure 36 As shown.

[0238] 3. Au@Ag stored for 8 hours was used as the enhancement substrate.

[0239] Add 50 μl of Au@Ag sol that has been stored for 8 hours to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant after protein removal from serum c + Au@Ag stored for 8 hours. Figure 37 As shown.

[0240] 4. Au@Ag stored for 24 hours was used as the enhancement substrate.

[0241] Add 50 μl of Au@Ag sol that has been stored for 24 hours to the measuring device, then add 50 μl of the extracted supernatant and mix thoroughly with a pipette. Detect the central region of the sample cup using a Raman spectrometer to obtain the Raman spectrum of the supernatant after protein removal from serum c + Au@Ag stored for 24 hours. Figure 38 As shown.

[0242] In this embodiment, by... Figure 36 , Figure 37 and Figure 38 Comparative analysis reveals the following conclusions: When Au@Ag stored for 24 hours was used as the enhancement substrate, the characteristic spectrum of the serum could no longer be effectively detected; however, when Au@Ag stored for 8 hours and freshly prepared Au@Ag were used as enhancement substrates, the characteristic spectrum of the serum could be detected, and the signal intensities of the two were similar. This result indicates that the storage time of Au@Ag has a significant impact on its enhancement performance. With prolonged storage time, the surface activity or structural stability of Au@Ag may gradually decrease, leading to a weakening of its enhancement effect. Au@Ag stored for 8 hours still maintains high performance, while it completely fails after 24 hours of storage. Therefore, in practical applications, it is recommended to control the storage time of Au@Ag to within 8 hours to ensure its stability as an enhancement substrate and the reliability of the detection results. Furthermore, freshly prepared Au@Ag remains the optimal choice, providing the best signal enhancement effect.

[0243] The above content is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and optimizations can be made without departing from the core principles of the present invention, and these adjustments should also fall within the protection scope of the present invention.

[0244] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A Raman spectroscopic analysis method for small molecule compounds, characterized in that, The method includes the following steps: a. Add Au@Ag to the sample to be tested and mix well to form a mixture; and b. The mixture is placed in a container containing an aluminum substrate for Raman detection.

2. The method as described in claim 1, characterized in that, The small molecule compounds are selected from the group consisting of organic or inorganic small molecules, including carbohydrates, amino acids, peptides, fatty acids, nucleotides, vitamins, minerals, alkaloids, aglycones, flavonoids, acids, bases, alcohols, esters, oxides, and / or amines.

3. The method as described in claim 1, characterized in that, The diameter of the Au@Ag is 10-100nm, preferably 10-50nm, and more preferably 10-30nm.

4. The method as described in claim 1, characterized in that, The Au@Ag has a characteristic absorption peak at 407±10nm.

5. The method as described in claim 1, characterized in that, The container containing an aluminum substrate includes: a container made of aluminum or a container formed by placing aluminum sheets or films inside the container.

6. The method as described in claim 1, characterized in that, When the method is used on serum samples, it includes the following steps: a. Add ammonium sulfate to the serum sample to precipitate proteins and separate the supernatant; b. Add Au@Ag to the supernatant to form a mixture; and c. The mixture is placed in a sample container containing an aluminum substrate for Raman detection.

7. The method as described in claim 6, characterized in that, The ammonium sulfate, used as a quality control material in the measurement system, exhibits a characteristic Raman signal at 970 cm⁻¹ ± 5 cm⁻¹.

8. A Raman spectroscopic analysis device for small molecule compounds, characterized in that, The device includes: A. Input module, the input module includes: (1) pre-processing the sample to be tested, and (2) mixing the processed sample with Au@Ag to form a mixture; B. A Raman detection module, wherein the Raman detection module is configured to: detect a mixture placed in a container containing an aluminum substrate using a Raman spectrometer; and C. Output module, wherein the output module is configured to output Raman characteristic spectral signals.

9. A kit for Raman spectroscopy analysis of small molecule compounds, characterized in that, The kit includes: (i) Colloidal gold reagent, silver nitrate reagent, and reducing agent used in the preparation of Au@Ag; and (ii) Containers containing an aluminum substrate.

10. The kit according to claim 9, characterized in that, The kit also includes (iii) ammonium sulfate reagent.

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