SERS (Surface Enhanced Raman Scattering) rapid detection and tracing method for bolete poison protein

Abstract

The invention discloses a bolete poison protein SERS (Surface Enhanced Raman Scattering) rapid detection and traceability method which comprises the following steps: (1) sucking a bolete solution containing poison protein by using a pipette, and dropwise adding the bolete solution on the surface of a small-size high-stability enhanced substrate; (2) placing the substrate in an ultrasonic instrument, carrying out ultrasonic drying, and then carrying out SERS signal acquisition; and (3) carrying out denoising and baseline correction pretreatment on the collected SERS spectral data, and carrying out dimensionality reduction visualization and traceability identification on the SERS spectral data obtained from boletes from different producing areas. The enhanced substrate with small size and high stability is used for SERS detection, the detection effect is stable, and the problem of unstable signal enhancement caused by coffee ring effect is solved, so that stable and rapid detection of bolete poisoning protein can be realized, and the method is suitable for rapid determination of a poisoning site. Meanwhile, the method has very high distinction degree on detection of bolete from different producing areas, and can meet the requirement of accurate traceability.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a SERS rapid detection and tracing method for bovine toxic protein. Background Art

[0002] Mushroom poisoning is a prominent public health problem worldwide and one of the main causes of foodborne poisoning in my country. Common species of mushroom poisoning include Boletus lanmo, Boletus edulis, Boletus edulis and Boletus edulis, which are mainly distributed in East China, South China and Southwest my country. Eating these mushrooms raw, improperly processed or consumed in excess can cause hallucinogenic poisoning, and related poisoning incidents occur frequently. Boletus toxin proteins derived from poisonous mushrooms. Can cause hallucinations and mental abnormalities. The main symptoms are: dizziness, hallucinations, delirium, mania, which can easily induce mental confusion, schizophrenia and persecution delusions. In severe cases, pulmonary edema, respiratory depression and coma may occur and lead to death, posing a huge threat to people's life and health.

[0003] At present, the existing detection methods for toxin protein detection are: (1) enzyme activity inhibition method; (2) immunoassay method; (3) liquid chromatography; (4) mass spectrometry; (5) biosensor assay method. Enzyme activity inhibition method is an indirect detection method. Immunoassay is simple, rapid and low-cost, but due to the influence of the matrix and extraction solvent, false positives and false negatives may occur. Liquid chromatography and mass spectrometry methods have high detection sensitivity, but require sophisticated instruments and are costly. Biosensor methods based on new biorecognition elements such as molecular imprinting and aptamers must precisely control the microenvironment in which they grow, and have high technical barriers. Therefore, the development of rapid detection and traceability methods for toxin proteins is urgent.

[0004] Surface-enhanced Raman spectroscopy (SERS) has the advantages of fingerprint recognition and highly sensitive detection, high detection efficiency, and rich spectral information. It has application value in the accurate detection and attribution of biological toxins in different scenarios. For example, the SERS-based sandwich immunoassay has been used to detect and distinguish active ricin from other homologous toxins, which can provide a portable and sensitive analytical strategy for toxin detection.

[0005] Poisoning or poisoning by biotoxin proteins has occurred frequently. Highly toxic biotoxin proteins represented by mushroom toxins, such as bolesatine protein (GenBank: AAB24601.1), have the characteristics of wide sources, complex structures, strong toxicity, and strong concealment. In addition, there is a lack of countermeasures against the cause. It is urgent to establish accurate identification technology from detection to traceability, fill the gap in scientific evidence tracking, and improve active protection capabilities. However, existing detection methods cannot meet actual needs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for SERS detection and traceability of boletus toxic protein. The present invention collects SERS spectra in boletus samples containing toxic proteins using a small-sized, highly stable enhanced substrate, preprocesses the SERS spectrum data, and reduces the dimension for visualization, thereby achieving SERS traceability identification of boletus from different origins. Specifically, the present invention is implemented using the following technical solutions:

[0007] A method for rapid detection and source tracing of bovine toxic protein SERS, comprising the following steps:

[0008] (1) Use a pipette to draw 5-10 µL of the boletus solution containing the toxic protein and drop it onto the surface of the small-sized, highly stable enhanced substrate;

[0009] (2) Place the substrate in an ultrasonicator for 5-7 min. After the solution dries, use a small Raman spectrometer (excitation light wavelength of 785 nm, integration time of 5-8 s, laser power of 100-300 mW) to collect SERS signals.

[0010] (3) The collected SERS spectral data were preprocessed by denoising and baseline correction, and the SERS spectral data obtained from boletus mushrooms from different origins were visualized and traced.

[0011] The preparation method of the small-sized high-stability enhanced substrate used in the present invention is as follows: take 5 μL of concentration (1.5-6.0)×10 11 The gold nanorod sol of 1000 μL is dripped onto a silicon wafer treated with hexadecyltrimethoxysilane, and placed in a 55-65°C oven for 8-12 min before being taken out to obtain a small-sized, highly stable enhanced substrate. The concentration of the gold nanorod sol is preferably (2.0-4.0)×10 11 Particles / μL.

[0012] By immersing the silicon wafer in hexadecyltrimethoxysilane to activate the silicon wafer surface, a hydrophobic interface can be formed on the silicon wafer surface. At the same time, it is placed in a 55-65°C oven for 8-12 min, especially in a 60°C oven for 10 min, so that the gold nanorods can form more hotspots on it, thereby facilitating the SERS signal detection of subsequent samples.

[0013] The inventors have found that poor control of substrate preparation conditions can easily lead to the coffee ring effect, such as excessive addition of sol and excessive size of the substrate after drying, which results in the coffee ring effect. When detecting a target, the signal enhancement in the area outside the coffee ring is unstable.

[0014] In addition, the present invention uses gold nanorod sol to prepare a small-sized, highly stable enhanced substrate, and the preparation method thereof is as follows:

[0015] (1) Preparation of seed solution: In a 28 ℃ constant temperature water bath, take 10 mL of 0.1 M hexadecyltrimethylammonium bromide solution in a 25 mL conical flask, add 0.1 mL of 25 mM chloroauric acid aqueous solution under magnetic stirring, and then quickly add 0.6 mL of freshly prepared 0.01 M sodium borohydride solution. Stir vigorously for 2 min and let stand for use;

[0016] (2) Preparation of growth medium: In a constant temperature water bath at 28 °C, place 10 mL of 0.1 M hexadecyltrimethylammonium bromide solution in a 25 mL conical flask, add 120 μL of 8 mM silver nitrate, 0.2 mL of 25 mM chloroauric acid, 0.1 mL of 2 M nitric acid, and 0.06 mL of 0.1 M ascorbic acid in sequence and mix well.

[0017] (3) Take 12 μL of the seed solution and add it to the growth medium. Mix well and let it stand for 24 hours.

[0018] The boletus described in the present invention is a commercial product, which can be wild, such as Yunnan wild boletus, including Yunnan Chuxiong boletus, Yunnan Simao boletus, and other edible mushrooms. The boletus solution is made by drying the boletus and crushing it into powder, and then preparing it into a concentration of 1×10 -9 -1×10 -3 g / mL aqueous solution preferably has a concentration of 1×10 -6 -1×10 -3 g / mL. The present invention uses homemade gold nanorod sol to prepare a small-sized, highly stable enhanced substrate, which has a small diameter (0.9-2.3 mm) and a large thickness (15-25 μm). It is well known to those skilled in the art that the thickness of the substrate commonly used in SERS detection is very small, about 2 μm, while the thickness of the substrate of the present invention can be about 20 μm, which makes SERS detection easier to apply. In addition, the small-sized, highly stable enhanced substrate of the present invention is simple to prepare and has low cost, and there is no coffee ring effect (such as Figure 8 ), the detection repeatability is high. The inventors found that if the size of the enhanced substrate is too large, the coffee ring effect will appear (such as Fig. 9 ), when detecting in the middle of the substrate, the SERS detection repeatability is poor; if the size is too small, the laser cannot be accurately focused on the substrate, causing detection difficulties.

[0019] The beneficial effects of the present invention are:

[0020] (1) The present invention realizes the rapid detection of bolesatine, a protein in the poisoning of boletus, and the target object detection can be completed within 20 minutes, which is suitable for rapid judgment at the poisoning scene.

[0021] (2) The present invention uses a self-made small-sized and highly stable SERS enhancement substrate for SERS detection, and the detection effect is stable (such as Figure 7 , RSD is less than 2%), overcoming the problem of unstable signal enhancement caused by the coffee ring effect.

[0022] (3) The present invention has a high degree of differentiation in the detection of boletus from different origins and can meet the needs of accurate traceability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SERS spectrum of the toxic protein from Chuxiong Boletus in Yunnan in Example 1.

[0024] Figure 2 This is the SERS spectrum of the toxic protein from Boletus edulis in Yunnan in Example 2.

[0025] Figure 3 This is the result of dimensionality reduction visualization processing of boletus spectra in Chuxiong, Yunnan and Simao, Yunnan in Example 3.

[0026] Figure 4 This is the result of spectral identification and SERS traceability of Boletus in Chuxiong, Yunnan and Simao, Yunnan in Example 3. In the figure, 30 and 31 refer to the number of samples, and the vertical axis on the right refers to the type of sample. The data set consists of 300 SERS data of Boletus randomly collected from Chuxiong and Simao, Yunnan, with a Raman shift range of 200-2000 cm -1 The training set and the test set were randomly allocated at a ratio of 7:3. The results showed that the final traceability accuracy reached 100%. The data detected by SERS can reliably trace boletus from different origins.

[0027] Figure 5 This is the SERS spectrum of the boletus of Comparative Example 1.

[0028] Figure 6 This is the SERS spectrum of the toxic protein in Comparative Example 2.

[0029] Figure 7 This is a detection stability test of the SERS enhanced substrate of the present invention.

[0030] Figure 8 1 is a SEM picture of the substrate without coffee ring effect of the present invention.

[0031] Fig. 9 This is a SEM image of the substrate that produces the coffee ring effect. DETAILED DESCRIPTION

[0032] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0033] The preparation method of the small-size high-stability enhanced substrate used in the present invention is: take a concentration of (2.0-4.0)×10 11 5 μL of gold nanorod sol with a density of 100 μL / μL is dripped onto a silicon wafer treated with hexadecyltrimethoxysilane, and placed in a 60°C oven for 10 min before being taken out to obtain a small-sized, highly stable enhanced substrate. The preparation method of the gold nanorod sol is as follows:

[0034] (1) Preparation of seed solution: In a 28 ℃ constant temperature water bath, take 10 mL of 0.1 M hexadecyltrimethylammonium bromide solution in a 25 mL conical flask, add 0.1 mL of 25 mM chloroauric acid aqueous solution under magnetic stirring, and then quickly add 0.6 mL of freshly prepared 0.01 M sodium borohydride solution. Stir vigorously for 2 min and let stand for use;

[0035] (2) Preparation of growth medium: In a constant temperature water bath at 28 °C, place 10 mL of 0.1 M hexadecyltrimethylammonium bromide solution in a 25 mL conical flask, add 120 μL of 8 mM silver nitrate, 0.2 mL of 25 mM chloroauric acid, 0.1 mL of 2 M nitric acid, and 0.06 mL of 0.1 M ascorbic acid in sequence and mix well.

[0036] (3) Take 12 μL of the seed solution and add it to the growth medium. Mix well and let it stand for 24 hours.

[0037] The present invention uses commercially available Yunnan Chuxiong bolete without toxic protein bolesatine and Yunnan Simao bolete without toxic protein bolesatine as raw materials to carry out the following examples and comparative examples, respectively, wherein the toxic protein bolesatine is synthesized by Qiangyao Biological Company.

[0038] Example 1

[0039] (1) Preparation of toxic protein in Chuxiong Boletus, Yunnan: add 1×10 -4 g / mL solution of Chuxiong Boletus in Yunnan was added with 10 µL of 2×10 -45 µL of the Yunnan Chuxiong Boletus solution containing the toxic protein bolesatine was taken with a pipette and dropped onto the surface of the small-sized high-stability enhanced substrate.

[0040] (2) The substrate was placed in an ultrasonicator for 5 min. After the solution dried, a small Raman spectrometer (excitation wavelength of 785 nm, integration time of 5 s, laser power of 100 mW) was used to collect SERS signals. The detection results were as follows: Figure 1 As shown in the figure, the characteristic peaks of Boletus are located at 960 and 1040 cm -1 The characteristic peak positions of the toxic protein are 1073, 1381, 1528, 1578 cm -1 .

[0041] (3) The collected SERS spectral data are preprocessed by denoising and baseline correction, and the SERS spectral data obtained by detecting boletus are visualized by dimensionality reduction and traceability identification as shown in Example 3.

[0042] Example 2

[0043] (1) Preparation of toxic protein in Boletus edulis in Yunnan: Add 1×10 -4 g / mL Yunnan Boletus Simao solution was added with 10 µL of 2×10 -4 M's toxic protein bolesatine solution is obtained to obtain a Yunnan Simao Boletus solution containing the toxic protein bolesatine. 10 µL of the Yunnan Simao Boletus solution containing the toxic protein bolesatine is taken with a pipette and dropped onto the surface of the small-sized high-stability enhanced substrate;

[0044] (2) The substrate was placed in an ultrasonicator for 7 min. After the solution dried, a small Raman spectrometer (excitation wavelength of 785 nm, integration time of 8 s, laser power of 300 mW) was used to collect SERS signals. The detection results were as follows: Figure 2 As shown;

[0045] The characteristic peaks of Boletus are at 960 and 1040 cm -1 The characteristic peak positions of the toxic protein are 893, 1144, 1381, 1528, and 1578 cm -1 .

[0046] (3) The collected SERS spectral data are preprocessed by denoising and baseline correction, and the SERS spectral data obtained by detecting boletus are visualized by dimensionality reduction and traceability identification as shown in Example 3.

[0047] Example 3

[0048] (1) Using the collected spectra, the SERS spectral data obtained from detecting boletus from different origins (Chuxiong, Yunnan, Simao, Yunnan) were processed for dimensionality reduction and visualization, such as Figure 3 As shown in the figure. The SERS spectral data obtained from different production areas, Chuxiong, Yunnan and Simao, Yunnan, were visualized by spectral dimension reduction using principal component analysis. Due to differences in geographical location and growth environment, the SERS spectral data of the two places are different. Through principal component analysis and dimensionality reduction, it can be found that there are obvious differences between the samples from the two places, which can be effectively distinguished.

[0049] (2) Through the difference in signals, SERS identification and traceability prediction of boletus from different origins (Chuxiong, Yunnan and Simao, Yunnan) were performed, such as Figure 4 The data set consists of 300 SERS data of randomly collected Boletus from Chuxiong and Simao, Yunnan, with a Raman shift range of 200-2000 cm -1 The training set and the test set were randomly allocated at a ratio of 7:3. The results showed that the final traceability accuracy reached 100%. The data detected by SERS can reliably trace boletus from different origins.

[0050] Comparative Example 1

[0051] SERS detection was performed on Chuxiong Boletus in Yunnan, which does not contain the toxic protein bolesatine. No SERS signal of the toxic protein was detected, and the obtained signal belongs to the SERS spectrum of Boletus. The experimental process is as follows:

[0052] (1) Use a pipette to draw a 1×10 -4 5 μL of the Yunnan Chuxiong Boletus solution was dropped onto the surface of the small-sized, highly stable enhanced substrate;

[0053] (2) The substrate was placed in an ultrasonicator for 5 min. After the solution dried, a small Raman spectrometer (excitation wavelength of 785 nm, integration time of 5 s, laser power of 100 mW) was used to collect SERS signals. The detection results were as follows: Figure 5 As shown in the figure, the characteristic peak positions of Boletus are 960, 1040, and 1300 cm -1 .

[0054] Comparative Example 2

[0055] The SERS detection of bolesatine, a toxic protein, was performed and the signal detected was the characteristic signal of the toxic protein.

[0056] (1) Use a pipette to draw a 2×10 -45 µL of bolesatine solution of M, a toxic protein, was dropped onto the surface of a small-sized, highly stable enhanced substrate;

[0057] (2) The substrate was placed in an ultrasonicator for 5 min. After the solution dried, a small Raman spectrometer (excitation wavelength of 785 nm, integration time of 5 s, laser power of 100 mW) was used to collect SERS signals. The detection results were as follows: Figure 6 As shown; the characteristic peak positions of the toxic protein are 893, 1073, 1144, 1266, 1381, 1496, 1528, 1578 cm -1 .

[0058] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

1. A method for rapid detection and traceability of bovine toxic protein by SERS, comprising the following steps: (1) Use a pipette to draw 5-10 µL of the boletus solution containing the toxic protein and drop it onto the surface of the small-sized, highly stable enhanced substrate; (2) Place the substrate in an ultrasonicator for 5-7 min. After the solution dries, use a small Raman spectrometer to collect SERS signals. (3) The collected SERS spectral data were preprocessed by denoising and baseline correction, and the SERS spectral data obtained from boletus mushrooms from different origins were visualized and traced.

2. The method for rapid detection and source tracing of boletus toxin protein by SERS according to claim 1, characterized in that: The concentration of the boletus solution was 1×10 -9 -1×10 -3 g / mL.

3. The method for rapid detection and source tracing of boletus toxin protein by SERS according to claim 2, characterized in that: The concentration of the boletus solution was 1×10 -6 -1×10 -3 g / mL.

4. The method for rapid detection and source tracing of boletus toxin protein by SERS according to claim 1, characterized in that: The small-size high-stability enhanced substrate has a size of 0.9-2.3 mm and a thickness of 15-25 μm.

5. The method for rapid detection and source tracing of bovine toxic protein by SERS according to claim 1, characterized in that: The preparation method of the small-sized highly stable enhanced substrate is as follows: 5 μL of gold nanorod sol is added dropwise on a silicon wafer treated with hexadecyltrimethoxysilane, and the mixture is placed in a 55-65° C. oven for 8-12 minutes and then taken out to obtain a small-sized highly stable enhanced substrate.

6. The SERS rapid detection and source tracing method of boletus toxin protein according to claim 5, characterized in that: The gold nanorod sol is prepared by the following method: (1) Preparation of seed solution: In a 28 ℃ constant temperature water bath, take 10 mL of 0.1 M hexadecyltrimethylammonium bromide solution in a 25 mL conical flask, add 0.1 mL of 25 mM chloroauric acid aqueous solution under magnetic stirring, and then quickly add 0.6 mL of freshly prepared 0.01 M sodium borohydride solution. Stir vigorously for 2 min and let stand for use; (2) Preparation of growth medium: In a 28 ℃ constant temperature water bath, add 10 mL of 0.1 M hexadecyltrimethylammonium bromide solution into a 25 mL conical flask, add 120 μL of 8 mM silver nitrate, 0.2 mL of 25 mM chloroauric acid, 0.1 mL of 2 M nitric acid, and 0.06 mL of 0.1 M ascorbic acid in sequence and mix well; (3) Take 12 μL of the seed solution and add it to the growth medium. Mix well and let it stand for 24 hours.

7. The method for rapid detection and source tracing of boletus toxin protein by SERS according to claim 5, characterized in that: The gold nanorod sol concentration is (1.5-6.0) × 10 11 Pieces / μL.

8. The method for rapid detection and source tracing of boletus toxin protein by SERS according to claim 7, characterized in that: The gold nanorod sol concentration is (2.0-4.0) × 10 11 Pieces / μL.

9. The method for rapid detection and source tracing of boletus toxin protein by SERS according to claim 1, characterized in that: The detection conditions of the Raman spectrometer are: the excitation light wavelength is 785 nm, the integration time is 5-8 s, and the laser power is 100-300 mW.

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