SERS (Surface Enhanced Raman Scattering) probe regulated and controlled by isomeride and application of SERS probe in detection of

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) probe regulated and controlled by an isomeride and application of the SERS probe in detection of various pathogens, and belongs to the technical field of biological detection. According to the invention, p-methoxybenzenethiol, m-methoxybenzenethiol, o-methoxybenzenethiol, p-fluorobenzenethiol, m-fluorobenzenethiol and o-fluorobenzenethiol with different Raman characteristic peaks are used as Raman report molecules, so that the Raman characteristic peaks can be prevented from overlapping; when six groups of probes (a p-methoxybenzenethiol probe, an m-methoxybenzenethiol probe, an o-methoxybenzenethiol probe, a p-fluorobenzenethiol probe, an m-fluorobenzenethiol probe and an o-fluorobenzenethiol probe) are respectively modified and mixed with pathogen antibodies, the obtained SERS probe can be used for synchronously detecting various pathogens; meanwhile, the enhancement effect of the Raman reporter molecule is good, so that multiple pathogens can be conveniently, synchronously, quickly and accurately detected; therefore, the method has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an isomer-regulated SERS probe and its application in the detection of various pathogens. Background Technology

[0002] Zoonotic infectious disease pathogens pose a serious threat to global public health and the economy. Currently, over 250 such pathogens have been identified globally, of which 89 are significantly harmful, and approximately 90 have been confirmed in my country, including rabies (with a near 100% fatality rate) and anthrax (capable of aerosol transmission). The harm caused by these pathogens manifests primarily in three ways: first, public health risks, such as the transnational spread of highly pathogenic avian influenza; second, economic losses in the livestock industry, such as the large-scale culling of animals due to brucellosis; and third, challenges in prevention and control, including accelerated pathogen mutation and increased antibiotic resistance. Therefore, there is an urgent need to develop low-cost, highly sensitive, rapid on-site detection technologies to effectively address transmission chains and biosafety risks.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a rapid, non-destructive spectroscopic technique. Through proper design, utilizing electromagnetic and chemical enhancement, the Raman signal of molecules can be increased by 10-1. 6 ~10 14 This technology promises to achieve single-molecule detection at a rate several times higher. Furthermore, it boasts advantages such as high sensitivity, high accuracy, fingerprint spectroscopy, and immunity to water molecule interference, making it particularly suitable for the analysis of biological samples and trace contaminants.

[0004] Electrohydrodynamics (EHD), a technique reported in recent years, offers advantages such as accelerating molecular collisions, improving reaction kinetics, and removing weakly bonded substances (e.g., non-specifically adsorbed substances). EHD involves applying alternating current to a pair of asymmetric microelectrodes, inducing two unevenly charged electric double layers and corresponding hydrodynamic forces on their surfaces. The intensity of these hydrodynamic forces can be altered by controlling the frequency and amplitude of the alternating current, thereby driving the liquid to move within a nanoscale range. Applying EHD technology to asymmetric gold microelectrode chips significantly improves the specificity and sensitivity of biomarker detection. Therefore, integrating EHD technology into SERS is of great significance for developing integrated detection platforms.

[0005] Raman reporter molecules are a core component of SERS probes, but currently common Raman reporter molecules generally suffer from overlapping Raman characteristic peaks and lack effective signal modulation strategies, making it difficult to distinguish signals in multi-target detection systems, thus limiting the ability to simultaneously detect multiple targets. Summary of the Invention

[0006] The purpose of this invention is to provide an isomer-regulated SERS probe and its application in the detection of various pathogens. This addresses the problem that existing Raman reporter molecules generally suffer from overlapping Raman characteristic peaks, preventing them from effectively detecting multiple targets simultaneously.

[0007] In a first aspect, the present invention provides an isomer-regulated SERS probe comprising six probes, each probe comprising a cuprous oxide core layer, gold nanoparticles deposited on the cuprous oxide core layer, a reporter molecule connected to the gold nanoparticles, and a silver shell layer encapsulating the reporter molecule; wherein the reporter molecules corresponding to the six probes are p-methoxybenzyl mercaptan, m-methoxybenzyl mercaptan, o-methoxybenzyl mercaptan, p-fluorobenzyl mercaptan, m-fluorobenzyl mercaptan, and o-fluorobenzyl mercaptan, respectively.

[0008] In this invention, the inventors discovered that using p-methoxybenzylthiol, m-methoxybenzylthiol, o-methoxybenzylthiol, p-fluorobenzylthiol, m-fluorobenzylthiol, and o-fluorobenzylthiol with different Raman characteristic peaks as Raman reporter molecules can avoid the overlap of Raman characteristic peaks. When the six groups of probes (p-methoxybenzylthiol probe, m-methoxybenzylthiol probe, o-methoxybenzylthiol probe, p-fluorobenzylthiol probe, m-fluorobenzylthiol probe, and o-fluorobenzylthiol probe) are modified and mixed with pathogen antibodies respectively, the resulting SERS probe can simultaneously detect multiple pathogens. At the same time, the above-mentioned Raman reporter molecules have good enhancement effects, which facilitates further simultaneous, rapid, and accurate detection of multiple pathogens.

[0009] In a second aspect, the present invention provides a method for preparing the SERS probe as described above, comprising the following steps: S1, preparing a cuprous oxide core layer; S2, depositing gold nanoparticles on the cuprous oxide core layer, and then connecting one of six reporter molecules to obtain six Cu2O@Au@ reporter molecule particles, the six reporter molecules being p-methoxybenzylthiol, m-methoxybenzylthiol, o-methoxybenzylthiol, p-fluorobenzylthiol, m-fluorobenzylthiol, and o-fluorobenzylthiol; S3, coating the six Cu2O@Au@ reporter molecule particles with a silver shell layer to obtain six sets of probes.

[0010] The method for preparing the SERS probe provided by this invention is simple, and the raw materials used are inexpensive and readily available, making it suitable for large-scale industrial production applications.

[0011] In some embodiments, step S1, preparing the cuprous oxide core layer includes: adding an alkaline solution and a reducing agent solution to a copper salt solution, followed by stirring and centrifugation to obtain the cuprous oxide core layer; wherein the concentration of the copper salt solution is 0.05-0.15M, preferably 0.1M; the concentration of the alkaline solution is 0.5-1.5M, preferably 1M; the concentration of the reducing agent solution is 0.1-0.3M, preferably 0.2M; and the volume ratio of the copper salt solution, alkaline solution, and reducing agent solution is (0.3-0.5):(2-3):(1.5-2.5), preferably 0.4:2.6:2.

[0012] In this invention, by controlling the concentrations of the copper salt solution, alkaline solution, and reducing agent solution, and ensuring that the volume ratio of the copper salt solution, alkaline solution, and reducing agent solution is within a specific range, a cuprous oxide core layer with moderate particle size and good performance can be obtained, which is convenient for subsequent applications.

[0013] In some embodiments, the copper salt solution includes a copper sulfate solution, the alkaline solution includes a sodium hydroxide solution, and the reducing agent solution includes an ascorbic acid solution; the stirring speed is 600-700 rpm, preferably 650 rpm; the stirring time is 5-15 min, preferably 10 min; the centrifugation includes centrifugation at 700-900 g (preferably 800 g) for 5-15 min, preferably 10 min.

[0014] It is understood that the types of copper salt solution, alkaline solution, and reducing agent solution can be conventionally selected according to actual usage needs, as long as a cuprous oxide core layer with appropriate particle size and good performance can be obtained. For example, in this invention, the copper salt solution preferably includes copper sulfate solution, the alkaline solution preferably includes sodium hydroxide solution, and the reducing agent solution preferably includes ascorbic acid solution.

[0015] In some embodiments, step S2, depositing gold nanoparticles on the cuprous oxide core layer and then attaching one of the six reporter molecules, includes: resuspending the cuprous oxide core layer in ultrapure water, then adding potassium chloroaurate solution for a first incubation, followed by adding hydroxylamine hydrochloride solution for a second incubation; finally, adding chitosan solution containing one of the six reporter molecules for a third incubation, and centrifuging to obtain six Cu2O@Au@reporter molecule particles; wherein the concentration of potassium chloroaurate solution is 15-25 mM, preferably 20 mM; the concentration of hydroxylamine hydrochloride solution is 15-25 mM, preferably 20 mM. The volume ratio of ultrapure water, potassium chloroaurate solution, and hydroxylamine hydrochloride solution is (180-220):(5-10):(10-20), preferably 200:7:15; the concentration of the six reporter molecules is 0.5-1.5 mM, preferably 1 mM; the concentration of chitosan solution is 0.5-1.5 mg / ml, preferably 1 mg / ml; the volume ratio of the six reporter molecules to chitosan solution is (3-7):(25-35), preferably 5:30; centrifugation includes centrifugation at 300-500g (preferably 400g) for 5-15 min, preferably 10 min.

[0016] In this invention, by controlling the concentrations of potassium chloroaurate solution and hydroxylamine hydrochloride solution, and by keeping the volume ratio of ultrapure water, potassium chloroaurate solution, and hydroxylamine hydrochloride solution within a specific range, gold nanoparticles can be uniformly deposited on the cuprous oxide core layer; furthermore, by controlling the concentrations of the six reporter molecules within a specific range, the reporter molecules can be completely linked.

[0017] In some implementations, the first incubation time is 2-4 minutes, preferably 3 minutes; the second incubation time is 8-12 minutes, preferably 10 minutes; and the third incubation time is 3-5 hours, preferably 4 hours.

[0018] Understandably, the incubation times for the first, second, and third incubations can be adjusted according to actual usage needs, as long as the gold nanoparticles are uniformly deposited on the cuprous oxide core layer and one of the six reporter molecules is fully connected to the gold nanoparticles.

[0019] In some embodiments, step S3, which involves coating the six types of Cu2O@Au@reporter molecules with a silver shell, includes: resuspending the six types of Cu2O@Au@reporter molecules in ultrapure water, adding silver nitrate solution, ammonia, and ascorbic acid solution, incubating, and centrifuging to obtain six sets of probes; wherein the concentration of the silver nitrate solution is 8-12 mM, preferably 10 mM; the concentration of the ascorbic acid solution is 40-60 mM, preferably 50 mM; the volume ratio of ultrapure water, silver nitrate solution, ammonia, and ascorbic acid solution is (180-220):(3-7):(2-4):(2-4), preferably 200:5:3:3; the incubation time is 15-25 min, preferably 20 min; and the centrifugation includes centrifuging at 300-500 g (preferably 400 g) for 5-15 min, preferably 10 min.

[0020] In this invention, by controlling the concentrations of silver nitrate solution and ascorbic acid solution, and within a specific range the volume ratio of ultrapure water, silver nitrate solution, ammonia water, and ascorbic acid solution, the silver shell can be completely coated, further improving the detection performance of the SERS probe.

[0021] In a third aspect, the present invention provides the application of SERS probes prepared as described above or by any of the above preparation methods in the detection of various pathogens.

[0022] The SERS probe provided by this invention can detect multiple pathogens simultaneously, rapidly, and accurately, and therefore has good application prospects.

[0023] In some implementation schemes, the application includes the following steps: 1) Six probe groups were modified with antibodies against Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci, respectively, to obtain the modified probe groups. These modified probe groups were then resuspended and mixed to obtain a mixed SERS probe solution. 2) The Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci antibodies were mixed and dropped onto an asymmetric gold substrate. After incubation and blocking, the modified asymmetric gold substrate was obtained. 3) The modified asymmetric gold substrate is immersed in the pathogen sample to be tested under an electric current condition for incubation. After the power is turned off, it is taken out and washed to obtain the asymmetric gold substrate after capturing the antigen; 4) The asymmetric gold substrate after capturing the antigen is immersed in the mixed SERS probe solution under an electric current condition for incubation. After the power is turned off, it is taken out and washed to obtain the spectral signal; the spectral signal is used to determine whether the pathogen sample to be tested is negative or positive for Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci.

[0024] In this invention, six probes are modified with antibodies against Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci, respectively. These probes are then resuspended and mixed to obtain a SERS probe solution. Furthermore, the Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci antibodies are mixed and modified onto an asymmetric gold substrate to obtain an electrohydrodynamically modified asymmetric gold substrate. In the presence of multiple pathogen antigens, the SERS probes, multiple pathogen antigens, and the electrohydrodynamically modified asymmetric gold substrate form a "sandwich" structure, thereby achieving simultaneous and accurate detection of six high-risk zoonotic pathogens: Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci. This multivariate detection method is simple to operate, highly specific, and has the potential for rapid and immediate detection of these six zoonotic pathogens.

[0025] Understandably, during the process of modifying the six probes with Toxoplasma gondii antibodies, Bacillus anthracis antibodies, Mycobacterium bovis antibodies, rabies virus antibodies, Brucella antibodies, and Chlamydia psittaci antibodies, the six probes can be randomly paired with the six antibodies for modification. For example, when one probe is paired with an antibody for modification, the other five probes are paired with the other five antibodies for modification.

[0026] In some embodiments, in step 1), the volume ratio of the six modified probe solutions contained in the mixed SERS probe solution is (0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5), preferably 1:1:1:1:1:1.

[0027] In this invention, the SERS probe can be used for the rapid detection of a variety of pathogens.

[0028] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention uses p-methoxybenzylthiol, m-methoxybenzylthiol, o-methoxybenzylthiol, p-fluorobenzylthiol, m-fluorobenzylthiol, and o-fluorobenzylthiol with different Raman characteristic peaks as Raman reporter molecules, which can avoid the overlap of Raman characteristic peaks. When the six groups of probes (p-methoxybenzylthiol probe, m-methoxybenzylthiol probe, o-methoxybenzylthiol probe, p-fluorobenzylthiol probe, m-fluorobenzylthiol probe, and o-fluorobenzylthiol probe) are modified and mixed with pathogen antibodies respectively, the resulting SERS probe can simultaneously detect multiple pathogens. At the same time, the enhancement effect of the above-mentioned Raman reporter molecules is good, which facilitates the simultaneous, rapid, and accurate detection of multiple pathogens. Therefore, it has good application prospects. Attached Figure Description

[0029] Figure 1This is an electron microscope image of the cuprous oxide core layer prepared in Example 1 of the present invention; Figure 2 This is an electron microscope image of the p-methoxybenzylthiol probe prepared in Example 1 of the present invention; Figure 3A The Raman spectra of the p-methoxybenzylthiol, m-methoxybenzylthiol, and o-methoxybenzylthiol probes prepared in Example 1 of this invention are shown below. Figure 3B The Raman spectra of the probes for p-fluorobenzenethiol, m-fluorobenzenethiol, and o-fluorobenzenethiol prepared in Example 1 of this invention are shown below. Figure 3C The Raman spectra of the probes for p-aminobenzylthiol, m-aminobenzylthiol, and o-aminobenzylthiol prepared in Example 1 of this invention are shown below. Figure 3D The Raman spectra of the p-hydroxybenzylthiol, m-hydroxybenzylthiol, and o-hydroxybenzylthiol probes prepared in Example 1 of this invention are shown below. Figure 3E The Raman spectra of the probes for p-bromophenylthiol, m-bromophenylthiol, and o-bromophenylthiol prepared in Example 1 of this invention are shown below. Figure 3F The Raman spectra of the p-chlorobenzyl mercaptan, m-chlorobenzyl mercaptan, and o-chlorobenzyl mercaptan probes prepared in Example 1 of this invention are shown below. Figure 3G The Raman spectra of the probes for p-carboxybenzyl mercaptan, m-carboxybenzyl mercaptan, and o-carboxybenzyl mercaptan prepared in Example 1 of this invention are shown below. Figure 4 The Raman spectra of the probes for p-methoxybenzyl mercaptan, m-methoxybenzyl mercaptan, o-methoxybenzyl mercaptan, p-fluorobenzyl mercaptan, m-fluorobenzyl mercaptan, and o-fluorobenzyl mercaptan prepared in Example 1 of this invention are shown below. Figure 5 This is a schematic diagram illustrating the principle of using isomer-regulated SERS probes to detect six pathogens in a sample in Embodiment 2 of the present invention. Figure 6A This is the Raman spectrum of the test sample containing Toxoplasma gondii in Example 2 of the present invention; Figure 6B This is the Raman spectrum of the test sample containing Bacillus anthracis in Example 2 of the present invention; Figure 6C This is the Raman spectrum of the test sample containing Mycobacterium bovis in Example 2 of the present invention; Figure 6D This is the Raman spectrum of the test sample containing rabies virus in Example 2 of the present invention; Figure 6E This is the Raman spectrum of the test sample containing Brucella in Example 2 of the present invention; Figure 6F This is the Raman spectrum of the test sample containing Chlamydia psittaci in Example 2 of the present invention; Figure 6G This is the Raman spectrum of a mixed sample containing Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci in Example 2 of the present invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0032] Example 1: Screening of Raman reporter molecules This embodiment detects and analyzes the Raman signals of probes (six groups of probes: p-methoxybenzylthiol probe, m-methoxybenzylthiol probe, o-methoxybenzylthiol probe, p-fluorobenzylthiol probe, m-fluorobenzylthiol probe, and o-fluorobenzylthiol probe) prepared from different Raman reporter molecules.

[0033] Specifically, the p-methoxybenzylthiol probe is first prepared, including the following steps: S1. Pour 35 mL of ultrapure water into an Erlenmeyer flask and stir at 770 rpm in a 37°C water bath. Then add 400 μL of 0.1 M copper sulfate solution and boil for 1 min. Next, add 2.6 mL of 1.0 M sodium hydroxide solution at 37°C, followed immediately by 2 mL of 0.2 M ascorbic acid solution. Continue the reaction at 650 rpm for 10 min, then centrifuge at 800 g for 10 min. Resuspend the precipitate in 200 μL of ultrapure water to obtain the cuprous oxide core layer solution (electron microscopy image shown). Figure 1 (As shown).

[0034] S2. Add 7 μL of 20 mM potassium chloroaurate solution to the cuprous oxide core layer solution in step S1, incubate for 3 min, then add 15 μL of 20 mM hydroxylamine hydrochloride solution and incubate for another 10 min to obtain a mixture; take 5 μL of 1 mM p-methoxybenzylthiol solution and add it to 30 μL of 1 mg / ml chitosan solution, shake and mix thoroughly, then add it to the above mixture, incubate for 4 h, centrifuge at 400 g for 10 min, discard the supernatant, and resuspend the precipitate in 200 μL of ultrapure water to obtain Cu2O@Au@p-methoxybenzylthiol reporter molecule particle solution.

[0035] S3. Add 5 μL of 1 mM silver nitrate solution, 3 μL of ammonia water, and 3 μL of 50 mM ascorbic acid solution to the Cu2O@Au@p-methoxybenzylthiol reporter particle solution obtained in step S2. After incubation for 20 min, centrifuge at 400 g for 10 min to obtain the p-methoxybenzylthiol probe (its electron micrograph is shown in Figure 1). Figure 2 (As shown).

[0036] Similarly, following the above preparation method, probes for m-methoxybenzyl mercaptan, o-methoxybenzyl mercaptan, p-fluorobenzyl mercaptan, m-fluorobenzyl mercaptan, o-fluorobenzyl mercaptan, p-aminobenzyl mercaptan, m-aminobenzyl mercaptan, o-aminobenzyl mercaptan, p-hydroxybenzyl mercaptan, m-hydroxybenzyl mercaptan, o-hydroxybenzyl mercaptan, p-bromobenzyl mercaptan, m-bromobenzyl mercaptan, o-bromobenzyl mercaptan, p-chlorobenzyl mercaptan, m-chlorobenzyl mercaptan, o-chlorobenzyl mercaptan, p-carboxybenzyl mercaptan, m-carboxybenzyl mercaptan, and o-carboxybenzyl mercaptan were obtained respectively.

[0037] Raman spectroscopy was performed on the twenty-one sets of probes prepared above, and the results are as follows: Figure 3A-3G As shown.

[0038] Furthermore, on Figure 3A-3G Raman signals of probes prepared from different Raman reporter molecules were analyzed. Raman reporter molecules with good enhancement effects and no overlapping characteristic peaks were selected. Finally, p-methoxybenzylthiol, m-methoxybenzylthiol, o-methoxybenzylthiol, p-fluorobenzylthiol, m-fluorobenzylthiol, and o-fluorobenzylthiol probes were chosen for subsequent detection of various pathogens. The Raman spectral detection results of the six selected probes are shown below. Figure 4 As shown.

[0039] from Figure 4 As can be seen, the Raman signal enhancement effect of the six probes is good, and there is no overlap of characteristic peaks.

[0040] Example 2: Detection of six pathogens using isomer-regulated SERS probes. This embodiment provides isomer-regulated SERS probes for the detection of six pathogens in a sample. The detection principle diagram is shown below. Figure 5 As shown.

[0041] Specifically, it includes the following steps: 1) The p-methoxybenzylthiol probe, m-methoxybenzylthiol probe, o-methoxybenzylthiol probe, p-fluorobenzylthiol probe, m-fluorobenzylthiol probe, and o-fluorobenzylthiol probe obtained in Example 1 were modified with antibodies. Taking the modification of the p-methoxybenzylthiol probe as an example, the specific steps were as follows: Prepare a 1 mg / mL Toxoplasma gondii antibody solution, mix the antibody solution with a 5 mM potassium carbonate solution at a volume ratio of 1:1, and add 200 μL to the p-methoxybenzylthiol probe. Then, incubate it overnight at 4°C, and finally add 20%... The probe was blocked in BSA at 4°C for 30 min, then centrifuged at 400 g for 10 min to obtain a p-methoxybenzylthiol probe modified with Toxoplasma gondii antibody. Similarly, following the same method, m-methoxybenzylthiol probes modified with Bacillus anthracis antibody, o-methoxybenzylthiol probes modified with Mycobacterium bovis antibody, p-fluorobenzylthiol probes modified with rabies virus antibody, m-fluorobenzylthiol probes modified with Brucella antibody, and o-fluorobenzylthiol probes modified with Chlamydia psittaci antibody were obtained. The modified probes were resuspended in 40 μL of ultrapure water, and the resuspended modified probe solutions were mixed to obtain a mixed SERS probe solution.

[0042] 2) Prepare a mixed antibody solution of 1 mg / mL with Toxoplasma gondii antibody, Bacillus anthracis antibody, Mycobacterium bovis antibody, rabies virus antibody, Brucella antibody, and Chlamydia psittaci antibody. Mix the mixed antibody solution with 5 mM potassium carbonate solution at a volume ratio of 1:1. Take 10 μL and drop it onto the asymmetric gold substrate. Then incubate it overnight at 4°C. Finally, block the asymmetric gold substrate in 20% BSA at 4°C for 30 min. After removing it and drying it, the modified asymmetric gold substrate is obtained.

[0043] 3) Immerse the asymmetric gold substrate in the test sample for 45 minutes under power (200mV, 1000HZ AC). After power is turned off, remove the substrate and wash it thoroughly with 1% BSA solution. The asymmetric gold substrate with captured antigen is obtained.

[0044] 4) Under power (200mV, 1000HZ AC), immerse the asymmetric gold substrate after capturing the antigen in the mixed SERS probe solution and incubate for 30 min. After power is turned off, remove the substrate, wash it thoroughly with 1% BSA solution, dry it, and place it in a Raman spectrometer to obtain the spectral signal. Based on the spectral signal, determine whether the sample tested is negative or positive for Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci.

[0045] The above method was used to test samples containing Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, Chlamydia psittaci, and mixed samples containing Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci. The results are as follows: Figure 6A-6G As shown.

[0046] from Figure 6A-6G As can be seen, when the sample to be tested is added, if one or more of the above six pathogens are present in the sample, the Raman spectrum of the corresponding probe can be obtained (pathogen positive). If there are no pathogens in the sample, the Raman spectrum of the corresponding probe cannot be obtained (pathogen negative). The Raman spectrum is used to determine whether the pathogen in the sample is negative or positive. The above results show that the detection method provided by the present invention can rapidly and accurately detect Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci.

[0047] In summary, the present invention uses p-methoxybenzylthiol, m-methoxybenzylthiol, o-methoxybenzylthiol, p-fluorobenzylthiol, m-fluorobenzylthiol, and o-fluorobenzylthiol with different Raman characteristic peaks as Raman reporter molecules, which can avoid the overlap of Raman characteristic peaks. When the six groups of probes (p-methoxybenzylthiol probe, m-methoxybenzylthiol probe, o-methoxybenzylthiol probe, p-fluorobenzylthiol probe, m-fluorobenzylthiol probe, and o-fluorobenzylthiol probe) are modified and mixed with pathogen antibodies respectively, the resulting SERS probe can simultaneously detect multiple pathogens.

[0048] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0049] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An isomer-regulated SERS probe, characterized in that, It includes six sets of probes, each set of probes including a cuprous oxide core layer, gold nanoparticles deposited on the cuprous oxide core layer, a reporter molecule connected to the gold nanoparticles, and a silver shell layer wrapped around the reporter molecule; The reporter molecules corresponding to the six groups of probes are p-methoxybenzyl mercaptan, m-methoxybenzyl mercaptan, o-methoxybenzyl mercaptan, p-fluorobenzyl mercaptan, m-fluorobenzyl mercaptan, and o-fluorobenzyl mercaptan, respectively.

2. A method for preparing a SERS probe as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of cuprous oxide core layer; S2. Deposit gold nanoparticles on the cuprous oxide core layer, and then connect one of the six reporter molecules to obtain six Cu2O@Au@reporter molecule particles, namely p-methoxybenzyl mercaptan, m-methoxybenzyl mercaptan, o-methoxybenzyl mercaptan, p-fluorobenzyl mercaptan, m-fluorobenzyl mercaptan, and o-fluorobenzyl mercaptan. S3. After coating the six types of Cu2O@Au@reporter molecules with a silver shell, six sets of probes were obtained.

3. The method for preparing the SERS probe according to claim 2, characterized in that, In step S1, the preparation of the cuprous oxide core layer includes: adding an alkaline solution and a reducing agent solution to a copper salt solution, followed by stirring and centrifugation to obtain the cuprous oxide core layer; Wherein, the concentration of the copper salt solution is 0.05-0.15M, the concentration of the alkaline solution is 0.5-1.5M, and the concentration of the reducing agent solution is 0.1-0.3M; the volume ratio of the copper salt solution, the alkaline solution, and the reducing agent solution is (0.3-0.5):(2-3):(1.5-2.5).

4. The method for preparing the SERS probe according to claim 3, characterized in that, The copper salt solution includes copper sulfate solution, the alkaline solution includes sodium hydroxide solution, and the reducing agent solution includes ascorbic acid solution; The stirring reaction is carried out at a speed of 600-700 rpm for 5-15 minutes.

5. The method for preparing a SERS probe according to claim 2, characterized in that, In step S2, depositing gold nanoparticles on the cuprous oxide core layer and then attaching one of the six reporter molecules to each of them includes: resuspending the cuprous oxide core layer in ultrapure water, then adding potassium chloroaurate solution for a first incubation, then adding hydroxylamine hydrochloride solution for a second incubation; finally adding chitosan solution containing one of the six reporter molecules for a third incubation, and centrifuging to obtain six Cu2O@Au@reporter molecule particles. Wherein, the concentration of the potassium chloroaurate solution is 15-25 mM, the concentration of the hydroxylamine hydrochloride solution is 15-25 mM; the volume ratio of ultrapure water, the potassium chloroaurate solution, and the hydroxylamine hydrochloride solution is (180-220):(5-10):(10-20). The concentrations of the six reporter molecules are all 0.5-1.5 mM, the concentration of the chitosan solution is 0.5-1.5 mg / ml, and the volume ratio of the six reporter molecules to the chitosan solution is (3-7):(25-35).

6. The method for preparing a SERS probe according to claim 5, characterized in that, The first incubation time is 2-4 minutes, the second incubation time is 8-12 minutes, and the third incubation time is 3-5 hours.

7. The method for preparing a SERS probe according to claim 2, characterized in that, In step S3, the silver shell layer encapsulating the six types of Cu2O@Au@reporter molecules includes: The six Cu2O@Au@ reporter molecules were resuspended in ultrapure water, and incubated with silver nitrate solution, ammonia, and ascorbic acid solution. After centrifugation, six sets of probes were obtained. The concentration of the silver nitrate solution is 8-12 mM, and the concentration of the ascorbic acid solution is 40-60 mM; the volume ratio of ultrapure water, the silver nitrate solution, the ammonia water, and the ascorbic acid solution is (180-220):(3-7):(2-4):(2-4); and the incubation time is 15-25 min.

8. The application of the SERS probe as described in claim 1 or the SERS probe prepared by any one of claims 2-7 in the detection of various pathogens.

9. The application according to claim 8, characterized in that, Includes the following steps: 1) Six groups of probes were modified with Toxoplasma gondii antibody, Bacillus anthracis antibody, Mycobacterium bovis antibody, rabies virus antibody, Brucella antibody, and Chlamydia psittaci antibody, respectively, to obtain the modified six groups of probes. The modified probes were then resuspended and mixed to obtain the mixed SERS probe solution. 2) Mix Toxoplasma gondii antibody, Bacillus anthracis antibody, Mycobacterium bovis antibody, rabies virus antibody, Brucella antibody, and Chlamydia psittaci antibody, and drop them onto an asymmetric gold substrate. After incubation and blocking, a modified asymmetric gold substrate is obtained. 3) The modified asymmetric gold substrate is immersed in the pathogen sample to be tested under an electric current condition and incubated. After the power is turned off, it is taken out and washed to obtain the asymmetric gold substrate after capturing the antigen. 4) The asymmetric gold substrate after capturing the antigen is immersed in the mixed SERS probe solution under an electric current condition for incubation. After the power is turned off, it is taken out, washed, and the spectral signal is obtained. Based on the spectral signal, it is determined whether the pathogen sample to be tested is negative or positive for Toxoplasma gondii, Bacillus anthracis, Mycobacterium bovis, rabies virus, Brucella, and Chlamydia psittaci.

10. The application according to claim 9, characterized in that, In step 1), the volume ratio of the six modified probe solutions contained in the mixed SERS probe solution is (0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5).

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