A sensor of a composite material and a method of manufacturing and use thereof

Abstract

The application discloses a composite material sensor and a preparation method and application thereof, and belongs to the field of composite material sensors.The composite material sensor is composed of an immune substrate and an immune probe.The immune substrate is prepared by combining ZIF-67 and VP through an in-situ growth method, and is loaded with a novel coronavirus antibody and a BSA protective layer on the surface.The immune probe is loaded with RhB Raman molecules, a novel coronavirus antibody and a BSA protective layer on the surface of BP NPs as a carrier.The composite material sensor has the advantages of a large specific surface area, which is beneficial to the better combination of ZIF-67 on the surface and the formation of a heterojunction, and is beneficial to the capture and adsorption of target molecules and has a good chemical enhancement effect.The novel coronavirus can be detected through an immune reaction.The application has the advantages of simple operation and low cost, and can be applied to large-scale rapid detection of novel coronavirus proteins in human saliva, blood and water samples and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of composite materials and nanotechnology, and particularly relates to a composite material sensor and a preparation method and use thereof. BACKGROUND

[0002] In recent years, surface-enhanced Raman scattering (SERS) technology has been widely used in ultra-sensitive detection of biological molecules, monitoring of chemical reactions, in-situ determination of pesticides, etc. due to its high sensitivity, non-invasiveness and information characteristic spectrum. Traditional SERS substrates are gold or silver nanomaterials, because they have strong localized surface plasmon resonance effect (LSRP) and have higher electromagnetic enhancement effect. However, under the irradiation of external laser, the local temperature of the electromagnetic hot spot region of the noble metal nanoparticle substrate increases, which can cause deformation of the metal nanostructure and affect the stability of the test. In addition, a biologically toxic surfactant is usually required in the preparation process. The above reasons limit the wide application of noble metal nanoparticle substrates in the field of biological medicine.

[0003] Metal-organic frameworks (MOFs) are generally easy to prepare and have high stability and good enrichment capacity. The semiconductor-like characteristics of MOF materials can cause electron migration during laser irradiation, thereby improving the SERS performance and having great potential in the field of SERS detection. Although MOF materials have certain SERS performance, the performance is generally not high. Some researchers have improved the SERS performance by introducing oxygen vacancies, but the complex preparation steps, great technical challenges, low precision and stability limit the wide application of MOF materials in the field of SERS.

[0004] The emergence of composite materials based on semiconductor materials opens up a new direction for the field of SERS detection. However, the reported cases of combining semiconductor materials and MOF materials are mostly applied in the field of photocatalysis. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a composite material sensor and a preparation method and use thereof to solve the problems existing in the prior art. The preparation method of the sensor is simple, has high sensitivity and strong selectivity, and can be applied to detect viruses in a sample.

[0006] The technical scheme adopted by the present application to solve the above technical problem is as follows:

[0007] A preparation method of a composite material sensor, comprising the following steps:

[0008] Step (1): preparation of an immune substrate

[0009] A certain amount of cobalt nitrate hexahydrate was added to a certain amount of methanol solution and stirred at room temperature to obtain a methanol solution containing Co(II). A certain amount of 2-methylimidazole was added to a certain amount of methanol solution and stirred at room temperature to obtain a methanol solution containing 2-methylimidazole. The methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly to obtain a methanol solution containing a complex of 2-methylimidazole and cobalt ions (abbreviated as ZIF-67). A 5 mm x 5 mm piece of purple phosphorus (abbreviated as VP) was placed into the above methanol solution containing ZIF-67, stirred slowly for a certain time, and then allowed to stand for a certain time. It was then taken out, washed three times with methanol solution and dried to obtain a VP@ZIF-67 surface-enhanced Raman scattering (SERS) substrate, abbreviated as VP@ZIF-67SERS substrate.

[0010] Take the VP@ZIF-67SERS substrate prepared in the above steps, add a certain amount of novel coronavirus antibody solution, and incubate at 4℃ for a certain time to obtain VP@ZIF-67SERS substrate with novel coronavirus antibody;

[0011] The surface of the VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus was washed with tris(hydroxymethyl)aminomethane buffer (TBS solution), phosphate buffer (PBS solution), and ultrapure water to remove excess antibodies against the novel coronavirus (hereinafter referred to as the washing step). A certain amount of bovine serum albumin (BSA) solution was added to the washed VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus, and the substrate was placed at room temperature for a certain period of time. The above washing step was repeated once to obtain a VP@ZIF-67SERS substrate with a bovine serum albumin protective layer containing antibodies against the novel coronavirus, which is the immune substrate.

[0012] The ligand in ZIF-67 is 2-methylimidazole, the metal ion is divalent cobalt ion, the space group is Fd-3m, and the unit cell parameter is... α=β=γ=90°, the crystal system is cubic.

[0013] Step (2): Preparation of immune probe dispersion

[0014] A certain amount of lumpy black phosphorus was ground into powder in a glove box. Then, a certain amount of black phosphorus powder and polyvinylpyrrolidone were weighed and added to anhydrous ethanol solution. The mixture was then placed in an ultrasonic bath for ultrasonic exfoliation to obtain a brown dispersion. Large pieces of black phosphorus were removed by centrifugation. The upper dispersion was taken, washed with methanol and ultrapure water, and the precipitate was collected. The collected precipitate was dispersed in ultrapure water to obtain a dispersion of black phosphorus nanosheets (BP NPs). A certain amount of BP NPs dispersion was taken, and a certain amount of Rhodamine B (RhB) Raman molecular aqueous solution was added. The mixture was placed at room temperature for a certain period of time for incubation to obtain the incubated BP NPs dispersion.

[0015] Take the BP NPs dispersion after centrifugation and incubation, remove the supernatant, and disperse the precipitate with PBS aqueous solution to obtain a dispersion of BP NPs with RhB Raman molecules attached. Add a certain amount of novel coronavirus antibody solution to the dispersion of BP NPs with RhB Raman molecules attached, and incubate at 4°C for a certain time to obtain a dispersion of BP NPs with RhB Raman molecules attached and novel coronavirus antibody attached. Then centrifuge the dispersion of BP NPs with RhB Raman molecules attached and novel coronavirus antibody attached, remove the supernatant, disperse the precipitate with PBS aqueous solution, add a certain amount of BSA solution, and incubate at room temperature for a certain time to obtain a dispersion of BP NPs with RhB Raman molecules attached and novel coronavirus antibody attached with bovine serum albumin protective layer. Then centrifuge the dispersion of BP NPs with RhB Raman molecules attached and novel coronavirus antibody attached with bovine serum albumin protective layer, remove the supernatant, and disperse the precipitate with PBS aqueous solution to obtain a dispersion of the precipitate, which is the immune probe dispersion.

[0016] The concentration of the RhB Raman molecule aqueous solution was 1.0 × 10⁻⁶. -2 Moles per liter.

[0017] Step (3): Fabrication of composite material sensors

[0018] A certain amount of the immune probe dispersion prepared in step (2) is dropped onto the immune substrate prepared in step (1), incubated at 37°C for a certain time, and then naturally dried to obtain a surface-enhanced Raman scattering (SERS) sensor, which is the sensor of the composite material.

[0019] Furthermore, the present invention also provides the use of the prepared composite material sensor for surface-enhanced Raman detection of novel coronavirus protein, specifically as follows: a certain amount of saliva containing novel coronavirus protein is dropped onto the dried composite material sensor, and then washed with TBS solution, PBS solution, and ultrapure water to remove unlinked novel coronavirus protein. After it is completely dried, its Raman signal is recorded at a laser wavelength of 532 nm using a Raman spectrometer to determine the relative concentration of novel coronavirus protein in the sample. This sensor is used for the detection of novel coronavirus protein in human saliva, blood, or water.

[0020] Compared with the prior art, the advantages of the present invention are as follows:

[0021] This invention discloses for the first time a composite material sensor, its preparation method, and its applications. The sensor's preparation process is simple, low-cost, and exhibits good biocompatibility. It requires no surfactants and can specifically capture viral proteins for high-throughput detection. Furthermore, ZIF-67, combined with a VP sheet and forming an array on the VP sheet, constitutes a specific "capture network," providing better retention and capture effects for patient saliva and blood samples. This invention can specifically capture SARS-CoV-2 proteins, and through a "sandwich structure," quantitative analysis of the virus can be performed by reading the Raman peaks of immune probes, making it applicable to the field of rapid on-site detection of SARS-CoV-2. Attached Figure Description

[0022] Figure 1 This is a scanning electron microscope image of the VP@ZIF-67SERS substrate prepared in Example 1 of this invention;

[0023] Figure 2 The image shows the surface-enhanced Raman spectrum of the sensor made of the composite material prepared in Example 1 of this invention for detecting novel coronavirus protein.

[0024] Figure 3 This is a scanning electron microscope image of the VP@ZIF-67SERS substrate prepared in Example 2 of the present invention;

[0025] Figure 4 The image shows the surface-enhanced Raman spectrum of the sensor made of the composite material prepared in Example 2 of this invention for detecting novel coronavirus protein.

[0026] Figure 5 This is a scanning electron microscope image of the VP@ZIF-67SERS substrate prepared in Example 3 of the present invention;

[0027] Figure 6 The image shows the surface-enhanced Raman spectrum of the sensor made of the composite material prepared in Example 3 of this invention for detecting novel coronavirus protein.

[0028] Figure 7 This is a scanning electron microscope image of the VP@ZIF-67SERS substrate prepared in Example 4 of the present invention;

[0029] Figure 8 The zeta potential results are for the immune probes prepared in Example 4 of this invention.

[0030] Figure 9 The image shows the surface-enhanced Raman spectrum of the sensor for detecting novel coronavirus protein using the composite material prepared in Example 4 of this invention.

[0031] Figure 10 This is a scanning electron microscope image of the VP@ZIF-67SERS substrate prepared in Example 5 of the present invention;

[0032] Figure 11 This is a surface-enhanced Raman spectroscopy (SMR) spectrum of the novel coronavirus protein detected by the sensor of the composite material prepared in Example 5 of this invention. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this disclosure. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0035] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this disclosure. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0036] This disclosure provides a composite material sensor, its preparation method, and its application in this embodiment. The composite material sensor consists of two parts: an immune substrate and an immune probe. The immune substrate is fabricated by combining ZIF-67 and VP in situ, and a novel coronavirus antibody and a BSA protective layer are loaded onto it. The immune probe uses BPNPs as a carrier and is loaded with RhB Raman molecules, a novel coronavirus antibody, and a BSA protective layer. The above-mentioned composite material sensor can effectively recognize the virus through an "immune reaction." The specific scheme is detailed below.

[0037] Example 1

[0038] This embodiment provides a composite material sensor, its preparation method, and its application, including the following steps:

[0039] Step (1): Preparation of the immune base:

[0040] 1.16 g of cobalt nitrate hexahydrate was added to 40 mL of methanol solution and stirred at room temperature for 12 hours to obtain a methanol solution containing Co(II). 0.65 g of 2-methylimidazole was added to 40 mL of methanol solution and stirred at room temperature for 10 minutes to obtain a methanol solution containing 2-methylimidazole. The methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly for 30 minutes to obtain a methanol solution containing a complex of 2-methylimidazole and cobalt ions (abbreviated as ZIF-67). A 5 mm x 5 mm piece of purple phosphorus (abbreviated as VP) was placed into the above methanol solution containing ZIF-67, stirred slowly for 12 hours, and then allowed to stand for 2 hours. After that, it was taken out, washed three times with methanol solution, and dried to obtain a VP@ZIF-67 surface-enhanced Raman scattering (SERS) substrate, abbreviated as VP@ZIF-67SERS substrate.

[0041] Take the VP@ZIF-67SERS substrate prepared in the above steps, and add 10 μL of novel coronavirus antibody solution (10 μL). -2 The solution was incubated at 4°C for 12 hours to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus. The surface of the VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus was washed with TBS solution, PBS solution, and ultrapure water to remove excess antibodies against the novel coronavirus (hereinafter referred to as the washing step). 10 μL of bovine serum albumin (BSA) solution was added to the washed VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus, and the substrate was placed at room temperature for 3 hours. The washing step was repeated once to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus with a bovine serum albumin protective layer, which is the immune substrate.

[0042] The ligand in ZIF-67 is 2-methylimidazole, the metal ion is divalent cobalt ion, the space group of ZIF-67 is Fd-3m, and the cell parameters are... α=β=γ=90°, the crystal system is cubic.

[0043] Step (2): Preparation of the immunoprobe dispersion:

[0044] 50 mg of lumpy black phosphorus was ground into powder in a glove box. Then, 30 mg of black phosphorus powder and 90 mg of polyvinylpyrrolidone were weighed and added to anhydrous ethanol solution (30 mL). The mixture was placed in an ultrasonic bath on ice for 4 hours to obtain a brown dispersion. The mixture was centrifuged at 10,000 rpm for 15 minutes (hereinafter referred to as centrifugation) to remove large pieces of black phosphorus. The supernatant dispersion was collected, washed with methanol and ultrapure water, and the precipitate was collected. The collected product was dispersed in ultrapure water at a concentration of 1 mg / mL to obtain a black phosphorus flake dispersion (BP NPs). 1 mL of the BP NPs dispersion was taken, and 10 μL of RhB Raman molecular aqueous solution (1.0 × 10⁻⁶) was added dropwise. -2 The BP NPs dispersion was incubated at room temperature for more than 12 hours (mol / L) to obtain a fully incubated BP NPs dispersion. Then, the incubated BP NPs dispersion was centrifuged, the supernatant was removed, and the precipitate was dispersed in PBS aqueous solution to obtain a dispersion of BP NPs with RhB Raman molecules attached. 20 μL of novel coronavirus antibody solution (10...) -2 (mg / mL) was added dropwise to the dispersion of RhB Raman molecules attached to BP NPs, and incubated at 4°C for 1.5 hours to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs was centrifuged, the supernatant was removed, the precipitate was dispersed with PBS aqueous solution, 10 μL of BSA solution was added, and incubated at room temperature for 1 hour to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer was centrifuged again, the supernatant was removed, and the precipitate was dispersed with PBS aqueous solution to a volume of 1 mL to obtain a dispersion of the precipitate, which is the immune probe dispersion.

[0045] Step (3): Fabrication of the composite material sensor:

[0046] Take 10 μL of the immune probe dispersion prepared in step (2) and drop it onto the immune substrate prepared in step (1). Incubate at 37°C for 2 hours until it is completely dry to obtain a surface-enhanced Raman scattering (SERS) sensor, which is the sensor of the composite material.

[0047] The sensor made from the above-prepared composite material is used for surface-enhanced Raman detection of novel coronavirus protein, specifically as follows: Take 10 μL of saliva containing novel coronavirus protein (10 μL... -2A 1 mg / mL sample was dropped onto the dried composite material sensor. Then, it was washed with TBS solution, PBS solution, and ultrapure water to remove unlinked novel coronavirus protein. After drying, the Raman signal was recorded at a laser wavelength of 532 nm using a Raman spectrometer to determine the relative concentration of novel coronavirus protein in the sample.

[0048] Figure 1 This shows a scanning electron microscope image of the VP@ZIF-67SERS substrate prepared in this embodiment. Figure 1 It can be seen that ZIF-67 particles are distributed on the surface of VP.

[0049] Figure 2 The surface-enhanced Raman spectroscopy (SMR) spectrum of the composite material sensor prepared in this embodiment for detecting novel coronavirus proteins is shown. Figure 2 It can be seen that this example is at 1646cm -1 The Raman signal intensity at that location is 542.

[0050] Example 2

[0051] This embodiment provides a composite material sensor, its preparation method, and its application, including the following steps:

[0052] Step (1): Preparation of the immune base:

[0053] 1.16 g of cobalt nitrate hexahydrate was added to 40 mL of methanol solution and stirred at room temperature for 12 hours to obtain a methanol solution containing Co(II). 1.31 g of 2-methylimidazole was added to 40 mL of methanol solution and stirred at room temperature for 10 minutes to obtain a methanol solution containing 2-methylimidazole. The methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly for 30 minutes to obtain a methanol solution containing a complex of 2-methylimidazole and cobalt ions (referred to as ZIF-67). A 5 mm x 5 mm piece of purple phosphorus (referred to as VP) was placed into the above methanol solution containing ZIF-67, stirred slowly for 12 hours, and then allowed to stand for 2 hours. After that, it was taken out, washed three times with methanol solution, and dried to obtain a VP@ZIF-67 surface-enhanced Raman scattering (SERS) substrate, abbreviated as VP@ZIF-67SERS substrate.

[0054] Take the VP@ZIF-67SERS substrate prepared in the above steps, and add 10 μL of novel coronavirus antibody solution (10 μL). -2The solution was incubated at 4°C for 12 hours to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus. The surface of the VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus was washed with TBS solution, PBS solution, and ultrapure water to remove excess antibodies against the novel coronavirus (hereinafter referred to as the washing step). 10 μL of bovine serum albumin (BSA) solution was added to the washed VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus, and the substrate was placed at room temperature for 3 hours. The washing step was repeated once to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus with a bovine serum albumin protective layer, which is the immune substrate.

[0055] The ligand in ZIF-67 is 2-methylimidazole, the metal ion is divalent cobalt ion, the space group of ZIF-67 is Fd-3m, and the cell parameters are... α=β=γ=90°, the crystal system is cubic.

[0056] Step (2): Preparation of the immunoprobe dispersion:

[0057] 50 mg of lumpy black phosphorus was ground into powder in a glove box. Then, 30 mg of black phosphorus powder and 90 mg of polyvinylpyrrolidone were weighed and added to anhydrous ethanol solution (30 mL). The mixture was placed in an ultrasonic bath on ice for 4 hours to obtain a brown dispersion. The mixture was centrifuged at 10,000 rpm for 15 minutes (hereinafter referred to as centrifugation) to remove large pieces of black phosphorus. The supernatant dispersion was collected, washed with methanol and ultrapure water, and the precipitate was collected. The collected product was dispersed in ultrapure water at a concentration of 1 mg / mL to obtain a black phosphorus flake dispersion (BP NPs). 1 mL of the BP NPs dispersion was taken, and 10 μL of RhB Raman molecular aqueous solution (1.0 × 10⁻⁶) was added dropwise. -2 The BP NPs dispersion was incubated at room temperature for more than 12 hours (mol / L) to obtain a fully incubated BP NPs dispersion. Then, the incubated BP NPs dispersion was centrifuged, the supernatant was removed, and the precipitate was dispersed in PBS aqueous solution to obtain a dispersion of BP NPs with RhB Raman molecules attached. 20 μL of novel coronavirus antibody solution (10...) -2(mg / mL) was added dropwise to the dispersion of RhB Raman molecules attached to BP NPs, and incubated at 4°C for 1.5 hours to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs was centrifuged, the supernatant was removed, the precipitate was dispersed with PBS aqueous solution, 10 μL of BSA solution was added, and incubated at room temperature for 1 hour to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer was centrifuged again, the supernatant was removed, and the precipitate was dispersed with PBS aqueous solution to a volume of 1 mL to obtain a dispersion of the precipitate, which is the immune probe dispersion.

[0058] Step (3): Fabrication of the composite material sensor:

[0059] Take 10 μL of the immune probe dispersion prepared in step (2) and drop it onto the immune substrate prepared in step (1). Incubate at 37°C for 2 hours. After it is completely dry, a surface-enhanced Raman scattering (SERS) sensor is obtained, which is the sensor of the composite material.

[0060] The sensor made from the above-prepared composite material is used for surface-enhanced Raman detection of novel coronavirus protein, specifically as follows: Take 10 μL of saliva containing novel coronavirus protein (10 μL... -2 A 1 mg / mL sample was dropped onto the dried composite material sensor. Then, it was washed with TBS solution, PBS solution, and ultrapure water to remove unlinked novel coronavirus protein. After drying, the Raman signal was recorded at a laser wavelength of 532 nm using a Raman spectrometer to determine the relative concentration of novel coronavirus protein in the sample.

[0061] Figure 3 The image shows a scanning electron microscope (SEM) image of the VP@ZIF-67SERS substrate prepared in this embodiment. From... Figure 3 It can be seen that ZIF-67 particles are more distributed on the surface of VP than in the previous example (i.e., Example 1).

[0062] Figure 4 The surface-enhanced Raman spectroscopy (SMR) spectrum of the composite material sensor prepared in this embodiment for detecting novel coronavirus proteins is shown. Figure 4 It can be seen that this example is at 1646cm -1 The Raman signal intensity at that location is 2631.

[0063] Example 3

[0064] This embodiment provides a composite material sensor, its preparation method, and its application, including the following steps:

[0065] Step (1): Preparation of the immune base:

[0066] 1.16 g of cobalt nitrate hexahydrate was added to 40 mL of methanol solution and stirred at room temperature for 12 hours to obtain a methanol solution containing Co(II). 1.97 g of 2-methylimidazole was added to 40 mL of methanol solution and stirred at room temperature for 10 minutes to obtain a methanol solution containing 2-methylimidazole. The methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly for 30 minutes to obtain a methanol solution containing a complex of 2-methylimidazole and cobalt ions (referred to as ZIF-67). A 5 mm x 5 mm piece of purple phosphorus (referred to as VP) was placed into the above methanol solution containing ZIF-67, stirred slowly for 12 hours, and then allowed to stand for 2 hours. After that, it was taken out, washed three times with methanol solution, and dried to obtain a VP@ZIF-67 surface-enhanced Raman scattering (SERS) substrate, abbreviated as VP@ZIF-67SERS substrate.

[0067] Take the VP@ZIF-67SERS substrate prepared in the above steps, and add 10 μL of novel coronavirus antibody solution (10 μL). -2 The solution was incubated at 4°C for 12 hours to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus. The surface of the VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus was washed with TBS solution, PBS solution, and ultrapure water to remove excess antibodies against the novel coronavirus (hereinafter referred to as the washing step). 10 μL of bovine serum albumin (BSA) solution was added to the washed VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus, and the substrate was placed at room temperature for 3 hours. The washing step was repeated once to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus with a bovine serum albumin protective layer, which is the immune substrate.

[0068] The ligand in ZIF-67 is 2-methylimidazole, the metal ion is divalent cobalt ion, the space group of ZIF-67 is Fd-3m, and the cell parameters are... α=β=γ=90°, the crystal system is cubic.

[0069] Step (2): Preparation of the immunoprobe dispersion:

[0070] 50 mg of lumpy black phosphorus was ground into powder in a glove box. Then, 30 mg of black phosphorus powder and 90 mg of polyvinylpyrrolidone were weighed and added to anhydrous ethanol solution (30 mL). The mixture was placed in an ultrasonic bath on ice for 4 hours to obtain a brown dispersion. The mixture was centrifuged at 10,000 rpm for 15 minutes (hereinafter referred to as centrifugation) to remove large pieces of black phosphorus. The supernatant dispersion was collected, washed with methanol and ultrapure water, and the precipitate was collected. The collected product was dispersed in ultrapure water at a concentration of 1 mg / mL to obtain a black phosphorus flake dispersion (BP NPs). 1 mL of the BP NPs dispersion was taken, and 10 μL of RhB Raman molecular aqueous solution (1.0 × 10⁻⁶) was added dropwise. -2 The BP NPs dispersion was incubated at room temperature for more than 12 hours (mol / L) to obtain a fully incubated BP NPs dispersion. Then, the incubated BP NPs dispersion was centrifuged, the supernatant was removed, and the precipitate was dispersed in PBS aqueous solution to obtain a dispersion of BP NPs with RhB Raman molecules attached. 20 μL of novel coronavirus antibody solution (10...) -2 (mg / mL) was added dropwise to the dispersion of RhB Raman molecules attached to BP NPs, and incubated at 4°C for 1.5 hours to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs was centrifuged, the supernatant was removed, the precipitate was dispersed with PBS aqueous solution, 10 μL of BSA solution was added, and incubated at room temperature for 1 hour to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer was centrifuged again, the supernatant was removed, and the precipitate was dispersed with PBS aqueous solution to a volume of 1 mL to obtain a dispersion of the precipitate, which is the immune probe dispersion.

[0071] Step (3): Fabrication of the composite material sensor:

[0072] Take 10 μL of the immune probe dispersion prepared in step (2) and drop it onto the immune substrate prepared in step (1). Incubate at 37°C for 2 hours. After it is completely dry, the surface-enhanced Raman scattering (SERS) sensor is the sensor of the composite material.

[0073] The sensor made from the above-prepared composite material is used for surface-enhanced Raman detection of novel coronavirus protein, specifically as follows: Take 10 μL of saliva containing novel coronavirus protein (10 μL... -2A 1 mg / mL sample was dropped onto the dried composite material sensor. Then, it was washed with TBS solution, PBS solution, and ultrapure water to remove unlinked novel coronavirus protein. After drying, the Raman signal was recorded at a laser wavelength of 532 nm using a Raman spectrometer to determine the relative concentration of novel coronavirus protein in the sample.

[0074] Figure 5 The image shows a scanning electron microscope (SEM) image of the VP@ZIF-67SERS substrate prepared in this embodiment. From... Figure 5 It can be seen that ZIF-67 particles are more distributed on the surface of VP than in the previous example (i.e., Example 2).

[0075] Figure 6 The surface-enhanced Raman spectroscopy (SMR) spectrum of the composite material sensor prepared in this embodiment for detecting novel coronavirus proteins is shown. Figure 6 It can be seen that this example is at 1646cm -1 The Raman signal intensity at that location is 3767.

[0076] Example 4

[0077] This embodiment provides a composite material sensor, its preparation method, and its application, including the following steps:

[0078] Step (1): Preparation of the immune base:

[0079] 1.16 g of cobalt nitrate hexahydrate was added to 40 mL of methanol solution and stirred at room temperature for 12 hours to obtain a methanol solution containing Co(II). 2.62 g of 2-methylimidazole was added to 40 mL of methanol solution and stirred at room temperature for 10 minutes to obtain a methanol solution containing 2-methylimidazole. The methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly for 30 minutes to obtain a methanol solution containing a complex of 2-methylimidazole and cobalt ions (referred to as ZIF-67). A 5 mm x 5 mm piece of purple phosphorus (referred to as VP) was placed into the above methanol solution containing ZIF-67, stirred slowly for 12 hours, and then allowed to stand for 2 hours. After that, it was taken out, washed three times with methanol solution, and dried to obtain a VP@ZIF-67 surface-enhanced Raman scattering (SERS) substrate, abbreviated as VP@ZIF-67SERS substrate.

[0080] Take the VP@ZIF-67SERS substrate prepared in the above steps, and add 10 μL of novel coronavirus antibody solution (10 μL). -2The solution was incubated at 4°C for 12 hours to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus. The surface of the VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus was washed with TBS solution, PBS solution, and ultrapure water to remove excess antibodies against the novel coronavirus (hereinafter referred to as the washing step). 10 μL of bovine serum albumin (BSA) solution was added to the washed VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus, and the substrate was placed at room temperature for 3 hours. The washing step was repeated once to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus with a bovine serum albumin protective layer, which is the immune substrate.

[0081] The ligand in ZIF-67 is 2-methylimidazole, the metal ion is divalent cobalt ion, the space group of ZIF-67 is Fd-3m, and the cell parameters are... α=β=γ=90°, the crystal system is cubic.

[0082] Step (2): Preparation of the immunoprobe dispersion:

[0083] 50 mg of lumpy black phosphorus was ground into powder in a glove box. Then, 30 mg of black phosphorus powder and 90 mg of polyvinylpyrrolidone were weighed and added to anhydrous ethanol solution (30 mL). The mixture was placed in an ultrasonic bath on ice for 4 hours to obtain a brown dispersion. The mixture was centrifuged at 10,000 rpm for 15 minutes (hereinafter referred to as centrifugation) to remove large pieces of black phosphorus. The supernatant dispersion was collected, washed with methanol and ultrapure water, and the precipitate was collected. The collected product was dispersed in ultrapure water at a concentration of 1 mg / mL to obtain a black phosphorus flake dispersion (BP NPs). 1 mL of the BP NPs dispersion was taken, and 10 μL of RhB Raman molecular aqueous solution (1.0 × 10⁻⁶) was added dropwise. -2 The BP NPs dispersion was incubated at room temperature for more than 12 hours (mol / L) to obtain a fully incubated BP NPs dispersion. Then, the incubated BP NPs dispersion was centrifuged, the supernatant was removed, and the precipitate was dispersed in PBS aqueous solution to obtain a dispersion of BP NPs with RhB Raman molecules attached. 20 μL of novel coronavirus antibody solution (10...) -2(mg / mL) was added dropwise to the dispersion of RhB Raman molecules attached to BP NPs, and incubated at 4°C for 1.5 hours to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs was centrifuged, the supernatant was removed, the precipitate was dispersed with PBS aqueous solution, 10 μL of BSA solution was added, and incubated at room temperature for 1 hour to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer was centrifuged again, the supernatant was removed, and the precipitate was dispersed with PBS aqueous solution to a volume of 1 mL to obtain a dispersion of the precipitate, which is the immune probe dispersion.

[0084] Step (3): Fabrication of the composite material sensor:

[0085] Take 10 μL of the immune probe dispersion prepared in step (2) and drop it onto the immune substrate prepared in step (1). Incubate at 37°C for 2 hours. After it is completely dry, a surface-enhanced Raman scattering (SERS) sensor is obtained, which is the sensor of the composite material.

[0086] The sensor made from the above-prepared composite material is used for surface-enhanced Raman detection of novel coronavirus protein, specifically as follows: Take 10 μL of saliva containing novel coronavirus protein (10 μL... -2 A 1 mg / mL sample was dropped onto the dried composite material sensor. Then, it was washed with TBS solution, PBS solution, and ultrapure water to remove unlinked novel coronavirus protein. After drying, the Raman signal was recorded at a laser wavelength of 532 nm using a Raman spectrometer to determine the relative concentration of novel coronavirus protein in the sample.

[0087] Figure 7 The image shows a scanning electron microscope (SEM) image of the VP@ZIF-67SERS substrate prepared in this embodiment. From... Figure 7 It can be seen that ZIF-67 particles are evenly distributed on the surface of VP.

[0088] Figure 8 The Zeta potential results of the immune probes prepared in this embodiment are shown. From Figure 8As can be seen, BP NPs are negatively charged (-26.4 mV), while RhB Raman molecules are positively charged (1.4 mV). After modification with RhB Raman molecules, the potential of BPNPs@RhB increased (24.8 mV), and the potential of BP NPs@RhB@novel coronavirus antibody@BSA increased significantly (-17.5 mV) because the novel coronavirus antibody@BSA carries a positive potential. This demonstrates the successful synthesis of the immune probe.

[0089] Figure 9 The surface-enhanced Raman spectroscopy (SMR) spectrum of the composite material sensor prepared in this embodiment for detecting novel coronavirus proteins is shown. Figure 9 It can be seen that this example is at 1646cm -1 The Raman signal intensity at that location is 6717.

[0090] Example 5

[0091] This embodiment provides a composite material sensor, its preparation method, and its application, including the following steps:

[0092] Step (1): Preparation of the immune base:

[0093] 1.16 g of cobalt nitrate hexahydrate was added to 40 mL of methanol solution and stirred at room temperature for 12 hours to obtain a methanol solution containing Co(II). 3.28 g of 2-methylimidazole was added to 40 mL of methanol solution and stirred at room temperature for 10 minutes to obtain a methanol solution containing 2-methylimidazole. The methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly for 30 minutes to obtain a methanol solution containing a complex of 2-methylimidazole and cobalt ions (referred to as ZIF-67). A 5 mm x 5 mm piece of purple phosphorus (referred to as VP) was placed into the above methanol solution containing ZIF-67, stirred slowly for 12 hours, and then allowed to stand for 2 hours. After that, it was taken out, washed three times with methanol solution, and dried to obtain a VP@ZIF-67 surface-enhanced Raman scattering (SERS) substrate, abbreviated as VP@ZIF-67SERS substrate.

[0094] Take the VP@ZIF-67SERS substrate prepared in the above steps, and add 10 μL of novel coronavirus antibody solution (10 μL). -2The solution was incubated at 4°C for 12 hours to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus. The surface of the VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus was washed with TBS solution, PBS solution, and ultrapure water to remove excess antibodies against the novel coronavirus (hereinafter referred to as the washing step). 10 μL of bovine serum albumin (BSA) solution was added to the washed VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus, and the substrate was placed at room temperature for 3 hours. The washing step was repeated once to obtain VP@ZIF-67SERS substrate containing antibodies against the novel coronavirus with a bovine serum albumin protective layer, which is the immune substrate.

[0095] The ligand in ZIF-67 is 2-methylimidazole, the metal ion is divalent cobalt ion, the space group of ZIF-67 is Fd-3m, and the cell parameters are... α=β=γ=90°, the crystal system is cubic.

[0096] Step (2): Preparation of the immunoprobe dispersion:

[0097] 50 mg of lumpy black phosphorus was ground into powder in a glove box. Then, 30 mg of black phosphorus powder and 90 mg of polyvinylpyrrolidone were weighed and added to anhydrous ethanol solution (30 mL). The mixture was placed in an ultrasonic bath on ice for 4 hours to obtain a brown dispersion. The mixture was centrifuged at 10,000 rpm for 15 minutes (hereinafter referred to as centrifugation) to remove large pieces of black phosphorus. The supernatant dispersion was collected, washed with methanol and ultrapure water, and the precipitate was collected. The collected product was dispersed in ultrapure water at a concentration of 1 mg / mL to obtain a black phosphorus flake dispersion (BP NPs). 1 mL of the BP NPs dispersion was taken, and 10 μL of RhB Raman molecular aqueous solution (1.0 × 10⁻⁶) was added dropwise. -2 The BP NPs dispersion was incubated at room temperature for more than 12 hours (mol / L) to obtain a fully incubated BP NPs dispersion. Then, the incubated BP NPs dispersion was centrifuged, the supernatant was removed, and the precipitate was dispersed in PBS aqueous solution to obtain a dispersion of BP NPs with RhB Raman molecules attached. 20 μL of novel coronavirus antibody solution (10...) -2(mg / mL) was added dropwise to the dispersion of RhB Raman molecules attached to BP NPs, and incubated at 4°C for 1.5 hours to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs was centrifuged, the supernatant was removed, the precipitate was dispersed with PBS aqueous solution, 10 μL of BSA solution was added, and incubated at room temperature for 1 hour to obtain a dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer; then, the dispersion of antibodies against RhB Raman molecules and the novel coronavirus attached to BP NPs with a bovine serum albumin protective layer was centrifuged again, the supernatant was removed, and the precipitate was dispersed with PBS aqueous solution to a volume of 1 mL to obtain a dispersion of the precipitate, which is the immune probe dispersion.

[0098] Step (3): Fabrication of the composite material sensor:

[0099] Take 10 μL of the immune probe dispersion prepared in step (2) and drop it onto the immune substrate prepared in step (1). Incubate at 37°C for 2 hours. After it is completely dry, a surface-enhanced Raman scattering (SERS) sensor is obtained, which is the sensor of the composite material.

[0100] The sensor made from the above-prepared composite material is used for surface-enhanced Raman detection of novel coronavirus protein, specifically as follows: Take 10 μL of saliva containing novel coronavirus protein (10 μL... -2 A 1 mg / mL sample was dropped onto the dried composite material sensor. Then, it was washed with TBS solution, PBS solution, and ultrapure water to remove unlinked novel coronavirus protein. After drying, the Raman signal was recorded at a laser wavelength of 532 nm using a Raman spectrometer to determine the relative concentration of novel coronavirus protein in the sample.

[0101] Figure 10 The image shows a scanning electron microscope (SEM) image of the VP@ZIF-67SERS substrate prepared in this embodiment. From... Figure 10 It can be seen that ZIF-67 particles are distributed in layers on the surface of VP.

[0102] Figure 11 The surface-enhanced Raman spectroscopy (SMR) spectrum of the composite material sensor prepared in this embodiment for detecting novel coronavirus proteins is shown. Figure 11 It can be seen that this example is at 1646cm -1 The Raman signal intensity at that location is 4779.

[0103] The present invention has been described in detail above with general description and specific embodiments. However, any modifications or improvements made without departing from the spirit of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A sensor made of composite materials, characterized in that, The sensor made of this composite material consists of two parts: an immune substrate and an immune probe. The immune substrate is made by combining ZIF-67 and VP, and loaded with a novel coronavirus antibody and a BSA protective layer. The immune probe uses BP NPs as a carrier and is loaded with RhB Raman molecules, a novel coronavirus antibody, and a BSA protective layer. Preparation of the immune substrate: A certain amount of cobalt nitrate hexahydrate was added to a certain amount of methanol solution and stirred at room temperature to obtain a methanol solution containing Co(II); a certain amount of 2-methylimidazole was added to a certain amount of methanol solution and stirred at room temperature to obtain a methanol solution containing 2-methylimidazole; the methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly to obtain a methanol solution containing a complex ZIF-67 of 2-methylimidazole and cobalt ions; a 5 mm x 5 mm purple phosphorus VP was placed into the above methanol solution containing ZIF-67, stirred slowly for a certain time, and then allowed to stand for a certain time. Then it was taken out, washed three times with methanol solution and dried to obtain VP@ZIF-67 surface-enhanced Raman scattering substrate - VP@ZIF-67 SERS substrate; Take the VP@ZIF-67 SERS substrate prepared in the above steps, add a certain amount of novel coronavirus antibody solution, and incubate at 4 ℃ for a certain time to obtain VP@ZIF-67 SERS substrate with novel coronavirus antibody; Washing steps: The surface of the VP@ZIF-67 SERS substrate containing the novel coronavirus antibody was washed sequentially with Tris(hydroxymethyl)aminomethane buffer (TBS), phosphate buffer (PBS), and ultrapure water to remove excess novel coronavirus antibody; a certain amount of bovine serum albumin (BSA) solution was added to the washed VP@ZIF-67 SERS substrate containing the novel coronavirus antibody, and the substrate was placed at room temperature for a certain period of time. The above washing steps were repeated once to obtain the VP@ZIF-67 SERS substrate containing the novel coronavirus antibody with a bovine serum albumin protective layer, which is the immune substrate. The VP is purple phosphorus; The BP NPs are black phosphorus nanosheets; The RhB is Rhodamine B; The BSA is bovine serum albumin; The ligand in ZIF-67 is 2-methylimidazole, the metal ion is divalent cobalt ion, the space group is Fd-3m, and the cell parameters are a = b = c = 17.064 Å. α = β = γ = 90°, the crystal system is cubic.

2. A method for preparing a composite material sensor as described in claim 1, characterized in that, The preparation method includes the following steps: Step (1) Preparation of immune substrate: A certain amount of cobalt nitrate hexahydrate was added to a certain amount of methanol solution and stirred at room temperature to obtain a methanol solution containing Co(II); A certain amount of 2-methylimidazole was added to a certain amount of methanol solution and stirred at room temperature to obtain a methanol solution containing 2-methylimidazole; The methanol solution containing 2-methylimidazole was slowly added to the methanol solution containing Co(II) and stirred slowly to obtain a methanol solution containing a complex ZIF-67 of 2-methylimidazole and cobalt ions; A 5 mm x 5 mm purple phosphorus VP was placed into the above methanol solution containing ZIF-67, stirred slowly for a certain time and then left to stand for a certain time, then taken out, washed three times with methanol solution and dried to obtain VP@ZIF-67 surface-enhanced Raman scattering substrate - VP@ZIF-67 SERS substrate; Take the VP@ZIF-67 SERS substrate prepared in the above steps, add a certain amount of novel coronavirus antibody solution, and incubate at 4 ℃ for a certain time to obtain a VP@ZIF-67 SERS substrate with novel coronavirus antibody; Washing steps: Wash the surface of the VP@ZIF-67 SERS substrate with novel coronavirus antibody in sequence with Tris(hydroxymethyl)aminomethane buffer (TBS) solution, phosphate buffer (PBS) solution, and ultrapure water to remove excess novel coronavirus antibody; Add a certain amount of bovine serum albumin (BSA) solution to the washed VP@ZIF-67 SERS substrate with novel coronavirus antibody, place at room temperature for a certain time, and repeat the above washing steps once to obtain a VP@ZIF-67 SERS substrate with novel coronavirus antibody with bovine serum albumin protective layer, which is the immune substrate; Step (2) Preparation of immune probe dispersion: A certain amount of blocky black phosphorus was ground into powder in a glove box. Then, a certain amount of black phosphorus powder and polyvinylpyrrolidone were weighed and added to anhydrous ethanol solution. The mixture was placed in an ultrasonic bath for ultrasonic peeling to obtain a brown dispersion. Large pieces of black phosphorus were removed by centrifugation. The upper dispersion was taken, washed with methanol and ultrapure water, and the precipitate was collected. The collected precipitate was dispersed in ultrapure water to obtain a dispersion of black phosphorus nanosheets BP NPs. A certain amount of BP NPs dispersion was taken, and a certain amount of Rhodamine B RhB Raman molecule aqueous solution was added. The mixture was placed at room temperature for a certain time for incubation to obtain a BP NPs dispersion after incubation. The BP NPs dispersion after centrifugation was taken, the supernatant was removed, and the precipitate was dispersed with PBS aqueous solution to obtain a dispersion of BP NPs with RhB Raman molecules attached. A certain amount of novel coronavirus antibody solution was added to the dispersion of BP NPs with RhB Raman molecules attached. The mixture was incubated at 4 ℃ for a certain time to obtain BP NPs. A dispersion of RhB Raman molecules and antibodies against the novel coronavirus was prepared by centrifuging BP NPs with a bovine serum albumin (BSA) protective layer. The supernatant was removed, and the precipitate was dispersed with PBS aqueous solution. A certain amount of BSA solution was added, and the mixture was incubated at room temperature for a certain period of time to obtain a dispersion of RhB Raman molecules and antibodies against the novel coronavirus on BP NPs with a bovine serum albumin (BSA) protective layer. The dispersion of the precipitate was then prepared by centrifugation, removing the supernatant, and dispersing the precipitate with PBS aqueous solution. This dispersion is the immune probe dispersion. Step (3) Preparation of the composite material sensor: Take a certain amount of the immune probe dispersion prepared in step (2) and drop it onto the immune substrate prepared in step (1), and incubate it at 37°C for a certain time; after it is completely dry, a surface-enhanced Raman scattering sensor is obtained, which is the composite material sensor.

3. The use of the composite material sensor according to claim 1, wherein the composite material sensor is used for the detection of novel coronavirus protein in human saliva, blood, or water, characterized in that, A certain amount of saliva containing the novel coronavirus protein was dropped onto the dried composite material sensor. Then, it was washed sequentially with TBS solution, PBS solution and ultrapure water to remove unlinked novel coronavirus protein. After drying, the Raman signal was recorded at a laser wavelength of 532 nm using a Raman spectrometer to determine the relative concentration of novel coronavirus protein in the sample.

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