Multi-channel virus detection SERS chip based on resonant cavity and preparation method thereof

Abstract

The invention discloses a multi-channel virus detection SERS chip based on a resonant cavity and a preparation method thereof, wherein the chip comprises a plurality of detection holes, each of which is composed of a plurality of V-shaped Au nanopore arrays, wherein Au nanoparticles are distributed in the Au nanopore arrays to form an enhanced structure of the Au nanopore resonant cavity; the method firstly plates an Al film on a substrate, then corrodes and oxidizes the Al film, then deposits an Au film on the corroded and oxidized Al film, and finally drips Au nanoparticles on the V-shaped Au nanopore array to obtain a SERS chip. The chip disclosed in the invention specifically captures virus particles through a size effect, and based on the resonant cavity enhancement effect, effectively excites the overall Raman signal of the virus, greatly improves the reproducibility of the Raman signal, and realizes the distinction between different viruses; the preparation method disclosed in the invention is simple to operate, highly repeatable, and can realize uniform preparation of a large area of ​​the chip.

Description

Technical Field

[0001] The invention belongs to the field of rapid detection of respiratory viruses, and in particular relates to a resonant cavity-based multi-channel virus detection SERS chip and a preparation method thereof. Background Art

[0002] Respiratory viruses are a major threat to human health. Usually, the clinical symptoms of respiratory virus infection are very similar, and it is difficult to determine the type of virus infected by simple symptom observation. At present, laboratory detection of respiratory viruses is mainly based on real-time fluorescent polymerase chain reaction (RT-PCR) to detect viral nucleic acid, which has many experimental steps and long detection time. More importantly, different primers need to be used for amplification of different viruses, making it difficult to achieve multiple detection of different respiratory viruses.

[0003] Surface enhanced Raman spectroscopy (SERS) technology can obtain the fingerprint spectrum of the virus, and can quickly, accurately and in real time detect and distinguish different virus molecules, and realize multi-link detection of different respiratory viruses. SERS chip is the core technology of surface enhanced Raman spectroscopy detection, and there is currently a lack of effective SERS chips for rapid detection of respiratory viruses.

[0004] The patent (CN111665356A) discloses a SERS-based virus detection method and device, which achieves rapid detection of viruses by fixing gold or silver sol-gel with SERS activity in a channel and using antibodies to specifically capture the virus. However, since the Raman enhancement hotspot of the sol-gel SERS substrate is very small (<10nm), which is very different from the size of common virus particles (>40nm), it is difficult to effectively stimulate the overall Raman signal of the virus, resulting in poor reproducibility of the virus's Raman signal, and it cannot be used for the detection of complex clinical systems; Patent (CN112326630B) discloses a SERS chip for the detection of new coronavirus and a preparation method thereof. The SERS chip has a multi-layer island structure, which can achieve rapid detection of the new coronavirus by capturing virus particles by adjusting nanopores, but the microstructure of the SERS chip is disordered, resulting in poor repeatability of the Raman signal and difficulty in distinguishing different viruses; Patent (CN111879753A) discloses a noble metal nanocone SERS substrate for detecting the new coronavirus and a preparation method thereof. The SERS substrate is a conical noble metal nanoarray, which can effectively capture virus particles and realize Raman detection, but the enhanced hotspot of the SERS substrate is mainly located at the tip of the nanocone. In addition, the substrate is prepared by a polystyrene self-assembly method, which often has many defects and is difficult to achieve large-area uniform preparation. Summary of the invention

[0005] In view of this, the present invention provides a multi-channel virus detection SERS chip based on a resonant cavity and a preparation method thereof. The chip effectively stimulates the overall Raman signal of the virus based on the enhancement effect of the resonant cavity, greatly improves the reproducibility of the Raman signal, and realizes the distinction between different viruses. The preparation method is simple to operate and has high repeatability, and can realize uniform preparation of a large area of ​​the chip.

[0006] To achieve this object, the present invention adopts the following technical scheme: a multi-channel virus detection SERS chip based on a resonant cavity, wherein the chip includes a plurality of detection holes, each of which is composed of a plurality of V-shaped Au nanohole arrays, and Au nanoparticles are distributed in the Au nanoholes to form an enhanced structure of the Au nanohole resonant cavity; and the diameter of the middle part of the Au nanohole is smaller than the minimum diameter of the virus to be detected.

[0007] Preferably, the diameter of the upper end of the V-shaped Au nanopore is in the range of 100 nm to 1000 nm, and the thickness of Au plated on the inner wall of the Au nanopore is in the range of 25 nm to 125 nm.

[0008] Preferably, the Au nanoparticles have a diameter of 30 to 80 nm.

[0009] A method for preparing a multi-channel virus detection SERS chip based on a resonant cavity, the method comprising:

[0010] S1: Al thin film is deposited on the surface of a smooth substrate with multiple grooves using magnetron sputtering technology;

[0011] S2: The substrate on which the Al film is deposited is corroded by a multi-step anodization technique to prepare an anodized aluminum with a V-shaped nanopore array on the surface of the Al film;

[0012] S3: Using magnetron sputtering technology to deposit Au thin film on the surface of anodized aluminum to form a V-shaped Au nanopore array;

[0013] S4: Synthesize Au nanoparticles using chemical synthesis method;

[0014] S5: dropping a certain volume of Au nanoparticles onto the surface of the V-shaped Au nanohole array to form a V-shaped Au nanoresonance cavity array enhancement structure.

[0015] Preferably, the substrate is a glass substrate, an aluminum substrate or an organic plastic substrate; the dimensions of the groove are 5 to 10 mm in length and width or diameter and 1 to 3 mm in depth, and each groove corresponds to a detection channel.

[0016] Preferably, the Al film has a thickness of 2 to 10 μm.

[0017] Preferably, the multi-step anodizing technique comprises the following steps:

[0018] 2.1: Immerse the substrate with the Al film deposited in an oxalic acid solution with a temperature of 2 to 5°C and a concentration of 0.1 to 0.5 M;

[0019] 2.2: Anodize the substrate with the Al film deposited using a constant potential instrument, apply a 30-60V DC voltage for 10-30 minutes, then gradually increase the applied voltage to 120-150V for 10-30 minutes, and then increase the applied voltage to 150-180V for 1-2 hours;

[0020] 2.3: Immerse the anodized substrate with the Al film deposited thereon in a mixed solution of 1.5 wt % chromic acid and 5 wt % phosphoric acid at a temperature of 40 to 60° C. and etch for 5 to 10 hours;

[0021] 2.4: Immerse the etched Al-film-deposited substrate in a phosphoric acid solution with a temperature of 10-40°C and a concentration of 4-10wt%, and perform secondary anodization on the Al-plated substrate using a constant potential instrument, applying a 120-150V DC voltage for 1-20 minutes.

[0022] Preferably, in step S3, the thickness of the Au film is 25-125 nm.

[0023] Preferably, the specific process of the chemical synthesis method is as follows: 80-200 ml of 200-350 μM chloroauric acid solution is heated to 100° C., and 8-20 ml of 30-60 mM sodium citrate is added and stirred thoroughly. After the solution turns wine red, the reaction is continued for about 30-60 minutes, and then cooled to room temperature to obtain Au nanoparticles.

[0024] Preferably, the certain volume in step S5 is 2-10 μl.

[0025] The beneficial effects of the present invention are:

[0026] (1) The multi-channel virus detection SERS chip based on the resonant cavity disclosed in the present invention can specifically capture viruses by utilizing the size effect, wherein virus particles whose size is larger than the diameter of the upper end of the V-shaped Au nanopore will be retained outside the pore, and impurity particles whose size is too small will fall into the bottom of the pore. Only virus particles whose particle size matches the V-shaped Au nanopore can enter the Raman enhancement region in the middle of the V-shaped Au nanopore, thereby achieving specific capture of virus particles and improving the recognition ability of virus Raman signals;

[0027] (2) The multi-channel virus detection SERS chip based on the resonant cavity disclosed in the present invention can more effectively excite the overall Raman signal of the virus based on the volume enhancement effect, because the virus particles will fall into the Au nano V-shaped hole as a whole, thereby greatly improving the reproducibility of the Raman signal and achieving the distinction between different viruses.

[0028] (3) The multi-channel virus detection SERS chip based on the resonant cavity disclosed in the present invention introduces Au nanoparticles into the Au nano V-shaped holes, which can cause the localized surface plasmons between the two to produce electromagnetic coupling, forming a resonance effect, and further improving the local electromagnetic field intensity, thereby obtaining a higher Raman enhancement effect and improving the detection sensitivity.

[0029] (4) The preparation method disclosed in the present invention is simple to operate and has high repeatability, and can achieve uniform preparation of chips over a large area. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the SERS chip structure in an embodiment of the present invention;

[0031] Figure 2 The SEM photos of the V-shaped Au nanopore array-Au nanoparticle resonant cavity SERS chip prepared by the method of the present invention in Example 1, where the upper end diameter of the V-shaped Au nanopore is 450 nm and the diameter of the Au nanoparticle is 30 nm, are at different magnification ratios;

[0032] Figure 3 This is a schematic diagram of the structure of a resonant cavity-based multi-channel virus detection SERS chip with 6 Raman detection channels prepared by the method of the present invention in Example 1;

[0033] In the figure: 1. Detection hole 1-1. V-shaped Au nanopore 1-2. Au nanoparticles 1-3. Virus particles. DETAILED DESCRIPTION

[0034] Those skilled in the art will appreciate that the embodiments described herein are intended to help readers understand the principles of the present invention, and should be understood that the protection scope of the present invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific variations and combinations that do not deviate from the essence of the present invention based on the technical revelations disclosed by the present invention, and these variations and combinations are still within the protection scope of the present invention.

[0035] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] A multi-channel virus detection SERS chip based on a resonant cavity, such as Figure 1 Shown and Figure 3As shown, the chip includes multiple detection holes 1, each of which is composed of a plurality of V-shaped Au nanopore 1-1 arrays. Au nanoparticles 1-2 are distributed in the Au nanopore array to form a resonance enhancement structure of the Au nanopore array, and the diameter of the middle part of the V-shaped Au nanopore is smaller than the minimum diameter of the virus to be detected. The size of the V-shaped Au nanopore 1-1 is generally determined according to the size of the detected virus particles 1-3. Here, the upper end diameter range of the V-shaped Au nanopore 1-1 is selected to be 100nm to 1000nm, the thickness of the Au plated inner wall of the Au nanopore is 25nm to 125nm, and the diameter of the Au nanoparticle 1-2 matched therewith is 30 to 80nm.

[0037] The above-mentioned V-shaped Au nanopore 1-1 array can specifically capture viruses by using the size effect. Virus particles 1-3 whose size is larger than the diameter of the upper end of the V-shaped Au nanopore 1-1 will be retained outside the hole, and impurity particles that are too small will fall into the bottom of the V-shaped Au nanopore 1-1. Only virus particles 1-3 whose particle size matches the V-shaped Au nanopore 1-1 can enter the Raman enhancement area in the middle of the V-shaped Au nanopore 1-1, thereby achieving specific capture of virus particles and improving the ability to identify virus Raman signals. At the same time, since the virus particles will fall into the interior of the Au nano V-shaped hole as a whole, based on the body enhancement effect, the structure can more effectively excite the overall Raman signal of the virus, thereby greatly improving the reproducibility of the Raman signal and achieving the distinction between different viruses. In addition, the introduction of Au nanoparticles into the Au nano V-shaped hole can cause the localized surface plasmons between the two to produce electromagnetic coupling, forming a resonance effect, further improving the local electromagnetic field intensity, thereby obtaining a higher Raman enhancement effect and improving the detection sensitivity.

[0038] A method for preparing a multi-channel virus detection SERS chip based on a resonant cavity, the method comprising:

[0039] Step 1: Depositing an Al film on a smooth substrate with multiple grooves by magnetron sputtering technology. The substrate is preferably any one of a glass substrate, an aluminum substrate or an organic plastic substrate. The groove size is 5 to 10 mm in length and width or diameter, and 1 to 3 mm in depth. Each groove corresponds to a detection channel. The thickness of the Al film is 2 to 10 μm.

[0040] Step 2: The substrate on which the Al film is deposited is corroded by a multi-step anodizing technique to prepare anodized aluminum with a V-shaped nanopore array on the surface of the Al film. The specific process is as follows:

[0041] 2.1: Immerse the substrate with the Al film deposited in an oxalic acid solution with a temperature of 2 to 5°C and a concentration of 0.1 to 0.5 M;

[0042] 2.2: Anodize the substrate with the Al film deposited using a constant potential instrument, apply a 30-60V DC voltage for 10-30 minutes, then gradually increase the applied voltage to 120-150V for 10-30 minutes, and then increase the applied voltage to 150-180V for 1-2 hours;

[0043] 2.3: Immerse the anodized substrate with the Al film deposited thereon in a mixed solution of 1.5 wt % chromic acid and 5 wt % phosphoric acid at a temperature of 40 to 60° C. and etch for 5 to 10 hours;

[0044] 2.4: Immerse the etched Al-film-deposited substrate in a phosphoric acid solution with a temperature of 10 to 40° C. and a concentration of 4 to 10 wt %, and perform secondary anodization on the Al-plated substrate using a constant potential instrument, applying a DC voltage of 120 to 150 V for 1 to 20 minutes;

[0045] Step 3: Using magnetron sputtering technology to deposit Au thin film on the surface of anodized aluminum to form a V-shaped Au nanopore array, the thickness of the Au film is 25 to 125 nm;

[0046] S4: Au nanoparticles were synthesized by chemical synthesis. The specific process was as follows: 80-200 ml of 200-350 μM chloroauric acid solution was heated to 100°C, and 8-20 ml of 30-60 mM sodium citrate was added and stirred thoroughly. After the solution turned into wine red, the reaction was continued for about 30-60 minutes, and then cooled to room temperature to obtain Au nanoparticles.

[0047] S5: dropping 2-10 μl of Au nanoparticles onto the surface of the V-shaped Au nanohole array to form a V-shaped Au nanoresonance cavity array enhancement structure.

[0048] Example 1

[0049] (1) An aluminum substrate was placed in a vacuum chamber. The aluminum substrate had six grooves with a diameter of 10 mm and a depth of 3 mm on its surface. The vacuum was drawn to a pressure of less than 5×10 -4 Pa, introduce argon gas and adjust the gas pressure to 0.5 Pa to deposit a 2 μm thick Al film. After sputtering, cool for 1 hour and take out the sample.

[0050] (2) The aluminum substrate on which the Al film was deposited was immersed in an oxalic acid solution with a temperature of 4°C and a concentration of 0.5M; then the Al-plated substrate was anodized using a potentiostat: a 40V DC voltage was applied and maintained for 20 minutes, the applied voltage was gradually increased to 140V and maintained for 20 minutes, and then the applied voltage was increased to 160V and maintained for 2 hours; subsequently, the anodized Al-plated substrate was immersed in a mixed solution of 1.5wt% chromic acid and 5wt% phosphoric acid at a temperature of 40°C and etched for 10 hours; finally, the etched Al-plated substrate was immersed in a phosphoric acid solution with a temperature of 10°C and a concentration of 4wt%, and the Al-plated substrate was anodized again using a potentiostat: a 140V DC voltage was applied and maintained for 15 minutes.

[0051] (3) Place the anodized substrate in a vacuum chamber and evacuate the vacuum to less than 5×10 -4 pa, introduce argon gas, adjust the gas pressure to 0.5Pa, deposit 25nm Au film, after sputtering, cool for 10min and take out the sample.

[0052] (4) 80 ml of 200 μM chloroauric acid solution was heated to 100°C, and 8 ml of 30 mM sodium citrate was added and stirred thoroughly. After the solution turned into wine red, the reaction was continued for about 30 minutes, and then cooled to room temperature to obtain Au nanoparticles with a diameter of about 30 nm.

[0053] (5) Take 2 μl of the above-mentioned Au particles with a diameter of 30 nm and drop them into the groove on the surface of the aluminum substrate on which the Au film was deposited in step 3. After drying, a Figure 3 The 6-channel SERS detection chip shown has a V-shaped hole Au nanoparticle resonant cavity structure.

[0054] Example 2

[0055] (1) Place a plastic substrate with a groove of 10 mm in diameter and 1 mm in depth into a vacuum chamber and evacuate the chamber to a pressure of <5×10 -4 Pa, introduce argon gas, adjust the gas pressure to 0.5Pa, deposit 5μm Al film, cool for 1h after sputtering, and take out the sample;

[0056] (2) The aluminum substrate deposited with the Al film is immersed in an oxalic acid solution with a temperature of 2°C and a concentration of 0.1M, and the Al-plated substrate is anodized using a constant potential instrument: a 30V DC voltage is applied for 30 minutes, the applied voltage is gradually increased to 120V for 30 minutes, and then the applied voltage is increased to 150V for 2 hours; then, the anodized Al-plated substrate is immersed in a mixed solution of 1.5wt% chromic acid and 5wt% phosphoric acid at a temperature of 50°C for etching for 8 hours; finally, the etched Al-plated substrate is immersed in a phosphoric acid solution with a temperature of 20°C and a concentration of 6wt%, and the Al-plated substrate is subjected to a secondary anodization treatment using a constant potential instrument: a 120V DC voltage is applied for 20 minutes;

[0057] (3) Place the anodized substrate in a vacuum chamber and evacuate the vacuum to less than 5×10 -4 pa, introduce argon and adjust the pressure to 0.5 Pa. Deposit 80nm Au film; after sputtering, cool for 10min and take out the sample;

[0058] (4) 150 ml of 280 μM chloroauric acid solution was heated to 100° C., and 15 ml of 45 mM sodium citrate was added and stirred thoroughly. After the solution turned into wine red, the reaction was continued for about 45 minutes, and then cooled to room temperature to obtain Au nanoparticles with a diameter of about 60 nm.

[0059] (5) Take 5 μl of the above-mentioned Au particles with a diameter of 60 nm and drop them into the grooves on the surface of the plastic substrate on which the Au film is deposited in step 3. After drying, a single-channel SERS detection chip with a V-shaped hole Au nanoparticle resonant cavity structure is formed.

[0060] Example 3

[0061] (1) Place a glass substrate with 12 grooves of 5 mm in length and width and 2 mm in depth into a vacuum chamber and evacuate the chamber to a vacuum of <5×10 -4 pa, introduce argon gas, adjust the gas pressure to 0.5Pa, deposit 8μm Al film, cool for 1h after sputtering, and take out the sample;

[0062] (2) The aluminum substrate on which the Al film was deposited was immersed in an oxalic acid solution with a temperature of 5°C and a concentration of 0.3M, and the Al-plated substrate was anodized using a constant potentiostat: a DC voltage of 60V was applied and maintained for 10 minutes, the applied voltage was gradually increased to 150V and maintained for 10 minutes, and then the applied voltage was increased to 180V and maintained for 1 hour; subsequently, the anodized Al-plated substrate was immersed in a mixed solution of 1.5wt% chromic acid and 5wt% phosphoric acid at a temperature of 60°C and etched for 5 hours; finally, the etched Al-plated substrate was immersed in a phosphoric acid solution with a temperature of 40°C and a concentration of 10wt%, and the Al-plated substrate was anodized again using a constant potentiostat: a DC voltage of 150V was applied and maintained for 1 minute.

[0063] (3) Place the anodized substrate in a vacuum chamber and evacuate the vacuum to less than 5×10 -4 pa, introduce argon and adjust the gas pressure to θ.5 Pa. Deposit 125 nm Au film, after sputtering, cool for 10 min and take out the sample.

[0064] (4) 200 ml of 350 μM chloroauric acid solution was heated to 100° C., and 20 ml of 60 mM sodium citrate was added and stirred thoroughly. After the solution turned into wine red, the reaction was continued for about 60 minutes, and then cooled to room temperature to obtain Au nanoparticles with a diameter of about 80 nm.

[0065] (5) Take 10 μl of the above-mentioned Au particles with a diameter of 80 nm and drop them into the grooves on the surface of the glass substrate on which the Au film is deposited in step 3. After drying, a 12-channel SERS detection chip with a V-shaped hole Au nanoparticle resonant cavity structure is formed.

Claims

1. A multi-channel virus detection SERS chip based on a resonant cavity, characterized in that: The chip includes multiple detection holes, each of which is composed of multiple V-shaped Au nanopore arrays. Au nanoparticles are distributed in the Au nanopores to form an enhanced structure of the Au nanopore resonant cavity; the diameter of the middle part of the Au nanopore is smaller than the minimum diameter of the virus to be detected, the upper end diameter range of the V-shaped Au nanopore is 100nm to 1000nm, and the thickness of the Au plated on the inner wall of the Au nanopore is 25nm to 125nm; the diameter of the Au nanoparticles is 30 to 80nm.

2. The resonant cavity-based multi-channel virus detection SERS chip according to claim 1, characterized in that: The diameter of the upper end of the V-shaped Au nanopore is in the range of 100nm to 1000nm, and the thickness of Au plated on the inner wall of the Au nanopore is in the range of 25nm to 125nm.

3. The multi-channel virus detection SERS chip based on resonant cavity according to claim 1, characterized in that: The Au nanoparticles have a diameter of 30 to 80 nm.

4. A method for preparing a resonant cavity-based multi-channel virus detection SERS chip as claimed in any one of claims 1 to 3, characterized in that: The method comprises: S1: Al thin film is deposited on the surface of a smooth substrate with multiple grooves using magnetron sputtering technology; S2: The substrate on which the Al film is deposited is corroded by a multi-step anodization technique to prepare an anodized aluminum with a V-shaped nanopore array on the surface of the Al film; S3: Using magnetron sputtering technology to deposit Au thin film on the surface of anodized aluminum to form a V-shaped Au nanopore array; S4: Synthesize Au nanoparticles using chemical synthesis method; S5: dropping 2 to 10 μl of Au nanoparticles onto the surface of the V-shaped Au nanohole array to form an enhanced structure of the V-shaped Au nanohole resonant cavity.

5. The method for preparing a resonant cavity-based multi-channel virus detection SERS chip according to claim 4, characterized in that: The substrate is a glass substrate, an aluminum substrate or an organic plastic substrate; the groove has a length and width or a diameter of 5 to 10 mm and a depth of 1 to 3 mm, and each groove corresponds to a detection channel.

6. The method for preparing a resonant cavity-based multi-channel virus detection SERS chip according to claim 4, characterized in that: The Al film has a thickness of 2 to 10 μm.

7. The method for preparing a resonant cavity-based multi-channel virus detection SERS chip according to claim 4, characterized in that: The multi-step anodizing technique comprises the following steps: 2.1: Immerse the substrate with the Al film deposited in an oxalic acid solution with a temperature of 2 to 5°C and a concentration of 0.1 to 0.5 M; 2.2: Use a constant potential instrument to anodize the substrate with the Al film deposited, load a 30-60V DC voltage for 10-30 minutes, then gradually increase the load voltage to 120-150V for 10-30 minutes, and then increase the load voltage to 150-180V for 1-2 hours; 2.3: Immerse the anodized substrate with the Al film deposited thereon in a mixed solution of 1.5 wt % chromic acid and 5 wt % phosphoric acid at a temperature of 40 to 60° C. and etch for 5 to 10 hours; 2.4: Immerse the etched Al-film-deposited substrate in a phosphoric acid solution with a temperature of 10-40°C and a concentration of 4-10wt%, and perform secondary anodization on the Al-plated substrate using a constant potential instrument, applying a 120-150V DC voltage for 1-20 minutes.

8. The method for preparing a resonant cavity-based multi-channel virus detection SERS chip according to claim 4, characterized in that: In the step S3, the thickness of the Au film is 25-125 nm.

9. The method for preparing a resonant cavity-based multi-channel virus detection SERS chip according to claim 4, characterized in that: The specific process of the chemical synthesis method is as follows: 80-200 ml of 200-350 μM chloroauric acid solution is heated to 100° C., and 8-20 ml of 30-60 mM sodium citrate is added and stirred sufficiently. After the solution turns wine red, the reaction is continued for 30-60 minutes, and then cooled to room temperature to obtain Au nanoparticles.

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