SERS biochip for oilseed rape and preparation method and application thereof

Abstract

The invention relates to the technical field of nanotechnology, detection technology and biosensing, and discloses an SERS biochip for oilseed rape and a preparation method and application thereof.The SERS biochip for oilseed rape comprises a substrate, an Ag nano film is evaporated on the surface of the substrate, and the substrate is a single-pass porous anodic aluminum oxide template with the surface modified with SiO2 microspheres. The SERS biochip for the oilseed rape is simple in manufacturing process, lower in cost and beneficial to large-scale application, and has repeatability, stability and sensitivity at the same time.

Description

Technical Field

[0001] The invention relates to the fields of nanotechnology, detection technology and biosensor technology, and in particular to a SERS biochip for rapeseed and a preparation method and application thereof. Background Art

[0002] Pesticide residues are directly related to food safety and environmental pollution. Rapeseed, as the main source of edible vegetable oil and plant protein, occupies an important position in agricultural products. Therefore, it is very important to test rapeseed crops for pesticides. Existing testing methods are expensive and time-consuming, and cannot meet the actual needs of pesticide residue testing.

[0003] Molecular Raman spectroscopy has been widely used in molecular identification in biochemistry and many other fields due to its high sensitivity and good specificity, because it can provide unique spectral information of various substances by capturing the inelastic scattering of incident light. However, Raman spectroscopy has the characteristics of low sensitivity and susceptibility to external interference, and the original spectrum is weak and difficult to be detected. This can be solved by surface enhanced Raman spectroscopy (SERS) technology.

[0004] However, the existing SERS biochip production process for rapeseed is usually long and expensive, which is not conducive to rapid detection and large-scale application. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems in the prior art that the production process of SERS biochips for rapeseed is usually long and the cost is high, which is not conducive to rapid detection and large-scale application. A SERS biochip for rapeseed and its preparation method and application are provided. The production process of the SERS biochip for rapeseed is simple, the cost is lower, it is conducive to large-scale application, and it has repeatability, stability and sensitivity.

[0006] In order to achieve the above object, the present invention provides a SERS biochip for rapeseed, the SERS biochip for rapeseed comprising a substrate, a Ag nanofilm is evaporated on the surface of the substrate, and a SiO nanofilm is modified on the surface of the substrate. 2 Single-pass porous anodized aluminum template for microspheres.

[0007] Preferably, the thickness of the Ag nanofilm is 20 to 40 nm;

[0008] The SiO 2 The diameter of the microspheres is 150-250 nm.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned SERS biochip for rapeseed, comprising the following steps:

[0010] (1) SiO 2The microsphere suspension is centrifuged, and the supernatant is removed to obtain a lower layer product, and the lower layer product is spin-coated on the surface of a pretreated single-pass porous anodized aluminum template, and then allowed to stand to obtain a substrate;

[0011] (2) A Ag nanofilm is evaporated on the surface of the substrate to obtain a SERS biochip for rapeseed.

[0012] Preferably, in step (1), the SiO 2 The concentration of the microsphere suspension is 2 to 3 wt%;

[0013] The centrifugal conditions include: a centrifugal speed of 4000-6000 r / min, and a centrifugal time of 4-6 min.

[0014] Preferably, in step (1), the ratio of the amount of the lower layer product to the surface area of ​​the pretreated single-pass porous anodized aluminum template on which the lower layer product is spin-coated is 90-120 μL: 1 cm 2 ;

[0015] The spin coating conditions include: a spin coating speed of 650 to 800 r / min, a spin coating time of 1.5 to 3 min, and a spin coating temperature of 20 to 25° C.

[0016] Preferably, in step (1), the pretreated single-pass porous anodized aluminum template is prepared by ultrasonically cleaning the single-pass porous anodized aluminum template for 4 to 6 minutes and then drying it at 70 to 90° C. for 4 to 6 minutes.

[0017] Preferably, in step (1), the standing conditions include: a standing temperature of 20 to 25° C. and a standing time of 2 to 5 min.

[0018] Preferably, in step (2), the evaporation conditions include: evaporation power of 90 to 110 W, vacuum degree of 1 to 1.2 × 10 -3 Pa.

[0019] A third aspect of the present invention provides an application of the above-mentioned SERS biochip for rapeseed in pesticide detection.

[0020] Preferably, the pesticide comprises rhodamine, acetamiprid, carbendazim and myclobutanil.

[0021] The beneficial effects of the present invention are:

[0022] 1. Using porous anodic aluminum oxide (AAO) with matrix arrangement and vertical arrangement as a template, SiO is deposited in its loose porous structure based on the planar self-assembly method. 2 Active particles, and then assemble silver nanoparticles into an ordered array structure. SiO 2The array structure formed in the loose nanoporous structure of AAO can increase the surface area and the distribution density of active hot spots on the substrate, thereby working together with Ag nanoparticles to significantly improve the Raman surface enhancement effect. The detection of pesticides can reach below 0.01 mg / L, and it has good uniformity, stability and repeatability.

[0023] 2. The complex components of common rapeseed pesticides such as rhodamine, acetamiprid, carbendazim and myclobutanil have been detected, and the characteristic peaks of different substances can be accurately characterized, providing a reliable method for the application of Raman-enhanced biological substrates in biochemical analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart of the method for making a SERS biochip for rapeseed of the present invention;

[0025] Figure 2 This is a spectrum result diagram of the effect of the thickness of the Ag nanofilm on the SERS biochip for rapeseed of the present invention;

[0026] Figure 3 SiO 2 Spectral results of the effect of microspheres on the Raman enhancement of SERS biochip for rapeseed;

[0027] Figure 4 This is a spectrum of Rhodamine 6G detected by the SERS biochip for rapeseed of the present invention;

[0028] Figure 5 The uniformity analysis of the attribution peaks measured by the SERS biochip for rapeseed of the present invention;

[0029] Figure 6 The invention provides a repeatability test for the SERS biochip for rapeseed;

[0030] Figure 7 The Raman spectra of the rapeseed SERS biochip of the present invention for detecting different concentrations of acetamiprid;

[0031] Figure 8 This is a Raman spectrum diagram of the rapeseed SERS biochip for detecting acetamiprid, carbendazim and myclobutanil. DETAILED DESCRIPTION

[0032] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings and examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0033] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0034] In addition, the technical solutions between the various embodiments provided by the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0035] Example 1

[0036] The present invention provides a SERS biochip for rapeseed, which comprises a substrate, a Ag nanofilm is evaporated on the surface of the substrate, and a SiO nanofilm is modified on the surface of the substrate. 2 Single-pass porous anodized aluminum template for microspheres.

[0037] Because single-pass porous anodic aluminum oxide is formed by low-cost ordered anodic oxidation based on pure aluminum, it has a matrix-arranged and vertically arranged nanoporous structure and unique mechanical, chemical and electrical properties. This makes single-pass porous anodic aluminum oxide a versatile template for making reproducible, low-cost and cost-effective SERS substrates.

[0038] In the preferred embodiment of the present invention, the thickness of the Ag nanofilm is 20 to 40 nm; 2 The diameter of the microspheres is 150-250 nm.

[0039] In this embodiment, the thickness of the Ag nanofilm is 30 nm, and the SiO 2 The diameter of the microspheres is 200 nm.

[0040] Example 2

[0041] A method for preparing a SERS biochip for rapeseed according to Example 1, as follows Figure 1 As shown, the following steps are included:

[0042] (1) SiO 2 The microsphere suspension is centrifuged, and the supernatant is removed to obtain a lower layer product, and the lower layer product is spin-coated on the surface of a pretreated single-pass porous anodized aluminum template, and then allowed to stand to obtain a substrate;

[0043] (2) Ag nanofilm is evaporated on the surface of the substrate to obtain a SERS biochip for rapeseed.

[0044] In the present invention, the SiO 2 The microsphere suspension is commercially available and is made by mixing SiO 2 The microspheres were mixed with ethanol, in which SiO 2 The concentration of the microsphere suspension is 2-3 wt%.

[0045] In this embodiment, the SiO 2 The concentration of the microsphere suspension was 2.5 wt%.

[0046] In step (1) of the present invention, since the conventionally purchased SiO 2 The microsphere suspension concentration is low, and if the concentration is low, it will lead to the subsequent SiO 2 The microsphere deposition effect is not good, so it is necessary to 2 The microsphere suspension is concentrated to increase its concentration. Therefore, the present invention uses a centrifuge to concentrate SiO 2 The microsphere suspension was centrifuged and the supernatant was removed for the purpose of concentration. The lower layer product obtained was concentrated SiO 2 Microsphere suspension.

[0047] Furthermore, in step (1), the centrifugal conditions include: a centrifugal speed of 4000 to 6000 r / min, and a centrifugal time of 4 to 6 min.

[0048] In this embodiment, the centrifugal speed is 5000 r / min and the centrifugal time is 5 min.

[0049] In step (1) of the present invention, the single-pass porous anodized aluminum template has a model of SP450-400-2000, an outer contour size of 20 mm×20 mm, and a thickness of 2 μm. It is cut into a size of 10 mm×10 mm using a utility knife and then pretreated to obtain a pretreated single-pass porous anodized aluminum template; wherein the pretreated single-pass porous anodized aluminum template is prepared by ultrasonically cleaning the single-pass porous anodized aluminum template for 4 to 6 minutes and then drying it at 70 to 90° C. for 4 to 6 minutes.

[0050] In this embodiment, the pretreated single-pass porous anodized aluminum template is prepared by ultrasonically cleaning the cut single-pass porous anodized aluminum template with deionized water for 5 minutes and then drying it on a drying table at 80° C. for 5 minutes.

[0051] In the specific case of step (1) of the present invention, after obtaining the lower layer product, use a pipette to take out 100 μL of the lower layer product and drop it onto the surface of the pretreated single-pass porous anodized aluminum template to ensure that the lower layer product evenly covers the surface, and then place it in a constant temperature environment (20-25°C), and use a coating machine to spin-coat the lower layer product onto the surface of the pretreated single-pass porous anodized aluminum template.

[0052] In a preferred embodiment, in step (1), the ratio of the amount of the lower layer product to the surface area of ​​the pretreated single-pass porous anodized aluminum template on which the lower layer product is spin-coated is 90 to 120 μL: 1 cm 2 .

[0053] The ratio of the amount of the lower layer product to the surface area of ​​the pretreated single-pass porous anodized aluminum template on which the lower layer product is spin-coated is 90-120 μL: 1 cm 2 It means that the lower layer product is evenly spin-coated on one side of the pretreated single-pass porous anodized aluminum template, and the ratio of the amount of the lower layer product spin-coated on the surface to the area of ​​the surface is 90-120 μL: 1 cm 2 .

[0054] For example, in this embodiment, 100 μL of the lower layer product is used, and the length and width specifications of the pretreated single-pass porous anodized aluminum template used are 10 mm×10 mm. All of this 100 μL of the lower layer product is dripped on the surface (single side) of the pretreated single-pass porous anodized aluminum template and spin-coated. In this embodiment, the ratio of the amount of the lower layer product used to the surface area of ​​the pretreated single-pass porous anodized aluminum template on which the lower layer product is spin-coated is 100 μL: 1 cm 2 .

[0055] The spin coating conditions include: a spin coating speed of 650 to 800 r / min, a spin coating time of 1.5 to 3 min, and a spin coating temperature of 20 to 25° C. (constant temperature environment).

[0056] In this embodiment, the spin coating speed is 700 r / min, the spin coating time is 2 min, and the spin coating temperature is 20-25°C.

[0057] After the spin coating is completed in the present invention, the pre-treated single-pass porous anodized aluminum template coated with the lower layer product is taken down from the spin coater and allowed to stand in a constant temperature environment. The purpose of the standing still is to allow the solvent in the lower layer product to evaporate. The standing still conditions include: the standing still temperature is 20-25°C and the standing still time is 2-5 minutes.

[0058] In this embodiment, the standing temperature is 20-25° C., and the standing time is 3 minutes.

[0059] In step (1) of the present invention, a horizontal self-assembly method is used to assemble SiO 2 The microspheres self-assemble into an ordered structure based on the horizontal self-assembly method. 2The microspheres form an orderly arrangement structure during the evaporation of the solvent and are assembled into the inner wall of the nanoscale microporous structure of the single-pass porous anodized aluminum template for surface modification, resulting in a structure with a high surface area. 2 The microspheres were deposited in the pores of the single-pass porous anodized aluminum template, and some were attached to the surface.

[0060] Further, after the standing in step (1) is completed, a substrate is obtained, i.e., a substrate having a surface modified with SiO 2 Single-pass porous anodized aluminum template for microspheres.

[0061] After obtaining the substrate, the present invention uses a vacuum evaporation coating system to sputter Ag nanoparticles on the surface of the substrate in step (2), that is, to evaporate an Ag nanofilm on the surface of the substrate to obtain a SERS biochip for rapeseed. The specific steps include: preheating a ZHDS400z vacuum thermal evaporation coating system, placing the substrate, placing an Ag metal target, and setting evaporation process parameters. The evaporation conditions include: an evaporation power of 90 to 110 W, a vacuum degree of 1 to 1.2 × 10 -3 Pa, and then turned on the ZHDS400z vacuum thermal evaporation coating system for automatic evaporation, and evaporated Ag nanofilm on the surface of the substrate to obtain a SERS biochip for rapeseed (the vacuum thermal evaporation coating system heats the Ag metal target into nanoscale particles, and then deposits them according to the set thickness, and automatically stops when the specified Ag nanofilm thickness is reached).

[0062] In this embodiment, the evaporation power is 100W and the vacuum degree is 1×10 -3 Pa.

[0063] The present invention deposits Ag metal particles on the surface of the substrate through a vacuum evaporation coating system, and finally forms a rapeseed SERS biochip with a high enhancement effect. The overall manufacturing process is simple, the production efficiency is high, the unit cost is low, and it has repeatability and stability. The rapeseed SERS biochip prepared by this method can achieve rapid, accurate and stable detection of various chemical substances.

[0064] Example 3

[0065] According to the method of Example 2, rapeseed SERS biochips with Ag nanofilm thickness of 50 nm and 70 nm were prepared respectively (that is, the preparation method is the same as that of Example 2, except that the thickness of the Ag nanofilm is 50 nm and 70 nm respectively), and the effect of Ag nanofilms of different thicknesses on the performance of rapeseed SERS biochips was tested. The specific operation is as follows: the laser micro-Raman spectrometer power is set to 30 mw, the excitation light wavelength is 785 nm, the laser power is 30 mw, the integration time is 10 s, the integration times are 2 times, and the spectrum acquisition wavelength range is set to 100 cm -1 ~2000cm-1 The concentration was 1×10 -4 mol / L Rhodamine 6G solution was respectively added dropwise to the surface of the rapeseed SERS biochip of Example 1, the Ag nanofilm with a thickness of 50 nm, and the Ag nanofilm with a thickness of 70 nm, to collect Raman spectra, wherein 10 points were randomly selected on each rapeseed SERS biochip, and a spectrum was collected for each point to obtain 10 spectra, and then the 10 spectra were averaged to obtain the average spectrum of the rapeseed SERS biochip, and the collected spectra were analyzed by stacking diagram, as shown in FIG. Figure 2 As shown, according to Figure 2 It can be seen that in the range of Ag nanofilm thickness of 30nm to 70nm, as the thickness of the Ag nanofilm increases, the intensity of the average Raman scattering spectrum shows a significant decreasing trend, and the rapeseed SERS biochip with a Ag nanofilm thickness of 30nm has the highest peak response intensity.

[0066] Example 4

[0067] Verify SiO 2 Effect of microspheres on the performance of rapeseed SERS biochip: SiO-free microspheres were prepared according to the method of Example 2. 2 The difference between the microsphere rapeseed SERS biochip and the pretreated single-pass porous anodized aluminum template is that the Ag nanofilm is directly evaporated on the pretreated single-pass porous anodized aluminum template; then the laser micro-Raman spectrometer power is set to 30mw, the excitation light wavelength is 785nm, the laser power is 30mw, the integration time is 10s, the integration times are 2 times, and the spectrum acquisition wavelength range is set to 100cm -1 ~2000cm -1 The concentration was 1×10 -4 mol / L Rhodamine 6G solution was added dropwise to the rapeseed SERS biochip and the SiO-free 2 The surface of the rapeseed SERS biochip of the microsphere is used for Raman spectrum collection, wherein 10 points are randomly selected on each rapeseed SERS biochip, and the spectrum is collected at each point to obtain 10 spectra, and then the 10 spectra are averaged to obtain the average spectrum of the rapeseed SERS biochip, and the collected spectra are analyzed by stacking diagram, such as Figure 3 As shown, AAO-SiO 2 -Ag represents the detection result of the rapeseed SERS biochip in Example 1, and AAO-Ag represents the detection result of the rapeseed SERS biochip without SiO 2 The detection results of rapeseed microspheres using SERS biochip, according to Figure 3 It can be seen that the present invention utilizes SiO 2 The porous loose nanopore structure of single-pass anodic aluminum oxide (AAO) can be modified by microspheres, which can significantly enhance the Raman signal reflection intensity of the substrate.

[0068] Example 5

[0069] The rapeseed SERS biochip of Example 1 was placed on a glass slide, and a 1×10 -4 mol / L Rhodamine 6G solution, set the laser micro-Raman spectrometer power to 30mw, the excitation light wavelength to 785nm, the laser power to 30mw, the integration time to 10s, the number of integrations to 2 times, and the spectrum acquisition wavelength range to 100cm -1 ~2000cm -1 , start collecting the Raman spectrum characteristic signals of Rhodamine 6G solution, randomly select 10 points on the chip, measure 10 spectra for each point as a group, and get 10 groups of spectra in total. Then average the 10 spectra in each group to get 10 average spectra, and use waterfall chart to visualize and analyze the 10 average spectra. The results are as follows Figure 4 As shown in the figure, through the visualization analysis of ten sets of data, the results show that each set of data clearly presents the typical Raman characteristic peaks of the rhodamine 6G probe molecule, these characteristic peaks have obvious Raman shifts, and the signal intensity distribution is uniform, without significant fluctuations or inconsistencies. This phenomenon shows that the enhancement effect of the rapeseed SERS biochip of the present invention at multiple detection points maintains a certain uniformity, thereby ensuring the stability and consistency of the Raman signal;

[0070] The 1506 cm -1 The attribution peak is used as the basis for stability detection. For the 10 average spectra obtained above, the peak at 1506 cm -1 The intensity values ​​at the locations were obtained to obtain 10 intensity values. The RSD values ​​of these 10 intensity values ​​were calculated to evaluate the uniformity of the SERS biochip for rapeseed. Figure 5 As shown, according to Figure 5 It can be seen that the average intensity of the 10 Raman spectra measured is 1506cm -1 The calculated RSD value at the attribution peak is 19.75%, which confirms that the SERS biochip for rapeseed of the present invention has uniformity during the detection process and can support stable spectral detection.

[0071] Example 6

[0072] A concentration of 1×10 -4mol / L of Rhodamine 6G solution was used to collect Raman spectra, wherein 10 points were randomly selected on the rapeseed SERS biochip, and a spectrum was collected at each point to obtain 10 spectra, and then the 10 spectra were averaged to obtain the average spectrum of the rapeseed SERS biochip, and the collected spectra were stacked for analysis, and then the rapeseed SERS biochip was clamped with tweezers, and the chip surface was cleaned in running deionized water for 1 minute, and then a 1×10 -4 mol / L of Rhodamine 6G solution, Raman spectrum collection, and randomly select 10 points on the cleaned rapeseed SERS biochip, collect spectra at each point, and obtain 10 spectra, and then average the 10 spectra to obtain the average spectrum of the cleaned rapeseed SERS biochip, and perform stacking analysis on the collected spectra to evaluate their repeatability. The results are as follows Figure 6 As shown by Figure 6 As shown, the SERS biochip for rapeseed can still obtain good Raman signals after cleaning, and its signal intensity and characteristic peak remain stable without significant decline or change. This experiment shows that the SERS biochip for rapeseed of the present invention has good repeatability and stability, can maintain stable performance after multiple cleanings and reuses, and is suitable for long-term and multiple experimental operations.

[0073] Example 7

[0074] Six concentrations of acetamiprid pesticide solutions of 1000 mg / L, 100 mg / L, 10 mg / L, 1 mg / L, 0.1 mg / L, and 0.01 mg / L were added to the rapeseed SERS biochip of Example 1, and the laser micro-Raman spectrometer power was set to 30 mw, the excitation light wavelength was 785 nm, the laser power was 30 mw, the integration time was 10 s, the integration number was 2 times, and the spectrum acquisition wavelength range was set to 100 cm -1 ~2000cm -1 , collect the corresponding Raman spectral characteristic signals, where each concentration is detected in a grid format with a step size of 500μm, and 100 spectra are detected for each concentration. These 100 spectra are averaged to obtain the average spectrum of each concentration, and its detection sensitivity is evaluated. The results are as follows Figure 7 As shown, according to Figure 7It can be seen that the SERS biochip for rapeseed of the present invention can effectively detect the Raman characteristic peak of acetamiprid in six concentration gradients. Even at the lowest concentration of 0.01 mg / L, the characteristic peak can still be clearly observed, and the peak value and concentration show a relatively regular change trend. As the concentration of the pesticide solution increases, the peak value of the characteristic peak increases significantly. The chip has a sensitive characterization capability for the pesticide concentration, which also proves that the chip has a high Raman enhancement effect and sensitivity under low concentration conditions. These results show that the SERS biochip for rapeseed of the present invention has good detection sensitivity, can effectively capture the characteristic signals of pesticide molecules at extremely low concentrations, and provide effective tools and technical support for pesticide residue detection.

[0075] Example 8

[0076] Three commonly used pesticides (acetamiprid, carbendazim and myclobutanil) were diluted and prepared into 1000 mg / L standard solutions, and the standard solutions of acetamiprid, carbendazim and myclobutanil pesticides were added to the rapeseed SERS biochip of Example 1, and the laser micro-Raman spectrometer power was set to 30 mw, the excitation light wavelength was 785 nm, the laser power was 30 mw, the integration time was 10 s, the integration number was 2 times, and the spectrum acquisition wavelength range was set to 100 cm -1 ~2000cm -1 , collect the corresponding Raman spectral characteristic signals, where each pesticide is detected in a grid format with a step size of 500μm, and 100 spectra are detected for each pesticide. These 100 spectra are averaged to obtain the average spectrum of each pesticide, and its specificity for detecting pesticides is evaluated. The results are as follows Figure 8 As shown, according to Figure 8 It can be seen that the Raman spectrum of acetamiprid can be seen at 650cm -1 、849cm -1 、1129cm -1 、1294cm -1 Obvious characteristic peaks were observed at 647 cm -1 、1040cm -1 、1267cm -1 、1318cm -1 Obvious characteristic peaks were observed at 648 cm -1 、742cm -1 、1090cm -1 、1447cm -1 Obvious characteristic peaks were observed at , indicating that the rapeseed SERS biochip of the present invention has a high degree of specificity in detecting pesticides, and provides a fast and efficient analytical method for biochemical analysis.

[0077] It should be understood that parts not elaborated in detail in this specification belong to the prior art.

[0078] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A SERS biochip for rapeseed, characterized in that: The SERS biochip for rapeseed comprises a substrate, a Ag nanofilm is evaporated on the surface of the substrate, and the substrate is a single-pass porous anodized aluminum template with SiO2 microspheres modified on the surface.

2. The SERS biochip for rapeseed according to claim 1, characterized in that: The thickness of the Ag nanofilm is 20 to 40 nm; The diameter of the SiO2 microspheres is 150-250 nm.

3. The method for preparing a SERS biochip for rapeseed according to claim 1 or 2, characterized in that: The following steps are involved: (1) centrifuging the SiO2 microsphere suspension, removing the supernatant to obtain a lower layer product, spin-coating the lower layer product on the surface of a pretreated single-pass porous anodized aluminum template, and then allowing it to stand to obtain a substrate; (2) A Ag nanofilm is evaporated on the surface of the substrate to obtain a SERS biochip for rapeseed.

4. The preparation method according to claim 3, characterized in that: In step (1), the concentration of the SiO2 microsphere suspension is 2-3 wt%. The centrifugal conditions include: a centrifugal speed of 4000-6000 r / min, and a centrifugal time of 4-6 min.

5. The preparation method according to claim 3, characterized in that: In step (1), the ratio of the amount of the lower layer product to the surface area of ​​the pretreated single-pass porous anodized aluminum template on which the lower layer product is spin-coated is 90-120 μL: 1 cm 2 ; The spin coating conditions include: a spin coating speed of 650 to 800 r / min, a spin coating time of 1.5 to 3 min, and a spin coating temperature of 20 to 25° C.

6. The preparation method according to claim 3 or 5, characterized in that: In step (1), the pretreated single-pass porous anodized aluminum template is prepared by ultrasonically cleaning the single-pass porous anodized aluminum template for 4 to 6 minutes and then drying it at 70 to 90° C. for 4 to 6 minutes.

7. The preparation method according to claim 3, characterized in that: In step (1), the standing conditions include: a standing temperature of 20 to 25° C. and a standing time of 2 to 5 minutes.

8. The preparation method according to claim 3, characterized in that: In step (2), the evaporation conditions include: evaporation power of 90 to 110 W, vacuum degree of 1 to 1.2 × 10 -3 Pa.

9. Use of the SERS biochip for rapeseed according to claim 1 or 2 in pesticide detection.

10. The use according to claim 9, characterized in that: The pesticides include rhodamine, acetamiprid, carbendazim and myclobutanil.

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