A sers-based aspergillus detection kit and a preparation and detection method thereof

Abstract

The application discloses an aspergillus niger detection kit based on SERS and a preparation and detection method thereof. The kit comprises a SERS nanolabel, and the structure of the SERS nanolabel is that a bionic gold nanoflower is used as a gold core, and a Raman active dye and aspergillus niger specific aptamer are modified on the surface of the gold core. The kit has high sensitivity, specificity, reproducibility, uniformity and stability, can collect Raman spectrum through a portable Raman spectrum equipment, can be used for analyzing trace aspergillus niger in a sample, and the detection limit is lower than 10 CFU / mL. Compared with a traditional bacterial culture method, the kit has advantages in operation simplicity, detection period and analysis cost. The kit is suitable for food safety monitoring.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine analysis and detection, and particularly relates to an Aspergillus niger detection kit based on SERS, and a preparation method and a detection method of the kit. BACKGROUND

[0002] Aspergillus niger is a core functional microorganism in the food fermentation industry, and is widely used in Pu'er tea fermentation and soy sauce koji making. The annual output of related products reaches tens of thousands to millions of tons. Recent studies have found that Aspergillus niger is also used for wastewater treatment. Although the fungus has been granted GRAS safety certification, recent studies have revealed that 83% of 69 industrial Aspergillus niger strains can produce fumonisin B2 (FB2), 33% can synthesize ochratoxin A (OTA), and 26% of the strains have the ability to synthesize both toxins. This potential dual toxin-producing property and the contradiction with its GRAS status highlight the urgency of establishing an efficient detection system. Therefore, establishing a rapid and accurate Aspergillus niger pollution detection technology has become a key issue to ensure the safety of fermented foods.

[0003] Current fungal detection techniques are divided into traditional microbiological methods and modern instrumental analysis techniques. Traditional culture method, as the gold standard, relies on isolation culture and morphological identification, although it is low-cost, but it needs 3-7 days cycle, which is difficult to meet the rapid detection demand. Modern techniques include molecular detection (such as PCR), immunoassay (ELISA), and mass spectrometry, flow cytometry and other physical and chemical means, although they have high sensitivity, but are limited by professional operation and high equipment cost. Therefore, developing an Aspergillus niger screening technology with rapid response (<1 hour), simple operation and suitable for on-site detection has become an important research direction to break through the existing technical barriers.

[0004] Surface-enhanced Raman scattering (SERS) technology innovates microbial detection with single-molecule-level sensitivity and fingerprint spectrum characteristics, and its core applications include two modes: label-free detection directly captures bacterial characteristic spectrum to construct "chemical identity card", but is limited by endogenous molecule activity (detection limit > 10³ CFU / mL) and spectral overlap between strains; labeled detection uses gold / silver nano substrate to combine Raman reporter molecules (such as 4-mercaptobenzoic acid), and uses antibodies / aptamers to achieve specific capture, and the sensitivity is improved to 1-10 CFU / mL. Current technical breakthroughs need to focus on the development of ultra-high enhancement factor substrates, and need to solve key problems such as hotspot density optimization, signal stability and anti-matrix interference. SUMMARY

[0005] The application aims to provide an Aspergillus niger detection kit based on SERS and a preparation method and a detection method thereof.

[0006] The technical scheme adopted by the application is:

[0007] The application provides an Aspergillus niger detection kit based on SERS, wherein the kit comprises a SERS nanolabel, and the structure of the SERS nanolabel is that a biomimetic gold nanoflower (AuNFs) is used as a gold core, and a Raman active dye and an Aspergillus niger specific aptamer are modified on the surface of the gold core.

[0008] SEQ ID NO. 1:

[0009] 5' -SH -C6-CGTTTGGGCGGTATGAGTTCGGGGGTATACCGCAG-3'.

[0010] Further, the kit further comprises a silicon wafer, and the silicon wafer refers to a single crystal <100> P doped silicon wafer with a resistivity in the range of 1-15 Ω.

[0011] Further, the particle size of the gold core is 50-100 nm.

[0012] Further, the Raman active dye is 4-ethynylaniline (4-EBZN).

[0013] Further, the preparation method of the gold core is:

[0014] (1) adding an aqueous chloroauric acid solution into a three-necked round-bottom flask and heating to boiling; (2) under vigorous stirring, adding an aqueous trisodium citrate solution into the solution in step (1) at a constant speed, continuously stirring, heating to boiling and maintaining for 15-20 min, completing phase equilibrium under constant temperature conditions, cooling to room temperature (25℃) to form a colloid, and obtaining a stable dispersed gold seed (AuNPs) solution;

[0015] (3) Take 0.1-0.5 mM (preferably 0.25 mM) chloroauric acid aqueous solution in a three-necked round-bottom flask, and inject hydrochloric acid, gold seed solution, silver nitrate aqueous solution and L-ascorbic acid aqueous solution in sequence under stirring, and react at 20-30℃ and 100-150 rpm until the solution color becomes gray blue (preferably the reaction time is 30s); then add sodium dodecyl sulfate aqueous solution for surface modification to ensure the colloidal stability of the nanoparticles, and obtain gold core (denoted as AuNFs), which is stored at 4℃ in the dark for later use.

[0016] Further, the concentration of the chloroauric acid aqueous solution in step (1) is 0.5-5 mM (preferably 1 mM), and the concentration of the trisodium citrate aqueous solution in step (2) is 5g / L-15g / L (preferably 10g / L), and the volume ratio of the trisodium citrate aqueous solution to the chloroauric acid aqueous solution is 1:20-5:20 (preferably 3:20).

[0017] Further, in step (3), the concentration of hydrochloric acid is 0.5-5 M (preferably 1 M), the concentration of silver nitrate aqueous solution is 1-5 mM (preferably 2 mM), the concentration of L-ascorbic acid aqueous solution is 50-200 mM (preferably 100 mM), and the concentration of sodium dodecyl sulfate aqueous solution is 5g / L-15g / L (preferably 10g / L); the volume ratio of the chloroauric acid aqueous solution to hydrochloric acid, gold seed solution, silver nitrate aqueous solution, L-ascorbic acid aqueous solution and sodium dodecyl sulfate aqueous solution is 100:(0.05-0.5):(0.5-5):(0.5-5):(0.1-1):(0.1-1), preferably 100:0.1:1:1:0.5:0.5.

[0018] Further, the method for modifying the surface of the gold core with Raman active dye is as follows: dropwise add Raman active dye ethanol solution to the gold core at room temperature (25℃), and react at 20-30℃ for 0.1-2h (preferably 25℃ and 1h), centrifuge (preferably 1500-7500 rpm for 10 min), and disperse the precipitate in ultrapure water to obtain gold core modified with Raman active dye (denoted as AuNFs@4-EBZN).

[0019] Further, the concentration of the Raman active dye ethanol solution is 0.5-15 mM (preferably 10 mM), and the volume ratio of the Raman active dye ethanol solution to the gold core is 1:5-20, preferably 1:10.

[0020] Further, the preparation method of the SERS nanolabel is as follows:

[0021] The ultra-pure water solution of the Aspergillus niger specific aptamer is incubated with the gold core modified with the Raman active dye in a constant temperature shaker at 28°C and 180 rpm for 0.1-2h (preferably 0.25h), and separated by low-speed centrifugation (preferably 1500-7500 rpm for 20 min). The precipitate is suspended in ultra-pure water to obtain the SERS nanolabel (denoted as AuNFs@4-EBZN-Apt), which is stored at 4°C in the dark.

[0022] Further, the concentration of the ultra-pure water solution of the Aspergillus niger specific aptamer is 2.5-15 μM, preferably 7.5 μM; and the volume ratio of the ultra-pure water solution of the Aspergillus niger specific aptamer to the gold core modified with the Raman active dye is 1:5-15 (preferably 1:10).

[0023] The present application also provides a detection method for the SERS-based Aspergillus niger detection kit, which comprises the following steps:

[0024] 1) vortex mixing the sample to be tested with the SERS nanolabel, and incubating at 28°C and 180 rpm for 0.1-2h (preferably 0.75h) to complete the biological binding reaction;

[0025] 2) drop the reaction solution of step 1) on the surface of a silicon wafer, and dry at room temperature; the silicon wafer refers to a single crystal <100> P-doped silicon wafer with a resistivity in the range of 1-15 Ω;

[0026] 3) collect the SERS signal under the condition of excitation wavelength 785 nm and laser power 200 mW using a portable Raman spectrometer; establish a semi-log calibration curve of the negative logarithm value of the Aspergillus niger solution concentration (-logC, X axis) and the characteristic peak intensity of the Raman active dye (the characteristic peak of 4-EBZN is at 1596 cm -1 ); and obtain the concentration of Aspergillus niger in the sample to be tested by linear regression analysis.

[0027] Further, the volume ratio of the sample to be tested to the SERS nanolabel in step 1) is 1:1-10, preferably 1:5.

[0028] Further, the calibration curve in step 3) is prepared as follows: incubate different concentrations of Aspergillus niger ATCC 16404 solution with the SERS nanolabel at 28°C and 180 rpm for 0.1-2h, drop the reaction solution on the surface of a silicon wafer, and dry at room temperature; collect the SERS signal using a portable Raman spectrometer, and plot a calibration curve with the negative logarithm value of the Aspergillus niger solution concentration (-logC) as the abscissa and the characteristic peak intensity of the Raman active dye as the ordinate.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] (1) The SERS-based Aspergillus niger detection kit of the present application can be used for the analysis of trace Aspergillus niger in samples, and the detection limit is 2.56 CFU / mL-1.47 CFU / mL.

[0031] (2) The enhancement substrate of the SERS nanolabel in the kit is biomimetic gold nanoflower (AuNFs), which has high stability and provides a guarantee for reliable detection of Aspergillus niger.

[0032] (3) The kit can collect Raman spectrum through a portable Raman spectrum device, which provides the possibility for on-site rapid detection.

[0033] (4) The kit has high sensitivity, and verification in complex food matrices (Pu'er tea, soy sauce and drinking water) shows high recovery rate, and the linear correlation coefficient R² is greater than 0.99.

[0034] (5) The kit has high specificity and can specifically detect Aspergillus niger from bacterial liquid containing Aspergillus niger, Escherichia coli O157:H7, Salmonella enteritidis, Pseudomonas aeruginosa and Staphylococcus aureus interference strains.

[0035] (6) The kit has high reproducibility, uniformity and stability. Reproducibility test shows that the relative standard deviation (RSD) values of the characteristic peak intensities of 1179, 1596 and 2045 cm -1 are 3.2%, 3.8% and 3.5%, respectively; in the uniformity test, 10 independent sites were detected, and the RSD values of each peak were all less than 4.3%, confirming that the substrate surface distribution is well homogenized; further stability experiments show that the RSD of the 1596 cm -1 peak intensity is only 1.94% after storage at 4°C for 30 days.

[0036] (7) Compared with the traditional bacterial culture method, the detection method of the kit has advantages in operation simplicity, detection period, analysis cost, etc. The traditional Aspergillus niger detection often uses plate counting method, and the detection period usually needs 3 to 5 days, while the method established in the present application can be completed within 30 minutes from sample processing to result output, significantly shortening the detection time. In terms of cost, although the traditional method has low material cost, it has high demand for human resources; in contrast, the single detection of the method of the present application has lower cost advantage in terms of reagent and human resource investment. In terms of detection performance, the detection limit of the traditional method is usually between 10-100 CFU / mL, while the detection limit of the method of the present application in actual samples is less than 10 CFU / mL, showing higher sensitivity. It is suitable for food safety monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The schematic diagram for the preparation of the Aspergillus niger detection kit.

[0038] Figure 2 The schematic diagram for the operation of the Aspergillus niger detection kit.

[0039] Figure 3 The TEM image of AuNPs, scale bar 20 nm.

[0040] Figure 4 The TEM image of AuNFs, scale bar 20 nm.

[0041] Figure 5 The size distribution diagram of AuNPs.

[0042] Figure 6 The size distribution diagram of AuNFs.

[0043] Figure 7 The ultraviolet-visible absorption spectrum and optical photograph of AuNPs, AuNFs, AuNFs@4-EBZN and AuNFs@4-EBZN-Apt.

[0044] Figure 8 The Raman spectrum of AuNFs, 4-EBZN, AuNFs@4-EBZN and AuNFs@4-EBZN-Apt.

[0045] Figure 9 The Zeta potential measurement results of AuNFs, AuNFs@4-EBZN and AuNFs@4-EBZN-Apt (n=3).

[0046] Figure 10 The detection of AuNFs on 10 -9 The Raman spectrum of M 4-EBZN and 10 -2 The conventional Raman spectrum comparison of M 4-EBZN.

[0047] Figure 11 The X-ray photoelectron spectroscopy (XPS) full spectrum comparison diagram of AuNFs, 4-EBZN and AuNFs@4-EBZN.

[0048] Figure 12 The C 1s orbital spectrum of AuNFs@4-EBZN.

[0049] Figure 13 The Au 4f orbital spectrum of AuNFs@4-EBZN.

[0050] Figure 14 The N 1s orbital spectrum of AuNFs@4-EBZN.

[0051] Figure 15 EDS elemental mapping of AuNFs@4-EBZN.

[0052] Figure 16 EDS elemental mapping of AuNFs@4-EBZN-Apt.

[0053] Figure 17 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0054] Figure 18 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction. Figure 17 SERS nanotags at 1596 cm -1 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0055] Figure 19 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0056] Figure 20 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction. Figure 19 SERS nanotags at 1596 cm -1 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0057] Figure 21 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0058] Figure 22 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction. Figure 21 SERS nanotags at 1596 cm -1 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0059] Figure 23 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0060] Figure 24 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction. Figure 23 SERS nanotags at 1596 cm -1 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0061] Figure 25 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0062] Figure 26 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction. Figure 25 SERS nanotags at 1596 cm -1 Raman spectra of SERS nanotags prepared by different 4-EBZN concentration and gold core reaction.

[0063] Figure 27 Flow chart for the detection kit of A. niger.

[0064] Figure 28 Raman spectra of A. niger incubated with SERS nanotags at different concentrations.

[0065] Figure 29 The linear relationship between the intensity of the characteristic peak of Figure 28 and the logarithm of the concentration of A. niger (Log 10 [C]).

[0066] Figure 30 Schematic diagram of the selectivity and specificity evaluation of SERS nanotags.

[0067] Figure 31 Raman spectra of A. niger incubated with SERS nanotags at the same site for 10 times.

[0068] Figure 32 The intensity of the characteristic peak at 1596 cm Figure 31 and the RSD value of 10 times of detection at the same site. -1

[0069] Figure 33 Raman spectra of A. niger incubated with SERS nanotags at 10 different sites.

[0070] Figure 34 The intensity of the characteristic peak at 1596 cm Figure 33 and the RSD value of 10 times of detection at different sites. -1

[0071] Figure 35 Raman spectra of A. niger incubated with SERS nanotags and stored at 4℃ for 30 days.

[0072] Figure 36 The intensity of the characteristic peak at 1596 cm Figure 35 and the RSD value of continuous detection in -1

[0073] Figure 37 Correlation curve of SERS method and plate counting method for detecting A. niger in Pu'er tea samples.

[0074] Figure 38 Correlation curve of SERS method and plate counting method for detecting A. niger in soy sauce samples.

[0075] Figure 39 Correlation curve of SERS method and plate counting method for detecting A. niger in drinking water samples.

[0076] ​​​Figure 40 Raman spectra of Pu'er tea samples after adding different concentrations of Aspergillus niger.

[0077] Figure 41 Raman spectra of soy sauce samples after adding different concentrations of Aspergillus niger. Figure 40 Linear regression curve of logarithm of Aspergillus niger concentration in the middle and intensity of characteristic peak at 1596 cm -1

[0078] Figure 42 Raman spectra of drinking water samples after adding different concentrations of Aspergillus niger.

[0079] Figure 43 Linear regression curve of logarithm of Aspergillus niger concentration in the middle and intensity of characteristic peak at 1596 cm -1

[0080] Figure 44 Linear regression curve of logarithm of Aspergillus niger concentration in the middle and intensity of characteristic peak at 1596 cm -1

[0081] Figure 45 Linear regression curve of logarithm of Aspergillus niger concentration in the middle and intensity of characteristic peak at 1596 cm -1 DETAILED DESCRIPTION Figure 44 The application will be further described below in conjunction with specific embodiments, but the protection scope of the application is not limited to this:

[0082] The application will be further described below in conjunction with specific embodiments, but the protection scope of the application is not limited to this:

[0083] The ultrapure water in the embodiments of the application refers to water with a resistivity of 18 MΩ*cm (25℃).

[0084] The silicon wafer in the embodiments of the application refers to a single crystal <100> P doped silicon wafer with a resistivity in the range of 1-15 Ω and a size of 5 mm*5 mm*0.6 mm.

[0085] Example 1, preparation of SERS nanotags

[0086] As shown in the following scheme, the structure of the SERS nanotag is a biomimetic gold nanoflower (AuNFs) as a gold core, and a Raman active dye and Aspergillus niger specific aptamer are modified on the surface of the gold core, and the specific preparation process is as follows: Figure 1 S1, the preparation method of the gold core is:

[0087] (1) Take 40 mL of an aqueous solution (1 mM) of chloroauric acid (HAuCl4*3H2O) in a three-necked round-bottom flask, and the three-necked round-bottom flask is equipped with a condensation reflux device, and then the solution is heated to boiling.

[0088]

[0089] ​​​​​(2) Under vigorous stirring, 6 mL of 10 g / L trisodium citrate (C6H5Na307·2H20) aqueous solution was quickly added into the solution of step (1), and the solution was kept at 100°C for 15-20 min, and then was cooled to room temperature (25°C) to obtain 46 mL of gold seed (AuNPs) solution with a particle size of 15-30 nm.

[0090] (3) 200 mL of low-concentration chloroauric acid aqueous solution (0.25 mM) was taken in a three-necked round-bottom flask, and the following reagents were sequentially injected: hydrochloric acid (200 μL, 1 M), gold seed solution (2 mL), silver nitrate (AgN03) aqueous solution (2 mL, 2 mM), and L-ascorbic acid (L-AA) aqueous solution (1 mL, 100 mM). Under the driving of magnetic stirring at 800 rpm, the solution was reacted at 25°C for 30 s until the color of the solution changed to gray blue. After the reaction was completed, 1 mL of sodium dodecyl sulfate aqueous solution (10 g / L) was added into the three-necked round-bottom flask under stirring to perform surface modification, so as to ensure the colloidal stability of the nanoparticles, and 206.2 mL of biomimetic gold nanoflower (AuNFs) solution, i.e., gold core solution, was obtained, which had a particle size of 50-100 nm and was stored at 4°C in the dark for later use.

[0091] S2, the preparation method of the gold core solution with surface-modified Raman active dye was as follows:

[0092] At room temperature (25°C, static), 100 μL of 4-EBZN ethanol solution with a concentration of 10 mM was added dropwise into 1 mL of the gold core solution prepared in step S1, and was uniformly mixed and reacted for 1 h. After centrifugation (7500 rpm, 10 min), the precipitate was redispersed in 1 mL of ultrapure water to obtain 1 mL of gold core solution with surface-modified Raman active dye, which was denoted as AuNFs@4-EBZN.

[0093] S3, the preparation method of the SERS nanolabel was as follows:

[0094] 100 μL of Aspergillus niger specific aptamer solution (7.5 μM, solvent: ultrapure water) was incubated with 1 mL of the gold core solution prepared in step S2 in a constant-temperature shaker (180 rpm, 28°C) for 15 min. After centrifugation (1500 rpm, 20 min), the precipitate was redispersed in 1 mL of ultrapure water to obtain 1 mL of gold core solution with surface-modified Raman active dye and Aspergillus niger specific aptamer, i.e., SERS nanolabel, which was denoted as AuNFs@4-EBZN-Apt and was stored at 4°C.

[0095] The nucleotide sequence (SEQ ID NO. 1) of the Aspergillus niger specific aptamer was as follows:

[0096] 5'-SH-C6-CGTTTGGGCGGTATGAGTTCGGGGGTATACCGCAG-3'

[0097] Example 2, Characterization of SERS Nanotags

[0098] 1. Transmission electron microscopy (TEM) characterization

[0099] The solutions of AuNPs and AuNFs prepared by the method of Example 1 were respectively subjected to TEM detection, and the results are shown in Figure 3 and Figure 4 The particle size distribution is shown in Figure 5 and Figure 6 AuNPs exhibited uniform spherical morphology, and the particle size was 15-30 nm; AuNFs had dendritic protruding structures on the surface, and the particle size was 50-100 nm.

[0100] 2. Ultraviolet-visible absorption spectrum

[0101] The solutions of AuNPs, AuNFs, AuNFs@4-EBZN and AuNFs@4-EBZN-Apt prepared by the method of Example 1 were directly subjected to ultraviolet-visible absorption spectrum detection at 400-800 nm using an ultraviolet-visible absorption spectrometer (UV-1800, Shimadzu, Japan), and the results are shown in Figure 7 The local surface plasmon resonance (LSPR) peaks of AuNPs and AuNFs were respectively located at 521 nm (corresponding to wine red) and 655 nm (corresponding to gray blue). At the same time, due to the successful modification of 4-EBZN on the surface of AuNFs, the absorption peak of AuNFs@4-EBZN showed red shift compared with that of AuNFs, and the decrease in absorbance was due to the loss of part of AuNFs in the process of removing excess 4-EBZN by centrifugation. Compared with AuNFs@4-EBZN, the absorption peak of AuNFs@4-EBZN-Apt also showed red shift and the absorbance decreased, which was attributed to the combination of aptamer with AuNFs@4-EBZN through Au-S bond, resulting in red shift of the absorption peak and decrease in absorbance, proving that the Aspergillus niger aptamer had been successfully combined.

[0102] 3. Raman spectrum

[0103] 10 μL of the solutions of AuNPs, AuNFs, AuNFs@4-EBZN and AuNFs@4-EBZN-Apt prepared by the method of Example 1 were directly dropped on a silicon wafer, and after air-drying at room temperature (25°C), Raman spectrum detection was performed using a portable optical fiber probe Raman spectrometer (ZOLIX, Beijing, China, model FI-FO785) with a 785 nm laser (power 500 mW, collection time 2 s), and the results are shown inFigure 8 AuNFs' Raman spectrum has no 4-EBZN characteristic peak, while 4-EBZN characteristic peak appears at 1596 cm -1 after the combination of AuNFs and 4-EBZN, indicating that AuNFs and 4-EBZN are successfully combined. When the aptamer is combined with AuNFs@4-EBZN, the intensity of 4-EBZN characteristic peak decreases due to the additional effect of the aptamer on the surface, verifying the occurrence of the combination event.

[0104] 4. Potential test

[0105] The solutions of AuNFs, AuNFs@4-EBZN and AuNFs@4-EBZN-Apt prepared by the method of Example 1 were directly subjected to zeta potential test by using Zeta potential analysis module (Malvern Zetasizer ZS90) system, and the results are shown in Figure 9 The average zeta potentials of AuNFs, AuNFs@4-EBZN and AuNFs@4-EBZN-Apt are -41.4 mV, -20.7 mV and -41.7 mV, respectively.

[0106] 5. Gold core enhancement factor calculation

[0107] At room temperature (25℃, standing) environment, 100 μL of 4-EBZN ethanol solution with a concentration of 10 -9 mol / L was added dropwise into 1 mL of gold core solution prepared in step S1 of Example 1, and reacted for 1 h. After centrifugation (7500 rpm, 10 min), the precipitate was redispersed in 1 mL of ultrapure water to obtain 1 mL of gold core solution modified with Raman active dye, which was subjected to Raman spectrum detection by the method of step 3, as shown in Figure 10 The Raman spectrum of 4-EBZN ethanol solution with a concentration of 10 -2 mol / L not combined with gold core was directly detected by the method of step 3, and the enhancement factor was calculated according to the formula. The enhancement factor of gold core solution prepared by 4-EBZN modified AuNFs reached 2.37 x 10 8 The "hot spot" formed by the needle-like branches and coupling regions on the surface of AuNFs can significantly enhance the electromagnetic field strength, thereby improving the Raman signal response.

[0108] The calculation formula of the enhancement factor is:

[0109] EF=(I SERS ×C BULK ) / (I BULK ×C SERS )

[0110] Wherein, I SERS is the Raman signal intensity of 4-EBZN modified AuNFs, I -9Gold core solutions prepared by 4-EBZN-modified AuNFs were heated at 1596 cm⁻¹. -1 Raman intensity at I BULK It is 10 -2 mol / L 4-EBZN ethanol solution at 1596 cm⁻¹ -1 Raman intensity at point C BULK The molecular concentration of 4-EBZN in the gold core solution prepared by 4-EBZN modification with AuNFs is 10. -9 mol / L; C SERS The molecular concentration of 4-EBZN in the 4-EBZN ethanol solution is 10. -2 mol / L.

[0111] 6. XPS Spectrum

[0112] The solutions of AuNFs and AuNFs@4-EBZN prepared by the method in Example 1, as well as the 4-EBZN ethanol solution, were lyophilized into powders. The XPS spectra were then detected using X-ray photoelectron spectroscopy (Kratos AXIS Ultra DLD, Shimadzu, Japan), and the results are shown in Figure 11. It was observed that AuNFs mainly contain gold and silver. The XPS spectrum of AuNFs@4-EBZN indicated the presence of gold, silver, carbon, and nitrogen. The C 1s orbital spectrum, Au 4f orbital spectrum, and N 1s orbital spectrum of AuNFs@4-EBZN are shown below. Figure 12-14 As shown, the deconvolution peaks at 83.48 eV and 87.15 eV correspond to Au 4f7 / 2 and Au 4f5 / 2, respectively. The deconvolution peak at 399.12 eV corresponds to a different CN feature peak. The appearance of the Au-C feature peak (284.15 eV) can be attributed to the successful coupling of 4-EBZN with AuNFs, and the deconvolution peak at 285.67 eV corresponds to the C≡C feature peak.

[0113] 7. EDS Layered Diagram

[0114] Solutions of AuNFs@4-EBZN and AuNFs@4-EBZN-Apt prepared directly using the method in Example 1 were analyzed using an energy-dispersive X-ray spectroscopy (EDS mapping, accelerating voltage 200 kV). The results are shown in [Figure 1]. Figure 15 and Figure 16 EDS layered images of AuNFs@4-EBZN and AuNFs@4-EBZN-Apt show that C and N elements are distributed relatively abundantly and uniformly on the AuNFs surface, indicating that 4-EBZN has been successfully modified onto the surface of gold nanoflowers. S and P elements are concentrated on the surface of gold nanoflowers, indicating that the aptamer has been successfully modified onto the surface of gold nanoflowers.

[0115] Example 3, Optimization of synthesis parameters of SERS nanotags

[0116] 1. Optimization of 4-EBZN concentration

[0117] The concentration of 4-EBZN in step S2 of Example 1 was changed to 0.5, 1, 5, 10, 15 mM respectively, and other operations were the same, SERS nanotags were prepared respectively, and Raman spectrum detection was carried out by the method of Example 2, and the results are shown in Figure 17 , and the relationship between the characteristic peak intensity at 1596 cm -1 and the concentration of 4-EBZN is shown in Figure 18 . The results show that adding 100 μL of 10 mM 4-EBZN solution (final concentration 2 mM) to 1 mL of AuNFs solution can ensure the best balance between molecular coverage and signal stability.

[0118] 2. Optimization of reaction time of 4-EBZN with gold core

[0119] The reaction time of 4-EBZN with gold core in step S2 of Example 1 was changed to 0, 15, 30, 60, 120 min respectively, and other operations were the same, SERS nanotags were prepared respectively, and Raman spectrum detection was carried out by the method of Example 2, and the results are shown in Figure 19 , and the relationship between the characteristic peak intensity at 1596 cm -1 and the reaction time is shown in Figure 20 . The results show that 60 min is the best reaction time of 4-EBZN with gold core.

[0120] 3. Optimization of aptamer concentration

[0121] The aptamer concentration in step S3 of Example 1 was changed to 2.5, 5, 7.5, 10, 15 mM respectively, and other operations were the same, SERS nanotags were prepared respectively, and Raman spectrum detection was carried out by the method of Example 2, and the results are shown in Figure 21 , and the relationship between the characteristic peak intensity at 1596 cm -1 and the aptamer concentration is shown in Figure 22 . The results show that 7.5 μM is the best aptamer concentration.

[0122] 4. Optimization of reaction time of aptamer with gold core

[0123] The reaction time of aptamer with gold core in step S3 of Example 1 was changed to 0, 15, 30, 60, 120 min respectively, and other operations were the same, SERS nanotags were prepared respectively, and Raman spectrum detection was carried out by the method of Example 2, and the results are shown in Figure 23 , and the relationship between the characteristic peak intensity at 1596 cm -1The relationship between the intensity of the characteristic peak and the reaction time between the aptamer and the gold nucleus is shown in the figure. Figure 24 The results showed that the optimal reaction time between the aptamer and the gold nucleus was 15 min.

[0124] 5. Optimization of incubation time between SERS nanotags and Aspergillus niger solution

[0125] The incubation time of *Aspergillus niger* with AuNFs@4-EBZN-Apt also significantly affected the SERS signal. 250 μL of SERS nanotags prepared by the method in Example 1 were mixed with 50 μL of *Aspergillus niger* ATCC 16404 bacterial culture prepared by the method in Example 5 (1.0 × 10⁻⁶). 9 The samples (CFU / mL) were incubated at 28°C for 0, 15, 30, 60, and 120 min, respectively, and Raman spectroscopy was performed using the method described in Example 2. The results are shown in [Figure 1]. Figure 25 As shown, 1596 cm -1 The relationship between the characteristic peak intensity and incubation time is shown in the figure. Figure 26 The results showed that the optimal incubation time between the SERS nanotags and Aspergillus niger solution was 45 min.

[0126] Example 4: Detection method of Aspergillus niger detection kit based on SERS

[0127] like Figure 2 and Figure 27 As shown, the detection method of the SERS-based Aspergillus niger detection kit includes the following steps:

[0128] (1) Mix 50 μL of the bacterial culture to be tested with 250 μL of the SERS nanotag prepared by the method in Example 1 in a 1.5 mL centrifuge tube by vortexing and incubating at 28 °C and 180 rpm for 45 min to complete the biobinding reaction;

[0129] (2) Take 10 μL of the reaction solution and drop it onto the surface of the silicon wafer, then dry it at room temperature;

[0130] (3) SERS signal acquisition was performed using the portable fiber optic probe Raman spectrometer described in Example 2. The negative logarithm (-logC, X-axis) of the Aspergillus niger concentration and the Raman reactive dye at 1596 cm⁻¹ were established according to Example 5. -1 The semi-logarithmic calibration curve of the characteristic peak intensity (Y-axis) at ( Figure 29 The concentration of Aspergillus niger in the test bacterial solution was obtained by linear regression analysis.

[0131] Example 5: SERS Nanotag Sensitivity Assessment

[0132] 50 μL of different concentrations (10 0 10 1 102 10 3 10 4 10 5 A CFU / mL Aspergillus niger ATCC 16404 bacterial suspension was mixed with 250 μL of SERS nanotags prepared by the method in Example 1. The reaction and detection were performed using the method in Example 4. The results are shown in […]. Figure 28 As shown, 1596 cm -1 The negative logarithmic relationship between Raman intensity and Aspergillus niger concentration at a given location. Figure 29 As shown. Figure 28-29 It can be seen that 4-EBZN is at 1596 cm⁻¹ -1 The characteristic peak intensity at the location showed a good linear relationship with the logarithm of Aspergillus niger concentration, with linear correlation coefficients R² all greater than 0.99.

[0133] Preparation method of Aspergillus niger ATCC 16404 bacterial suspension: Pick a small amount of Aspergillus niger ATCC 16404 spores from a -80 ℃ environment and inoculate them in the center of a fresh solid PDA medium plate. Incubate at 28 ℃ for 5-7 days until a black spore layer forms. Scrape off 0th generation spores with an inoculation loop and draw a "Z" pattern on the surface of three petri dishes. Avoid picking the bottom layer hyphae; focus on spores. Incubate at 28 ℃ in the dark for 5 days, stopping when the slant is covered with black spores. Take 1st generation spores and prepare a spore suspension with sterile physiological saline. Inoculate 100 μL of the spore suspension onto a solid PDA medium plate and spread evenly using a disposable spreader. Incubate at 28 ℃ in the dark for 2-3 days, stopping when the plate is covered with black spores. Take 2nd generation spores and prepare a spore suspension with sterile physiological saline. 100 μL of second-generation spore suspension was inoculated onto a solid PDA medium plate, spread evenly using a disposable spreader, and incubated at 28°C in the dark for 2-3 days until the plate was covered with black spores. Third-generation spores were collected and a spore suspension was prepared using sterile physiological saline. The spore suspension was centrifuged at 8000 r / min for 5 min, the supernatant was removed, and the spores were redispersed in 1 mL of sterile physiological saline. The suspension was serially diluted 10-fold, and the total colony count at the appropriate concentration gradient was determined using the traditional plate count method to determine the original colony count. Three parallel experiments were performed. The suspension was then adjusted to 1.0 × 10⁻⁶. 9 CFU / mL.

[0134] Preparation of solid PDA culture medium: Weigh 47.0g of commercially available PDA culture medium, heat and dissolve it in 1000mL of purified water, and autoclave at 121℃ for 20 minutes.

[0135] Example 6: Evaluation of the selectivity and specificity of SERS nanotags

[0136] Preparation of E. coli 0157:H7 bacterial solution: E. coli 0157:H7 strain ATCC 35150 was inoculated into liquid broth medium and cultured in an incubator with constant temperature oscillation at 37°C with oxygen for 24 h at 150 rpm. The collected bacteria were centrifuged at 8000 rpm for 5 min at 4°C, washed with sterile normal saline for 3 times, and then the bacterial precipitate was resuspended into sterile normal saline. The bacterial suspension was serially diluted with 10-fold concentration gradient, and the total number of colonies at the appropriate concentration gradient was determined by traditional plate counting method to determine the number of colonies of the original bacterial solution, and three parallel experiments were performed. The bacterial suspension was adjusted to 1.0 x 10 9 CFU / mL.

[0137] Preparation of liquid nutrient broth medium: 18.0 g of commercially available nutrient broth medium was weighed, heated and dissolved in 1000 mL of purified water, and autoclaved at 121°C for 20 min.

[0138] Preparation of solid nutrient broth medium: 28.5 g of commercially available nutrient broth agar medium was weighed, heated and dissolved in 1000 mL of purified water, and autoclaved at 121°C for 20 min.

[0139] Using the same preparation method of E. coli 0157:H7 bacterial solution, the bacterial suspensions of Salmonella enteritidis strain CMCC (B) 50335, Pseudomonas aeruginosa ATCC 10145, and Staphylococcus aureus strain ATCC 6538 were prepared, and the bacterial concentrations were all 1.0 x 10 9 CFU / mL.

[0140] The Aspergillus niger (ATCC 16404) bacterial solution was prepared by the same method as in Example 5, and the bacterial concentration was 1.0 x 10 9 CFU / mL.

[0141] Preparation of MIX1 mixed bacterial solution: 200 μL of each of the above E. coli 0157:H7, Salmonella enteritidis CMCC (B) 50335, Pseudomonas aeruginosa ATCC 10145, and Staphylococcus aureus ATCC 6538 bacterial solutions were mixed to obtain MIX1 mixed bacterial solution.

[0142] Preparation of MIX2 mixed bacterial solution: 200 μL of each of the above E. coli 0157:H7, Salmonella enteritidis CMCC (B) 50335, Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 6538, and Aspergillus niger ATCC 16404 bacterial solutions were mixed to obtain MIX2 mixed bacterial solution.

[0143] The above bacterial solutions were detected by the method of Example 4, and the Raman signal intensity at 1596 cm -1 is shown in Table 1. Figure 30 .

[0144] Figure 30 The results show that compared with E. coli O157:H7, Salmonella enteritidis, Pseudomonas aeruginosa, Staphylococcus aureus and MIX1 (a mixture of the other four bacteria without Aspergillus niger), Aspergillus niger and MIX2 (a mixture of five bacteria) all show obvious Raman signals at 1596 cm -1 This phenomenon highlights the excellent specificity and selectivity of the SERS-based Aspergillus niger detection kit of the present application in detecting Aspergillus niger in a complex pathogenic bacteria mixture.

[0145] Example 7, evaluation of reproducibility, uniformity and stability of SERS nanotags

[0146] 1. Reproducibility

[0147] 50 μL of Aspergillus niger ATCC 16404 bacterial solution (1.0 x 10 9 CFU / mL) prepared by the method of Example 5 was vortexed with 250 μL of SERS nanotags prepared by the method of Example 1 in a 1.5 mL centrifuge tube, and incubated at 28°C, 180 rpm for 45 min to complete the biological binding reaction.

[0148] (2) 10 μL of the reaction solution was added dropwise onto the surface of a silicon wafer and dried at room temperature; the surface of the silicon wafer was signal-collected using the portable Raman spectrometer described in Example 2, and 10 repeated samplings were performed at the same point, and the results are shown in Figure 31 The relative standard deviation (RSD) of the intensity of the 1596 cm⁻¹ characteristic peak was calculated, and the results are shown in Figure 32 , and the performance of the probe was evaluated comprehensively.

[0149] The reproducibility test showed that the relative standard deviation (RSD) values of the intensities of the 1179, 1596 and 2045 cm -1 characteristic peaks were 3.2%, 3.8% and 3.5%, respectively.

[0150] 2. Uniformity

[0151] The 10 repeated samplings at the same point in Step 1 above were replaced by 10 samplings at different points, and the other operations were the same, and the results are shown in Figure 33 and Figure 34 In the uniformity test, the 10 independent site detections showed that the RSD values of each peak were all less than 4.3%, confirming that the substrate surface distribution was well homogenized.

[0152] 3. Stability

[0153] The reaction solution in (1) of Step 1 above was stored at 4°C for 30 days, respectively, and sampled every day for detection by the portable Raman spectrometer described in Example 2 and calculation of RSD, with the results shown in Table 1 below. Figure 35 and Figure 36 .

[0154] The stability experiment showed that the RSD of the peak intensity of 1596 cm -1 was only 2.16% after storage at 4°C for 30 days.

[0155] The above results provide key methodological support for the field application of the SERS-based Aspergillus detection kit of the present application, and the excellent performance thereof is due to the stable signal output mechanism under the synergistic action of the aptamer-nanoprobe.

[0156] Example 8, Detection of Actual Sample 1

[0157] 1. Spiked recovery rate

[0158] Commercially available Pu'er tea was selected as the matrix to construct an Aspergillus contamination model. The Pu'er tea was ground and passed through an 80-mesh sieve, 2 g of Pu'er tea powder was added to a centrifuge tube, 10 mL of physiological saline was added to soak at room temperature for 10 min, and the solid impurities were removed by centrifugation (3500 rpm, 10 min). The supernatant was sterilized by high-pressure steam, and then diluted with 9 times the volume of physiological saline. 10 mL of the diluted supernatant was added to the Aspergillus liquid prepared by the method of Example 5 to make the final concentration 2.1 x 10 1 ~2.1 x 10 4 CFU / mL, as the test bacterial liquid, and the Aspergillus concentration was detected by the method of Example 4. The traditional plate counting method was used for comparison.

[0159] As shown in Table 1, the spiked recovery rate of Aspergillus in the Pu'er tea matrix was 96.10%~104.90%, as shown in Figure 37, the detected Aspergillus concentration in the Pu'er tea was highly correlated with the results of the plate counting method (r 普洱茶 = 0.9999), and no statistically significant difference was observed at all test concentrations (p 普洱茶 = 0.7694).

[0160] It is confirmed that the SERS-based Aspergillus detection kit of the present application is adaptable to complex food matrices. These results show that the method is feasible for the detection of Aspergillus in practical applications.

[0161] Table 1

[0162]

[0163] 2. Sensitivity

[0164] To further evaluate the sensitivity of SERS nanotags in real samples, the final concentration of Aspergillus niger in step 1 above was changed to 2.1 × 10⁻⁶. 0 ~2.1×10 5 CFU / mL, with all other procedures remaining the same, Raman spectra were detected using the method described in Example 4. Results are shown below. Figure 40 As shown. 4-EBZN at 1596 cm -1 The intensity of the characteristic peak at the concentration increased in a dose-dependent manner with increasing Aspergillus niger concentration, and showed a good linear relationship with the logarithm of concentration (lg C). Figure 41 In the Pu-erh tea matrix, the linear equation is y = 1069.9x + 4251.2 (R² = 0.9942), and the LOD is 1.85 CFU / mL.

[0165] Example 9, Detection of actual sample 2

[0166] 1. Spike recovery rate

[0167] A commercially available soy sauce was selected as the matrix to construct a *Aspergillus niger* contamination model. A 10 mL soy sauce sample was centrifuged (3500 rpm, 10 min) to remove solid impurities. After autoclaving, the supernatant was diluted with 9 times its volume of physiological saline. 10 mL of the diluted supernatant was then added to the *Aspergillus niger* culture prepared according to the method in Example 5, bringing the final concentration to 1.6 × 10⁻⁶. 1 ~1.6×10 4 The concentration of Aspergillus niger was determined using the method described in Example 4, with CFU / mL as the test bacterial solution. Comparison was performed using the conventional plate count method.

[0168] As shown in Table 1, the recoveries of Aspergillus niger in the soy sauce matrix ranged from 95.13% to 104.44%. As shown in Figure 38, the concentration of Aspergillus niger in the soy sauce was highly correlated with the results of the plate count method (r... 酱油 =0.9991), and no statistically significant differences were observed at any of the tested concentrations (p=0.9991). 酱油 = 0.9905). This confirms the adaptability of SERS nanotags to complex food matrices. These results demonstrate the feasibility of this method for detecting Aspergillus niger in practical applications.

[0169] 2. Sensitivity

[0170] To further evaluate the sensitivity of SERS nanotags in real samples, the final concentration of Aspergillus niger in step 1 above was changed to 1.6 × 10⁻⁶. 0 ~1.6×10 5 CFU / mL, with all other procedures remaining the same, Raman spectra were detected using the method described in Example 4. Results are shown below. Figure 42 As shown. Experimental results show that 4-EBZN at 1596 cm⁻¹-1 The intensity of the characteristic peak at the concentration increased in a dose-dependent manner with increasing Aspergillus niger concentration, and showed a good linear relationship with the logarithm of concentration (lg C). Figure 43 In the soy sauce matrix, the linear equation is y = 1154.4x + 2690.8 (R² = 0.9914), and the LOD is 2.56 CFU / mL.

[0171] Example 10, Detection of actual sample 3

[0172] 1. Spike recovery rate

[0173] A *Aspergillus niger* contamination model was constructed using commercially available drinking water as the substrate. 10 mL of drinking water was centrifuged (3500 rpm, 10 min) to remove solid impurities. After autoclaving, 10 mL of the supernatant was added to the *Aspergillus niger* culture prepared according to the method in Example 5, bringing the final concentration to 2.9 × 10⁻⁶. 1 ~2.9×10 4 The concentration of Aspergillus niger was determined using the method described in Example 4, with CFU / mL as the test bacterial solution. Comparison was performed using the conventional plate count method.

[0174] As shown in Table 1, the recovery rate of Aspergillus niger in drinking water substrates reached 95.66%–100.24%. Figure 39 As shown, the bacterial concentration detected in drinking water is highly correlated with the results of plate counting (r 饮用水 =0.9999), and no statistically significant differences were observed at any of the tested concentrations (p=0.9999). 饮用水 =0.8658). This confirms the adaptability of SERS nanotags to complex food matrices. These results demonstrate the feasibility of this method for detecting Aspergillus niger in practical applications.

[0175] 2. Sensitivity

[0176] To further evaluate the sensitivity of SERS nanotags in real samples, the final concentration of Aspergillus niger in step 1 above was changed to 2.9 × 10⁻⁶. 0 ~2.9×10 5 CFU / mL, with all other procedures remaining the same, Raman spectra were detected using the method described in Example 4. Results are shown below. Figure 44 As shown. Experimental results show that 4-EBZN at 1596 cm⁻¹ -1 The intensity of the characteristic peak at the concentration increased in a dose-dependent manner with increasing Aspergillus niger concentration, and showed a good linear relationship with the logarithm of concentration (lg C). Figure 45 In drinking water matrix, the linear equation is y = 834.03x + 5797.1 (R²). 2 = 0.997), LOD is 1.47 CFU / mL.

[0177] The difference of sensitivity of three systems is caused by matrix inhibition effect caused by complex components in sample, which verifies the strong compatibility of SERS nanolabel to food matrix.

[0178] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any partial change on the basis of the formula and process should be within the protection scope of the present application.

Claims

1. A SERS-based Aspergillus detection kit, characterized by, The kit comprises a SERS nanolabel, the structure of the SERS nanolabel is biomimetic gold nanoflower as a gold core, and a Raman active dye and an Aspergillus niger specific aptamer are modified on the surface of the gold core; the nucleotide sequence of the Aspergillus niger specific aptamer is shown in SEQ ID NO. 1, and the Raman active dye is 4-ethynylaniline.

2. The SERS-based Aspergillus niger detection kit according to claim 1, wherein, The particle size of the gold core is 50-100 nm.

3. The SERS-based Aspergillus niger detection kit according to claim 1, wherein, The preparation method of the gold core is as follows: (1) an aqueous chloroauric acid solution is added to a three-necked round-bottom flask, and heated to boiling; (2) under vigorous stirring, an aqueous trisodium citrate solution is added to the solution in step (1) at a constant speed, and heated to boiling under continuous stirring for 15-20 min, and phase equilibrium is completed under constant temperature, and the solution is cooled to room temperature to form a colloid, and a stable dispersed gold seed solution is obtained; (3) 0.1-0.5 mM of an aqueous chloroauric acid solution is taken in a three-necked round-bottom flask, and under stirring, hydrochloric acid, the gold seed solution, an aqueous silver nitrate solution and an aqueous L-ascorbic acid solution are sequentially added, and the solution is reacted at 20-30 DEG C and 100-150 rpm until the solution color turns to gray blue; then an aqueous sodium dodecyl sulfate solution is added for surface modification to ensure the colloidal stability of the nanoparticles, and a gold core is obtained.

4. The SERS-based Aspergillus niger detection kit of claim 3, wherein, In step (1), the concentration of the aqueous chloroauric acid solution is 0.5-5 mM, and in step (2), the concentration of the aqueous trisodium citrate solution is 5 g / L-15 g / L, and the volume ratio of the aqueous trisodium citrate solution to the aqueous chloroauric acid solution is 1:20-5:20; in step (3), the concentration of the hydrochloric acid is 0.5-5 M, the concentration of the aqueous silver nitrate solution is 1-5 mM, the concentration of the aqueous L-ascorbic acid solution is 50-200 mM, and the concentration of the aqueous sodium dodecyl sulfate solution is 5 g / L-15 g / L; the volume ratio of the aqueous chloroauric acid solution to the hydrochloric acid, the gold seed solution, the aqueous silver nitrate solution, the aqueous L-ascorbic acid solution and the aqueous sodium dodecyl sulfate solution is 100:0.05-0.5:0.5-5:0.5-5:0.1-1:0.1-1.

5. The SERS-based A. niger detection kit of claim 1, wherein, The method for modifying the Raman active dye on the surface of the gold core is as follows: under room temperature, an ethanol solution of the Raman active dye is added dropwise to the gold core, and the reaction is carried out at 20-30 DEG C for 0.1-2 h, and after centrifugation, the precipitate is dispersed in ultrapure water to obtain the gold core with the surface modified Raman active dye.

6. The SERS-based A. niger detection kit of claim 5, wherein, The concentration of the ethanol solution of the Raman active dye is 0.5-15 mM, and the volume ratio of the ethanol solution of the Raman active dye to the gold core is 1:5-20; and the volume ratio of the ethanol solution of the Raman active dye to ultrapure water is 1:5-20.

7. The SERS-based A. niger detection kit of claim 1, wherein, The preparation method of the SERS nanolabel is as follows: The ultrapure aqueous solution of the Aspergillus niger specific aptamer and the gold core with the surface modified Raman active dye are incubated in a constant-temperature shaker at 28 DEG C and 180 rpm for 0.1-2 h, and then separated by centrifugation, and the precipitate is suspended in ultrapure water to obtain the SERS nanolabel.

8. The SERS-based Aspergillus niger detection kit according to claim 7, wherein, The concentration of the ultrapure aqueous solution of the Aspergillus niger specific aptamer is 2.5-15 muM; and the volume ratio of the ultrapure aqueous solution of the Aspergillus niger specific aptamer to the gold core with the surface modified Raman active dye is 1:1-10.

9. A detection method of the SERS-based Aspergillus detection kit according to claim 1, characterized by, The method comprises the following steps: 1) The test bacteria solution is mixed with the SERS nanolabel by vortexing, and the biological binding reaction is completed by incubating at 28℃ and 180 rpm for 0.1-2h; 2) The reaction solution of step 1) is dropped on the surface of a silicon wafer and dried at room temperature; 3) The SERS signal is collected by a portable Raman spectrometer at an excitation wavelength of 785 nm and a laser power of 200 mW; the concentration of Aspergillus niger in the test bacteria solution is obtained by linear regression analysis based on the semi-log calibration curve of the negative logarithmic value of the Aspergillus niger solution concentration and the characteristic peak intensity of the Raman active dye.

10. The method of claim 9, wherein, The volume ratio of the test bacteria solution to the SERS nanolabel in step 1) is 1:1-10.

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