SERS (Surface Enhanced Raman Scattering) aptamer sensor based on double-layer core-satellite magnetic gold nanostructure and method for detecting biogenic amine in food by using SERS aptamer sensor

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) aptamer sensor based on a double-layer core-satellite magnetic gold nanostructure and a method for detecting biogenic amine in food by using the SERS aptamer sensor. The Fe3O4 (at) Au MNPs nanoparticles are combined with a nucleic acid aptamer, and a specific magnetic capture probe is obtained; the method comprises the following steps: mixing gold nanoparticle solutions with different particle sizes with 4-MBN, incubating to obtain a'biological silence zone 'SERS tag, and combining the'biological silence zone' SERS tag with pretreated sulfydryl modified c-DNA to obtain a specific SERS signal probe; and oscillating and incubating the specific SERS signal probe solution and the specific magnetic capture probe solution to obtain the SERS aptamer sensor based on the double-layer core-satellite magnetic gold nanostructure. According to the method, a'biological silence zone 'SERS label is combined with a magnetic induction technology, so that the problems of poor anti-interference performance and poor accuracy during detection of trace components in a complex matrix by SERS are solved, and high-efficiency detection of tryptamine in biogenic amine is realized.

Description

Technical Field

[0001] This invention relates to the field of food detection technology, specifically to a SERS aptamer sensor based on a double-core-satellite magnetic gold nanostructure and a method for detecting biogenic amines, especially tryptophan, in food. Background Technology

[0002] Biogenic amines are low-molecular-weight nitrogen-containing organic compounds, mainly produced through enzymatic decarboxylation of free tryptophan or proteins catalyzed by microbial enzymes. Tryptophan is widely found in protein-rich foods and fermented products, such as seafood, meat, dairy products, and vinegar. Consuming foods with high concentrations of tryptophan can cause migraines, rashes, nausea, and high blood pressure. Current techniques for detecting tryptophan mainly include high-performance liquid chromatography (HPLC), optical sensor methods, adsorption-stripping voltammetry, HPLC-tandem mass spectrometry, and fluorescence sensors. However, these methods still suffer from drawbacks such as complexity, high cost, cumbersome operation, and long detection times. Therefore, developing a rapid, sensitive, and accurate method for detecting biogenic amines, especially tryptophan, in food is crucial. Surface-enhanced Raman scattering (SERS) technology has attracted much attention due to its excellent "fingerprint" molecular pattern recognition capability (the spectrum directly reflects the vibrational and rotational signals of the molecular structure), high sensitivity (enabling single-molecule detection), rapid response (signal response can be obtained within seconds or tens of seconds), and ease of operation. The core of the SERS enhancement effect lies in the design of the enhancement substrate. SERS hotspots are generated through localized surface plasmon resonance of metallic nanostructures, which can significantly enhance the Raman signal intensity of analytes adsorbed on the substrate surface. Currently, commonly used SERS substrate materials mainly include spherical noble metal nanoparticles, such as gold nanoparticles (AuNPs), silver nanoparticles (Ag NPs), and Au@Ag core-shell nanoparticles. In addition, nanoparticles with nano-interstices, metal tips, and rough surfaces, such as nanostars, nanotubes, and nanoflowers, as well as controllable aggregates composed of various plasmon nanoparticles (including dimers, polymers, and core-satellite nanostructures), can all generate higher SERS activity.

[0003] In recent years, plasmonic nanoparticle components have been extensively studied in the field of SERS detection due to their powerful and tunable optical properties. Among them, Au-NPs plasmonic nanoparticles based on DNA-driven self-assembly technology have been developed to construct high-yield dimers, trimers, and other oligomers with regular geometries, and have been successfully used to detect several important biomarkers and small molecule biomolecules. This plasmonic nanoparticle assembly not only significantly improves optical performance but also exhibits high detection sensitivity and excellent resistance to environmental interference, greatly enhancing detection sensitivity and stability. Notably, the DNA-driven magnetically induced core-satellite self-assembly strategy has significant advantages in constructing plasmonic nanoparticles, providing a promising technical method for modulating changes in SERS signals. These core-satellite nanostructures typically consist of individual Au or Ag nanoparticles (NPs) distributed on the surface of a magnetic core substrate, thereby generating a controlled number of SERS hotspots. SERS intensity is proportional to the number of hotspots, and the number of hotspots in the core-satellite nanostructure depends on the number of satellites attached to the core and the composition of the structure. Therefore, using multilayer nanostructures as satellites to further increase the number of hotspots may be an effective method to improve SERS enhancement. To date, the most commonly used magnetic core nanostructure is Fe3O4@AuNP, which not only provides rapid signal separation and enrichment capabilities but also offers DNA binding sites, exhibiting excellent SERS performance. However, for detecting small molecules (such as tryptamine) in complex substrates like food, the characteristic Raman peaks of the target analyte easily overlap due to interference from other substances, increasing the difficulty of SERS analysis and quantification. Emerging SERS tagging technology cleverly combines the SERS effect of plasmonic nanoparticles with the unique fingerprint signal of Raman reporter molecules, achieving quantitative detection by detecting the specific SERS signal of the reporter molecule in the presence of the target. To a certain extent, this significantly reduces the influence of coexisting interfering substances on Raman detection. It is worth noting that Raman reporter molecules in the "fingerprint" region are still partially affected by signal interference caused by overlapping spectral peaks, while "biologically silent" Raman reporter molecules in the 1800–2800 cm⁻¹ region... -1 The region exhibits pure characteristic peaks, virtually unaffected by background signals from biological materials. "Bio-silenced" region SERS detection technology has proven to be an interference-free detection technique. Therefore, combining DNA-driven magnetically induced self-assembly technology and "bio-silenced" SERS tagging technology may be an effective method for achieving highly stable and interference-resistant SERS detection. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a SERS aptamer sensor based on a dual-core-satellite magnetic gold nanostructure and a method for detecting biogenic amines in food. This invention overcomes the problems of low accuracy and weak anti-interference ability of SERS in detecting trace substances under complex substrates by employing a novel dual-core-satellite structure manufacturing strategy, thereby enabling accurate and efficient detection of tryptamines among biogenic amines.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a SERS aptamer sensor based on a bilayer core-satellite magnetic gold nanostructure. The aptamer-modified Fe3O4@Au MNPs magnetic SERS nanomaterial is used as a tryptamine capture probe, and two SERS tags Au NPs with different particle sizes and modified with c-DNA and 4-MBN are used as SERS signal probes for indirect detection of tryptamine, thus constructing a "core-satellite" type magnetically induced SERS sensor. The SERS aptamer sensor based on a bilayer core-satellite magnetic gold nanostructure was prepared by the following method: (1) Preparation of specific Fe3O4 capture probe: Fe3O4@Au MNPs nanoparticles were incubated with 5' amino-terminal modified nucleic acid aptamers at room temperature with shaking to obtain specific Fe3O4 capture probe solution; (2) Preparation of specific SERS signal probes: Two solutions of gold nanoparticles with different particle sizes were mixed and incubated with 4-mercaptobenzonitrile to obtain a “biosilent zone” SERS tag. Then, the tag was incubated with pretreated 5' end thiol-modified c-DNA at room temperature to obtain specific SERS signal probe-1 solution and specific SERS signal probe-2 solution. (3) The specific SERS signal probe-1 solution and specific SERS signal probe-2 solution prepared in step (2) are shaken and incubated with the specific magnetic capture probe solution prepared in step (1) to obtain a SERS aptamer sensor based on a double-layer core-satellite magnetic gold nanostructure.

[0006] Preferably, in step (1), the Fe3O4@Au MNPs nanoparticles are prepared by the following method: 1) FeCl3·6H2O was dissolved in ethylene glycol solution, PVP was added, and the mixture was heated and stirred under nitrogen. After adding sodium acetate, the mixture was reacted at high temperature to obtain Fe3O4 nanoparticles. 2) Take another HAuCl4·4H2O solution and sodium citrate solution, add NaBH4 solution, and age at room temperature to obtain gold seed solution; 3) Add PEI aqueous solution to cover the surface of Fe3O4 nanoparticles with PEI, then add gold seed solution and sonicate to obtain Fe3O4-Au MNPs seeds; 4) Fe3O4-Au MNPs seeds were dispersed in HAuCl4 solution, and then reacted with hydroxylamine hydrochloride solution and PVP to obtain Fe3O4@Au MNPs.

[0007] Preferably, in step (1), the 5' amino-modified nucleic acid aptamer is an amino-modified tryptophan with the sequence: 5'-SH-(CH2)6-AGCAGCACAGAGGTCAGATGTATGAACGATTTACTCGTA-CAGACGACACTTATCATTTGCCCTATGCGTGCTACCGTGAA-3' (as shown in SEQ ID No. 1).

[0008] Preferably, in step (2), the two gold nanoparticle solutions with different particle sizes are a gold nanoparticle solution with a particle size of 25 nm and a gold nanoparticle solution with a particle size of 60 nm.

[0009] Preferably, the solution of gold nanoparticles with a particle size of 25 nm is prepared by the following method: Mix ultrapure water and 0.4 wt% tetrachloroauric acid solution, stir at a constant speed, heat to boiling, then add 1 wt% trisodium citrate solution, continue heating and stirring until the solution turns red, stop heating and continue stirring for 30 min to obtain the solution; The solution of gold nanoparticles with a particle size of 60 nm was prepared by the following method: After heating a 0.01wt% HAuCl4 solution to 120℃, add a 1% trisodium citrate solution. After heating and observing the color change from colorless to red, stop heating and continue stirring for 20 min to obtain the final product.

[0010] Preferably, in step (2), the sequence of the thiol-modified c-DNA is: C1: 5'-SH-(CH2)6-TTTTTTTTTTAAATCGTTCATACATCTG (as shown in SEQ ID No. 2); C2: 5'-SH-(CH2)6-TTTTTTTTTTTGATAAGTGTCGTCTG (as shown in SEQ ID No. 3).

[0011] Preferably, a solution of gold nanoparticles with a particle size of 25 nm is incubated with thiol-modified c-DNA with sequence C1 to obtain a specific SERS signal probe-1 solution; a solution of gold nanoparticles with a particle size of 60 nm is incubated with thiol-modified c-DNA with sequence C2 to obtain a specific SERS signal probe-2 solution.

[0012] Preferably, in step (3), the volume ratio of the specific Fe3O4 capture probe solution, the SERS signal probe-1 solution, and the SERS signal probe-2 solution is 50:120:120.

[0013] Preferably, in step (3), the temperature of the shaking incubation is 37 ℃ and the time is 120 min.

[0014] A second aspect of the present invention provides the application of a SERS aptamer sensor based on a dual-core-satellite magnetic gold nanostructure in the detection of tryptophan.

[0015] A third aspect of the present invention provides a method for detecting tryptophan in biogenic amines based on a dual-core-satellite magnetic gold nanostructure SERS aptamer sensor, comprising the following steps: (1) A series of tryptophan standard solutions with different concentration gradients were added to the SERS aptamer sensor based on a double-core-satellite magnetic gold nanostructure to obtain a mixture; the mixture was magnetically separated, washed, and then dropped onto a glass slide, and the reading was recorded at 2226 cm⁻¹. -1 Raman intensity at the location; (2) With 2226cm -1 The Raman intensity at a given point is plotted on the ordinate, and the negative logarithm of the tryptamine standard solution concentration is plotted on the abscissa to create a working curve. The tryptamine content in the test solution is then detected based on the working curve.

[0016] Preferably, the method for preparing the test solution is as follows: Sodium chloride was added to the sample to be tested until completely dissolved, followed by the addition of diethyl ether. The mixture was stirred vigorously. The ether layer was transferred, and the aqueous phase was extracted again. After drying the extract with nitrogen, the residue was dissolved in acetonitrile and filtered through a 0.22 μm filter membrane to obtain the solution to be tested.

[0017] Preferably, the detection range of the method is 0.001-100 mg / L; the limit of detection is 0.40 × 10⁻⁶ mg / L. -3 mg / L.

[0018] The beneficial effects of this invention are: (1) This invention employs a two-layer core satellite structure manufacturing strategy, using nucleic acid aptamers to construct the SERS sensor. The SERS tag formed by the two layers of Au nanostructures ensures that the core satellite component exhibits a very robust and tunable SERS signal. Simultaneously, the sensing strategy based on the "bioquiescent" region provides more reliable results, improving the anti-interference capability, accuracy, and sensitivity of tryptamine detection. (Based on 2226 cm⁻¹) -1The Raman intensity at the target analyte concentration was linearly correlated with the concentration of the target analyte, establishing a novel and sensitive detection method. The linear range for detecting tryptophan using the bilayer core-satellite magnetic gold nanostructure SERS aptamer sensor method of this invention was 0.001-100 mg / L, with a limit of detection of 0.40 × 10⁻⁶ mg / L. -3 mg / L is sufficient to meet the needs of trace detection of tryptamine in biogenic amines in actual samples.

[0019] (2) The present invention is based on a dual-core-satellite magnetic gold nanostructure SERS aptamer sensor with good anti-interference, sensitivity and selectivity, and can be widely used for the rapid determination of tryptophan in a variety of food samples. Attached Figure Description

[0020] Figure 1 (a) Raman spectra at different incubation times; (b) Time-optimized working curves; Figure 2 (a) Raman spectra under different incubation ratios; (b) Working curves for ratio optimization; Figure 3 (a) Raman spectra of tryptamine standard solutions of different concentrations; (b) Tryptamine working curve; Figure 4 Selectivity experiment diagram of this sensor; Figure 5 : Reproducibility test diagram of this sensor; Figure 6 : Schematic diagram of the principle of this sensor for detecting tryptamine. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] As introduced in the background section, current detection of tryptamine mainly relies on instrumental methods, but these methods still suffer from drawbacks such as complexity, high cost, cumbersome operation, and long detection time. SERS technology offers high sensitivity, rapid response, and ease of operation, making it suitable for trace detection of various substances. Furthermore, Au-NPs plasma nanodevices combined with DNA assembly technology not only enhance signal intensity but also offer advantages such as high sensitivity and excellent resistance to environmental interference.

[0023] Based on this, the purpose of this invention is to provide a SERS aptamer sensor based on a bilayer core-satellite magnetic gold nanostructure. This invention fabricates a highly stable and interference-free bilayer core-satellite SERS aptamer sensor for detecting low concentrations of tryptophan in food samples. First, aptamer-modified Fe3O4@Au nanoparticles are used as the core structure and tryptophan recognition probe. Then, a DNA cross-linking-based self-assembly strategy is used to fabricate a bilayer satellite structure of Au NPs with high regional controllability to construct the SERS tag. Using two specially designed DNA sequences, a base complementarity strategy based on DNA and aptamers is used to form the core satellite assembly, wherein a 25nm Au NP modified with 4-MBN serves as the outer satellite layer, and a 60nm Au NP modified with 4-MBN serves as the inner satellite layer. The SERS tag formed by the bilayer Au nanostructure ensures that the core satellite assembly exhibits a very robust and tunable SERS signal. When tryptophan is present, it preferentially binds to the aptamer on the recognition probe, causing the bilayer SERS tag carrying 4-MBN to separate. The detection principle is illustrated in the diagram below. Figure 6 As shown, ultra-low concentrations of tryptamine in food can be detected using a strong SERS signal. A sensing strategy based on "bioquiescent" regions can provide more reliable results, thus enabling reliable and accurate detection. Simultaneously, this novel two-layer core satellite structure fabrication strategy provides a new method for constructing advanced, controllable self-assembly superstructures and their applications in rapid detection.

[0024] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0025] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0026] Example 1: Fabrication of a SERS aptamer sensor based on a bilayer core-satellite magnetic gold nanostructure (1) Preparation of specific Fe3O4 capturing probe Preparation of superparamagnetic Fe3O4 nanospheres: 2 mmol of FeCl3·6H2O was dissolved in 20 mL of ethylene glycol and magnetically stirred in a beaker. After 30 min, 2 g of PVP was added to the solution, and the mixture was continuously stirred and heated at 120 °C under nitrogen protection to obtain a transparent solution. After 1 hour, 1.5 g of sodium acetate was added to the solution, and heating was stopped. After further vigorous stirring for 30 min, the obtained homogeneous solution was transferred to a 25 mL stainless steel autoclave lined with Teflon, sealed, and heated at 200 °C. After reacting for 12 hours, the autoclave was cooled to room temperature. The obtained product was washed three times with ethanol and water, and dried under vacuum at 60 °C for 12 h to obtain Fe3O4 nanospheres.

[0027] Preparation of AuNPs: 0.5 mL of 24.3 mM HAuCl4·4H2O solution was added to 50 mL of H2O and stirred vigorously. Then, 1.5 mL of 1 wt% sodium citrate aqueous solution was added. After 1 min, 0.5 mL of 0.075 wt% NaBH4 solution was added. Stirring was continued for 12 h to obtain 3 nm AuNPs.

[0028] Preparation of Fe3O4@Au MNPs seeds: 0.1 g of the prepared Fe3O4 nanospheres were added to 10 mL of a 1 mg / mL PEI aqueous solution and sonicated for 15 min. During this process, PEI gradually covered the surface of the Fe3O4 nanospheres, yielding Fe3O4@PEI MNPs. After washing 5 times, the Fe3O4@PEI MNPs were added to 50 mL of Au NPs and sonicated for 30 min to form Fe3O4@Au MNPs seeds.

[0029] 10 mg of Fe3O4@Au MNPs seeds were dispersed in 50 mL of 0.1 mM HAuCl4 solution. Then, 0.5 mL of 100 mg / mL hydroxylamine hydrochloride solution was rapidly added under strong sonication at 30 °C. After 5 min, 300 mg of PVP was added, and the mixture was sonicated for 15 min. The final product was separated by a magnet and washed three times with deionized water to obtain Fe3O4@Au MNPs.

[0030] Preparation of specific Fe3O4 capture probe: Add 25 μL (0.01 mM) of amino-modified nucleic acid aptamer (sequence shown in SEQ ID No.1), and shake the mixture at 37 °C for 12 hours to obtain specific Fe3O4 capture probe solution.

[0031] (2) Preparation of specific SERS signal probes Synthesis of 60 nm AuNPs: 100 mL of 0.01 wt% HAuCl4 solution was heated to 120 °C, and then 1.0 mL of 1 wt% trisodium citrate solution was added. After observing the color change from colorless to red, heating was stopped and stirring was continued for 20 min. A 60 nm AuNPs solution was obtained.

[0032] Synthesis of 25 nm AuNPs: 48.75 mL of ultrapure water and 1.25 mL of 0.4 wt% HAuCl4 solution were added to a flask. The mixture was stirred at a constant speed and heated to boiling for 7-8 min. Then, 0.8 mL of 1 wt% trisodium citrate solution was quickly added to each of the two flasks. The mixture was heated and stirred continuously. The solution in the flask changed from colorless to blue and then to red. Heating was stopped and stirring was continued for 30 min to obtain a 25 nm AuNPs solution.

[0033] Preparation of SERS signal probes: Take 5 mL of 25 nm AuNPs solution and 5 mL of 60 nm AuNPs solution, respectively, and incubate with 3 μL of 100 mM 4-MBN for 60 min. After centrifugation at 9000 rpm for 10 min, reconstitute with 1 mL of BB (diamino blue). Add 1 μL of 100 mM nucleic acid aptamer / c-DNA solution (C1 added to the 25 nm AuNPs solution, and C2 added to the 60 nm AuNPs solution, prepared by Shanghai Bioengineering Co., Ltd.). Incubate at 37℃ for 12 h, centrifuge at 9000 rpm for 10 min to remove excess nucleic acid aptamers / c-DNA, and reconstitute with 1 mL of BB solution. The 25 nm SERS signal probe solution and the 60 nm SERS signal probe solution are obtained, and are designated as SERS signal probe-1 solution and SERS signal probe-2 solution, respectively.

[0034] (3) Sensor ratio optimization The specific Fe3O4 capture probe solution prepared in step (1), the SERS signal probe-1 solution prepared in step (2), and the SERS signal probe-2 solution were added to a 5 mL centrifuge tube in volume ratios of 50:20:20; 50:40:40; 50:80:80; 50:100:100; 50:120:120; 50:150:150 (unit: μL). The mixture was incubated at 37°C for 12 h. After incubation, the mixture was magnetically separated and washed to obtain the sensor. The sensor was dispersed in 1 mL of BB buffer and then dropped onto a glass slide. A 2226 cm⁻¹ reading was recorded. -1 Raman intensity at the location.

[0035] Figure 2 It shows 2226 cm -1Raman signal intensity. It can be seen that the Raman signal intensity reaches a high value when the ratio is 50:120:120, and the Raman signal intensity is similar to the previous ratio when the ratio is 50:150:150. Therefore, the ratio of 50:120:120 was chosen for subsequent experiments.

[0036] (4) Optimization of sensor incubation time Using the optimized ratio described above, the specific Fe3O4 capture probe prepared in step (1), and the SERS signal probe-1 and SERS signal probe-2 prepared in step (2) were added to a 5 mL centrifuge tube and incubated at 37 °C for 30 min, 60 min, 90 min, 120 min, and 150 min, respectively. After incubation, the mixture was magnetically separated, washed, and then dropped onto a glass slide. The reading was recorded at 2226 cm⁻¹. -1 Raman intensity at the location.

[0037] Figure 1 It shows 2226 cm -1 Raman signal intensity was measured. It can be seen that the Raman signal intensity reaches a high value when the incubation time is 120 min, and the Raman signal intensity is similar to that at 120 min when the incubation time is 150 min. Therefore, an incubation time of 120 min was chosen for subsequent experiments.

[0038] The specific Fe3O4 capture probe prepared in step (1), the SERS signal probe-1 and SERS signal probe-2 prepared in step (2) were mixed at a ratio of 50:120:120 and incubated at 37 °C for 120 min to obtain a sensor for subsequent experiments.

[0039] Example 2: Establishment of a method for detecting tryptophan based on a dual-core-satellite magnetic gold nanostructure SERS aptamer sensor (1) A series of tryptamine standard solutions with varying concentrations (0.001 mg / L, 0.01 mg / L, 0.1 mg / L, 1 mg / L, 10 mg / L, and 100 mg / L) were added to the sensor prepared in Example 1 to obtain a mixture; the mixture was magnetically separated, washed, and then dropped onto a glass slide, and the reading was recorded at 2226 cm⁻¹. -1 Raman intensity at the location; (2) With 2226cm -1 The Raman intensity at a given point is plotted on the ordinate, and the negative logarithm of the tryptamine standard solution concentration is plotted on the abscissa to create a working curve. The tryptamine content in the test solution is then detected based on the working curve.

[0040] The working curve is: y = -3313.3lg(c) + 21977 (R) 2 =0.9996).

[0041] Depend on Figure 3 It can be seen that the linear range for the detection of tryptamine using this method is 0.001-100 mg / L, and the limit of detection is 0.40 × 10⁻⁶ mg / L. - 3 mg / L.

[0042] Example 3: Repeatability test of the method for detecting tryptophan based on a dual-core-satellite magnetic gold nanostructure SERS aptamer sensor To evaluate the repeatability of tryptamine signal detection based on the sensor, Raman spectra were acquired at 15 randomly selected regions on the sensor, and the data were recorded at 2226 cm⁻¹. -1 Raman intensity at that location. For example... Figure 5 As shown, the Raman spectra of 15 randomly selected regions at 2226 cm⁻¹ -1 The relative standard deviation (RSD) of the Raman intensity was 0.92%, indicating that the developed SERS aptamer sensor based on a double-core-satellite magnetic gold nanostructure has good signal repeatability.

[0043] Example 4: Specificity test of the method for detecting tryptophan using a double-core-satellite magnetic gold nanostructure SERS aptamer sensor To evaluate the specificity of the sensor in detecting tryptophan, standard solutions of tryptophan and its structural analogues, including histamine (0.1 mg / L), propionamide (0.1 mg / L), tyramine (0.1 mg / L), and o-phenylenediamine (0.1 mg / L), were added to the base sensor at specific concentrations. After magnetic separation and washing, the mixture was dropped onto a glass slide, and the readings were recorded at 2226 cm⁻¹. -1 Raman intensity at the location.

[0044] Figure 4 It shows 2226 cm -1 The Raman intensity at 2226 cm⁻¹ indicates that this method has good specificity for detecting tryptamine, where A: tryptamine; B: propionamide; C: tyramine; D: histamine; E: o-phenylenediamine. For tryptamine analogues, 2226 cm⁻¹ is the optimal value. -1 The Raman signal intensity at the same location is similar to that of the sensor with tryptamine, but significantly reduced compared to the sensor with tryptamine. The results indicate that the presence of interfering substances has almost no impact on the recognition probe's ability to identify tryptamine, and the SERS aptamer sensor based on the bilayer core-satellite magnetic gold nanostructure exhibits high tryptamine detection specificity.

[0045] Application examples (1) Preparation of tryptamine standard series solutions: Take 0.10 mL, 0.25 mL, 0.50 mL, 1.0 mL, 1.50 mL, 2.50 mL and 5.0 mL of 100 mg / L tryptamine standard mixed solution respectively, put them into 10 mL volumetric flasks, dilute to the mark with 0.1 mol / L hydrochloric acid solution, mix well, so that the concentrations are 1.0 mg / L, 2.5 mg / L, 5.0 mg / L, 10.0 mg / L, 15.0 mg / L, 25.0 mg / L and 50.0 mg / L respectively, and prepare fresh before use.

[0046] (2) Derivatization of reagents: Accurately measure 1.0 mL of 52-degree baijiu 1, 44-degree baijiu 2, total acid 6g / 100mL white vinegar, and total acid 4.5g / 100mL rice vinegar samples into 15 mL plastic centrifuge tubes. Add 1 mL of saturated sodium bicarbonate solution, 100 μL of sodium hydroxide solution (1 mol / L), and 1 mL of 10 mg / mL dansyl chloride derivatizing reagent in sequence. Vortex mix for 1 min, then place in a 60 ℃ constant temperature water bath for 15 min for derivatization. Remove and add 100 μL of sodium glutamate solution to each, shake to mix, and react at 60 ℃ for 15 min. Remove and cool to room temperature. Add 1 mL of ultrapure water to each centrifuge tube, vortex mix for 1 min, remove acetone (about 1 mL) by nitrogen blowing under a 40 ℃ bath, add 0.5 g of sodium chloride, vortex until completely dissolved, then add 5 mL of diethyl ether, vortex for 2 min, and after standing and layering, transfer the upper organic phase (diethyl ether layer) to a 15 mL plastic centrifuge tube. The aqueous phase (lower layer) was extracted again in a 1 mL centrifuge tube, and the two ether extracts were combined and dried under nitrogen in a 40 °C water bath. 1 mL of acetonitrile was added and the mixture was shaken to dissolve the residue. The solution was then filtered through a 0.22 μm syringe filter to obtain the test solution. The tryptamine standard and the actual sample were measured using high-performance liquid chromatography (HPLC). The measurement results were recorded, and a standard curve of the tryptamine standard was plotted to obtain the tryptamine concentration in the sample.

[0047] (3) Incubate 100 μL of the test solution with the same amount of the sensor prepared in Example 1 at room temperature by gentle shaking for 3 h. Collect the precipitate by magnetic separation, wash it twice with BB buffer, and then disperse it in 100 μL of BB buffer. Finally, transfer it to a glass slide and record the precipitate at 2226 cm⁻¹ using a Raman spectrometer. -1 Raman signal at the location.

[0048] (4) According to the standard curve drawn in Example 2, the concentrations of tryptamine in samples 1, 2, white vinegar and rice vinegar were 1.69 mg / L, 7.816 mg / L, 1.862 mg / L and 1.629 mg / L, respectively.

[0049] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A SERS aptamer sensor based on a dual-core-satellite magnetic gold nanostructure, characterized in that, Using aptamer-modified Fe3O4@Au MNPs magnetic SERS nanomaterials as tryptamine capture probes, and using two different particle sizes of SERS tags Au NP modified with c-DNA and 4-MBN as SERS signal probes for indirect detection of SERS signals by tryptamine, a "nuclear-satellite" type magnetically induced SERS sensor was constructed. The SERS aptamer sensor based on a dual-core-satellite magnetic gold nanostructure was prepared by the following method: (1) Preparation of specific Fe3O4 capture probe: Fe3O4@Au MNPs nanoparticles were incubated with 5' amino-terminal modified nucleic acid aptamers at room temperature with shaking to obtain specific Fe3O4 capture probe solution; (2) Preparation of specific SERS signal probes: Two solutions of gold nanoparticles with different particle sizes were mixed and incubated with 4-mercaptobenzonitrile to obtain "biosilent region" SERS tags. Then, they were incubated with pretreated 5' end thiol-modified c-DNA at room temperature to obtain specific SERS signal probe-1 solution and specific SERS signal probe-2 solution. (3) The specific SERS signal probe-1 solution and specific SERS signal probe-2 solution prepared in step (2) are shaken and incubated with the specific magnetic capture probe solution prepared in step (1) to obtain a SERS aptamer sensor based on a double-layer core-satellite magnetic gold nanostructure.

2. The SERS aptamer sensor based on a dual-core-satellite magnetic gold nanostructure according to claim 1, characterized in that, In step (1), the Fe3O4@Au MNPs nanoparticles are prepared by the following method: 1) FeCl3·6H2O was dissolved in ethylene glycol solution, PVP was added, and the mixture was heated and stirred under nitrogen. After adding sodium acetate, the mixture was reacted at high temperature to obtain Fe3O4 nanoparticles. 2) Take another HAuCl4·4H2O solution and mix it with sodium citrate solution, then add NaBH4 solution and age at room temperature to obtain gold seed solution; 3) Add PEI aqueous solution to cover the surface of Fe3O4 nanoparticles with PEI, then add gold seed solution and sonicate to obtain Fe3O4-Au MNPs seeds; 4) Fe3O4-Au MNPs seeds were dispersed in HAuCl4 solution, and then reacted with hydroxylamine hydrochloride solution and PVP to obtain Fe3O4@Au MNPs.

3. The SERS aptamer sensor based on a dual-layer core-satellite magnetic gold nanostructure according to claim 1, characterized in that, In step (1), the 5' amino-modified nucleic acid aptamer is an amino-modified tryptophan with the following sequence: 5'-SH-(CH2)6-AGCAGCACAGAGGTCAGATGTATGAACGATTTACTCGTA-CAGACGACACTTATCATTTGCCCTATGCGTGCTACCGTGAA-3' (as shown in SEQ ID No.1).

4. The SERS aptamer sensor based on a dual-core-satellite magnetic gold nanostructure according to claim 1, characterized in that, In step (2), the two gold nanoparticle solutions with different particle sizes are a gold nanoparticle solution with a particle size of 25 nm and a gold nanoparticle solution with a particle size of 60 nm.

5. The SERS aptamer sensor based on a dual-core-satellite magnetic gold nanostructure according to claim 4, characterized in that, The solution of gold nanoparticles with a particle size of 25 nm was prepared by the following method: Mix ultrapure water and 0.4 wt% tetrachloroauric acid solution, stir at a constant speed, heat to boiling, then add 1 wt% trisodium citrate solution, continue heating and stirring until the solution turns red, stop heating and continue stirring for 30 min to obtain the solution; The solution of gold nanoparticles with a particle size of 60 nm was prepared by the following method: After heating a 0.01wt% HAuCl4 solution to 120℃, add a 1% trisodium citrate solution. After heating and observing the color change from colorless to red, stop heating and continue stirring for 20 min to obtain the final product.

6. The SERS aptamer sensor based on a dual-layer core-satellite magnetic gold nanostructure according to claim 1, characterized in that, In step (2), the sequence of the thiol-modified c-DNA is as follows: C1: 5'-SH-(CH2)6-TTTTTTTTTTAAATCGTTCATACATCTG (as shown in SEQ ID No. 2); C2: 5'-SH-(CH2)6-TTTTTTTTTTTGATAAGTGTCGTCTG (as shown in SEQ ID No. 3).

7. The application of the SERS aptamer sensor based on a bilayer core-satellite magnetic gold nanostructure as described in any one of claims 1 to 6 in the detection of tryptamine.

8. A method for detecting tryptamine in biogenic amines using a SERS aptamer sensor based on a double-layer core-satellite magnetic gold nanostructure as described in any one of claims 1 to 6, characterized in that, Includes the following steps: (1) A series of tryptophan standard solutions with different concentration gradients were added to the SERS aptamer sensor based on a double-core-satellite magnetic gold nanostructure to obtain a mixture; the mixture was magnetically separated, washed, and then dropped onto a glass slide, and the reading was recorded at 2226 cm⁻¹. -1 Raman intensity at the location; (2) With 2226cm -1 The Raman intensity at a given point is plotted on the ordinate, and the negative logarithm of the tryptamine standard solution concentration is plotted on the abscissa to create a working curve. The tryptamine content in the test solution is then detected based on the working curve.

9. The method according to claim 8, characterized in that, The method for preparing the test solution is as follows: Add sodium chloride to the sample to be tested until it is completely dissolved, then add diethyl ether and stir the mixture vigorously; transfer the ether layer and extract the aqueous phase again; After drying the extract with nitrogen, the residue was dissolved in acetonitrile and filtered through a 0.22 μm filter membrane to obtain the test solution.

10. The method according to claim 8, characterized in that, The detection range of the method is 0.001-100 mg / L; the limit of detection is 0.40 × 10⁻⁶ mg / L. -3 mg / L.

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