Bimetallic SERS nanoprobes based on "bioquiescent" regions, their preparation methods, and applications.

Abstract

This invention discloses a bimetallic SERS nanoprobe based on a "bioquiescent" region, its preparation method, and its applications. The sensor uses AuNS@4-MBN@Ag-aptamer as the nanoprobe and Fe3O4@AuNPs-cDNA (FA-cDNA) as the magnetic substrate. In this sensor, FA-cDNA and AuNS@4-MBN@Ag-aptamer form a magnetic complex, FA-cDNA-aptamer-AuNS@4-MBN@Ag, through base complementarity pairing. The SERS sensor established using this bimetallic nanoprobe in a "bioquiescent" region has significant practical application value in the detection of trace CPF in complex matrix environments.

Description

Technical Field

[0001] This invention relates to the field of materials, specifically to a bimetallic SERS nanoprobe based on a "bioquitous" region, its preparation method, and its application. Background Technology

[0002] Chlorpyrifos (CPF) is an organophosphorus pesticide widely used in agricultural pest control. It effectively eliminates pests by inhibiting cholinesterase activity, disrupting nerve conduction. However, the overuse of CPF has caused serious environmental and health problems, including harm to non-target organisms and neurotoxicity and genotoxicity in humans through ingestion. Various countries and regions have implemented strict regulations on CPF, establishing maximum residue limits (MRLs) or banning regulations. Several analytical techniques for CPF detection have been applied, such as gas chromatography-tandem mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and fluorescence spectroscopy. Although traditional methods offer excellent sensitivity and accuracy, they are limited by high cost, large equipment size, and low flexibility. Therefore, developing an economical, rapid, sensitive, and accurate method for on-site monitoring of agricultural product safety is crucial.

[0003] SERS (Sequencing-Effect Transient Array) has gained increasing attention in fields such as food safety, environmental monitoring, plant physiology research, and biological cell imaging due to its advantages including low reagent consumption, cost-effectiveness, molecular fingerprinting characteristics, and high SERS signal response mediated by "hotspots." Continuous improvements have significantly enhanced its ability to accurately identify and detect trace substances by combining recognition elements such as nucleic acid aptamers, antibodies, and specific chemicals with various SERS substrates (nanospheres, nanoflowers, nanostars, and nanowires). However, practical applications still face challenges, particularly the interference of complex organic components on the detection of target substances. It is necessary to explore a SERS signal probe that balances sensitivity to target substances with immunity to organic background interference.

[0004] In recent years, indirect detection using SERS probes has gained popularity in ultra-trace material analysis. Sensitive and interference-resistant SERS nanotags typically employ noble metal nanomaterials with optimal surface plasmon properties, specific recognition elements, and suitable signal molecules as tags. Attaching reporter molecules with large Raman scattering cross-sections to the surface of SERS nanomaterials can significantly amplify the Raman signal response. The detection process relies on the signal response of the SERS tag to the target molecule, rather than the intrinsic fingerprint of the target molecule. "Molecular fingerprint" reporter molecules such as 4-MBA, DTNB, and PMBA are chosen because of their stable chemical structures, broad Raman scattering cross-sections, and strong adhesion to nanoparticle surfaces through the interaction of thiol groups with Au and Ag. However, the Raman characteristic peaks of most reporter molecules are located in the 600–1800 cm⁻¹ range. -1Within this range, it may overlap with complex background interference, complicating effective detection. Therefore, in the "biological silence" region (1800–2800 cm⁻¹), -1 Molecules exhibiting Raman characteristic signal peaks, such as 4-mercaptobenzonitrile (4-MBN), Prussian blue, and 4-ethylbenzenethiol derivatives, are favored for their applications, as this helps avoid interference from complex organic backgrounds. Among them, 4-MBN can form stable covalent bonds with Au and Ag, and its nitrile group and benzene ring have large Raman scattering cross sections. Furthermore, it exhibits a strong Raman scattering peak at 2228 cm⁻¹. -1 It exhibits a stable Raman characteristic signal. Although a series of tag molecules with similar properties to 4-MBN have been developed and explored, their applications remain very limited. Attaching Raman reporter tags to noble metal nanoparticles with high Raman-enhancing activity in the "biosilent" region is one of the promising strategies for reducing interference from complex organic backgrounds. Summary of the Invention

[0005] This invention addresses the aforementioned technical problems by providing a bimetallic SERS nanoprobe based on a "bioquiescent" region, its preparation method, and its application.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The preparation method of bimetallic SERS nanoprobes based on the "bioquitous" region includes the following steps:

[0008] (1) Preparation of bimetallic SERS nanoprobes in the “bioquitous” region: 4-mercaptobenzonitrile (4-MBN) was loaded onto the surface of gold nanostars (AuNSs) via Au-S bonds; AuNS@4-MBN@Ag was synthesized via ascorbic acid (AA) reduction; AuNS@4-MBN@Ag-aptamer was prepared by loading chlorpyrifos (CPF) aptamer onto the surface of AuNS@4-MBN@Ag via Ag-S bonds;

[0009] (2) Preparation of magnetic substrate: Magnetic Fe3O4 was synthesized as the core material of magnetic substrate; Fe3O4@PEI was first prepared by layer-by-layer electrostatic self-assembly and then Fe3O4@AuNPs (FA); aptamer complementary strand (cDNA) was loaded on the surface of AuNPs through Au-S bond to realize the preparation of FA-cDNA.

[0010] (3) The bimetallic SERS nanoprobes of the “biosilent” region prepared in step (1) are mixed and incubated with the magnetic substrate FA-cDNA prepared in step (2) to obtain a SERS sensor based on the reporter molecule of the “biosilent” region.

[0011] In the above preparation method, the synthesis method of Au seeds is to heat HAuCl4·3H2O solution to boiling, quickly add sodium citrate solution to the boiling solution, and continue heating and stirring for 10 to 30 minutes to obtain Au seed solution;

[0012] The method for synthesizing AuNSs involves heating a HAuCl4·3H2O solution to 20–40°C while stirring, then adding Aueseds solution, followed by sodium citrate solution and hydroquinone solution; after stirring, centrifugation is performed, and finally the solution is dispersed in deionized water to obtain the AuNSs solution.

[0013] The synthesis method of AuNS@4-MBN@Ag-aptamer includes the following steps:

[0014] (1) Add 4-MBN solution to AuNSs solution and mix well, then add Triton-X-100 solution and mix well again before centrifugation;

[0015] (2) The precipitate obtained after centrifugation was dispersed in deionized water, and ascorbic acid AA solution and AgNO3 solution were added sequentially under sonication. Then Tween-20 solution was added and stirred and centrifuged again to disperse the precipitate in PBS solution to obtain AuNS@4-MBN@Ag solution.

[0016] (3) Incubate AuNS@4-MBN@Ag solution with CPF aptamer, then centrifuge to remove unbound aptamer, and finally resuspend in PBS solution to obtain AuNS@4-MBN@Ag-aptamer solution.

[0017] In the technical solution of this invention, the preparation method of FA-cDNA includes the following steps:

[0018] Fe3O4 nanoparticles were ultrasonically dispersed uniformly in PEI solution, and after washing, the Fe3O4@PEI nanoparticles were resuspended in deionized water to obtain Fe3O4@PEI nanoparticle solution; then, AuNPs solution and Fe3O4@PEI nanoparticle solution were mixed, washed and dried to obtain Fe3O4@AuNPs (FA).

[0019] The FA solution and cDNA solution were incubated in PBS for 3–5 h. The washed precipitate was resuspended to obtain a FA-cDNA solution with a concentration of 0.4 mg / mL.

[0020] In the above preparation method, the synthesis method of Au seeds is as follows: the mass concentration of HAuCl4·3H2O solution is 0.01-0.03%, the mass concentration of sodium citrate solution is 0.5-1.5%, and the volume ratio of HAuCl4·3H2O solution to sodium citrate solution is 100:1-10;

[0021] In the method for synthesizing AuNSs, the mass concentration of HAuCl4·3H2O solution is 0.01-0.03%, the mass concentration of sodium citrate solution is 0.5-1.5%, and the concentration of hydroquinone solution is 20-50 mM.

[0022] The volume ratio of HAuCl4·3H2O solution, Au seeds solution, sodium citrate solution and hydroquinone solution is 95-105:1-5:1-5:10-30;

[0023] In the synthesis method of AuNS@4-MBN@Ag-aptamer:

[0024] In step (1), the concentration of the 4-MBN solution is 1–10 mM, and the mass concentration of Triton-X-100 is 0.5–1.5%.

[0025] The volume ratio of 4-MBN solution, AuNSs solution and Triton-X-100 solution is 5-15:1000:5-15;

[0026] In step (2), the concentration of AA solution is 10-80 mM, the concentration of AgNO3 solution is 10-80 mM, the mass concentration of Tween-20 is 0.5-1.5%, and the concentration of AuNPs solution is 50-150 μg / mL.

[0027] The volume ratio of AuNPs solution, AA solution, AgNO3 solution, Tween-20 solution and PBS solution is 1000:5-15:5-15:5-15:1000.

[0028] In step (3), the volume ratio of AuNS@4-MBN@Ag solution, CPF aptamer solution and PBS solution is 1000:30 to 80:1000;

[0029] The concentration of the CPF aptamer solution was 0.8 μM.

[0030] In the above preparation method: the mass ratio of Fe3O4 nanoparticles to PEI is 1:1 to 8; the concentration of Fe3O4@PEI nanoparticle solution is 0.5 to 1 mg / mL; the concentration of AuNPs solution is 50 to 150 μg / mL; and the volume ratio of AuNPs solution to Fe3O4@PEI nanoparticle solution is 2 to 6:1.

[0031] In the above preparation method: the concentration of FA solution is 0.2-1 mg / mL, the concentration of cDNA is 0.5-5 μM, and the volume ratio of FA solution to cDNA solution is 5-15:1.

[0032] In the above preparation method: the volume ratio of FA-cDNA solution and AuNS@4-MBN@Ag-aptamer solution in step (3) is 1:1 to 5.

[0033] In the above preparation method: aptamer, 5′-SH-(CH2)6-CCTGCCACGCTCCGCAAGCTTAGGGTTA CGCCTGCAGCGATTCTTGATCGCGCTGCTGGTAATCCTTCTTTAAGCTTGGCACCCGC ATCGT-3′;

[0034] cDNA, 5′-SH-(CH2)6-ACGATGGCGGGTGCCAAGCTTAAAGAAGGAT-3′.

[0035] A bimetallic SERS nanoprobe based on the "bioquiescent" region was prepared using the method described above.

[0036] In the technical solution of this invention, the bimetallic SERS nanoprobe based on the "bioquitous" region prepared by the method has good application prospects for the accurate and rapid quantitative analysis of harmful substances in complex food and environmental matrices.

[0037] Establishment of standard curves

[0038] First, 1 mg / mL CPF acetonitrile solution was diluted to different concentrations with 10% ethanol. Then, 20 μL CPF was added to 1 mL AuNS@4-MBN@Ag-aptamer and gently mixed for 30 min. Next, 0.5 mL FA-cDNA was added to the mixture and incubated at 37 °C for 30 min. The magnetic conjugate was washed twice with deionized water using an external magnet and then dispersed in 0.5 mL of deionized water. A 2228 cm⁻¹ laser was recorded at an integration time of 264.5 mW and 1 s. -1 Raman signal intensity at the location.

[0039] Relative Raman intensity calculation formula: y = ΔI = (I 2228cm -1 )0–(I 2228cm -1 ), of which (I 2228cm -1 )0 and (I 2228cm -1() represents the blank group and the test group at 2228cm -1 Raman intensity at the location.

[0040] Establish C based on the formula CPF Linear relationship with y: y = k LgC CPF +b, where k and b are the slope and intercept of the standard curve, respectively.

[0041] SERS detection of CPF in actual samples

[0042] (1) Wheat Analysis

[0043] Mix 2g of wheat with 5mL of deionized water and 5mL of acetonitrile, then grind thoroughly in a mortar. Filter the slurry through double-layered gauze, centrifuge at 12000rpm for 8min, and then filter through a 0.22μm filter. Add different concentrations of CPF to the wheat sample for SERS detection.

[0044] (2) Apple Analysis

[0045] Chop 20g of fresh tissue and homogenize it in a fruit blender. Centrifuge the resulting apple juice at 12000rpm for 8min and filter using a 0.22μm filter. Add different concentrations of CPF to the apple juice samples for SERS detection.

[0046] The beneficial effects of this invention are:

[0047] (1) This invention prepares a high-performance surface-enhanced Raman scattering (SERS) nanoprobe (AuNS@4-MBN@Ag-aptamer), using a star-shaped bimetallic core-shell structure as the main Raman signal enhancement material, and 4-mercaptobenzonitrile (4-MBN) as the reporter molecule in the "biosilent" region (at 2228 cm⁻¹). -1(1) The nanoprobe has significant Raman characteristic peaks and exhibits good Raman signal enhancement effect and excellent resistance to signal interference from the "molecular fingerprint" region. (2) This invention introduces the "biosilent" region nanoprobe into the SERS aptamer sensor for ultrasensitive and interference-free detection of chlorpyrifos (CPF). The sensor uses AuNS@4-MBN@Ag-aptamer as the nanoprobe and Fe3O4@AuNPs-cDNA (FA-cDNA) as the magnetic substrate. In this sensor, FA-cDNA and AuNS@4-MBN@Ag-aptamer form a magnetic complex of FA-cDNA-aptamer-AuNS@4-MBN@Ag through base complementary pairing. When CPF is present in the mixed system, the binding of the nanoprobe and the magnetic substrate is hindered because CPF preferentially binds to the nanoprobe, which causes the Raman signal intensity of the magnetic complex to decrease with increasing CPF concentration. Under optimal SERS detection parameters, this sensor exhibits good selectivity and repeatability, with a linear detection range of 2.5 × 10⁻⁶. 2 Up to 5.0×10 4 pg / mL, LOD is 220.35 pg / mL (S / N = 3).

[0048] (3) The present invention uses this aptamer sensor to detect actual samples, and the results show that the spiked recoveries of wheat and apple are 89.61% to 107.33% (RSD ≤ 14.55%). Therefore, the SERS sensor based on the bimetallic nanoprobe in the "bioquiescent" region has important practical application significance in the detection of trace CPF in complex matrix environments. Attached Figure Description

[0049] Figure 1 The present invention relates to a detection principle diagram of an ultrasensitive, interference-free detection sensor based on a bimetallic SERS nanoprobe in a "bioquitous" region.

[0050] Figure 2 This is a TEM image of AuNS@4-MBN@Ag obtained in Example 1 of the present invention.

[0051] Figure 3 This is an evaluation of the SERS effect of the AuNS@4-MBN@Ag nanoprobe in Example 1 of the present invention.

[0052] Figure 4 Establishment of the standard curve. (a) 2228 cm⁻¹ corresponding to different CPF concentrations. -1 (a) SERS signal strength at 2228 cm⁻¹; (b) 2228 cm⁻ -1 Linear calibration curve of Raman signal intensity versus CPF concentration at the location.

[0053] Figure 5Evaluation of the anti-interference properties of 4-MBN. (a) Raman spectra of AuNS@4-MBN and 4-NBT@AuNS@4-MBN; (b) Raman spectra of AuNSs@4-MBN and 4-NBT@AuNSs@4-MBN at 2228 cm⁻¹. -1 Raman signal intensity at the location.

[0054] Figure 6 Selectivity evaluation of CPF detection method based on bimetallic SERS nanoprobes in the "bioquitous" region.

[0055] Figure 7 Reproducibility evaluation of CPF detection method based on bimetallic SERS nanoprobes in the "bioquitous" region. Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto:

[0057] The chlorpyrifos (CPF) aptamer and complementary strand (cDNA) were synthesized and purified by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China), and their sequences are as follows:

[0058] Aptamer, 5′-SH-(CH2)6-CCTGCCACGCTCCGCAAGCTTAGGGTTACGCCTGCAGCGA TTCTTGATCGCGCTGCTGGTAATCCTTTCTTTAAGCTTGGCACCCGCATCGT-3′;

[0059] cDNA, 5′-SH-(CH2)6-ACGATGGCGGGTGCCAAGCTTAAAGAAGGAT-3′.

[0060] Example 1: Fabrication of an ultrasensitive, interference-free detection sensor based on a bimetallic SERS nanoprobe in a "bioquitous" region

[0061] 1. Preparation of bimetallic SERS nanoprobes in the "bioquiescent" region

[0062] (1) Synthesis of gold seeds

[0063] Take 100 mL of HAuCl4·3H2O solution (0.01%, w / v), stir vigorously at 120 °C and heat to boiling. Then, quickly add 3 mL of 1% (w / v) sodium citrate solution to the boiling solution. After continuing heating and stirring for 20 min, the gold seed solution is obtained.

[0064] (2) Synthesis of gold nanostars (AuNSs)

[0065] AuNSs were synthesized using a seed-mediated growth method. 98 mL of HAuCl4·3H2O (0.01%, w / v) was heated to 30 °C with gentle stirring. Then, 2 mL of the prepared gold seed solution was added. Subsequently, 3 mL of 1% (w / v) sodium citrate was added. After 30 s, 20 mL of 30 mM hydroquinone solution was rapidly added. After vigorous stirring for 30 min, the mixture was centrifuged at 8000 rpm for 20 min, repeated twice, and finally dispersed in 50 mL of deionized water to obtain the AuNSs solution.

[0066] (3) Synthesis of AuNS@4-MBN@Ag

[0067] 10 μL of 6 mM 4-MBN solution was added to 1 mL of AuNSs solution. After vortexing for 2 h at room temperature, 10 μL of 1% (w / v) Triton-X-100 was added, and the mixture was stirred for 5 min. The mixture was then centrifuged at 8000 rpm for 15 min, repeated twice, and the precipitate was resuspended in 1 mL of deionized water. Then, 10 μL each of 50 mM ascorbic acid (AA) solution and 50 mM AgNO3 solution were added sequentially under sonication. After stirring for 5 min, 10 μL of 1% (w / v) Tween-20 was added, and the mixture was stirred for 10 min. After centrifugation at 7500 rpm for 10 min, the precipitate was dispersed in 1 mL of PBS solution (10 mM, pH = 7.2) to obtain a highly SERS-active AuNS@4-MBN@Ag solution. Figure 2 TEM image; Figure 3 (Results of SERS activity evaluation).

[0068] (4) Preparation of AuNS@4-MBN@Ag-aptamer

[0069] Incubate 1 mL of AuNS@4-MBN@Ag solution with 50 μL of 0.8 μM aptamer for 4 h, centrifuge at 7000 rpm for 10 min to remove unbound aptamer, and finally resuspend in 1 mL of PBS solution to obtain AuNS@4-MBN@Ag-aptamer.

[0070] 2. Preparation of magnetic substrates

[0071] (1) Synthesis of Fe3O4

[0072] 335 mg FeCl3·6H2O was dissolved in 10 mL (CH2OH)2 and stirred for 30 min. Then, 250 mg PEG6000 and 675 mg NaOAc were added. After complete dissolution, the mixture was transferred to a high-pressure reactor and heated at 210 °C for 12 h. The reaction product was washed three times each with ethanol and deionized water, and then dried under vacuum at 60 °C overnight to obtain Fe3O4.

[0073] (2) Preparation of Fe3O4@AuNPs (FA)

[0074] First, 20 mg of Fe3O4 nanoparticles were ultrasonically dispersed in 40 mL of 2 mg / mL PEI solution for 2 hours, and then washed four times with deionized water. The resulting Fe3O4@PEI nanoparticles were resuspended in 40 mL of deionized water. Then, AuNPs solution and Fe3O4@PEI solution were ultrasonically mixed at a volume ratio of 4:1 for 30 min. After washing three times with deionized water, the nanoparticles were vacuum dried at 60 °C for 6 h.

[0075] (3) Preparation of FA-cDNA

[0076] 0.5 mL of FA (0.4 mg / mL) and 50 μL of 1.5 μM cDNA were incubated in PBS for 4 h. After washing 3 times, the precipitate was resuspended in 0.5 mL of PBS to obtain the FA-cDNA solution (0.4 mg / mL).

[0077] 3. Construction of an ultrasensitive, interference-free detection sensor based on bimetallic SERS nanoprobes in the "bioquiescent" region

[0078] 0.5 mL of FA-cDNA (0.4 mg / mL) was added to 1 mL of AuNS@4-MBN@Ag-aptamer solution and incubated at 37 °C for 30 min. The magnetic conjugate was washed twice with deionized water using an external magnet and then dispersed in 0.5 mL of deionized water. A 2228 cm⁻¹ laser was recorded at a laser power of 264.5 mW and an integration time of 1 s. -1 The Raman signal intensity of the blank group at the location.

[0079] Example 2: Establishment of an ultrasensitive and interference-free CPF detection method based on a bimetallic SERS nanoprobe in a "bioquitous" region.

[0080] (1) Dilute 1 mg / mL CPF acetonitrile solution to different concentrations with 10% ethanol solution. Then, add 20 μL CPF to 1 mL AuNS@4-MBN@Ag-aptamer and gently shake for 30 min. Add 0.5 mL FA-cDNA to the mixture and shake at 37 °C for 30 min. Wash the magnetic hybrid twice with deionized water using an external magnet, and then disperse it in 0.5 mL of deionized water. Record 2228 cm⁻¹ at a laser power of 264.5 mW and an integration time of 1 s. -1 Raman signal intensity at the location.

[0081] (2) with 2228cm -1 The relative Raman signal intensity at a given location is plotted on the ordinate, and the logarithm of the CPF concentration is plotted on the abscissa to create a working curve. The CPF content in the test solution is then detected based on the working curve.

[0082] Relative signal strength calculation formula: y = ΔI = (I 2228cm -1 )0–(I 2228cm -1 ), of which (I 2228cm -1 )0 and (I 2228cm -1 () represents the blank group and the test group at 2228cm -1 Raman intensity at the location.

[0083] Establish C based on the formula CPF Linear relationship with y: Y = 3727.70Lg C CPF –7555.29(R 2 =0.986)( Figure 4 The linear range is 2.5 × 10⁻⁶. 2 Up to 5.0×10 4 The limit of detection was 220.35 pg / mL.

[0084] Example 3: Evaluation of the anti-interference ability of CPF detection method based on bimetallic SERS nanoprobes in the "bioquitous" region (1) SERS detection was performed using a mixture of AuNS@4-NBT / 4-MBN and AuNS@4-MBN, and the anti-interference ability of 2228 cm⁻¹ was compared. -1 Raman signal intensity at the location.

[0085] (2) Although in the "molecular fingerprint" region (800-1800 cm) -1 The Raman spectra of 4-MBN and the organic reagent 4-NBT overlap, but the 2228 cm⁻¹ peak is the most significant. -1 The signal strength at that location is unaffected. Figure 5The results show that this SERS nanoprobe based on 4-MBN as a signal source can achieve a signal intensity of 2228 cm⁻¹. -1 The Raman signal at that location has good anti-interference capability.

[0086] Example 4: Selection and reproducibility evaluation of CPF detection method based on bimetallic SERS nanoprobes in the "bioquitous" region

[0087] (1) Selectivity tests were performed using 50 ng / mL glyphosate, imidacloprid, acetamiprid, thiamethoxam, and dinotefuran, as well as a mixture of multiple pesticides containing 10 ng / mL CPF, respectively, in place of 10 ng / mL CPF. Reproducibility was evaluated by analyzing 10 independent FA / AuNS@4-MBN@Ag hybrids and preparing 20 independent test solutions containing 10 ng / mL CPF.

[0088] (2) Figure 6 As shown, this aptamer sensor exhibits significant specificity, showing a strong signal response to CPF and CPF-containing mixtures, while the response to other pesticides remains weak even at high concentrations. Therefore, this CPF detection method based on a bimetallic SERS nanoprobe in a "bioquiescent" region demonstrates good selectivity.

[0089] (3) Figure 7 As shown, the FA / AuNS@4MBN@Ag hybrid at 2228 cm⁻¹ -1 The RSD of the signal intensity at the initial location was 3.84%, while the RSD of the detection result containing CPF test solution was 5.61%. Therefore, the CPF detection method based on the bimetallic SERS nanoprobe in the "bioquitous" region has good reproducibility.

[0090] Application example:

[0091] (1) Wheat and apples were selected as actual samples for CPF detection. For wheat samples, 2g of wheat was mixed with 5mL of deionized water and 5mL of acetonitrile, and then thoroughly ground in a mortar. The slurry was filtered through double-layered gauze, centrifuged at 12000rpm for 8min, and then filtered through a 0.22μm filter. Different concentrations of CPF were added to the wheat samples for SERS detection. For apple samples, 20g of fresh tissue was chopped and homogenized in a fruit crusher. The resulting apple juice was centrifuged at 12000rpm for 8min and filtered through a 0.22μm filter. Different concentrations of CPF were added to the apple juice samples for SERS detection. The 2228cm² readings for different samples were recorded. -1 The Raman signal intensity at the location is calculated, and the relative Raman signal intensity is determined.

[0092] (2) The spiked recoveries of CPF in wheat and apple samples were calculated based on the standard working curve plotted in Example 2, which were 89.61%–107.33% and 91.62%–102.35% respectively (RSD≤14.55%).

[0093] This invention discloses a bimetallic SERS nanoprobe based on a "bioquiescent" region, its preparation method, and its applications. The sensor uses AuNS@4-MBN@Ag-aptamer as the nanoprobe and Fe3O4@AuNPs-cDNA (FA-cDNA) as the magnetic substrate. In this sensor, FA-cDNA and AuNS@4-MBN@Ag-aptamer form a magnetic complex, FA-cDNA-aptamer-AuNS@4-MBN@Ag, through base complementarity pairing. The SERS sensor established using this bimetallic nanoprobe in a "bioquiescent" region has significant practical application value in the detection of trace CPF in complex matrix environments.

Claims

1. A method for preparing bimetallic SERS nanoprobes based on "bioquiescent" regions, characterized in that: The steps of this method are as follows: S1: Preparation of bimetallic SERS nanoprobes in the "bioquitous" region: 4-mercaptobenzonitrile (4-MBN) was loaded onto the surface of gold nanostars (AuNSs) via Au-S bonds; AuNS@4-MBN@Ag was synthesized via ascorbic acid AA reduction; AuNS@4-MBN@Ag-aptamer was prepared by loading chlorpyrifos CPF aptamer onto the surface of AuNS@4-MBN@Ag via Ag-S bonds; The synthesis method of AuNS@4-MBN@Ag-aptamer includes the following steps: (1) Add 4-MBN solution to AuNSs solution and mix well, then add Triton-X-100 solution and mix well again before centrifugation; (2) The precipitate obtained after centrifugation was dispersed in deionized water, and ascorbic acid AA solution and AgNO3 solution were added sequentially under sonication. Then Tween-20 solution was added and stirred and centrifuged again to disperse the precipitate in PBS solution to obtain AuNS@4-MBN@Ag solution. (3) Incubate AuNS@4-MBN@Ag solution with CPF aptamer, then centrifuge to remove unbound aptamer, and finally resuspend in PBS solution to obtain AuNS@4-MBN@Ag-aptamer solution; S2: Preparation of magnetic substrate: Magnetic Fe3O4 was synthesized as the core material of the magnetic substrate; Fe3O4@PEI was first prepared by layer-by-layer electrostatic self-assembly, and then Fe3O4@AuNPs were prepared; aptamer complementary strand cDNA was loaded on the surface of AuNPs through Au-S bonds to achieve the preparation of FA-cDNA. The preparation method of Fe3O4@AuNPs is as follows: Fe3O4 nanoparticles are ultrasonically dispersed evenly in PEI solution, and after washing, the Fe3O4@PEI nanoparticles are resuspended in deionized water to obtain Fe3O4@PEI nanoparticle solution; then, the AuNPs solution and Fe3O4@PEI nanoparticle solution are mixed evenly, washed and dried to obtain Fe3O4@AuNPs. S3: The bimetallic SERS nanoprobes in the "biosilent" region prepared in step S1 are mixed and incubated with the magnetic substrate FA-cDNA prepared in step S2 to obtain an SERS sensor based on the "biosilent" region reporter molecule. Among them, the aptamer is 5′-SH-(CH2)6-CCTGCCACGCTCCGCAAGCTTAGGGTTACGCCTGCAGCGATTCTTGATCGCGCTGCTGGTAATCCTTCTTTAAGCTTGGCACCCGCATCGT-3′. cDNA, 5′-SH-(CH2)6-ACGATGGCGGGTGCCAAGCTTAAAGAAGGAT-3′.

2. The preparation method according to claim 1, characterized in that: The method for synthesizing Au seeds is to heat HAuCl4·3H2O solution to boiling, quickly add sodium citrate solution to the boiling solution, and continue heating and stirring for 10~30 min to obtain Au seed solution. The method for synthesizing AuNSs involves heating a HAuCl4⋅3H2O solution to 20-40°C while stirring, then adding Au seeds solution, followed by sodium citrate solution and hydroquinone solution; after stirring, centrifugation is performed, and finally the solution is dispersed in deionized water to obtain the AuNSs solution.

3. The preparation method according to claim 1, characterized in that: The method for preparing FA-cDNA is to incubate the FA solution and cDNA solution in PBS for 3-5 h, and then resuspend the washed precipitate to obtain a FA-cDNA solution with a concentration of 0.4 mg / mL.

4. The preparation method according to claim 2, characterized in that: In the synthesis method of Au seeds: the mass concentration of HAuCl4·3H2O solution is 0.01~0.03%, the mass concentration of sodium citrate solution is 0.5~1.5%, and the volume ratio of HAuCl4·3H2O solution to sodium citrate solution is 100:1~10; In the method for synthesizing AuNSs, the mass concentration of HAuCl4⋅3H2O solution is 0.01~0.03%, the mass concentration of sodium citrate solution is 0.5~1.5%, and the concentration of hydroquinone solution is 20~50 mM; The volume ratio of HAuCl4⋅3H2O solution, Au seeds solution, sodium citrate solution and hydroquinone solution is 95~105:1~5:1~5:10~30; In the synthesis method of AuNS@4-MBN@Ag-aptamer: In step (1), the concentration of the 4-MBN solution is 1~10 mM, and the mass concentration of Triton-X-100 is 0.5~1.5%; The volume ratio of 4-MBN solution, AuNSs solution and Triton-X-100 solution is 5~15:1000:5~15; In step (2), the concentration of AA solution is 10~80 mM, the concentration of AgNO3 solution is 10~80 mM, the mass concentration of Tween-20 is 0.5~1.5%, and the concentration of AuNPs solution is 50~150 μg / mL. The volume ratio of AuNPs solution, AA solution, AgNO3 solution, Tween-20 solution and PBS solution is 1000:5~15:5~15:5~15:1000. In step (3), the volume ratio of AuNS@4-MBN@Ag solution, CPF aptamer solution and PBS solution is 1000:30~80:1000; The concentration of the CPF aptamer solution was 0.8 μM.

5. The preparation method according to claim 1, characterized in that: The mass ratio of Fe3O4 nanoparticles to PEI is 1:1~8; the concentration of Fe3O4@PEI nanoparticle solution is 0.5~1 mg / mL; the concentration of AuNPs solution is 50~150 μg / mL; and the volume ratio of AuNPs solution to Fe3O4@PEI nanoparticle solution is 2~6:

1.

6. The preparation method according to claim 1, characterized in that: The concentration of FA solution is 0.2~1 mg / mL, the concentration of cDNA is 0.5~5 μM, and the volume ratio of FA solution to cDNA solution is 5~15:

1.

7. The preparation method according to claim 1, characterized in that: In step S3, the volume ratio of FA-cDNA solution to AuNS@4-MBN@Ag-aptamer solution is 1:1~5.

8. A bimetallic SERS nanoprobe based on a "biologically silent" region, characterized in that... The probe was prepared using the method described in any one of claims 1 to 7.

9. The application of the bimetallic SERS nanoprobe based on the "bioquiescent" region as described in claim 8 in the quantitative analysis of harmful substances in complex food and environmental matrices.

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