Near-infrared fluorescent probe and application thereof in detection of aflatoxin B1

Abstract

The invention relates to the technical field of substance detection, in particular to a near-infrared fluorescent probe and application of the near-infrared fluorescent probe in aflatoxin B1 detection. According to the invention, the synthesis of the near-infrared nanoparticles is guided by using quantum chemical calculation software and molecular dynamic simulation, the near-infrared nanoparticles are prepared by a microemulsion method, and the near-infrared nanoparticles are uniform in particle size, good in stability and capable of emitting ultrahigh-brightness fluorescence signals; by coupling the near-infrared nano particle with an anti-aflatoxin B1 monoclonal antibody, the near-infrared nano particle can be used as a near-infrared fluorescence signal probe for detecting aflatoxin B1; due to the fact that the near-infrared nanoparticles can achieve short-wave excitation and have large Stokes shift, interference of exciting light and emitted light can be effectively eliminated, self-fluorescence disturbance is avoided, and the detection sensitivity of the near-infrared fluorescence signal probe prepared from the near-infrared nanoparticles can be greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of substance detection, and in particular to a near-infrared fluorescent probe and its application in the detection of aflatoxin B1. Background Art

[0002] Aflatoxin B1 (AFB1) is a strong carcinogen produced by Aspergillus flavus and Aspergillus parasiticus. Long-term exposure can seriously endanger health, especially liver damage. It causes gene mutations by binding to DNA, thereby inducing malignant tumors such as liver cancer, and may also lead to immunosuppression, growth retardation, and damage to the kidneys and digestive system.

[0003] AFB1 detection methods include high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), gas chromatography-mass spectrometry (GC-MS), and fluorescence immunochromatography (FICA), etc. Among them, HPLC has high sensitivity and good accuracy, but the equipment is expensive and the operation is complex; ELISA is simple to operate and low in cost, suitable for large-scale screening, but has low sensitivity; GC-MS has high sensitivity and specificity and can detect multiple toxins simultaneously, but has high cost and strict technical requirements; FICA has the advantages of simple operation, rapid detection, and suitability for grass-roots use, and has become one of the on-site rapid screening methods, but there are problems of low sensitivity and matrix interference. Summary of the Invention

[0004] Based on this, the present invention provides a near-infrared fluorescent probe and its application in the detection of aflatoxin B1, which solves at least one problem in the prior art.

[0005] In the first aspect, the present invention provides a preparation method of a near-infrared fluorescent probe, which comprises the following steps: Mix a compound shown in formula 1, a compound shown in formula 2, poly(maleic anhydride-alt-1-octadecene), and an organic solvent to obtain an oil-phase solution; Mix a surfactant and water to obtain an aqueous-phase solution; Mix the above oil-phase solution and aqueous-phase solution, and perform emulsification to obtain an emulsion; After removing the organic solvent in the above emulsion, perform centrifugal washing to obtain a solid substance; Hydrolyze the above solid substance in an alkaline solution, and wash with water to obtain near-infrared nanoparticles; Couple the above near-infrared nanoparticles with an anti-aflatoxin B1 monoclonal antibody to obtain a near-infrared fluorescent probe.

[0006] Among them, the compound shown in Formula 1 and the compound shown in Formula 2 have a high degree of energy level matching, endowing the near-infrared nanoparticles with excellent fluorescence properties. After the near-infrared nanoparticles are conjugated with anti-aflatoxin B1 monoclonal antibody, the detection of aflatoxin B1 has extremely high specificity and sensitivity.

[0007] In some alternative embodiments, the method for preparing the above-mentioned near-infrared fluorescent probe further comprises the following steps: Calculating the energy donor and energy acceptor molecules through the quantum chemistry calculation software ORCA to determine the compound shown in Formula 1 and the compound shown in Formula 2 for synthesizing the near-infrared nanoparticles; Guiding the synthesis of the near-infrared nanoparticles through the molecular dynamics simulation software GROMACS to determine the dosage ratio of the compound shown in Formula 1 and the compound shown in Formula 2.

[0008] In some alternative embodiments, the mass ratio of the compound shown in Formula 1, the compound shown in Formula 2, and poly(maleic anhydride-alt-1-octadecene) is 1-5:1:2-10. Preferably, the mass ratio of the compound shown in Formula 1, the compound shown in Formula 2, and poly(maleic anhydride-alt-1-octadecene) is 3-5:1:6-10. Most preferably, the mass ratio of the compound shown in Formula 1, the compound shown in Formula 2, and poly(maleic anhydride-alt-1-octadecene) is 3:1:8.

[0009] In some alternative embodiments, the above-mentioned organic solvent is dichloromethane, chloroform or carbon tetrachloride. Preferably, the above-mentioned organic solvent is chloroform.

[0010] In some alternative embodiments, the above-mentioned surfactant is sodium dodecyl sulfate or sodium dodecylbenzenesulfonate. Preferably, the above-mentioned surfactant is sodium dodecyl sulfate.

[0011] In some alternative embodiments, the volume ratio of the above-mentioned oil phase solution to the water phase solution is 1-5:8. Preferably, the volume ratio of the above-mentioned oil phase solution to the water phase solution is 3:8.

[0012] In some alternative embodiments, the above-mentioned alkali solution is sodium hydroxide solution.

[0013] In some alternative embodiments, the mass ratio of the above-mentioned near-infrared nanoparticles to the anti-aflatoxin B1 monoclonal antibody is 1:0.5-1. Preferably, the mass ratio of the above-mentioned near-infrared nanoparticles to the anti-aflatoxin B1 monoclonal antibody is 1:0.6-0.8. Most preferably, the mass ratio of the above-mentioned near-infrared nanoparticles to the anti-aflatoxin B1 monoclonal antibody is 1:0.72.

[0014] In a second aspect, the present invention provides a near-infrared fluorescent probe obtained by the method for preparing the above-mentioned near-infrared fluorescent probe.

[0015] In a third aspect, the present invention provides an application of the above near-infrared fluorescent probe, wherein the above near-infrared fluorescent probe is used for detecting aflatoxin B1.

[0016] In a fourth aspect, the present invention provides a test strip for detecting aflatoxin B1, which comprises the above near-infrared fluorescent probe.

[0017] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: The synthesis of near-infrared nanoparticles was guided by using quantum chemistry calculation software and molecular dynamics simulation. Near-infrared nanoparticles were obtained by the microemulsion method. The near-infrared nanoparticles have uniform particle size and good stability, and can emit ultra-high brightness near-infrared fluorescence signals. By conjugating the near-infrared nanoparticles with anti-aflatoxin B1 monoclonal antibody, it can be used as a near-infrared fluorescence signal probe for detecting aflatoxin B1. Since the near-infrared nanoparticles can achieve short-wave excitation and have a large Stokes shift, they can effectively eliminate the interference between the excitation light and the emission light, avoid self-fluorescence perturbation, and the near-infrared fluorescence signal probe prepared therefrom can greatly improve the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the detection of AFB1 by the near-infrared fluorescent probe in the embodiment of the present invention.

[0019] Figure 2 It shows the optimal molecular structure, HOMO energy level and LUMO energy level obtained by quantum chemistry calculation in the embodiment of the present invention.

[0020] Figure 3 It shows the system potential energy results obtained by using molecular dynamics simulation software in the embodiment of the present invention.

[0021] Figure 4 It shows the root mean square deviation results obtained by using molecular dynamics simulation software in the embodiment of the present invention.

[0022] Figure 5 It is a scatter plot of weak interaction analysis obtained by using molecular dynamics simulation software in the embodiment of the present invention.

[0023] Figure 6 It shows the visualization results of weak interactions obtained by using molecular dynamics simulation software in the embodiment of the present invention.

[0024] Figure 7 It shows the system total energy results obtained by using molecular dynamics simulation software in the embodiment of the present invention.

[0025] Figure 8Shows the simulation snapshot results of the system obtained by using molecular dynamics simulation software in the embodiments of the present invention.

[0026] Figure 9 Is the nuclear magnetic resonance hydrogen spectrum of the compound shown in Formula 1 in the embodiments of the present invention.

[0027] Figure 10 Is the high-resolution mass spectrum of the compound shown in Formula 1 in the embodiments of the present invention.

[0028] Figure 11 Is the electron microscopy image of the near-infrared nanoparticles in the embodiments of the present invention.

[0029] Figure 12 Shows the fluorescence intensities of the near-infrared nanoparticles (after adding the compound shown in Formula 1) and the nanoparticles prepared without adding the compound shown in Formula 1 (before adding the compound shown in Formula 1) in the embodiments of the present invention.

[0030] Figure 13 Shows the conjugation of the near-infrared nanoparticles with anti-AFB1 monoclonal antibody in the embodiments of the present invention.

[0031] Figure 14 Is the physical picture of the detection of AFB1 by the near-infrared fluorescence probe in the embodiments of the present invention.

[0032] Figure 15 Shows the detection limit results of the near-infrared fluorescence probe in the embodiments of the present invention.

[0033] Figure 16 Shows the specific detection results of the near-infrared fluorescence probe in the embodiments of the present invention. Detailed implementation manners

[0034] The concept and technical effects of the present invention will be clearly and completely described below to fully elaborate the purpose, solution and effects of the present invention.

[0035] According to the embodiments of the present invention, first, the synthesis of near-infrared nanoparticles is theoretically guided by quantum chemical calculations and molecular dynamics simulations, then the near-infrared nanoparticles are prepared by the microemulsion method, and then the near-infrared nanoparticles are conjugated with anti-aflatoxin B1 monoclonal antibody to obtain a near-infrared fluorescence probe. The use of the near-infrared fluorescence probe to detect aflatoxin B1 improves the specificity and sensitivity of the detection of aflatoxin B1.

[0036] Example 1 Using quantum chemistry calculation software, the molecular structures of the energy donor and the energy acceptor were optimized by density functional theory, and their HOMO energy levels and LUMO energy levels were calculated to prove their energy level matching degree, and at the same time, the initial structure for molecular dynamics simulation was obtained. Among them, the quantum chemistry calculation software used was ORCA, and the B3LYP functional and 6-311G(d,p) basis set were selected for the study; the calculation contents included molecular structure and frequency optimization, electrostatic potential mapping, HOMO and LUMO energy level calculation, and the energy level matching degree between the energy donor and the energy acceptor. Finally, the compound shown in Formula 1 was determined as the energy donor, and the compound shown in Formula 2 was determined as the energy acceptor.

[0037] As Figure 2 shown, the HOMO energy level of the compound shown in Formula 1 is -4.98 eV, and the LUMO energy level is -2.44 eV; the HOMO energy level of the compound shown in Formula 2 is -4.97 eV, and the LUMO energy level is -3.51 eV. In comparison, the compound shown in Formula 1 has a lower HOMO energy level, and the compound shown in Formula 2 has a lower LUMO energy level, indicating that the combination of the compound shown in Formula 1 and the compound shown in Formula 2 perfectly matches the conditions for fluorescence resonance energy transfer.

[0038] Example 2 The molecular dynamics simulation software GROMACS was used to verify the distance requirement for fluorescence resonance energy transfer between the compound shown in Formula 1 and the compound shown in Formula 2: First, a molecular dynamics simulation box with a size of 4 nm×4 nm×4 nm was built, and the molecules of the compound shown in Formula 1 and the compound shown in Formula 2 with a molecular number ratio of 1:1 after structural optimization were added to it, and then the spc216 water model was used for solvation treatment; the three-dimensional periodic boundary condition (PBC) was adopted for the simulation box, a cutoff distance of 1.2 nm was used for calculating the short-range electrostatic interaction and van der Waals interaction, the particle mesh Ewald method was used for the long-range electrostatic interaction (PME), and the LINCS algorithm was used to constrain the bonds involving hydrogen atoms; the steepest descent algorithm was used for energy minimization to relax the structure, and then a 10 ns equilibration phase (T = 300 K) simulation was carried out, and after equilibration, a 100 ns production run was carried out; the kinetic data of the last 50 ns were collected to analyze the stability, weak interaction and distance relationship of the system.

[0039] As Figure 3 shown, the analysis of the potential energy shows that the overall system is stable, providing a reliable guarantee for subsequent data analysis.

[0040] As Figure 4As shown, there is no drastic fluctuation in the root mean square deviation, indicating that the system structure has not changed significantly.

[0041] As Figure 5 and Figure 6 shown, the results of the weak interaction analysis of the system indicate that there are obvious van der Waals interactions in the system. The distance between the molecules of the compound shown in Formula 1 and the compound shown in Formula 2 is 5 nm - 7 nm, which perfectly matches the distance requirement for the occurrence of fluorescence resonance energy transfer.

[0042] Example 3 Use the molecular dynamics simulation software GROMACS to optimize the quantitative relationship between the compound shown in Formula 1 and the compound shown in Formula 2 during the synthesis of near-infrared nanoparticles by the microemulsion method: First, build a molecular dynamics simulation box with a size of 9 nm × 9 nm × 9 nm. Add the compound shown in Formula 1 and the compound shown in Formula 2 with an optimized molecular ratio of 3:1 into it, and then perform solvation treatment using the spc216 water model; adopt three-dimensional periodic boundary conditions (PBC) for the simulation box, use a cutoff distance of 1.2 nm when calculating short-range electrostatic interactions and van der Waals interactions, use the particle mesh Ewald method to handle long-range electrostatic interactions (PME), and use the LINCS algorithm to constrain the bonds involving hydrogen atoms; use the steepest descent algorithm to minimize the energy to relax the structure, and then perform a 10 ns equilibrium phase (T = 300 K) simulation. After equilibrium, perform a 100 ns production run; collect the kinetic data of the last 50 ns to analyze the stability, weak interactions, and distance relationship of the system.

[0043] The simulation results of the compound shown in Formula 1 and the compound shown in Formula 2 with a molecular ratio of 3:1 are as Figure 7 shown. The kinetic data of the last 50 ns indicate that the system has low energy and good stability.

[0044] Figure 8 Show the simulation snapshots of the system. It can be seen that the nanoparticles are significantly spherical and overall uniform and stable.

[0045] Example 4 Prepare near-infrared nanoparticles according to the following steps: Dissolve 0.003 g of the compound shown in Formula 1, 0.001 g of the compound shown in Formula 2, and 0.006 g of poly(maleic anhydride-alt-1-octadecene) in 1 mL of chloroform to obtain an oil-phase solution; dissolve 0.002 g of sodium dodecyl sulfate in 1 mL of deionized water to obtain an aqueous-phase solution; mix 0.15 mL of the oil-phase solution and 0.4 mL of the aqueous-phase solution, and obtain a crude emulsion by ultrasonic oscillation; place the crude emulsion in a cell disruptor and ultrasonically emulsify it for 5 min (on for 3.3 s, off for 4.4 s) at room temperature with an emulsification power of 80 w to obtain a microemulsion; redissolve the obtained microemulsion in 2 mL of ultrapure water, and then rotary evaporate it for 15 min at 0.08 MPa to remove chloroform in the microemulsion; after the rotary evaporation, centrifuge and wash the obtained microemulsion (13500 rpm, 25 min, 4 °C), wash it twice with ultrapure water, remove the supernatant to obtain a solid substance; redissolve the solid substance in 1 mL of sodium hydroxide solution (pH = 11), hydrolyze it overnight, and after the hydrolysis is completed, wash it twice with ultrapure water (13500 rpm, 25 min, 4 °C) to obtain near-infrared nanoparticles.

[0046] Among them, the compound shown in Formula 1 is obtained by the following method: heat a mixture of 4,7-bis(4-bromophenyl)-2,1,3-benzothiadiazole (0.5 mmol), N-(4-(1,2,2-triphenylethynyl)phenyl)anthracen-1-amine (1.5 mmol), Cs2CO3 (1.14 g, 3.5 mmol), Pd(OAc)2 (11.2 mg, 0.05 mmol), P(t-Bu)3 (30.3 mg, 0.15 mmol) and toluene (40 mL), react at 40 °C for 2 hours; then heat the reaction mixture at 110 °C for 24 hours, cool to room temperature, add water (30 mL) and chloroform (300 mL) to the reaction mixture and separate the organic layer, and then wash the organic layer with brine; dry the organic layer with anhydrous MgSO4, then evaporate the organic layer to dryness under reduced pressure, and finally purify the crude product by column chromatography on silica gel using n-hexane / toluene as the eluent to obtain the compound shown in Formula 1. The 1H NMR spectrum and high-resolution mass spectrum of the compound shown in Formula 1 are as Figure 9 and Figure 10 shown.

[0047] The compound shown in Formula 2 was prepared according to the method described in the prior art literature (Incorporation of Planar Blocks into Twisted Skeletons: Boosting Brightness of Fluorophores for Bioimaging beyond 1500 Nanometer[J].ACS Nano, 2020, 14(10):14228-14239.DOI:10.1021 / acsnano.0c07527.).

[0048] Figure 11 It is the electron microscopy image of the prepared near-infrared nanoparticles. It can be seen that the near-infrared nanoparticles have uniform particle sizes, about 210 nm.

[0049] Figure 12 It is the fluorescence intensity of the near-infrared nanoparticles prepared in this example (after adding the compound shown in Formula 1) and the nanoparticles prepared without adding the compound shown in Formula 1 (before adding the compound shown in Formula 1). It can be seen that due to the FRET phenomenon occurring between the molecules of the compound shown in Formula 1 and the compound shown in Formula 2, the near-infrared fluorescence signal of the near-infrared nanoparticles prepared in this example at 940 nm increased by 12.36 times.

[0050] Example 5 Prepare the near-infrared fluorescence probe according to the following steps: Add 10 mL of PBS (phosphate buffer, pH = 7.0, 0.01 M) to a 25 mL clean flask. Under stirring conditions, add 1 mg of the near-infrared nanoparticles prepared in Example 4, 1000 μg of EDC (1-ethyl-3-(dimethylamino)propylcarbodiimide hydrochloride), and 720 μg of anti-AFB1 monoclonal antibody. Stir at 37 °C for 60 min, then add 1 mL of 10% (w / v) BSA (bovine serum albumin) solution, and block at room temperature for 1 h to obtain the near-infrared fluorescence labeling reaction solution; centrifuge the above reaction solution at 13500 rpm for 25 min, and redisperse the precipitate in 2 mL of probe complex solution (PBS buffer, pH = 7.4, 0.01 M, containing 25 wt% sucrose, 1 wt% BSA, 0.5 wt% polyethylene glycol 2000, 0.5 wt% Tween 20) to obtain the near-infrared fluorescence probe.

[0051] As Figure 13 shown, after the near-infrared nanoparticles were conjugated with anti-AFB1 monoclonal antibody, the particle size increased from 210 nm to 230 nm, and the potential increased from -47 mV to -25 mV, indicating the successful conjugation of the near-infrared nanoparticles with anti-AFB1 monoclonal antibody.

[0052] Example 6 Prepare a test strip for detecting aflatoxin B1 according to the following steps: Spray the near-infrared fluorescent probe prepared in Example 5 on the conjugate pad at a spraying amount of 5 μL / cm, and vacuum dry at 37°C for 2 h; spray the AFB1 complete antigen (0.4 mg / mL) and goat anti-mouse secondary antibody (1 mg / mL) on the nitrocellulose membrane as the test line (T line) and the quality control line (C line) respectively, and dry the nitrocellulose membrane at 65°C for 12 h; paste the nitrocellulose membrane, the conjugate pad, and the absorbent paper on the PVC bottom plate in sequence, and use an automatic strip cutter to cut it into test strips with a width of 3.9 mm, and dry at room temperature to obtain a test strip for detecting aflatoxin B1.

[0053] Example 7 Detect AFB1 in soy sauce samples according to the following steps: Prepare 8 portions of 60 μL soy sauce samples, add AFB1 to 7 of them respectively, so that the AFB1 concentrations in the 8 soy sauce samples are 0 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1000 ng / mL respectively; add these soy sauce samples to the sample addition holes of the test strips (prepared according to the method of Example 6) placed horizontally, after reacting for 15 min, use a test strip reader to read the fluorescence intensities of the T and C lines of the test strip. If both the T and C lines of the test strip show color, it is a negative result; if the T line of the test strip does not show color and the C line shows color, it is a positive result; if the C line does not show a fluorescent band, the detection is invalid.

[0054] Figure 14 shows the fluorescence changes of the T and C lines with concentration during the test strip detection, indicating that the near-infrared fluorescent probe can achieve highly sensitive detection when used in immunochromatographic test strips for detecting AFB1.

[0055] Example 8 Evaluate the detection sensitivity of the near-infrared fluorescent probe according to the following steps: Dilute the AFB1 antigen stock solution with a concentration of 100 ng / mL to a series of concentration gradients (0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1000 ng / mL), take 60 μL of the near-infrared fluorescent probe and add it to 300 μL of the spiked soy sauce sample, incubate at 37°C for 2 min, and then take 60 μL and drop it into the sample hole of the test strip (prepared according to the method of Example 6), and read the fluorescence intensity I of the T line after the chromatographic reaction for 15 min T and the fluorescence intensity I of the C lineC ; With I T / I C as the ordinate and the logarithm of the AFB1 concentration as the abscissa, plot the quantitative standard curve for detecting AFB1 with the near-infrared fluorescence probe, and investigate the sensitivity and linear range of the quantitative detection.

[0056] The results are as Figure 15 shown. The lowest detection limit for detecting AFB1 with the test strip is 0.01 ng / mL, and the detection linear range is 0.01 ng / mL - 1000 ng / mL.

[0057] Example 9 Evaluate the detection specificity of the near-infrared fluorescence probe according to the following steps: Prepare solutions of AFB1 (1 ng / mL), FB1 (fumonisin B1, 1 ng / mL), ZEN (zearalenone, 1 ng / mL), OTA (ochratoxin A, 1 ng / mL), and DON (vomitoxin, 1 ng / mL) with PBS buffer (0.01 M, pH 7.4); use the test strip (prepared according to the method of Example 6) to detect the mycotoxins in these solutions, and compare the I T / I C values of each solution to evaluate the specificity of the test strip.

[0058] The results are as Figure 16 shown. There is no obvious cross-reaction between the test strip for detecting AFB1 and other common mycotoxins, and it has good specificity.

[0059] As mentioned above, it is only the preferred embodiment of the present invention. The present invention is not limited to the above-mentioned embodiments. As long as it achieves the technical effects of the present invention by the same or equivalent means, it should fall within the protection scope of the present invention. Within the protection scope of the present invention, various different modifications and changes can be made to its technical solutions and / or embodiments.

Claims

1. A preparation method of a near-infrared fluorescent probe, characterized in that, It includes the following steps: Mix the compound shown in Formula 1, the compound shown in Formula 2, poly(maleic anhydride-alt-1-octadecene), and an organic solvent to obtain an oil-phase solution; Mix a surfactant and water to obtain an aqueous-phase solution; Mix the oil-phase solution and the aqueous-phase solution, and carry out emulsification to obtain an emulsion; After removing the organic solvent from the emulsion, carry out centrifugal washing to obtain a solid substance; Hydrolyze the solid substance in an alkaline solution, and wash with water to obtain near-infrared nanoparticles; Couple the near-infrared nanoparticles with anti-aflatoxin B1 monoclonal antibody to obtain a near-infrared fluorescent probe.

2. The method according to claim 1, wherein The preparation method of the near-infrared fluorescent probe further includes the following steps: Calculate the energy donor and energy acceptor molecules through the quantum chemistry calculation software ORCA to determine the compound shown in Formula 1 and the compound shown in Formula 2 for synthesizing near-infrared nanoparticles; Guide the synthesis of near-infrared nanoparticles through the molecular dynamics simulation software GROMACS to determine the dosage ratio of the compound shown in Formula 1 and the compound shown in Formula 2.

3. The method according to claim 1, characterized in that The mass ratio of the compound shown in Formula 1, the compound shown in Formula 2, and poly(maleic anhydride-alt-1-octadecene) is 1-5:1:2-10.

4. The method according to claim 1, characterized in that The organic solvent is dichloromethane, chloroform or carbon tetrachloride.

5. The method according to claim 1, characterized in that The surfactant is sodium dodecyl sulfate or sodium dodecylbenzenesulfonate.

6. The method according to claim 1, characterized in that, The volume ratio of the oil-phase solution to the aqueous-phase solution is 1-5:

8.

7. The method according to claim 1, characterized in that, The mass ratio of the near-infrared nanoparticles to the anti-aflatoxin B1 monoclonal antibody is 1:0.5-1.

8. A near-infrared fluorescent probe, characterized in that, It is obtained by the preparation method of the near-infrared fluorescent probe according to any one of claims 1-7.

9. Use of the near-infrared fluorescent probe according to claim 8, characterized in that, The near-infrared fluorescent probe is used for detecting aflatoxin B1.

10. A test strip for detecting aflatoxin B1, characterized in that, It includes the near-infrared fluorescent probe according to claim 8.

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