A near-infrared fluorescent probe and its application in the detection of aflatoxin B1

Abstract

The present invention relates to the field of substance detection technology, and more particularly to a near-infrared fluorescent probe and its application in the detection of aflatoxin B1. In the present invention, quantum chemical calculation software and molecular dynamics simulation are used to guide the synthesis of near-infrared nanoparticles, and near-infrared nanoparticles are prepared by a microemulsion method. The near-infrared nanoparticles have uniform particle size, good stability, and can emit ultra-high brightness fluorescent signals. By coupling the near-infrared nanoparticles with anti-aflatoxin B1 monoclonal antibodies, they can be used as near-infrared fluorescent signal probes for the detection of aflatoxin B1. Because the near-infrared nanoparticles can achieve short-wave excitation and have a large Stokes shift, they can effectively eliminate interference between excitation light and emitted light, avoiding autofluorescence disturbances. The near-infrared fluorescent signal probe prepared therefrom can significantly improve detection sensitivity.

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 potent carcinogen produced by Aspergillus flavus and Aspergillus parasiticus. Long-term exposure can cause serious health hazards, particularly liver damage. It binds to DNA and triggers gene mutations, which can lead to malignancies such as liver cancer. It can also cause immunosuppression, developmental delays, 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). HPLC offers high sensitivity and accuracy, but the equipment is expensive and the operation is complex. ELISA is easy to operate and low-cost, making it suitable for large-scale screening, but its sensitivity is relatively low. GC-MS has high sensitivity and specificity and can detect multiple toxins simultaneously, but it is expensive and requires strict technical skills. FICA offers advantages such as ease of operation, rapid detection, and suitability for grassroots use, making it a popular method for rapid on-site screening. However, it suffers from 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 at least solves one problem in the prior art.

[0005] In a first aspect, the present invention provides a method for preparing a near-infrared fluorescent probe, comprising the following steps:

[0006] Mixing the compound represented by Formula 1, the compound represented by Formula 2, poly(maleic anhydride-alt-1-octadecene), and an organic solvent to obtain an oil phase solution;

[0007]

[0008]

[0009] mixing a surfactant and water to obtain an aqueous phase solution;

[0010] The oil phase solution and the aqueous phase solution are mixed and emulsified to obtain an emulsion;

[0011] After removing the organic solvent from the emulsion, centrifugation and washing are performed to obtain a solid substance;

[0012] hydrolyzing the solid substance in an alkaline solution and washing with water to obtain near-infrared nanoparticles;

[0013] The near-infrared nanoparticles and anti-aflatoxin B1 monoclonal antibodies are coupled to obtain a near-infrared fluorescent probe.

[0014] The compound represented by Formula 1 and Formula 2 has a high degree of energy level matching, endowing the near-infrared nanoparticles with excellent fluorescence properties. When coupled with an anti-aflatoxin B1 monoclonal antibody, the near-infrared nanoparticles exhibit extremely high specificity and sensitivity for the detection of aflatoxin B1.

[0015] In some optional embodiments, the method for preparing the near-infrared fluorescent probe further comprises the following steps:

[0016] The energy donor and energy acceptor molecules were calculated using the quantum chemical calculation software ORCA to determine the compound shown in Formula 1 and the compound shown in Formula 2 for synthesizing near-infrared nanoparticles;

[0017] The molecular dynamics simulation software GROMACS was used to guide the synthesis of near-infrared nanoparticles, and the dosage ratio of the compound represented by Formula 1 and the compound represented by Formula 2 was determined.

[0018] In some optional embodiments, the mass ratio of the compound represented by Formula 1, the compound represented by Formula 2, and the poly(maleic anhydride-alt-1-octadecene) is 1-5:1:2-10. Preferably, the mass ratio of the compound represented by Formula 1, the compound represented by Formula 2, and the poly(maleic anhydride-alt-1-octadecene) is 3-5:1:6-10. Most preferably, the mass ratio of the compound represented by Formula 1, the compound represented by Formula 2, and the poly(maleic anhydride-alt-1-octadecene) is 3:1:8.

[0019] In some optional embodiments, the organic solvent is dichloromethane, chloroform or carbon tetrachloride. Preferably, the organic solvent is chloroform.

[0020] In some optional embodiments, the surfactant is sodium lauryl sulfate or sodium dodecylbenzene sulfonate. Preferably, the surfactant is sodium lauryl sulfate.

[0021] In some optional embodiments, the volume ratio of the oil phase solution to the water phase solution is 1-5:8. Preferably, the volume ratio of the oil phase solution to the water phase solution is 3:8.

[0022] In some optional embodiments, the alkaline solution is a sodium hydroxide solution.

[0023] In some optional embodiments, the mass ratio of the near-infrared nanoparticles to the anti-aflatoxin B1 monoclonal antibody is 1:0.5-1. Preferably, the mass ratio of the near-infrared nanoparticles to the anti-aflatoxin B1 monoclonal antibody is 1:0.6-0.8. Most preferably, the mass ratio of the near-infrared nanoparticles to the anti-aflatoxin B1 monoclonal antibody is 1:0.72.

[0024] In a second aspect, the present invention provides a near-infrared fluorescent probe, which is obtained by the above-mentioned preparation method of the near-infrared fluorescent probe.

[0025] In a third aspect, the present invention provides an application of the above-mentioned near-infrared fluorescent probe, wherein the above-mentioned near-infrared fluorescent probe is used to detect aflatoxin B1.

[0026] In a fourth aspect, the present invention provides a test strip for detecting aflatoxin B1, which includes the above-mentioned near-infrared fluorescent probe.

[0027] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects:

[0028] Quantum chemical calculation software and molecular dynamics simulation were used to guide the synthesis of near-infrared nanoparticles, and near-infrared nanoparticles were prepared by the microemulsion method. The near-infrared nanoparticles have uniform particle size, good stability, and can emit ultra-high brightness near-infrared fluorescence signals. By coupling the near-infrared nanoparticles with anti-aflatoxin B1 monoclonal antibodies, they can be used as near-infrared fluorescence signal probes 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 and avoid self-fluorescence disturbances. The near-infrared fluorescence signal probes prepared from them can greatly improve the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the principle of detecting AFB1 using a near-infrared fluorescent probe in an embodiment of the present invention.

[0030] Figure 2 The optimal molecular structure and HOMO and LUMO energy levels obtained by quantum chemical calculations in the embodiments of the present invention are shown.

[0031] Figure 3 The figure shows the system potential energy results obtained using molecular dynamics simulation software in the embodiment of the present invention.

[0032] Figure 4 The root mean square deviation results obtained using molecular dynamics simulation software in the embodiments of the present invention are shown.

[0033] Figure 5This is a scatter plot of weak interaction analysis obtained using molecular dynamics simulation software in an embodiment of the present invention.

[0034] Figure 6 The figure shows the visualization results of weak interactions obtained using molecular dynamics simulation software in an embodiment of the present invention.

[0035] Figure 7 The total energy of the system obtained using molecular dynamics simulation software in the embodiment of the present invention is shown.

[0036] Figure 8 The snapshot results of the system simulation obtained using molecular dynamics simulation software in the embodiment of the present invention are shown.

[0037] Figure 9 1 is the hydrogen nuclear magnetic resonance spectrum of the compound represented by formula 1 in the embodiment of the present invention.

[0038] Figure 10 This is a high-resolution mass spectrum of the compound represented by Formula 1 in the embodiment of the present invention.

[0039] Figure 11 This is an electron microscope image of the near-infrared nanoparticles in the embodiment of the present invention.

[0040] Figure 12 The fluorescence intensity of the near-infrared nanoparticles in the embodiment of the present invention (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) is shown.

[0041] Figure 13 The diagram shows the coupling of near-infrared nanoparticles and anti-AFB1 monoclonal antibodies in an embodiment of the present invention.

[0042] Figure 14 This is a real picture of the near-infrared fluorescent probe detecting AFB1 in an embodiment of the present invention.

[0043] Figure 15 The detection limit results of the near-infrared fluorescent probe in the examples of the present invention are shown.

[0044] Figure 16 The specific detection results of the near-infrared fluorescent probe in the embodiment of the present invention are shown. DETAILED DESCRIPTION

[0045] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effects of the present invention.

[0046] According to an embodiment of the present invention, the synthesis of near-infrared nanoparticles is first theoretically guided by quantum chemical calculations and molecular dynamics simulations, and then the near-infrared nanoparticles are prepared by a microemulsion method. Then, the near-infrared nanoparticles are coupled with anti-aflatoxin B1 monoclonal antibodies to obtain a near-infrared fluorescent probe. Aflatoxin B1 is detected using the near-infrared fluorescent probe, thereby improving the specificity and sensitivity of detecting aflatoxin B1.

[0047] Example 1

[0048] Using quantum chemical calculation software, density functional theory was used to optimize the molecular structures of the energy donor and energy acceptor. Their HOMO and LUMO energy levels were calculated to demonstrate energy level matching, and initial structures were obtained for molecular dynamics simulations. ORCA was used, using the B3LYP functional and the 6-311G (d, p) basis set. The calculations included molecular structure and frequency optimization, electrostatic potential mapping, HOMO and LUMO energy level calculations, and energy level matching between the energy donor and energy acceptor. Ultimately, the compound shown in Formula 1 was determined to be the energy donor, and the compound shown in Formula 2 was determined to be the energy acceptor.

[0049]

[0050]

[0051] like Figure 2 As shown, the HOMO energy level of the compound represented by Formula 1 is -4.98 eV and the LUMO energy level is -2.44 eV; the HOMO energy level of the compound represented by Formula 2 is -4.97 eV and the LUMO energy level is -3.51 eV. In comparison, the compound represented by Formula 1 has a lower HOMO energy level, and the compound represented by Formula 2 has a lower LUMO energy level, indicating that the combination of the compound represented by Formula 1 and the compound represented by Formula 2 perfectly matches the conditions for fluorescence resonance energy transfer.

[0052] Example 2

[0053] The molecular dynamics simulation software GROMACS was used to verify the distance requirement for fluorescence resonance energy transfer between the compound represented by Formula 1 and the compound represented by Formula 2:

[0054] First, a molecular dynamics simulation box of size 4 nm × 4 nm × 4 nm was built, into which the compound represented by Formula 1 and the compound represented by Formula 2 with a molecular number ratio of 1:1 after structural optimization were added. Then, the spc216 water model was used for solvation treatment. Three-dimensional periodic boundary conditions (PBC) were applied to the simulation box, and a cutoff distance of 1.2 nm was used for calculating short-range electrostatic interactions and van der Waals interactions. The particle mesh Ewald method was used to treat long-range electrostatic interactions (PME), and the LINCS algorithm was used to constrain bonds involving hydrogen atoms. The steepest descent algorithm was used for energy minimization to relax the structure, followed by a 10 ns equilibrium phase (T = 300 K) simulation, and a 100 ns production run after equilibrium. The final 50 ns of kinetic data were collected to analyze the stability, weak interactions, and distance relationships of the system.

[0055] like Figure 3 As shown, the analysis of potential energy shows that the system is overall stable, providing a reliable guarantee for subsequent data analysis.

[0056] like Figure 4 As shown, there is no drastic fluctuation in the RMS deviation, indicating that the architecture has not changed significantly.

[0057] like Figure 5 and Figure 6 As shown, the results of weak interaction analysis of the system indicate that there is an obvious van der Waals interaction in the system, and the distance between the molecules of the compound represented by Formula 1 and the compound represented by Formula 2 is 5 nm-7 nm, which perfectly matches the distance requirement for fluorescence resonance energy transfer to occur.

[0058] Example 3

[0059] The molecular dynamics simulation software GROMACS was used to optimize the quantitative relationship between the compound represented by Formula 1 and the compound represented by Formula 2 when synthesizing near-infrared nanoparticles by the microemulsion method:

[0060] First, a molecular dynamics simulation box with a size of 9 nm×9 nm×9 nm was built, and the compound represented by Formula 1 and the compound represented by Formula 2 with a molecular number ratio of 3:1 after structural optimization were added into it. Then, the spc216 water model was used for solvation treatment. Three-dimensional periodic boundary conditions (PBC) were applied to the simulation box, and a cutoff distance of 1.2 nm was used for calculating short-range electrostatic interactions and van der Waals interactions. The particle mesh Ewald method was used to treat long-range electrostatic interactions (PME), and the LINCS algorithm was used to constrain bonds involving hydrogen atoms. The steepest descent algorithm was used for energy minimization to relax the structure, followed by a 10 ns equilibrium phase (T = 300 K) simulation, and a 100 ns production run after equilibrium. The final 50 ns of kinetic data were collected to analyze the stability, weak interactions, and distance relationships of the system.

[0061] The simulation results of the compound represented by formula 1 and the compound represented by formula 2 with a molecular ratio of 3:1 are shown in FIG. Figure 7 As shown in Figure 3, the kinetic data of the last 50 ns indicate that the system has low energy and good stability.

[0062] Figure 8 The simulation snapshots of the system are shown, showing that the nanoparticles are obviously spherical and overall uniform and stable.

[0063] Example 4

[0064] Prepare near-infrared nanoparticles as follows:

[0065] 0.003 g of the compound represented by formula 1, 0.001 g of the compound represented by formula 2, and 0.006 g of poly(maleic anhydride-alt-1-octadecene) were dissolved in 1 mL of chloroform to obtain an oil phase solution. 0.002 g of sodium dodecyl sulfate was dissolved in 1 mL of deionized water to obtain an aqueous phase solution. 0.15 mL of the oil phase solution and 0.4 mL of the aqueous phase solution were mixed and ultrasonically vibrated to obtain a crude emulsion. The crude emulsion was ultrasonically emulsified in a cell disruptor at room temperature for 5 min (on 3.3 s, off 4.4 s) at an emulsification power of 80 W to obtain a microemulsion. The obtained microemulsion was redissolved in 2 mL of ultrapure water and then rotary evaporated at 0.08 MPa for 15 min to remove the chloroform in the microemulsion. After the rotary evaporation, the obtained microemulsion was centrifuged (13500 rpm, 25 min, 4°C), washed twice with ultrapure water, and the supernatant was removed to obtain a solid. The solid was redissolved in 1 mL of sodium hydroxide solution (pH 7.0). = 11), hydrolyzed overnight, and washed twice with ultrapure water (13500 rpm, 25 min, 4 °C) to obtain near-infrared nanoparticles.

[0066] The compound represented by Formula 1 was obtained by the following method: a mixture of 4,7-bis(4-bromophenyl)-2,1,3-benzothiadiazole (0.5 mmol), N-(4-(1,2,2-triphenylvinyl)phenyl)anthracene-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) was heated at 40°C for 2 hours; the reaction mixture was then heated at 110°C for 24 hours, cooled to room temperature, water (30 mL) and chloroform (300 mL) were added to the reaction mixture, the organic layer was separated, and the organic layer was then washed with brine; the organic layer was dried over anhydrous MgSO4, and then evaporated to dryness under reduced pressure. Finally, the crude product was purified by column chromatography on silica gel using n-hexane / toluene as the eluent to obtain the compound represented by Formula 1. The H NMR spectrum and high-resolution mass spectrum of the compound shown in Formula 1 are as follows: Figure 9 and Figure 10 shown.

[0067] The compound represented by 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.).

[0068] Figure 11 This is an electron microscope image of the prepared near-infrared nanoparticles. It can be seen that the particle size of the near-infrared nanoparticles is uniform, about 210 nm.

[0069] Figure 12 : This is the fluorescence intensity of the near-infrared nanoparticles prepared in this example (after adding the compound represented by Formula 1) and the nanoparticles prepared without adding the compound represented by Formula 1 (before adding the compound represented by Formula 1). It can be seen that due to the FRET phenomenon between the molecules of the compound represented by Formula 1 and the compound represented by Formula 2, the near-infrared fluorescence signal of the near-infrared nanoparticles prepared in this example at 940 nm increased by 12.36 times.

[0070] Example 5

[0071] Prepare the near-infrared fluorescent probe as follows:

[0072] 10 mL of PBS (phosphate buffer, pH = 7.0, 0.01 M) was added to a 25 mL clean flask. 1 mg of the near-infrared nanoparticles prepared in Example 4, 1000 μg of EDC (1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride), and 720 μg of anti-AFB1 monoclonal antibody were added under stirring. The mixture was stirred at 37°C for 60 min, and then 1 mL of 10% (w / v) BSA (bovine serum albumin) solution was added. The mixture was blocked at room temperature for 1 h to obtain a near-infrared fluorescent marker reaction solution. The reaction solution was centrifuged at 13500 rpm for 25 min, and the precipitate was redispersed in 2 mL of probe reconstitution solution (PBS buffer, pH = 7.4, 0.01 M, containing 25 wt% sucrose, 1 wt% BSA, 0.5 wt% polyethylene glycol 2000, and 0.5 wt% Tween 20) to obtain a near-infrared fluorescent probe.

[0073] like Figure 13 As shown in the figure, after the near-infrared nanoparticles were coupled with the 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 that the near-infrared nanoparticles were successfully coupled with the anti-AFB1 monoclonal antibody.

[0074] Example 6

[0075] Follow these steps to prepare aflatoxin B1 test strips:

[0076] The near-infrared fluorescent probe prepared in Example 5 was sprayed onto a conjugate pad at a spraying amount of 5 μL / cm and dried in vacuo at 37°C for 2 h. AFB1 complete antigen (0.4 mg / mL) and goat anti-mouse secondary antibody (1 mg / mL) were sprayed onto a nitrocellulose membrane as a test line (T line) and a quality control line (C line), respectively. The nitrocellulose membrane was dried at 65°C for 12 h. The nitrocellulose membrane, conjugate pad, and absorbent paper were sequentially adhered to a PVC base plate and cut into test strips with a width of 3.9 mm using an automatic strip cutter. The strips were dried at room temperature to obtain test strips for detecting aflatoxin B1.

[0077] Example 7

[0078] Follow the steps below to detect AFB1 in soy sauce samples:

[0079] Eight 60 μL soy sauce samples were prepared. AFB1 was added to seven of them, resulting in AFB1 concentrations of 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. These soy sauce samples were added to the sample wells of a horizontally placed test strip (prepared according to the method of Example 6). After a 15-minute reaction, the fluorescence intensity of the T and C lines of the test strip was read using a test strip reader. If both the T and C lines of the test strip showed color, the result was negative; if the T line did not show color but the C line did, the result was positive; if the C line did not show a fluorescent band, the test was invalid.

[0080] Figure 14 The fluorescence of T and C lines changes with concentration during test strip detection, indicating that the near-infrared fluorescent probe can achieve high-sensitivity detection when used in immunochromatographic test strips to detect AFB1.

[0081] Example 8

[0082] Follow these steps to evaluate the detection sensitivity of the near-infrared fluorescent probe:

[0083] A 100 ng / mL AFB1 antigen stock solution was diluted 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). 60 μL of the near-infrared fluorescent probe was added to 300 μL of spiked soy sauce sample and incubated at 37°C for 2 min. Subsequently, 60 μL was added dropwise to the sample well of the test strip (prepared according to the method of Example 6). After 15 min of chromatography reaction, the T-line fluorescence intensity I was read. T and C-line fluorescence intensity I C ; with I T / I C The vertical axis is the logarithm of the AFB1 concentration and the horizontal axis is the quantitative standard curve of the near-infrared fluorescent probe for detecting AFB1 to investigate the sensitivity and linear range of the quantitative detection.

[0084] The results are as follows Figure 15 As shown in the figure, the minimum detection limit of the test strip for AFB1 is 0.01 ng / mL, and the linear range of detection is 0.01 ng / mL-1000 ng / mL.

[0085] Example 9

[0086] Follow these steps to evaluate the detection specificity of the near-infrared fluorescent probe:

[0087] 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) solutions were prepared in PBS buffer (0.01 M, pH 7.4); the mycotoxins in these solutions were detected using test strips (prepared according to the method of Example 6), and the mycotoxin concentrations of each solution were compared. T / I C Value, to evaluate the specificity of the test strip.

[0088] The results are as follows Figure 16 As shown in the results, the test strips showed no obvious cross-reaction with other common mycotoxins and had good specificity.

[0089] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any method that achieves the technical effects of the present invention by the same or equivalent means shall fall within the scope of protection of the present invention. Within the scope of protection of the present invention, various modifications and variations of the technical solutions and / or implementation methods are possible.

Claims

1. A method for preparing a near-infrared fluorescent probe, characterized in that: The following steps are involved: Mixing the compound represented by Formula 1, the compound represented by Formula 2, poly(maleic anhydride-alt-1-octadecene), and an organic solvent to obtain an oil phase solution; mixing a surfactant and water to obtain an aqueous phase solution; mixing the oil phase solution and the aqueous phase solution, and emulsifying them to obtain an emulsion; After removing the organic solvent from the emulsion, centrifugation and washing are performed to obtain a solid substance; hydrolyzing the solid matter in an alkaline solution and washing with water to obtain near-infrared nanoparticles; The near-infrared nanoparticles are coupled with anti-aflatoxin B1 monoclonal antibodies to obtain a near-infrared fluorescent probe; Wherein, the compound represented by formula 1 is an energy donor, and the compound represented by formula 2 is an energy acceptor.

2. The method according to claim 1, characterized in that The preparation method of the near-infrared fluorescent probe further comprises the following steps: The energy donor and energy acceptor molecules were calculated using the quantum chemical calculation software ORCA to determine the compound shown in Formula 1 and the compound shown in Formula 2 for synthesizing near-infrared nanoparticles; The molecular dynamics simulation software GROMACS was used to guide the synthesis of near-infrared nanoparticles, and the dosage ratio of the compound represented by Formula 1 and the compound represented by Formula 2 was determined.

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

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

5. The method according to claim 1, wherein 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 water 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 The near-infrared fluorescent probe is obtained by the preparation method of any one of claims 1 to 7.

9. The use of the near-infrared fluorescent probe according to claim 8, characterized in that: The near-infrared fluorescent probe is used to detect aflatoxin B1.

10. A test strip for detecting aflatoxin B1, characterized in that: The method comprises the near-infrared fluorescent probe according to claim 8.

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