A Raman beacon COF nanoflower, its preparation method and application

Abstract

This application belongs to the technical field of mycotoxin detection technology, specifically relating to a Raman beacon COF nanoflower, its preparation method, and its application. The method involves dissolving trialdehyde phloroglucinol, o-toluidine, and polyvinylpyrrolidone in methanol, ultrasonically mixing, adding a catalyst for reaction, adding benzaldehyde after the reaction, allowing the mixture to stand, centrifuging, washing, and redissolving to obtain TpDBD-COF nanoflowers. These nanoflowers are then dissolved in a mixed solution of ethanol and water, followed by the addition of AgNO3 and ultrasonic adsorption. After adsorption, trisodium citrate is added for heating reaction, centrifuging, washing, and redissolving to obtain the Raman beacon COF nanoflowers. The COF nanoflowers prepared by this invention exhibit a highly enhanced and stable Raman signal, and their surface groups have the advantage of being directly used for biomolecule coupling, specifically for the sensitive detection of T-2 toxin in samples.

Description

Technical Field

[0001] This application belongs to the field of fungal toxin detection technology, specifically relating to a Raman beacon COF nanoflower, its preparation method, and its application. Background Technology

[0002] Raman scattering is a spectroscopic phenomenon based on inelastic collisions between photons and molecules. When a beam of light of a specific wavelength illuminates an analyte, a characteristic fingerprint spectrum corresponding to the vibrational energy levels of the molecules is generated in the scattered light, enabling qualitative and quantitative analysis of the substance. Similar to fluorescence signals, which depend on the intensity of the excitation light, the intensity of the Raman signal is also positively correlated with the power of the incident laser. Compared to colorimetric beacons, Raman beacons offer stronger resistance to background interference.

[0003] However, traditional Raman signal probes, such as certain organic dye molecules, have limited Raman signal intensity and are prone to photobleaching and photothermal degradation under prolonged laser irradiation, leading to signal attenuation and affecting the stability and reproducibility of the analysis. Polymer materials such as COF nanomaterials are often used as Raman beacon loading matrices; their large rigid planar structure and polarizability indicate their inherent potential for Raman scattering peaks. However, few studies have evaluated the Raman energies of COF nanomaterials themselves. Therefore, optimizing the design of Raman signal probes to achieve extremely high enhancement factors while ensuring signal reproducibility and stability has become a new challenge and an urgent problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a Raman beacon COF nanoflower, its preparation method, and its application. Specifically, the following technical solution is adopted:

[0005] In a first aspect, the present invention provides a method for preparing Raman beacon COF nanoflowers, comprising the following steps:

[0006] Trialdehyde resorcinol, o-toluidine and polyvinylpyrrolidone were dissolved in methanol, ultrasonically mixed, and a catalyst was added to carry out the reaction. After the reaction was completed, benzaldehyde was added and the reaction was allowed to continue to stand. After the reaction was completed, the mixture was centrifuged, washed and reconstituted to obtain TpDBD-COF nanoflowers.

[0007] The TpDBD-COF nanoflowers were dissolved in a mixed solution of ethanol and water, and then AgNO3 was added. The mixture was ultrasonically adsorbed, and after the adsorption was completed, trisodium citrate was added for heating reaction. After the reaction was completed, the mixture was centrifuged, washed, and reconstituted to obtain the Raman beacon COF nanoflowers.

[0008] As a further preferred embodiment, the mass ratio of the trialdehyde phloroglucinol, o-toluidine, and polyvinylpyrrolidone is 0.4-0.8:0.42-0.84:100-500. More preferably, the mass ratio of the trialdehyde phloroglucinol, o-toluidine, and polyvinylpyrrolidone is 0.8:0.84:500.

[0009] As a further preferred embodiment, the mass ratio of AgNO3 to trisodium citrate is 3-13:15-20. More preferably, the mass ratio of AgNO3 to sodium citrate is 13:20.

[0010] As a further preferred embodiment, the catalyst is acetic acid.

[0011] As a further preferred embodiment, the heating reaction temperature is 60℃-100℃, and the reaction time is 20min-40min.

[0012] Secondly, the present invention provides a Raman beacon COF nanoflower, which is prepared by the above-described preparation method.

[0013] The Raman beacon COF nanoflowers (diameter 100 nm-1000 nm) prepared by this invention have nano-Ag particles on their surface that can be directly coupled with proteins. The nanoflowers have strong Raman signals and low background interference peaks. When applied to immunochromatographic test strips, they can quantitatively detect the concentration of analytes in samples and have the characteristics of high detection sensitivity.

[0014] Thirdly, the present invention provides an immunochromatographic test strip, including a base plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper that are sequentially overlapped and pasted on the base plate;

[0015] The glass fiber pad is coated with the probe prepared by the above-mentioned Raman beacon COF nanoflower and the antibody to be labeled.

[0016] As a further preferred embodiment, the antibody to be labeled is a monoclonal antibody, polyclonal antibody, nanobody, or phage expression antibody.

[0017] Fourthly, the present invention provides the application of the above-mentioned Raman beacon COF nanoflower or the above-mentioned immunochromatographic test strip in the detection of fungal toxins.

[0018] As a further preferred embodiment, the fungal toxin includes T-2 toxin.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention prepares an Ag@TpDBD-COF nanoflower based on its own Raman signal. This Raman beacon microstructure has a highly enhanced and stable Raman signal, and its surface groups have the advantage of being directly used for biomolecule coupling. It solves the problems of photobleaching and photothermal degradation of traditional Raman beacon probe dye molecules, as well as the frequent background interference in applications. It enables highly sensitive detection of analytes in samples on test strips, specifically for the sensitive detection of T-2 toxin in samples. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The diagram shown is a schematic diagram of the molecular structure of the TpDBD-COF nanoflowers prepared in Example 1.

[0023] Figure 2 The image shown is a scanning electron microscope image of TpDBD-COF and Ag@TpDBD-COF prepared in Example 1;

[0024] Figure 3 The figure shows the XPS orbital full spectrum of TpDBD-COF and Ag@TpDBD-COF prepared in Example 1, and the Ag 3d orbital spectrum in Ag@TpDBD-COF.

[0025] Figure 4 The image shows the infrared spectra of TpDBD-COF and Ag@TpDBD-COF prepared in Example 1.

[0026] Figure 5 The image shown is a scanning electron microscope image of TpDBD-COF and Ag@TpDBD-COF prepared in Example 2;

[0027] Figure 6 The Raman spectra of the nitrocellulose membrane, TpDBD-COF, Ag@TpDBD-COF, and Au@TpDBD-COF prepared in Example 1 are shown.

[0028] Figure 7 The image shown is a physical picture of the Raman COF beacon test strip prepared in Example 1 applied to the detection of T-2 toxin;

[0029] Figure 8 The figure shows the standard curve of the Raman COF beacon test strip prepared in Example 1 applied to the detection of T-2 toxin;

[0030] Figure 9 The image shown is a scanning electron microscope image of TpDBD-COF synthesized using reactants in Comparative Example 1 and Comparative Example 2 with different proportions.

[0031] Figure 10 The image shown is a scanning electron microscope image of TpDBD-COF synthesized using different solvents in Example 1 and Comparative Example 3.

[0032] Figure 11 The image shown is a physical picture of the Raman COF beacon test strip prepared in Example 1, testing its stability. Detailed Implementation

[0033] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0034] Example 1

[0035] A method for preparing Raman beacon COF nanoflowers (Ag@TpDBD-COF) specifically includes the following steps:

[0036] (1) Accurately weigh 0.8 mg of trialdehyde phloroglucinol (Tp), 0.84 mg of o-toluidine (DBD), and 500 mg of polyvinylpyrrolidone (PVP), dissolve them in 10 mL of methanol, sonicate and mix well, then add 400 μL of acetic acid as a catalyst, react at room temperature for 4 h, add 100 μL of benzaldehyde as a reaction quencher and end-capping agent, let stand for 1 h, centrifuge the reaction solution at 10000 rpm for 12 min, discard the supernatant, wash several times with acetonitrile until the supernatant is clear, redissolve the washed precipitate in 1 mL of ultrapure water, store at 4℃ to obtain TpDBD-COF nanoflowers (the schematic diagram of its structure is shown in Figure 1). Figure 1 (as shown)

[0037] (2) Take the above TpDBD-COF nanoflower solution, add 1 mL of ultrapure water, 0.4 mL of methanol, add 13 mg of AgNO3, sonicate for 20 min, then add 15 mg of trisodium citrate, react at 95℃ for 25 min, centrifuge the reaction solution at 8000 rpm for 10 min, discard the supernatant, wash several times with ultrapure water until the supernatant is clear, redissolve the washed precipitate in 1 mL of ultrapure water, store at 4℃ to obtain Ag@TpDBD-COF nanoflower.

[0038] The Ag@TpDBD-COF nanoflowers prepared above were characterized by scanning electron microscopy, infrared spectroscopy, and XPS. The results are as follows: Figure 2 , Figure 3 , Figure 4 As shown in the figure, the TpDBD-COF nanoflowers exhibit good morphology and dispersibility, with a particle size of approximately 200 nm-300 nm. The Ag@TpDBD-COF structure shows good Ag reduction, effectively encapsulating the TpDBD-COF nanoflower structure, and exhibits good dispersibility in aqueous solution. The infrared spectrum mainly reveals the bonding structure information in the compound, with visible structures located at 1540 cm⁻¹. -1 The characteristic C=C double bond infrared absorption peak generated by COF synthesis, and the 1630 cm⁻¹ peak. -1 The absorption peak of the C=O double bond in the COF indicates that Ag@TpDBD-COF was synthesized well. The characteristic double peak of Ag was observed in the XPS results, indicating the successful modification of Ag in Ag@TpDBD-COF.

[0039] Example 2

[0040] A method for preparing Raman beacon COF nanoflowers (Ag@TpDBD-COF) is described. The specific preparation process is similar to that of Example 1, except that the amounts of trialdehyde phloroglucinol (Tp) and o-toluidine (DBD) are changed to 0.4 mg and 0.42 mg, respectively, the amount of polyvinylpyrrolidone (PVP) is changed to 100 mg, and the amount of AgNO3 is changed to 3 mg. All other processes remain unchanged, and Ag@TpDBD-COF nanoflowers are obtained.

[0041] Transmission electron microscopy image of the obtained product is shown below. Figure 5 As shown, the results indicate that the synthesized TpDBD-COF nanoflowers have good morphology and dispersibility, with a particle size of about 200 nm. They are well dispersed in aqueous solution. After silver modification, the silver particles of Ag@TpDBD-COF nanoflowers are evenly distributed and located at the center of the nanoflowers, demonstrating good results.

[0042] Example 3

[0043] Raman spectra were determined using the Ag@TpDBD-COF nanoflowers prepared in Example 1. The specific measurement process is as follows:

[0044] TpDBD-COF nanoflower solutions and Ag@TpDBD-COF nanoflower solutions were dropped onto a nitrocellulose membrane, and Raman spectra were measured using an ultrafast imaging micro-Raman spectrometer (Thermo Fisher, DXR3xi) under the following conditions: excitation wavelength 785 nm, laser power 20 mW, exposure time 4 s, 10 scans, and a 50 μm gap. The measured Raman spectra are shown below. Figure 6 As shown.

[0045] The results showed that Ag@TpDBD-COF at 1604 cm⁻¹ -1 The nitrocellulose membrane exhibits a high Raman peak with no significant interference peaks in the near band, making it a suitable quantitative peak. However, the nitrocellulose membrane displays a single peak that coincides with that of Ag@TpDBD-COF, indicating that this Raman peak is easily interfered with and cannot be used for quantitative analysis. TpDBD-COF, on the other hand, shows an extremely low peak height, indicating that Ag modification significantly enhances its Raman signal, with a calculated enhancement factor reaching 10. 6 about.

[0046] Example 4

[0047] An application of a test strip based on the preparation of Raman beacon Ag@TpDBD-COF nanoflowers for the detection of T-2 toxin using an immunomarker competitive assay.

[0048] An immunochromatographic test strip includes a base plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane, and absorbent paper that are sequentially overlapped and pasted onto the base plate.

[0049] (1) Preparation of nitrocellulose membrane:

[0050] T-2 whole antigen and anti-mouse antibody were coated onto a nitrocellulose membrane: the concentration of T-2 whole antigen was diluted to 1 mg / mL with 0.01M pH 7.5 PBS (phosphate buffer), and the resulting solution was sprayed onto the membrane as a test line; the concentration of anti-mouse antibody was diluted to 0.5 mg / mL, and the resulting solution was sprayed onto the membrane as a control line. The spray volume for both lines was 0.74 μL / cm. The test line was 10 mm away from the top edge of the membrane, and the two lines were 5 mm apart. The membrane was dried at 37°C for 12 h and stored in a desiccator for later use.

[0051] (2) Preparation of Raman beacon Ag@TpDBD-COF nanoflower probe:

[0052] 100 μL of the prepared Raman beacon Ag@TpDBD-COF nanoflower was added to 900 μL of 0.1M PBS buffer, and 4 μL of 1M hydrochloric acid was added to adjust the pH. Then, 5 μg of T-2 monoclonal antibody was added, mixed well, and incubated for 30 min. After that, 100 μL of 1% casein blocking agent was added, and incubated for 30 min. The precipitate was collected by centrifugation and dispersed and reconstituted with 100 μL of PBS to prepare the Raman beacon Ag@TpDBD-COF nanoflower probe. The probe was sprayed onto a glass fiber pad at a volume of 3 μL / cm, vacuum dried for 2 h, and stored in a desiccator for later use.

[0053] (3) Establishment of standard curve based on Raman beacon Ag@TpDBD-COF nanoflower T-2 toxin immunochromatographic test strip:

[0054] To prepare the standard curve for the test strips: Paclobutrazol was spiked into the negative matrix at concentrations of 0, 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL.

[0055] Figure 7 The image shows the T-2 toxin immunochromatographic test strip based on the Raman beacon Ag@TpDBD-COF nanoflowers of this invention applied to the detection of T-2. The detection limit of the test strip shows the corresponding Raman signals at different concentrations. As the concentration of T-2 toxin increases, the Raman signal gradually weakens, indicating that the application of the Raman beacon Ag@TpDBD-COF nanoflowers based on their own Raman signal as a signal source on the test strip is effective in detecting T-2 toxin. Figure 8 The standard curve equation based on the Raman beacon Ag@TpDBD-COF nanoflower T-2 toxin immunochromatographic test strip was presented, and the detection limit was calculated to be 0.082 ng / mL based on negative samples.

[0056] Example 5

[0057] The recovery rate of T-2 toxin spiked in wheat matrix was determined using the Raman beacon Ag@TpDBD-COF nanoflower T-2 toxin immunochromatographic test strip prepared in Example 4. The specific detection process is as follows:

[0058] The difference from Example 4 is that a negative wheat sample extract was used as the sample matrix. First, 1 g of wheat sample powder was weighed, spiked with different concentrations, and then extracted directly with 5 mL of 0.1 M PBS buffer. After thorough extraction by shaking, the sample was centrifuged at 10000 rpm for 10 min. 100 μL of the extract was then directly analyzed using the Raman beacon-based Ag@TpDBD-COF nanoflower T-2 toxin immunochromatographic test strip prepared in Example 3. Raman signals were acquired using a DXR3xi micro Raman spectrometer (Thermo Fisher, USA) with a laser power of 20 mW, an exposure time of 4 s, 10 scans, and a 50 μm slit. The concentration values ​​were calculated using the standard curve established in Example 4, and the recovery rate was calculated based on the spiked amount. Precision was calculated by measuring parallel samples, with all other parameters and steps remaining unchanged. The detected recovery rates are shown in Table 1.

[0059] Table 1. Spike recovery rate of wheat negative samples

[0060]

[0061] As shown in the table above, the recovery rate is between 70% and 120%, and the test precision is less than 8.87, which meets the methodological requirements. This indicates that the established Raman beacon-based Ag@TpDBD-COF nanoflower T-2 toxin immunochromatographic test strip has practical application value.

[0062] Example 6

[0063] An immunochromatographic test strip based on Raman beacon Ag@TpDBD-COF nanoflower T-2 toxin was used to evaluate the stability of T-2 toxin in wheat matrix. The specific detection process is as follows:

[0064] The specific process is similar to that in Example 4, except that the established immunochromatographic test strip was used to perform 16 repeated determinations on wheat samples spiked with 10 μg / Kg T-2 toxin. The extraction procedure and test parameters were the same as in Example 4, and the results are as follows: Figure 11 As shown, the relative standard deviation (RSD) of the 16 measured results was only 4.18%, which indicates that the established Raman beacon-based Ag@TpDBD-COF nanoflower T-2 toxin immunochromatographic test strip has excellent repeatability and application value.

[0065] Comparative Example 1

[0066] A method for preparing Raman beacon COF nanoflowers (Ag@TpDBD-COF) is described. The specific preparation process is similar to that of Example 1, except that the amounts of trialdehyde phloroglucinol (Tp) and o-toluidine (DBD) are changed to 0.2 mg and 0.3 mg, respectively, while the rest of the process remains unchanged, to obtain Ag@TpDBD-COF nanoflowers.

[0067] Comparative Example 2

[0068] A method for preparing Raman beacon COF nanoflowers (Ag@TpDBD-COF) is described. The specific preparation process is similar to that of Example 1, except that the amounts of trialdehyde phloroglucinol (Tp) and o-toluidine (DBD) are changed to 1.0 mg and 1.2 mg, respectively, while the rest of the process remains unchanged, to obtain Ag@TpDBD-COF nanoflowers.

[0069] Transmission electron micrographs of the products obtained in Comparative Example 1 and Comparative Example 2 are shown below. Figure 9 As shown in the figure. The results indicate that when the dosage is 0.2 mg / 0.3 mg (meaning the dosages of trialdehyde phloroglucinol (Tp) and o-toluidine (DBD) are 0.2 mg and 0.3 mg, respectively), the yield is extremely low and it is difficult to enrich by centrifugation and washing, thus lacking the significance for further modification. When the dosage is 1.0 mg / 1.2 mg (meaning the dosages of trialdehyde phloroglucinol (Tp) and o-toluidine (DBD) are 1.0 mg and 1.2 mg, respectively), transmission electron microscopy reveals that the sample is difficult to disperse, and large particles (particle size > 2 μm) visible to the naked eye appear during the reaction process, indicating extremely poor dispersibility and making further application difficult.

[0070] Comparative Example 3

[0071] A method for preparing Raman beacon COF nanoflowers (Ag@TpDBD-COF) is described. The specific preparation process is similar to that of Example 1, except that dichloromethane and chloroform are used instead of methanol as the synthesis solvent. All other processes remain unchanged to obtain Ag@TpDBD-COF nanoflowers.

[0072] Transmission electron microscopy image of the obtained product is shown below. Figure 10 As shown, the results indicate that dichloromethane and chloroform, when used as solvents, produce products with irregular spherical shapes, and it is difficult to completely wash away polyvinylpyrrolidone (PVP) from the products, resulting in blurred results in the electron microscope and the presence of obvious colloidal complexes. Therefore, methanol is the optimal solvent for the preparation process of this invention.

[0073] Comparative Example 4

[0074] A method for preparing Raman beacon COF nanoflowers (Au@TpDBD-COF) is disclosed. The specific preparation process is similar to that of Example 1, except that AgNO3 in Example 1 is replaced with HAuCl4 to synthesize Au@TpDBD-COF nanoflowers. The Raman spectra of the Au@TpDBD-COF nanoflowers were then measured, and the results are as follows: Figure 6 As shown, the results indicate that the signal is basically consistent with that of Ag@TpDBD-COF nanoflowers, but its intensity is significantly lower than that of Ag@TpDBD-COF nanoflowers. Therefore, it can be concluded that the Ag@TpDBD-COF nanoflowers prepared by this invention have the best effect.

[0075] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing a Raman beacon COF nanoflower, characterized in that, The method comprises the following steps: TpDBD-COF nanoflowers are obtained by dissolving tri-aldehyde-based phloroglucinol, o-tolidine and polyvinylpyrrolidone in methanol, ultrasonic mixing, adding a catalyst for reaction, adding benzaldehyde for continued standing reaction after the reaction is completed, centrifugation, washing, redissolving, and the like; The Raman beacon COF nanoflowers are obtained by dissolving the TpDBD-COF nanoflowers in a mixed solution of ethanol and water, then adding AgNO3, ultrasonic adsorption, adding trisodium citrate for heating reaction after the adsorption is completed, centrifugation, washing, redissolving, and the like; The mass ratio of the tri-aldehyde-based phloroglucinol, o-tolidine and polyvinylpyrrolidone is 0.4-0.8:0.42-0.84:100-500. The mass ratio of the AgNO3 and trisodium citrate is 3-13:15-20.

2. The production method according to claim 1, characterized by, The catalyst is acetic acid.

3. The preparation method according to claim 1, characterized in that, The heating reaction is performed at a temperature of 60-100℃ for 20-40 min.

4. A Raman beacon COF nanoflower, characterized in that, The method is prepared by any one of claims 1-3.

5. An immunochromatographic test strip, characterized by, The method comprises a bottom plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and a water absorption paper which are sequentially overlapped and pasted on the bottom plate; The glass fiber pad is coated with the probe prepared from the Raman beacon COF nanoflowers of claim 4 and the antibody to be labeled.

6. The immunochromatographic test strip according to claim 5, characterized in that, The antibody to be labeled is a monoclonal antibody, a polyclonal antibody, a nanobody or a phage expressed antibody.

7. The Raman beacon COF nanoflowers of claim 4 or the immunochromatography test strip of any one of claims 5-6 are applied in detection of a mycotoxin.

8. Use according to claim 7, characterized in that, The mycotoxin comprises T-2 toxin.

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