Fluorescence-labeled Prussian blue nano-particles as well as preparation method and application thereof

Abstract

The invention relates to a fluorescently-labeled Prussian blue nano-particle as well as a preparation method and application thereof, the fluorescently-labeled Prussian blue nano-particle is prepared, and a carboxyl functional group is reserved on the surface of the nano-particle and is used for being subsequently coupled with an antigen or antibody molecule through a covalent bond. The nano material can provide colorimetric and fluorescent dual-mode detection functions, is beneficial to enhancing the detection sensitivity of an immunochromatography technology, and meanwhile, retains the detection convenience. The material is simple in preparation method, low in cost and easy for large-scale production and application.

Description

Technical Field

[0001] The invention belongs to the technical field of immunochromatographic detection, and particularly relates to fluorescently labeled Prussian blue nanoparticles and a preparation method and application thereof. Background Art

[0002] Immunochromatography, an advanced technology for detecting targets based on the specific binding of antigens and antibodies, has demonstrated its broad application potential and value in a variety of fields, including medical diagnosis and inspection and quarantine. Immunolabeling plays a crucial role in the preparation of immunochromatographic test strips. This technology cleverly attaches markers that produce highly sensitive detection signals to antigen or antibody molecules, enabling the precise capture and detection of target antibodies or antigens.

[0003] At present, among the many detection methods of immunolabeling technology, colorimetry and fluorescence detection are undoubtedly the two most commonly used methods. The colorimetric method, whose iconic marker is colloidal gold, is popular because it is easy to use, low cost, and the test results are intuitive and visible, without relying on special instruments and equipment. However, colloidal gold labeling technology also has certain limitations. It mainly binds to antigens or antibodies by physical adsorption. This binding method is relatively unstable and easily causes the label to fall off, thereby affecting the accuracy and sensitivity of the detection. In addition, the colorimetric method can only provide qualitative or semi-quantitative test results, which cannot meet the needs of accurate quantitative analysis of the target.

[0004] Compared to colorimetric methods, fluorescent markers exhibit higher detection sensitivity and quantification. Fluorescent markers emit specific fluorescent signals, which can be accurately captured by specialized fluorescence detection equipment, allowing for quantitative analysis of the target. However, the high sensitivity of fluorescence detection also presents corresponding challenges. On the one hand, specialized fluorescence detection equipment is expensive, increasing the cost of testing; on the other hand, its operation and maintenance require specialized knowledge, hindering its convenience.

[0005] Therefore, the organic combination of colorimetry and fluorescence detection has become an attractive research direction. This combination is expected to take into account the convenience of colorimetry and the high sensitivity and quantification of fluorescence detection, and realize complementary advantages. However, achieving this goal is not easy. Colorimetry and fluorescence detection have significant differences in principles, marker selection, detection equipment, etc. How to effectively integrate the two while maintaining the simplicity and ease of use of colorimetry and the high sensitivity and quantification of fluorescence detection is a difficult problem that requires in-depth research and exploration. At the same time, it is also necessary to consider how to reduce detection costs and improve detection accuracy and stability to meet the diverse needs in practical applications. Summary of the Invention

[0006] Technical Problem Solved: This invention provides fluorescently labeled Prussian blue nanoparticles, their preparation method, and applications. The resulting particles exhibit a blue color and emit fluorescence. They also possess free surface carboxyl groups, which can be used to covalently label antibody / antigen molecules to form stable complexes, thereby improving the stability of immunochromatographic detection techniques.

[0007] Technical solution: A method for preparing fluorescently labeled Prussian blue nanoparticles, comprising the following steps: (1) mixing bovine serum albumin and Prussian blue nanoparticles (PBNPs) in water, adjusting the pH to 8-10, wherein the mass ratio of the bovine serum albumin to the Prussian blue nanoparticles is (50-10):1; (2) adding ethanol dropwise to the above solution, wherein the mass ratio of the added ethanol to the albumin is (150-450:1); after the addition of ethanol, adding glutaraldehyde solution, wherein the concentration of the glutaraldehyde solution is 2.5%-25%, and the mass ratio of the glutaraldehyde solution to the albumin is 100:1-500:1, stirring for 8-24 hours, and centrifuging for purification; (3) adding a fluorescent molecule with an isothiocyanate group to the above product aqueous solution, wherein the mass ratio of the Prussian blue nanoparticles to the fluorescent molecule in the aqueous solution is 10:(1-3), reacting for 1-8 hours, centrifuging for purification, and freeze-drying to obtain fluorescently labeled Prussian blue nanoparticles.

[0008] The Prussian blue nanoparticles in step (1) are Prussian blue nanoparticles prepared using citric acid as a stabilizer, and have a particle size of 20-150 nm.

[0009] The mass ratio of bovine serum albumin to Prussian blue nanoparticles in step (1) is 25:1.

[0010] The preferred mass ratio of ethanol to albumin in step (2) is 450:1.

[0011] The fluorescent molecule with an isothiocyanate group in step (3) is fluorescein isothiocyanate or rhodamine isothiocyanate.

[0012] The centrifugal speed in step (3) is 22,000 rpm and the time is 10 minutes.

[0013] The preferred mass ratio of the fluorescent molecules to the Prussian blue nanoparticles in step (3) is 1:10.

[0014] The fluorescent-labeled Prussian blue nanoparticles prepared by the above preparation method.

[0015] Application of the fluorescently labeled Prussian blue nanoparticles in the preparation of an immunochromatographic detection kit.

[0016] An immunochromatographic detection kit contains the above-mentioned fluorescent-labeled Prussian blue nanoparticles as a dual-mode immunochromatographic detection marker.

[0017] Beneficial Effects: This invention prepares fluorescently labeled Prussian blue nanoparticles, with reserved carboxyl groups on the nanoparticle surface for subsequent covalent coupling to antigen or antibody molecules. This nanomaterial provides both colorimetric and fluorescent dual-mode detection, enhancing the sensitivity of immunochromatographic techniques while maintaining ease of detection. The material's preparation method is simple, cost-effective, and amenable to large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Transmission electron microscopy (TEM) images of Prussian blue nanoparticles (PBNPs) (left) and fluorescent molecule-modified Prussian blue nanoparticles (PBNPs@BSA-FITC) (right).

[0019] Figure 2 Dynamic light scattering spectra of Prussian blue nanoparticles (PBNPs) and fluorescent molecule-modified Prussian blue nanoparticles (PBNPs@BSA-FITC).

[0020] Figure 3 UV-visible absorption spectra of Prussian blue nanoparticles (PBNPs) and fluorescent molecule-modified Prussian blue nanoparticles (PBNPs@BSA-FITC).

[0021] Figure 4 Comparison of the absorption spectra sensitivity of Prussian blue nanoparticles (PBNO) and colloidal gold.

[0022] Figure 5 Comparison of the absorption and fluorescence sensitivities of Prussian blue nanoparticles modified with fluorescent molecules. DETAILED DESCRIPTION

[0023] Example 1

[0024] 1) Weigh 2 mM potassium ferrocyanide and dissolve it in 20 mL of water to prepare a solution, which is called Solution A.

[0025] 2) Weigh 1 mM citric acid and add it to solution A, recording it as solution B;

[0026] 3) Weigh 2 mM ferric chloride solution and dissolve it in 20 mL of water, denoted as solution C;

[0027] 4) Weigh 1 mM citric acid and add it to Solution C, recording it as Solution D;

[0028] 5) Mix solution B and solution D and stir for 30 minutes, then stop heating;

[0029] 6) The mixed solution was dialyzed and purified to obtain Prussian blue nanoparticles ( Figure 1 ).

[0030] Example 2

[0031] 1) Weigh 250 mg of bovine serum albumin, add 10 mg of Prussian blue nanoparticles, dissolve in 25 mL of purified water, and stir;

[0032] 2) adjusting the pH to 9 using 1 M sodium hydroxide solution;

[0033] 3) Add 80 mL of ethanol dropwise to the solution;

[0034] 4) Then, 30 μL of 2.5 wt.% glutaraldehyde aqueous solution was added and stirred overnight;

[0035] 5) centrifugation at 22,000 rpm to separate and purify the precipitate;

[0036] 6) Add 2 mL of water to the precipitate and add 1 mg of fluorescein isothiocyanate (FITC) and react for 2 h;

[0037] 7) 22000 rpm high-speed centrifugation to separate and purify, and obtain FITC-modified Prussian blue nanoparticles ( Figure 2 ).

[0038] Example 3

[0039] Steps 1)-5) are the same as in Example 1;

[0040] 6) Add 2 mL of water to the precipitate and add 1 mg of rhodamine isothiocyanate and react for 2 h;

[0041] 7) 22000 rpm high-speed centrifugation separation and purification to obtain isothiocyanate rhodamine modified Prussian blue nanoparticles (attached Figure 3 ).

[0042] Example 4

[0043] The absorption spectra of Prussian blue nanoparticles and colloidal gold were detected and their sensitivity was compared.

[0044] The absorption spectrum can show the color development of nanoparticles, so the absorption spectrum was measured to compare the colorimetric sensitivity of Prussian blue nanoparticles and colloidal gold.

[0045] 1) preparing solutions of Prussian blue nanoparticles and colloidal gold at concentrations of 0.1, 0.05, 0.025, 0.0125, and 0.00625 mg / mL, respectively;

[0046] 2) measuring its UV-visible absorption spectrum;

[0047] 3) analyzing the absorbance;

[0048] 4) The results showed that the absorbance of Prussian blue nanoparticles at the same concentration was much greater than that of colloidal gold, indicating that Prussian blue nanoparticles as a chromogenic group have higher sensitivity than colloidal gold ( Figure 4 ).

[0049] Example 5

[0050] The absorption and fluorescence spectra of Prussian blue nanoparticles modified with fluorescent molecules were detected, and the sensitivity of colorimetry and fluorescence detection was compared.

[0051] 1) preparing fluorescence spectrum modified Prussian blue nanoparticle solutions at concentrations of 0.05, 0.025, 0.0125, and 0.00625 mg / mL of Prussian blue nanoparticles, respectively;

[0052] 2) measuring its absorption spectrum and fluorescence spectrum;

[0053] 3) Spectral analysis: The results show that as the solution concentration decreases, the absorbance of the absorption spectrum gradually decreases. When the concentration reaches 0.00312 mg / mL, the absorbance is close to 0, indicating that the color of the solution can no longer be detected by the naked eye. The fluorescence intensity first increases and then decreases with the concentration. At a concentration of 0.00312 mg / mL, the fluorescence intensity is still clearly discernible, indicating that fluorescence detection has better sensitivity than colorimetry ( Figure 5 ).

Claims

1. A method for preparing fluorescently labeled Prussian blue nanoparticles, characterized in that: The preparation steps are as follows: (1) mixing bovine serum albumin and Prussian blue nanoparticles (PBNPs) in water, adjusting the pH to 8-10, wherein the mass ratio of the bovine serum albumin to the Prussian blue nanoparticles is (50-10):1; (2) adding ethanol dropwise to the above solution, wherein the mass ratio of the added ethanol to the albumin is (150-450):1, and after the addition of ethanol, adding glutaraldehyde solution, wherein the concentration of the glutaraldehyde solution is 2.5%-25%, and the mass ratio of the glutaraldehyde solution to the albumin is 100:1-500:1, stirring for 8-24 hours, and centrifuging for purification; (3) adding a fluorescent molecule with an isothiocyanate group to the above product aqueous solution, wherein the mass ratio of the Prussian blue nanoparticles to the fluorescent molecule in the aqueous solution is 10:(1-3), reacting for 1-8 hours, centrifuging for purification, and freeze-drying to obtain fluorescently labeled Prussian blue nanoparticles.

2. The method for preparing fluorescently labeled Prussian blue nanoparticles according to claim 1, wherein: The Prussian blue nanoparticles described in step (1) are Prussian blue nanoparticles prepared using citric acid as a stabilizer, and have a particle size of 20-150 nm.

3. The method for preparing fluorescently labeled Prussian blue nanoparticles according to claim 1, wherein: The mass ratio of bovine serum albumin to Prussian blue nanoparticles in step (1) is 25:

1.

4. The method for preparing fluorescently labeled Prussian blue nanoparticles according to claim 1, wherein: The preferred mass ratio of ethanol to albumin in step (2) is 450:

1.

5. The method for preparing fluorescently labeled Prussian blue nanoparticles according to claim 1, characterized in that: The fluorescent molecule with an isothiocyanate group in step (3) is fluorescein isothiocyanate or rhodamine isothiocyanate.

6. The method for preparing fluorescently labeled Prussian blue nanoparticles according to claim 1, characterized in that: The centrifugal speed in step (3) is 22,000 rpm and the time is 10 minutes.

7. The method for preparing fluorescently labeled Prussian blue nanoparticles according to claim 1, characterized in that: The preferred mass ratio of the fluorescent molecules to the Prussian blue nanoparticles in step (3) is 1:

10.

8. Fluorescently labeled Prussian blue nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the fluorescently labeled Prussian blue nanoparticles according to claim 8 in the preparation of an immunochromatographic detection kit.

10. An immunochromatographic detection kit, characterized in that: The immunochromatographic detection dual-mode marker contained therein is the fluorescent-labeled Prussian blue nanoparticles described in claim 8.

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