Tetrahedral DNA-rare earth fluorescent nanoparticle composite probe as well as preparation method and application thereof

Abstract

The invention discloses a tetrahedral DNA-rare earth fluorescent nanoparticle composite probe and a preparation method and application thereof, and relates to the technical field of nanomaterials, the composite probe comprises rare earth fluorescent nanoparticles, the surface of which is fixed with an antibody and streptavidin; one vertex of the tetrahedral DNA framework is modified with biotin, and the tetrahedral DNA framework is modified on the surface of the rare earth fluorescent nanoparticle through specific binding between the biotin and the streptavidin. The composite probe can be used for preparing a detection reagent, detection test paper or a detection kit for time-resolved fluorescence immunoassay so as to improve the detection specificity and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent probe, in particular to a tetrahedral DNA-rare earth fluorescent nanoparticle composite probe and a preparation method and application thereof. BACKGROUND

[0002] Time-resolved fluoroimmunoassay (TRFIA) is widely used in biomedical detection due to its high sensitivity and low background interference. Rare earth fluorescent nanoparticles have advantages such as long fluorescence lifetime and large stokes shift, and are ideal fluorescent labeling materials. The surface modification of the nanoparticles is usually random chemical coupling or physical adsorption using EDC / NHS, which is difficult to control the orientation and spacing of antibodies / nucleic acid probes on the particle surface, and is prone to Fab site pointing to the inside of the particle, active site being shielded, or probe conformation being limited due to multi-point crosslinking, thereby causing the proportion of effective binding sites to decrease, the apparent affinity to decrease, and finally the signal intensity to be insufficient, the specificity to decrease, and the detection repeatability to be poor.

[0003] Meanwhile, at the multiple interfaces of nitrocellulose membrane (NC membrane), polystyrene / PMMA particles and sample matrix (serum, plasma, whole blood, etc.), non-specific adsorption of proteins, lipids and heterophilic antibodies and other substances is prone to occur; and the surface chemical irregularity (such as hydrophobic patches, uneven charge) caused by random coupling will further amplify this effect, leading to blank background rising, signal-to-noise ratio decreasing and batch difference increasing, and the traditional blocking agent / buffer system is difficult to fully inhibit such non-specific adsorption without sacrificing the specific binding efficiency. In addition, although there is a tetrahedral DNA framework (TDF) that can realize the ordered fixation of biomolecules through vertex modification, improve the probe binding efficiency, and reduce non-specific adsorption. However, the efficient and standardized coupling method of TDF and europium nanoparticles (Eu-NP) has not been established, making it difficult to realize large-scale preparation and clinical application. SUMMARY

[0004] The present application aims to provide a tetrahedral DNA-rare earth fluorescent nanoparticle composite probe and a preparation method thereof, which can be used for preparing detection reagents, test papers or test kits for time-resolved fluorescence immunoassay, so as to improve the detection specificity and stability.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] In a first aspect, the present application provides a tetrahedral DNA-rare earth fluorescent nanoparticle composite probe, comprising: a rare earth fluorescent nanoparticle, the surface of which is fixed with an antibody and streptavidin; and a tetrahedral DNA framework, one vertex of which is modified with biotin, and the tetrahedral DNA framework is modified on the surface of the rare earth fluorescent nanoparticle through specific binding between the biotin and the streptavidin.

[0007] In the composite probe of the present application, the streptavidin on the surface of the rare earth fluorescent nanoparticle forms a point-to-point anchoring with the biotin on the vertex of the TDF, so that the TDF is modified on the surface of the particle in a single-point directional manner; the TDF, as a rigid three-dimensional framework, forms a nanoscale steric hindrance layer on the surface of the particle and provides a continuous negative charge and hydration layer, thereby inhibiting close contact through steric hindrance and electrostatic repulsion, reducing secondary aggregation between particles and stabilizing the surface potential of the particles; the TDF is charged and regularly distributed, which can homogenize the surface energy and weaken non-specific adsorption, while not interfering with the recognition conformation of the luminescent core and the surface antibody of the particle, thereby maintaining the optical output and specific recognition function. Thus, the composite probe is endowed with higher signal purity and structural stability; when used in time-resolved fluorescence immunoassay, the detection specificity and stability can be effectively improved, and the composite probe also has excellent water phase dispersibility and long-term storage stability.

[0008] In some preferred embodiments, the tetrahedral DNA framework is formed by four single-stranded DNAs self-assembled through base complementary pairing; wherein only the 5' end of one DNA chain contains a 4nt spacer sequence, and the 5' end of the DNA chain is modified with the biotin. Biotin modification is only performed on the 5' end of one DNA chain, so that the TDF is directionally combined with the streptavidin at one vertex, thereby avoiding the generation of multi-point adhesion on the surface of the particle and reducing the shielding of the antibody recognition site, so as to improve the specificity. The TDF is a rigid three-dimensional framework, which forms a nanoscale steric hindrance layer on the surface of the particle and provides a continuous negative charge, thereby inhibiting non-specific adsorption and close contact between particles, reducing the background and improving the specificity. The above directional connection and regular distribution make the modification on the surface of the particle more consistent, the particles are less likely to aggregate, the signal is more stable, and the fluctuation during storage and use is smaller, thereby reflecting better detection stability.

[0009] In some preferred embodiments, the edge length of the tetrahedral DNA framework is 20 bp, the edge length is about 6.8 nm, and the height is about 5.5 nm.

[0010] In some preferred embodiments, the sequences of the four single-stranded DNAs are as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4, respectively, which can form a stable tetrahedral framework structure through self-assembly, and have good structural consistency and biological adaptability.

[0011] In some preferred embodiments, the mass ratio of the antibody to the tetrahedral DNA framework is 1:1. This ratio outperforms other ratios in multiple aspects of optical and analytical performance. Both emission and excitation spectra confirm that this ratio achieves the strongest luminescence performance with the highest emission intensity at 615 nm and the strongest excitation response at 365 nm, showing statistically significant improvement over other ratios. Mechanistically, the superior performance from this mass ratio can be attributed to the moderate density of TDF modification. Insufficient modification would provide limited stabilization, while excessive TDF loading can cause steric hindrance effects, inter-particle bridging, and partial excitation shielding, thus reducing efficiency. In contrast, this mass ratio provides a balance in which TDF provides strong electrostatic repulsion and structural stabilization while maintaining high excitation accessibility and minimizing fluorescence self-quenching, while also facilitating spatial separation of nanoparticles to reduce non-radiative energy transfer, thus enhancing quantum efficiency. Overall, at this mass ratio, superior luminescence and excitation efficiency, improved quantum yield, enhanced dispersion stability, and extended dynamic range are combined. These synergistic advantages highlight its potential as a robust biosensing probe, especially for quantitative detection applications in time-resolved fluorescence immunoassays that require both high sensitivity and wide-range detection.

[0012] In some preferred embodiments, the rare earth fluorescent nanoparticles have a particle size of 250-350 nm and preferably comprise europium complexes, which are suitable for matching coupling with TDF structures to form a composite probe system, while having excellent fluorescence performance and biocompatibility.

[0013] In some preferred embodiments, the antibody is an interleukin-6 (IL-6) antibody. More preferably, the IL-6 antibody is WL15. Different antibody configurations were compared using IL-6 standard antigen, and it was found that WL15 is more suitable as a coating antibody in the composite probe.

[0014] In a second aspect, the application provides a preparation method of the above-mentioned tetrahedral DNA-rare earth fluorescent nanoparticle composite probe, comprising the following steps:

[0015] (1) fixing an antibody and streptavidin on the surface of the rare earth fluorescent nanoparticles;

[0016] (2) modifying the tetrahedral DNA framework on the surface of the rare earth fluorescent nanoparticles through specific binding between streptavidin and biotin to obtain a tetrahedral DNA-rare earth fluorescent nanoparticle composite probe.

[0017] In some embodiments, to obtain a highly efficient streptavidin-modified rare earth fluorescent nanoparticle-antibody conjugate, the step (1) can specifically comprise the following steps:

[0018] (11) centrifuging the suspension of the surface carboxyl-containing rare earth fluorescent nanoparticles, removing the supernatant, adding a first buffer solution, and redispersing to obtain a first redispersion system;

[0019] (12) adding an activating agent to the first redispersion system, performing a first rotational mixing reaction to obtain an activated system;

[0020] (13) adding an antibody and streptavidin to the activated system, performing a second rotational mixing reaction to immobilize the antibody and streptavidin on the surface of the rare earth fluorescent nanoparticles, removing the supernatant by centrifugation, adding a second buffer solution, and redispersing to obtain a second redispersion system;

[0021] (14) adding a blocking stabilizer to the second redispersion system, performing a third rotational mixing reaction to block unreacted active sites, removing the supernatant by centrifugation, adding a third buffer solution, and redispersing to obtain a suspension of streptavidin-modified rare earth fluorescent nanoparticle-antibody conjugates.

[0022] The suspension of the surface carboxyl-containing rare earth fluorescent nanoparticles can contain synthetic residues, unreacted small molecule stabilizers or salt ions on the surface, which can interfere with the subsequent activation reaction and reduce the carboxyl coupling efficiency. Therefore, in step (11), the original dispersion medium is removed by centrifugation, and a suitable buffer solution is used to redisperse the particles, so that the particles are uniformly dispersed, the surface carboxyl groups are fully exposed, and the subsequent carboxyl coupling efficiency is improved. The solid content of the suspension of the surface carboxyl-containing rare earth fluorescent nanoparticles can be 1% to 5%, for example, the suspension of the surface carboxyl-containing nanoparticles containing europium complexes with a solid content of 1% to 5%, wherein the dispersion medium is composed of 90% by volume of a borate-borax buffer solution (BBS) with a pH of 7.0 and 10% by volume of a 10% by mass bovine serum albumin (BSA) solution.

[0023] In step (12), the addition of the activating agent can activate the carboxyl groups to form an ester-activated intermediate, and the rotational mixing ensures uniform reaction.

[0024] In step (13), the antibody and streptavidin are added simultaneously, wherein the antibody can be adsorbed on the surface of the particles, and the streptavidin can specifically covalently couple with the activated carboxyl groups. Mixing can make the antibody and streptavidin uniformly contact the surface of the particles, and after the reaction, centrifugation and redispersion can remove the unbound antibody and streptavidin to avoid non-specific adsorption. In this way, the antibody and streptavidin are uniformly and stably bound to the surface of the particles to form a functionalized surface that can recognize antigens and biotin.

[0025] In step (14), the addition of the blocking stabilizer can fill the un-coupled active sites on the surface of the particles, and mixing ensures that the blocking stabilizer uniformly fills the surface of the particles. Finally, centrifugation and re-dissolution can obtain a stable conjugate suspension, preventing non-specific adsorption during subsequent combination with TDF, and improving the specificity and stability of the system.

[0026] The centrifugation and re-dissolution process in the above steps ensures that the reaction substances completely act on the surface of the particles, while removing excess reaction substances or by-products. This sequence of steps not only ensures efficient use of the coupling sites, but also avoids non-specific reactions, thereby obtaining rare earth fluorescent nanoparticle-streptavidin conjugates with high coupling efficiency and long-term stability.

[0027] In order to further improve the modification efficiency, the following process parameters can be met in each step:

[0028] In steps (11) to (14), the use of a borate-borate buffer (BBS) with a pH of 6-6.5 as the first buffer can further improve the carboxyl activation efficiency; the use of a borate-borate buffer with a pH of 7 as the second and third buffers is conducive to the structural stability of streptavidin and facilitates the reaction with activated carboxyl groups. Thus, the activation efficiency and the stability of streptavidin can be well balanced, making the coupling reaction more effective.

[0029] In order to completely settle the rare earth fluorescent nanoparticles, ensure the removal of free small molecules or by-products, and avoid excessive speed or long time causing particle aggregation or tight settling, which is not conducive to subsequent uniform re-dissolution, the speed during centrifugation can be 10000 rpm-15000 rpm for 5 min-15 min. More preferably, the speed is 13000 rpm-14000 rpm for 8 min-12 min. Thus, the particle dispersion is better, the reaction system is cleaner, and the coupling efficiency is improved.

[0030] Suitable ultrasonic dispersion power and time not only facilitate breaking up of particle agglomeration, making the particles uniformly dispersed, providing high surface area active carboxyl sites, and facilitating uniform binding of streptavidin, but also avoid excessive power or long time causing damage to the surface of the particles or degradation of the fluorescent properties. Preferably, the power of ultrasonic dispersion is 70 W-90 W for 1 min-5 min. More preferably, the time of ultrasonic dispersion in steps (11) and (13) can be 1 min-3 min; in step (14), the time can be 3 min-5 min.

[0031] In steps (12) to (14), the temperature of the first, second and third rotation mixing reactions is set to room temperature (e.g. 20-25°C) to ensure that the streptavidin structure is not destroyed; and the reaction time is set to ensure complete reaction and prevent streptavidin degradation, with the first rotation mixing reaction time being 25-35 minutes, the second rotation mixing reaction time being 1-3 hours, and the third rotation mixing reaction time being 0.5-1 hour.

[0032] In step (12), the activator comprises 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a mass ratio of 1:1, and both are dissolved in a boric acid-borax buffer solution with a pH of 6-6.5. The combination of EDC and NHS can generate a more stable ester activator. At the same time, an acidic system is beneficial to prolong the life of the activation intermediate. Thus, the coupling efficiency is improved and the side reaction is reduced.

[0033] In step (13), the mass ratio of the rare earth fluorescent nanoparticles, the antibody and the streptavidin is optimized to obtain a proper and uniform functional site density on the particle surface, which avoids insufficient target capture caused by low loading and avoids conformational crowding, shielding and multi-point crosslinking caused by high loading, so as to maintain the activity and orientation of the antibody and provide sufficient but not saturated anchoring sites for biotin-streptavidin binding; at the same time, the non-specific adsorption and local hydrophobic patches are reduced, the non-specific background is reduced, the cost caused by excessive reagents is avoided, and the specificity, sensitivity and stability of the detection are improved as a whole. Preferably, the mass ratio of the rare earth fluorescent nanoparticles, the antibody and the streptavidin is (10-100):1:1.

[0034] In step (14), the blocking stabilizer is preferably a 5%-15% (mass fraction) aqueous solution of bovine serum albumin (BSA), which can block the unreacted active sites while not affecting the binding sites of streptavidin, reduce non-specific adsorption, and improve the purity and stability of the conjugate.

[0035] In some preferred embodiments, in order to realize efficient modification of the tetrahedral DNA framework, step (2) can specifically comprise the following steps:

[0036] (21) mixing the tetrahedral DNA framework solution with the streptavidin-modified rare earth fluorescent nanoparticle-antibody conjugate suspension to incubate, so that the biotin and the streptavidin specifically bind to obtain a crude system containing tetrahedral DNA framework-modified rare earth fluorescent nanoparticles;

[0037] (22) Centrifuging the crude system to remove the supernatant containing excess tetrahedral DNA framework, and then resuspending the precipitate to obtain a tetrahedral DNA-rare earth fluorescent nanoparticle composite probe solution.

[0038] In step (21), the biotin at the end of the TDF specifically binds to the streptavidin on the surface of the particles through high-affinity binding, ensuring that the TDF can be modified on the surface of the particles in a directional and specific manner, without shielding the recognition sites of the surface antibodies and reducing random adhesion, reducing non-specific binding on the interface and improving apparent specificity. In step (22), free and loosely adsorbed TDF is removed by centrifugation, and the suspension is resuspended with a buffer, so that only the ordered shell is retained, avoiding free TDF acting as an “adsorber” or causing inter-particle bridging during detection, further reducing background and cross-reaction, while stabilizing the inter-particle spacing and particle size distribution, reducing the read drift caused by desorption and rearrangement. These two steps cooperate to ensure a single and uniform recognition interface on the surface of the particles, improving detection specificity and stability.

[0039] In some preferred embodiments, to ensure efficient assembly of the TDF, the four single-stranded DNAs are mixed in a Tris-HCl buffer with a pH of 7.5-8.5 at an equimolar ratio, heated and annealed at 90-100°C for 10-15 min, and then quickly cooled to 2-6°C, to obtain a stable and single-configuration tetrahedral DNA framework solution, facilitating subsequent modification steps.

[0040] To further improve the modification efficiency and stability, the following process parameters can be met in each step:

[0041] In step (21), the temperature of the mixed incubation is set to room temperature, and the time is 0.5-1.5 h. The room temperature is conducive to maintaining the stability of the SA and TDF configuration and improving the binding efficiency; and the appropriate time is conducive to sufficient coupling and reduces the risk of non-specific adsorption. The concentration of the tetrahedral DNA framework solution can be 2-3 μM.

[0042] In step (22), when the crude system is centrifuged, the PBST with a pH of 6.5-7.5 is used to centrifuge at 10,000-15,000 rpm for 5-15 min, so that the modified particles can be reliably settled, and the free or loosely adsorbed TDF and impurities are retained in the supernatant, thereby purifying the interface, reducing background and cross-reaction, and improving detection specificity; at the same time, this pH range takes into account the conformation and charged state of the TDF and SA, avoiding desorption or inactivation caused by extreme pH. Resuspension with PBS with a pH of 6.5-7.5 stabilizes the surface potential and hydration layer of the particles, reduces re-aggregation and signal drift, and further improves detection stability.

[0043] In a third aspect, the application provides the tetrahedral DNA-rare earth fluorescent nanoparticle composite probe prepared by the above method, or the application of the composite probe in preparing a detection reagent, test paper or kit for time-resolved fluorescence immunoassay.

[0044] Compared with the prior art, the technical scheme of the application has the following beneficial effects:

[0045] The application utilizes the specific binding between streptavidin and biotin to orientally modify a tetrahedral DNA framework (TDF) on the surface of a rare earth fluorescent nanoparticle; the rigid three-dimensional skeleton of the TDF forms a nanoscale steric hindrance layer on the particle surface and provides sustained negative electricity, which can effectively inhibit non-specific adsorption and particle close contact, reduce background and cross reaction, at the same time, does not shield the recognition site of the surface antibody, maintains the consistency of specific recognition and optical reading, so that when the composite probe is used for preparing a detection reagent, test paper or kit for time-resolved fluorescence immunoassay, the detection specificity and detection stability can be improved, and the antibody / nucleic acid composite probe can be fixed on the surface of Eu-NP in an ordered and oriented manner, so as to improve the effective binding efficiency and signal utilization rate.

[0046] Further, by reasonably designing and controlling the preparation steps and key parameters, the uniformity and interface purity of TDF anchoring can be improved, non-specific adsorption can be significantly reduced, and the signal-to-noise ratio and detection repeatability can be improved; the whole preparation method is simple, the parameters are controllable, is suitable for large-scale preparation and is compatible with various detection platforms; and in time-resolved fluorescence detection, high sensitivity and high stability are shown. BRIEF DESCRIPTION OF DRAWINGS

[0047] The following drawings detail the exemplary embodiments disclosed in the application. Those skilled in the art will understand that these embodiments are non-limiting, exemplary embodiments, and the drawings are only for the purpose of illustration and description, and are not intended to limit the scope of the application, and other ways of embodiments can also achieve the same purpose as the application. It should be understood that the drawings are not drawn to scale. Among them:

[0048] Figure 1 The polyacrylamide gel electrophoresis analysis diagram of the TDF prepared for the embodiment 2 of the application;

[0049] Figure 2 The AFM diagram of the TDF prepared for the embodiment 2 of the application;

[0050] Figure 3 The dispersion image of the Cy3-labeled Eu-NP-WL15&TDF composite probe, the Eu-NP-WL15&TDF composite probe and the Eu-NP-WL15 probe;

[0051] Figure 4Figure 1 is a morphological characterization chart of the probe nanoparticles; wherein a is the SEM chart of the carboxyl-modified Eu-NPs, b is the SEM chart of the Eu-NP-WL15 & SA, c is the SEM chart of the Eu-NP-WL15 & TDF composite probe, d is the TEM chart of the carboxyl-modified Eu-NPs, e and f are the TEM charts of the Eu-NP-WL15 & SA;

[0052] Figure 5 Figure 2 is a spectral scanning chart of the composite probes of Examples 3-7 of the present application, the Eu-NP-WL15 probe of Comparative Example 1, and the Eu-NPs; wherein a is the original emission scanning chart of different configurations under 615 nm emission; b is the original excitation scanning chart of different configurations under 365 nm excitation; c is the quantum efficiency scanning spectrum of No. 4 composite probe, Eu-NP-WL15 probe, and blank cuvette;

[0053] Figure 6 Figure 3 is a schematic diagram of the correlation analysis results of the immunochromatographic detection method of No. 4 composite probe and the Beckman reference method. DETAILED DESCRIPTION

[0054] The technical solutions of the present application will be described in detail below with reference to the accompanying drawings and specific examples. It should be noted that the present application is not limited to the structures or methods listed herein, and can also be changed. Unless otherwise defined, all technical and scientific terms used in the specification have the same meaning as understood by those skilled in the art of the technology to which the present application belongs. The terms used in the specification are only for the purpose of describing the specific examples and are not intended to limit the present application. Unless otherwise specified, the reagents and raw materials used can be purchased through commercial channels. The experimental methods in the following examples are not specified, and are selected according to conventional methods and conditions, or according to the product instructions.

[0055] 0.1M Borax stock solution: Take a 1L clean reagent bottle and place it on an electronic balance. Then weigh the chemicals shown in Table 1 on an analytical balance and pour them into the reagent bottle. After complete dissolution and mixing, 0.1M borax stock solution is obtained.

[0056] Table 1: Chemicals and amounts

[0057]

[0058] 0.4M Boric acid stock solution: Take a 1L clean reagent bottle and place it on an electronic balance. Then weigh the chemicals shown in Table 2 on an analytical balance and pour them into the reagent bottle. After complete dissolution and mixing, 0.4M borax stock solution is obtained.

[0059] Table 2: Chemicals and amounts

[0060]

[0061] 0.05M BBS pH=7.0: Take a 200 mL clean reagent bottle on the electronic balance, skinning. Add 1.27 g of 0.1M borax mother liquor, 18.73 g of 0.4M boric acid mother liquor, 140 g of Wahaha pure water, mix well to get it. Other pH value of BBS can be adjusted by adding drugs.

[0062] 10% mass fraction of BSA solution: take a clean 50 mL centrifuge tube on the electronic balance, skinning. Then use an analytical balance to weigh the chemicals shown in Table 3 and pour them into the centrifuge tube, mix well until completely dissolved, then store at 4°C, do not freeze.

[0063] Table 3: Drugs and usage

[0064]

[0065] 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) were purchased from Sigma (Kawasaki, Kanagawa, Japan). Streptavidin (SA) was purchased from Yeason Biotech (Shanghai) Co., Ltd. (Shanghai, China). IL-6 monoclonal antibody (WL15) was purchased from Shanghai Taihui Biological Technology Co., Ltd. (Shanghai, China).

[0066] Reference M. Liu, Y. Li, M. Xu, F. Li, Time-domain stepwise encoding based on a stepped photon emission material, Chem Commun (Camb), 58 (2022) 13927-13930, to prepare a 1% solid content carboxyl modified Eu-NP suspension:

[0067] (1) Synthesis of europium complex

[0068] The following synthetic route was used to prepare Eu(TTA)3(TPPO)2:

[0069]

[0070] The specific method is: in 10 milliliters of ethanol, add a solution of TPPO ligand (190 milligrams) and TTA ligand (220 milligrams), then add EuCl3·6H2O (120 milligrams, dissolved in 1.25 milliliters of water), adjust the pH to 5-6, stir and reflux heat in an oil bath for 8 hours. After cooling to room temperature, the crude precipitate is washed with water and ethanol three times. The residue is dried, and about 400 milligrams of europium complex is finally obtained.

[0071] The europium complex, PdOEP and SO were dispersed in a good solvent for the three, to configure the organic phase stock solution, ready for use.

[0072] (2) Preparation of carboxyl-modified Eu-NP suspension

[0073] The synthetic route of carboxyl-modified Eu-NP nanoparticles (HOOC-Eu-NP) is as follows:

[0074]

[0075] The carboxyl-modified Eu-NP nanoparticles were prepared by a conventional wet chemical encapsulation method (emulsification / solvent displacement). Carboxyl cross-linked polystyrene nanoparticles (PE) were used as nanocarriers, and europium complex (Eu) was encapsulated in the nanocarriers to form carboxyl-modified Eu-NP nanoparticles. Specifically, the carboxyl cross-linked polystyrene nanoparticles were dispersed in water to obtain a PE aqueous dispersion. Under stirring, the organic phase stock solution described above was slowly added to the PE aqueous dispersion to form a transient emulsion. Under stirring and mild ultrasonic conditions, the organic phase was gradually diffused, and the hydrophobic components were transferred into the polystyrene nanoparticles. The organic solvent was removed by natural evaporation to obtain a uniform pink nanoparticle suspension. Free small molecules and residual organic solvents were removed by centrifugation-redispersion, and the supernatant was washed until it was colorless. An inert aqueous stabilizer was used to resuspend the carboxyl-modified Eu-NP (which can also be simplified as Eu-NP) suspension to a solid content of 1%.

[0076] Example 1

[0077] Coupling of Eu-NP with antibody WL15, SA

[0078] The carboxyl-modified Eu-NP was coupled with antibody WL15, SA according to the EDC / NHS chemical method to obtain SA-modified Eu-NP-WL15 conjugate (Eu-NP-WL15 & SA), and the specific method is as follows:

[0079] 1) Ball washing

[0080] 400 μL of carboxyl-modified Eu-NP suspension (300 nm, 1% solid content) was centrifuged at a speed of 13400 rpm for 10 min, and after removing the supernatant, it was redispersed in 80 μL of boric acid-borax buffer (BBS, 0.05 M, pH 6.0) under ultrasonic treatment (80 W, 2 min) to obtain a first redispersion system.

[0081] 2) Activation

[0082] To the first reconstitution system, 40 μg of EDC (10 mg / mL, dissolved in 0.05 M BBS pH 6.0) and 40 μg of NHS (10 mg / mL, dissolved in 0.05 M BBS pH 6.0) were added and stirred gently at room temperature for 30 min to obtain an activation system.

[0083] 3) Coupling

[0084] To the activation system, 40 μg of antibody WL15 and 40 μg of SA were added and mixed by rotation at room temperature for 2 h. Then, the supernatant was removed by centrifugation at 13,400 rpm for 10 min, and 180 μL of 0.05 M BBS pH 7.0 was added to reconstitute the system. The reconstituted system was dispersed by ultrasonic treatment at 80 W for 2 min to obtain a second reconstitution system.

[0085] 4) Blocking

[0086] To the second reconstitution system, 20 μL of a 10% BSA solution was added to block the surface. The solution was mixed by rotation at room temperature for 1 h. Then, the supernatant was removed by centrifugation at 13,400 rpm for 10 min, and 180 μL of 0.05 M BBS pH 7.0 was added to reconstitute the system. The reconstituted system was dispersed by ultrasonic treatment at 80 W for 5 min to obtain an Eu-NP-WL15&SA suspension.

[0087] 5) Preservation

[0088] To the Eu-NP-WL15&SA suspension, 20 μL of a 10% BSA solution was added to block the unreacted carboxyl groups on the surface of the nanoparticles. The solution was stored at 4°C until use.

[0089] Example 2

[0090] Preparation of TDF

[0091] TDF was formed by self-assembly of four single-stranded DNAs (see Table 4 below) through base complementary pairing, wherein only strand 4 was a biotin-modified single-stranded DNA.

[0092] Table 4 Details of the four single-stranded DNA sequences

[0093]

[0094]

[0095] Four single-stranded DNAs in equimolar amount were mixed in 20 mM Tris-HCl buffer (containing 50 mM MgCl2, pH 8.0) with a final concentration of 2.5 μM, followed by annealing at 95 °C for 10 min, and then rapid cooling to 4 °C to complete self-assembly, to obtain a TDF solution. The formation of tetrahedral DNA framework structure was verified by 8% non-denaturing polyacrylamide gel (PAGE) electrophoresis (see Figure 1 ).

[0096] AFM characterization

[0097] The mica surface was modified with 30 μL of 0.5% APTES aqueous solution for 2 min. On the mica surface, 20 μL of 20 nM purified TDF solution and 30 μL of TM buffer were added and adsorbed for 5 min. The AFM equipment was Multimode 8 (Bruker) and the scanning mode was ScanAsyst. The AFM image of TDF was obtained as shown in Figure 2 From the AFM image, it can be observed that the TDF has the expected tetrahedral configuration, with clear features such as edges and corners, thus confirming that the TDF has successfully self-assembled into the expected structure. At the same time, it can also be seen from the AFM image that the TDF is uniformly dispersed in monomer form. Figure 2

[0098] Example 3

[0099] Preparation of composite probe

[0100] 80 μL of TDF solution was incubated with 20 μL of Eu-NP-WL15&SA suspension at room temperature for 1 hour to achieve high-affinity biotin-streptavidin coupling. After incubation, unbound TDF was removed by centrifugation (13400 rpm, 10 min, repeated twice) using PBST (1x, pH 7.4). The obtained precipitate was resuspended in 100 μL of PBS (pH 7.4) to obtain a tetrahedral DNA-rare earth fluorescent nanoparticle (Eu-NP-WL15&TDF) composite probe solution (No. 4 as shown in Table 5), and stored at 4 °C for further use.

[0101] Comparative Example 1

[0102] This comparative example provides a Eu-NP-WL15 probe, the difference between its preparation method and that of Eu-NP-WL15&SA in Example 1 is only that SA is not added during coupling in step 3).

[0103] TIRFM characterization

[0104] ​In the petri dish of plasma treatment, 50 μL of 40 nM sample was added, adsorbed for 1 min, and then imaged by total internal reflection fluorescence microscopy (Leica, DMi8 Infinity) at 405 nm (for Eu-NP excitation) and 550 nm (for Cy3 excitation) wavelengths, respectively, and dispersed images were taken at different storage times. The TIRFM images were analyzed using Image J (NIH) software.

[0105] Figure 3 The dispersed images of Cy3-labeled Eu-NP-WL15&TDF complex probe, Eu-NP-WL15&TDF complex probe, and Eu-NP-WL15 probe are shown. The successful binding of biotinylated TDF to streptavidin-modified Eu-NP was confirmed by the Cy3 fluorescence signal and colocalization in TIRFM imaging. Stability analysis showed that after four weeks of storage, the Eu-NP-WL15 probe aggregated, while the Eu-NP-WL15&TDF complex probe remained well dispersed, with a colocalization efficiency of >90%. This indicates that TDF modification significantly improves colloidal stability, preventing aggregation during storage.

[0106] Morphological characterization of nanoparticles

[0107] The surface morphology and structural integrity of the prepared probe nanoparticles were studied by SEM and TEM, with the following specific methods:

[0108] 10 μL of 1 nM sample was added to the carbon film, and adsorbed on the surface of the carbon film for 5 min. The carbon film was washed with Milli-Q water 3 times and dried at room temperature. Then the sample was imaged using Talo L120C G2 at an accelerating voltage of 120 kV, and a scanning electron microscope (GeminiSEM 360) at an accelerating voltage of 5 kV. Figure 4 SEM images of a) carboxyl-modified Eu-NP, b) Eu-NP-WL15&SA, c) Eu-NP-WL15&TDF complex probe, and TEM images of d) carboxyl-modified Eu-NP, e and f) Eu-NP-WL15&SA.

[0109] The SEM images show that the original carboxyl-modified Eu-NP exhibits a smooth spherical morphology and uniform particle size, about 300 nm Figure 4 After surface modification with IL-6 antibody and TDF, the nanoparticles maintain their spherical structure with clear boundaries, indicating that IL-6 antibody and TDF coupling did not cause aggregation or structural deformation Figure 4 TEM observation further confirms these results. Eu-NP shows uniform dispersion and consistent spherical structure (Figure 4 of d). In contrast, Eu-NP-WL15&SA shows monodispersed nanoparticles with clear edges and uniform size (e-f) of d). The uniformity of the structure after antibody modification is confirmed to be maintained. Taken together, SEM and TEM analysis indicates that surface functionalization with WL15&SA or WL15&TDF does not significantly change the morphology or monodispersity of Eu-NP, supporting their suitability for bio-detection applications. Figure 4

[0110] Examples 4-7

[0111] Referring to the preparation method of Example 3, the mass ratio of WL15 to TDF was changed to prepare No. 5, No. 6, No. 9, No. 10 composite probes as shown in Table 5.

[0112] Table 5 Composite probes of Examples 3-7

[0113]

[0114] Fluorescence spectral characterization

[0115] To evaluate whether TDF modification affects the luminescence properties of Eu-NP, the composite probes of Examples 3-7 (No. 4, No. 5, No. 6, No. 9, No. 10), the Eu-NP-WL15 probe of Comparative Example 1, and Eu-NP were tested using a FS5 fluorescence spectrometer (Edinburgh Instruments Ltd., Livingston, Scotland, UK) to obtain the spectral scan images as shown in Figure 5

[0116] Figure 5 a in Figure 1 is the original emission scan image of different configurations under 615 nm emission, and b is the original excitation scan image of different configurations under 365 nm excitation. All groups show a characteristic Eu 3+ emission peak centered at 615 nm, and the emission spectra remain consistent before and after TDF modification, with no obvious spectral shift observed, indicating that the intrinsic electronic transition of Eu 3+ ions is preserved, and TDF modification does not cause quenching or energy loss. The excitation spectra of all groups reach a maximum at 365 nm, with almost identical spectral shapes. Depending on the mass ratio, a small amplitude fluctuation in excitation efficiency is observed, but the characteristic excitation behavior is maintained. Among the tested ratios, the No. 4 composite probe produces the highest emission intensity and excitation efficiency. In summary, these results confirm that TDF modification does not change the basic optical properties of Eu-NP.

[0117] Figure 5 ​​The quantum efficiency scan spectra of No. 4 composite probe, Eu-NP-WL15 probe and blank cuvette (blank control) are shown in Figure 1. All samples exhibited characteristic Eu 3+ emission peak at 615 nm, confirming that the luminescence properties of the europium chelated core were preserved after bioconjugation. Compared with the Eu-NP-WL15 probe, No. 4 composite probe showed slightly enhanced emission intensity at 615 nm, while maintaining a low background comparable to the blank control. This indicates that the introduction of TDF did not quench the europium signal, but rather enhanced the effective signal output.

[0118] From a mechanistic point of view, the superior performance of No. 4 composite probe can be attributed to the moderate density of TDF modification. Insufficient modification would provide limited stabilization, while excessive TDF loading could cause steric effects, inter-particle bridging, and partial excitation shielding, thus reducing efficiency. In contrast, the equal proportion of WL15 and TDF provides a balance, in which the TDF shell provides strong electrostatic repulsion and structural stabilization, while maintaining high excitation accessibility and minimizing fluorescence self-quenching. The rigid TDF also helps to spatially separate the nanoparticles, reducing non-radiative energy transfer, thus enhancing quantum efficiency.

[0119] Time-resolved fluorescence immunoassay performance test

[0120] (1) Assembly, detection and evaluation of immunochromatographic test strip

[0121] The immunochromatographic test strip is composed of four parts, namely the sample pad, nitrocellulose (NC) membrane, absorbent paper and back plate. The NC membrane is pre-pasted on the back plate. IL-6 antibody (1.5 mg / mL) and goat anti-chicken IgG antibody (1 mg / mL) are loaded on the test line (T line) and control line (C line) of the NC membrane, respectively. The XYZ320 three-dimensional spotting platform of Biodot is used to fix the antibodies on the membrane, and the gold spraying amount is 1.00 μL / cm. The treated NC membrane is placed in an oven at 37°C overnight. Then the sample pad and absorbent paper are also pasted on the adhesive base. The assembled plate is cut into 4 mm wide strips, and the strips are loaded into matching plastic card boxes. The prepared probe (No. 4 composite probe or Eu-NP-WL15 probe) is fixed on the NC membrane, and the remaining steps are the same as above. The cut strips are assembled into chromatographic standard cards and tested. During testing, 100 μL of sample diluent (V diluent:V sample = 9:1) is added to the sample well, and after 10 minutes of reaction, the data is read using a fluorescence immunoassay instrument (FRIR-L102, Shanghai Taihui Biological Technology Co., Ltd.).

[0122] To determine the optimal linear range, the system evaluated all contiguous concentration intervals (> 3 points) according to the CLSI EP06 guidelines and selected the longest range that achieved a correlation coefficient R 2 > 0.99 in ordinary least squares (OLS) regression. This process ensures that the reported linear range reflects both statistical robustness and practical applicability, rather than relying solely on the entire calibration span. The limit of detection (LOD) and limit of quantitation (LOQ) were determined according to CLSI EP17-A2. The blank limit (LOB) was first determined as the mean blank signal plus 1.645 x the standard deviation of the blank. The limit of detection signal threshold was defined as LOB + 1.645 x the standard deviation of the lowest non-zero concentration. This threshold was converted to concentration units using the low segment regression curve fit to obtain the LODconc. The limit of quantitation (LOQ) was defined as the lowest concentration with a coefficient of variation (CV) of no more than 20% in replicate measurements.

[0123] (2) Performance comparison of Eu-NP-WL15&TDF complex probe and Eu-NP-WL15 probe

[0124] To compare the analytical performance of No. 4 complex probe and Eu-NP-WL15 probe in IL-6 detection, linear regression analysis was performed, and the results are shown in Table 6. The slope of No. 4 complex probe was 0.0001229, R 2 = 0.9609, while the slope of Eu-NP-WL15 probe was higher, 0.0002319, but R 2 = 0.9483 was slightly lower. The results showed that Eu-NP-WL15 probe provided higher sensitivity in unit concentration change, while No. 4 complex probe exhibited better linearity and fitting stability within the tested concentration range. The LOD of Eu-NP-WL15 probe was 12.4 pg / mL, and the LOQ was 41.3 pg / mL, while the LOD of No. 4 complex probe was lower, 8.7 pg / mL, and its LOQ was still 28.9 pg / mL. No. 4 complex probe performed better in noise suppression, thereby achieving a lower detection threshold with a lower slope, while Eu-NP-WL15 probe provided a steeper calibration curve within the low concentration range, which was beneficial for sensitive quantification when IL-6 was elevated early.

[0125] Table 6 Linear regression analysis results

[0126]

[0127]

[0128] In addition, the detection using No. 4 composite probe on the immunochromatographic test strip can be completed within 10 min, and the signal-to-noise ratio is significantly improved compared with Eu-NP-WL15 probe, and the repeatability is good.

[0129] Performance of composite probe in clinical samples

[0130] Seven clinical serum samples (provided by the hospital, IL-6 concentration range between 1.7 and 10009.83 pg / mL) were detected. As shown in Figure 6 The correlation analysis of the results of No. 4 composite probe immunochromatographic detection method and Beckman reference method showed an excellent linear relationship (R 2 = 0.9942, p < 0.0001), and the regression equation was Y = 1.046 * X + 26.76, indicating that the detection results of the two methods were highly consistent within the test concentration range.

[0131] It should be noted that the preferred embodiments of the present application are given in the specification and drawings of the present application, but the present application can be implemented in many different forms, and is not limited to the embodiments described in the specification. These embodiments are not additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, the above technical features continue to be combined with each other, forming various embodiments not listed above, which are considered to be within the scope of the present application. Furthermore, for those skilled in the art, the above description can be improved or changed, and all these improvements and changes should be within the scope of protection of the appended claims of the present application.

Claims

1. A tetrahedral DNA-rare earth fluorescent nanoparticle complex probe, characterized in that, Comprise: a rare earth fluorescent nanoparticle, the surface of which is fixed with an antibody and streptavidin; a tetrahedral DNA framework, one vertex of which is modified with biotin, and the tetrahedral DNA framework is modified on the surface of the rare earth fluorescent nanoparticle through specific binding between the biotin and the streptavidin.

2. The tetrahedral DNA-rare earth fluorescent nanoprobe complex probe according to claim 1, wherein, The tetrahedral DNA framework is formed by self-assembly of four single-stranded DNAs through base complementary pairing; wherein only the 5' end of one DNA chain contains a 4 nt spacer sequence, and the 5' end of the DNA chain is modified with the biotin.

3. The tetrahedral DNA-rare earth fluorescent nanoprobe complex of claim 2, wherein, The sequences of the four single-stranded DNAs are respectively shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO.

4.

4. The tetrahedral DNA-rare earth fluorescent nanoprobe complex according to any one of claims 1 to 3, wherein, The complex probe has at least one of the following characteristics: (a) the mass ratio of the antibody to the tetrahedral DNA framework is 1:1; (b) the particle size of the rare earth fluorescent nanoparticle is 250 nm to 350 nm, and the rare earth fluorescent nanoparticle contains an europium complex; (c) the antibody is an interleukin-6 antibody.

5. A method for preparing the tetrahedral DNA-rare earth fluorescent nanoparticle complex probe according to any one of claims 1 to 4, characterized in that, Comprise the following steps: (1) fixing an antibody and streptavidin on the surface of a rare earth fluorescent nanoparticle; (2) modifying a tetrahedral DNA framework on the surface of the rare earth fluorescent nanoparticle through specific binding between streptavidin and biotin to obtain a tetrahedral DNA-rare earth fluorescent nanoparticle complex probe.

6. The preparation method according to claim 5, characterized in that, The preparation method of the tetrahedral DNA framework comprises: mixing four single-stranded DNAs in a Tris-HCl buffer solution with a pH of 7.5 to 8.5 at an equimolar ratio, annealing treatment at 90°C to 100°C for 10 min to 15 min, and then rapidly cooling to 2°C to 6°C to obtain a tetrahedral DNA framework solution.

7. The production method according to claim 5 or 6, characterized by, The step (1) comprises the following steps: (11) centrifuging a suspension of rare earth fluorescent nanoparticles containing carboxyl groups on the surface, removing the supernatant, adding a first buffer solution for reconstitution, and ultrasonic dispersion to obtain a first reconstitution system; (12) adding an activating agent to the first reconstitution system, performing a first rotational mixing reaction to obtain an activated system; (13) adding an antibody and streptavidin to the activated system, performing a second rotational mixing reaction to fix the antibody and streptavidin on the surface of the rare earth fluorescent nanoparticle, removing the supernatant after centrifugation, adding a second buffer solution for reconstitution, and ultrasonic dispersion to obtain a second reconstitution system; (14) adding a blocking stabilizer to the second reconstitution system, performing a third rotational mixing reaction to block unreacted active sites, removing the supernatant after centrifugation, adding a third buffer solution for reconstitution, and ultrasonic dispersion to obtain a suspension of streptavidin-modified rare earth fluorescent nanoparticle-antibody conjugates.

8. The preparation method according to claim 7, characterized in that, The step (1) satisfies at least one of the following conditions: (A) the rotation speed during centrifugation is 10,000 rpm to 15,000 rpm for 5 min to 15 min; (B) the ultrasonic dispersion power is 70 W to 90 W for 1 min to 5 min; (C) the first buffer solution is a boric acid-borax buffer solution with a pH of 6 to 6.5, and the second buffer solution and the third buffer solution are boric acid-borax buffer solutions with a pH of 7. (D) the activator comprises 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a mass ratio of 1:1, and both are dissolved in a boric acid-borax buffer solution with a pH of 6-6.5; (E) the temperature of the first rotational mixing reaction is room temperature, and the time is 25 min-35 min; (F) the temperature of the second rotational mixing reaction is room temperature, and the time is 1 hour-3 hours; (G) the blocking stabilizer is a 5%-15% (mass fraction) aqueous solution of bovine serum albumin; (H) the temperature of the third rotational mixing reaction is room temperature, and the time is 0.5 hour-1 hour; (I) the solid content of the surface carboxyl-containing rare earth fluorescent nanoparticle suspension is 1%-5%; (J) the mass ratio of the rare earth fluorescent nanoparticles, the antibody, and the streptavidin is (10-100):1:

1.

9. The production method according to claim 5 or 6, characterized by, The step (2) comprises the following steps: (21) mixing and incubating the tetrahedral DNA framework solution with the streptavidin-modified rare earth fluorescent nanoparticle-antibody conjugate suspension to specifically bind biotin and streptavidin, to obtain a crude system containing tetrahedral DNA framework-modified rare earth fluorescent nanoparticles; (22) centrifuging the crude system, removing the supernatant containing excess tetrahedral DNA framework, and then resuspending the precipitate to obtain a tetrahedral DNA-rare earth fluorescent nanoparticle composite probe solution.

10. The method of claim 9, wherein, The step (2) satisfies at least one of the following conditions: (a) the temperature of the mixing and incubation is room temperature, and the time is 0.5 hour-1.5 hour; (b) when the crude system is centrifuged, a phosphate Tween buffer with a pH of 6.5-7.5 is used at 10,000 rpm-15,000 rpm for 5 min-15 min; and a phosphate buffer with a pH of 6.5-7.5 is used for resuspension; c) the concentration of the tetrahedral DNA framework solution is 2 μM-3 μM.

11. The tetrahedral DNA-rare earth fluorescent nanoparticle composite probe of any one of claims 1-4, or the composite probe prepared by the preparation method of any one of claims 5-10, for use in preparing a detection reagent, a test strip, or a kit for time-resolved fluorescence immunoassay.

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