High-identification, low-scattering quantum dot tubular assembly structure immunochromatography probe, preparation method and application thereof

Abstract

The application discloses a high-recognition and low-scattering quantum dot tubular assembly structure immunochromatography probe and a preparation method and application thereof, and first prepares a nickel-hydrazine complex nanorod, performs etching to obtain a one-dimensional hollow tubular silicon dioxide nanotube, performs mercapto-modification, constructs an oil-soluble tubular assembly through multi-layer assembly of quantum dots, transfers the assembly to an aqueous solvent through an efficient alkylsilane phase transfer strategy, deposits a thin and dense silicon oxide layer on the surface of the assembly to protect the assembly, and simultaneously performs carboxylation and coupling of a novel coronavirus antigen N protein antibody based on the silicon oxide layer to obtain a fluorescent probe. The one-dimensional hollow tubular material is used as a carrier of a signal tag, and due to double optimization of signal amplification effect and immunoreaction efficiency, the sensitivity of a lateral flow immunochromatography platform is improved, and important scientific basis is provided for early discovery, early treatment and early prevention and control of virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biological immunoassay technology, specifically relating to a high-recognition, low-scattering quantum dot tubular assembly structure immunochromatographic probe, its preparation method, and its application. Background Technology

[0002] Infectious diseases caused by viruses have become one of the most serious public health problems globally, resulting in significant losses of life and economic damage. Lateral flow immunochromatography (LFIA), as a mature and popular on-site detection method, offers advantages such as speed, portability, low cost, and user-friendliness, and is widely used for personal health self-testing. In traditional testing, colloidal gold nanoparticles (AuNPs) are widely used in colorimetric LFIA detection. However, colloidal gold test strips have limited sensitivity, a high false-negative rate, and weak quantitative ability, making it difficult to achieve highly sensitive detection. With the development of nanomaterials, many novel materials, such as upconversion luminescent materials and time-resolved fluorescent labeling materials, have been used as markers for LFIA, greatly promoting the development of LFIA and achieving highly sensitive detection of target analytes. However, this usually requires sophisticated instrument design, resulting in high complexity and cost. In contrast, quantum dots possess a series of unique optical properties, such as high fluorescence quantum yield, large molar extinction coefficient, and large Stokes shift, making them one of the ideal labeling materials for immunochromatographic techniques. Unfortunately, simply focusing on the assembly, amplification, and integration of signal units without considering immune recognition efficiency makes it difficult to achieve effective detection of low viral load antigens by LFIA. Therefore, we should explore the overall optimization of signal amplification and immune recognition efficiency from the perspective of signal tag shape, so as to obtain theories and methods to improve the sensitivity of LFIA.

[0003] Studies have shown that the intrinsic signal intensity of the signal probe and the efficiency of the immunoreaction with the capture antibody at the test site (T) are considered two important factors determining the sensitivity of LFIA. Especially when the target analyte concentration is close to the limit of detection (LOD), because the amount of target analyte is much less than the amount of antibody on the signal probe surface, all the target analyte in the sample solution is captured by the signal probe; it is generally assumed that the amount of analyte bound to each signal probe surface is the same. Therefore, in the LOD state, the binding affinity between the capture antibody sprayed on the T line and the formed signal probe-antigen immune complex is considered the most critical factor determining the sensitivity of LFIA. Since the pores of the nitrocellulose membrane (NC membrane) in the immunochromatographic platform can be approximated as a complex tubular channel, the dimensionality of the signal probe affects its throughput. Therefore, designing a probe of the correct dimensionality to match the tubular channels of the NC membrane, ensuring good flowability of the signal probe within the NC membrane pores, is beneficial for improving the recognition probability of the probe-antigen immune complex and the capture antibody. John C. Bischof's research group concluded through model simulations that, typically, when the probe size is >100 nm, the capture of the signal probe-antigen immune complex by the capture antibody at the test site is limited by the diffusion rate. Because traditional signal probes diffuse slowly from the bulk solution to the immobilized target on the nitrocellulose membrane (NC membrane), this introduces a significantly underestimated diffusion-related limitation at the immunoreaction kinetics level, which greatly weakens the sensitivity of the test strip. The probe designed in this invention possesses excellent diffusion rate and immunoreaction kinetics, which is beneficial for improving the immunorecognition efficiency of the signal probe-antigen immune complex and the capture antibody, playing a crucial role in enhancing the sensitivity of LFIA.

[0004] Currently, the mainstream carriers for commercial immunochromatographic probes are three-dimensional spherical templates with a size of 200-400 nm (such as polystyrene spheres and silica microspheres). These offer advantages such as good dispersibility, excellent stability, and ease of functionalization. Examples include polymer-embedded fluorescent microspheres (100-400 nm) developed by Bangs Labs in the US, and colored latex microspheres (200-300 nm) commonly used by immunochromatographic reagent companies such as Alltech Biotech and Wondfo Biotech in China. In academic research, Li et al. assembled fluorescent materials through electrostatic adsorption by modifying the surface of polystyrene spheres; Yu's research team assembled fluorescent materials into the internal cavities and surface of silica nanospheres through chemical bonding. Although these materials, as signal probes, can improve the sensitivity of LFIA to some extent, they still have significant performance limitations. This is because the pores of NC membranes are complex, and their size varies from tens to thousands of nanometers depending on the membrane type. For example, commonly used NC membrane types CN95 and CN140 have pore sizes of approximately 15 μm and 8 μm, respectively. Three-dimensional spherical structures are homogeneous structures. When traditional submicron-sized spheres pass through the intricate NC membrane tubular channels, they are simultaneously restricted in length, width, and height. Any dimension blocked by the channels leads to retention and blockage on the membrane, resulting in a lack of selective permeability. This drastically reduces the number of signal probe-antigen immune complexes participating in the immune reaction between the test strip and the captured antibody, significantly lowering the efficiency of the immune response. Furthermore, the portion of the signal probe-antigen immune complexes blocking the NC membrane tubular channels causes a high background signal on the test strip, reducing the signal-to-noise ratio. In addition, theoretical and experimental studies have shown that three-dimensional spherical structures have slower diffusion dynamics than one-dimensional tubular structures of the same size, resulting in lower recognition efficiency at the test site during the immune reaction.

[0005] From the perspective of signal amplification, most three-dimensional or two-dimensional materials inherently possess a thick medium, exhibiting a strong light scattering effect. When the fluorescence signal generated by the signal probe under ultraviolet excitation passes through this thick and non-uniform medium, some light deviates from its original propagation direction, scattering in all directions instead of directly reaching the target collection location. Since the human eye or smart devices receive fluorescence signals in a fixed direction, light scattering affects signal collection efficiency, leading to signal loss. Furthermore, light scattering caused by the thick silicon medium can also degrade signal quality. When light encounters this medium, it interacts, emitting weaker light (called wavelets) with the same frequency as the incident light in all directions. The uniformity and symmetry of this scattered light distribution depend on the relationship between the size of the scattering particles and the wavelength of the incident light. When the size of the scattering particles is much smaller than the wavelength of the incident light, molecular scattering or Rayleigh scattering occurs, resulting in a uniform and symmetrical scattered light distribution. However, when the size of the scattering particles is comparable to the wavelength of the incident light, Mie scattering occurs, resulting in an asymmetrical and complex intensity distribution of the scattered light. Therefore, these different types of scattering all affect signal quality. In summary, for the current mainstream carriers of LFIA, due to the inherent properties of their materials, they exhibit a strong light scattering effect, which reduces the signal amplification effect—something rarely reported in previous work.

[0006] To address the shortcomings of the aforementioned materials in signal amplification and immune recognition efficiency, this invention employs a one-dimensional hollow tubular material as a carrier for the signal probe. Its unique one-dimensional structure allows it to efficiently pass through the intricate NC membrane tubular channels with minimal cross-sectional dimension, ultimately reaching the test line and reacting with the capture antibody. Simultaneously, its unique one-dimensional hollow tubular structure enhances the diffusion rate of the probe, enabling a thorough immune recognition reaction with the capture antibody upon reaching the test line. The combined effect of these two factors improves the immune recognition efficiency of the signal probe-antigen immune complex and the capture antibody, which is of significant importance and value in effectively enhancing the sensitivity of LFIA. This invention uses a one-dimensional hollow tubular material as a carrier for the signal tag, reducing interference from light scattering effects. Furthermore, we can use chemical methods to controllably assemble quantum dots into the one-dimensional hollow tubular material, preparing composite nanostructures with high quantum dot loading to achieve better signal amplification. This dual optimization of signal amplification and immune reaction efficiency is beneficial for improving the sensitivity of the lateral flow immunochromatography platform, providing important scientific evidence for the early detection, treatment, and prevention of viral infections. Summary of the Invention

[0007] To address the limitations of existing commercially available submicron-level uniform morphology probes in signal amplification and immunorecognition efficiency, this application aims to provide a high-recognition, low-scattering quantum dot tubular assembly structure immunochromatographic probe, its preparation method, and applications. This invention develops a one-dimensional hollow tubular quantum dot controllable assembly labeling probe that integrates high immunorecognition efficiency, low scattering effect, and integrated signal amplification during assembly. Using hollow silica nanotubes as a carrier, this invention leverages the low light scattering effect and large specific surface area, employing an affinity polymer-mediated layer-by-layer assembly strategy to significantly improve the brightness of the labeling probe. Simultaneously, the probe exhibits excellent permeability and diffusion rate in NC films, enhancing immunoreaction efficiency.

[0008] This invention also constructs an immunochromatographic test strip with high quantitative sensitivity and low detection limit in the naked eye. First, well-defined nickel-hydrazine complex nanorods are formed through a precipitation reaction in reverse micelles, followed by silica coating using a sol-gel process. The silica deposition around the nickel-hydrazine complex stabilizes the complex, thereby replicating the rod-like structure. Finally, through selective etching, hollow silica nanotubes with high uniformity and high yield are generated after the complex dissolves. Then, quantum dots are densely and uniformly assembled on the surface of the hollow silica nanotubes using chemical bond-driven forces. Due to the low scattering effect of the hollow silica nanotubes and the affinity assembly driven by layer-by-layer coordination based on the polymer interlayer, the signal is maximized while maintaining the one-dimensional hollow tubular material structure, constructing a high-density quantum dot assembly and a high-fluorescence-intensity oil-phase assembly. The assembly is then transferred to an aqueous solvent using an efficient alkylsilane phase transfer strategy. A thin, dense silica layer is deposited on its surface to protect the assembly. Based on this, carboxylation is performed, and a fluorescent probe is obtained by conjugating a SARS-CoV-2 antigen N protein antibody. Finally, the fluorescent probe obtained by this method was used in a sandwich-type lateral flow immunochromatographic detection platform, and visualized and quantified by naked eye and smart device terminals, which has important significance and value for the early screening and diagnosis of SARS-CoV-2 antigen.

[0009] The technical solution adopted in this invention is as follows:

[0010] The method for preparing a high-recognition, low-scattering quantum dot tubular assembly structure immunochromatographic probe involves reacting Ni salt with hydrazine hydrate in the presence of a surfactant to form nickel-hydrazine complex nanorods. These nanorods are then coated with silica using a sol-gel process, followed by selective etching with hydrochloric acid to obtain one-dimensional hollow tubular silica nanotubes (SNTs). The SNTs are then thiolized to obtain SNTs-SH. Using SNTs-SH as a carrier and CdSe / CdS / ZnS red quantum dots (QDs) as signal tags, multilayer assembly of quantum dots is performed on the SNTs-SH carrier to construct an oil-soluble tubular assembly, dSi / tQD. Subsequently, a hydrolysis-condensation reaction is carried out on the surface of the tubular assembly using n-octyltrimethoxysilane (OTMS) to achieve phase transfer from the organic phase to the aqueous phase. Finally, the assembly is further processed by... The method utilizes tetraethyl orthosilicate (TEOS) hydrolysis to deposit a thin layer of silica to improve the stability of the internally assembled quantum dots. Then, the STQS-COOH is obtained by sequentially modifying the amino group with 3-aminopropyltriethoxysilane and the carboxyl group with succinic anhydride. The carboxyl-modified assembly STQS-COOH is coupled with a labeled antibody and blocked with a blocking solution containing BSA to finally obtain the probe.

[0011] Furthermore, the preparation method of the one-dimensional hollow tubular silica nanotubes (SNTs) specifically includes the following steps:

[0012] S1: Dissolve the surfactant Brij58 in cyclohexane, stir at 45-55℃, add Ni salt, then add hydrazine hydrate dropwise, stir for 2.8-3.2h to react Ni salt with hydrazine hydrate to form nickel-hydrazine complex nanorods;

[0013] S2: Then add diethylamine and TEOS to the system in step S1, and continue stirring the reaction for 1.5-2.5 hours to allow silica deposition to occur. After the reaction is complete, centrifuge and wash.

[0014] S3: The product obtained in step S2 is dispersed in a 0.8-1.2M hydrochloric acid solution and selectively etched by stirring at room temperature for 0.5-2 hours. The product is then collected by centrifugation and washed with water until the pH is neutral to obtain one-dimensional hollow tubular silica nanotubes (SNTs), which are then stored in anhydrous ethanol.

[0015] Further, in step S1, the surfactant is Brij58, and its concentration in cyclohexane is 0.5-0.6 g / mL. The molar ratio of hydrazine hydrate to Ni salt is 4-10:1, and the concentration of Ni salt in cyclohexane is 50-100 mmol / L.

[0016] Furthermore, in step S2, the volume ratio of diethylamine to TEOS is 0.4-0.6:1, and the volume ratio of TEOS in step S2 to the mole ratio of Ni salt in step S1 is 1:0.5-0.8. The volume unit is mL, and the mole unit is mmol.

[0017] Furthermore, the process of multilayer assembly of quantum dots on the template SNTs-SH is as follows:

[0018] 1) Assembly of the first layer of quantum dots: Take a toluene solution of CdSe / CdS / ZnS quantum dots, add the template SNTs-SH, and sonicate for 7-10 min to obtain a homogeneous solution. Then, centrifuge to collect the precipitate. After the residual toluene solvent in the precipitate evaporates, the assembly of the first layer of quantum dots, SNTs / QD1, is obtained. The mass ratio of the template SNTs-SH to CdSe / CdS / ZnS quantum dots is 1:0.8-1.0.

[0019] 2) Coating with aminated dendritic polymer: The assembled product obtained in step 1) is added to an ethanol solution containing aminated dendritic polyamide PAMA-NH2, sonicated for 7-10 min, and the supernatant is removed by centrifugation to obtain the nanocomposite SNTs / QD-PAMA with the outermost layer coated with amino groups. Then, the amino groups on its surface can coordinate with quantum dots to continue the assembly of quantum dots.

[0020] 3) Repeat steps 1)-2) above to achieve the alternating assembly of organic phase quantum dots and aminated dendritic polymer polyamide PAMA-NH2 in multiple layers, assembling a total of 2-4 quantum dot layers, preferably assembling 3 quantum dot layers, and finally obtaining the constructed oil-soluble assembly SNTs / tQD product.

[0021] Further, the process of conjugating the carboxyl-modified assembly with the labeled antibody and blocking it using a blocking buffer containing BSA is as follows: The carboxyl-modified assembly is added to pH 6.0 PB buffer, NHs and EDC are added, and it is incubated in a shaker for 20-40 min; then the product is collected by centrifugation; the product is resuspended in pH 7.4 PB buffer, the labeled antibody is added, and the reaction is carried out in a shaker for 2-3 h; after the reaction, it is centrifuged and blocked with pH 7.4 PB buffer containing 0.5-2% BSA for 1.5-3 h; then the tubular probe is collected by centrifugation, washed with pH 7.4 PB buffer, and stored in pH 7.4 PB buffer under cold storage.

[0022] This invention also provides an application of the aforementioned high-recognition, low-scattering quantum dot tubular assembly structure immunochromatographic probe, which is used to detect corresponding antigens, including the following steps:

[0023] S1 Immunochromatographic test strip construction: The immunochromatographic test strip includes a sample pad, an NC membrane and an absorbent pad assembled sequentially on a plastic base plate. The T line and C line of the NC membrane are coated with capture antibody and goat anti-mouse IgG antibody, respectively.

[0024] S2 Antigen Concentration Detection: The antigen is diluted with a PBS buffer (pH 7.4) containing 0.05-0.2% sodium caseinate, 0.1-1% BSA, 0.1-1% Tween 20, and 0.1-0.5% PVP360 to prepare a series of standard solutions with different antigen concentrations. The probe is added to the standard solutions and mixed thoroughly. The solutions are then dropped onto the sample pad of the test strip. Under capillary action, the probe flows through the NC membrane to the absorption pad. After 5-20 minutes, the test strip is photographed using a smartphone under 365nm UV excitation light to obtain images of the T-line and C-line. The R-value of the T-line image is read using ColorPicker software on the smartphone. A standard curve is plotted with the R-value on the ordinate and the antigen concentration on the abscissa.

[0025] Furthermore, the labeled antibody is SARS-CoV-2NP monoclonal antibody 2, the capture antibody is SARS-CoV-2NP monoclonal antibody 1, and the antigen is SARS-CoV-2NP antigen.

[0026] This invention, focusing on the probe itself, designs a probe that exhibits excellent throughput and superior immunoreaction kinetics in a lateral flow immunochromatography (LFIA) platform, thereby improving the efficiency of the LFIA immunoreaction. While reducing light scattering interference, the quantum dots are controllably assembled, significantly enhancing the signal intensity of individual materials. Due to the dual optimization of signal amplification and immunoreaction efficiency, this invention is beneficial for improving the sensitivity of LFIA, providing important scientific evidence for the early detection, treatment, and prevention of viral infections.

[0027] The beneficial effects achieved by this invention are:

[0028] 1) This invention forms nickel-hydrazine complex crystal nanorods in reverse micelles by end-capping with surfactants, and then obtains hollow silica nanotubes by silica coating and selective etching. Using these as nanocarriers and quantum dots as nano-signal units, the light scattering effect of hollow silica nanotubes and the high-density, uniform and controllable assembly of quantum dots are studied. From the perspective of signal amplification, the fluorescence intensity of the tubular assembly material is maximized, which effectively solves the problem that the light scattering phenomenon of other solid templates leads to a reduction in the fluorescence intensity of the material.

[0029] 2) This invention studies the immunocapture efficiency and immunoreaction kinetics of hollow tubular materials and solid spherical materials in solutions and test strips, and verifies that one-dimensional hollow tubular materials have greater advantages in immunocapture efficiency and immunoreaction kinetics than three-dimensional solid spherical materials. This provides important theoretical guidance and experimental basis for using them as immunocapture probes to improve the sensitivity of lateral flow immunochromatography platforms.

[0030] 3) This invention constructs a sandwich-type lateral flow immunochromatographic detection platform, which optimizes the design and synthesis of probes from the dimensions of signal amplification and immunodynamics, without introducing additional or complex operations and steps. It significantly improves the sensitivity of sandwich-type lateral flow immunochromatographic test strips in the simplest and most effective way, and is expected to be widely used in the fields of biodetection medicine and biosensing.

[0031] 4) Commercially available lateral flow immunochromatographic test strips primarily use colloidal gold or rare-earth fluorescent microspheres as labeled probes. However, due to their weak signal amplification, low immune capture efficiency, and slow immune reaction kinetics, these strips suffer from limited sensitivity, high false-negative rates, and weak quantitative capabilities, making high-sensitivity detection difficult. Therefore, there is an urgent need to develop a simple-to-synthesize, high-performance material as a labeled probe for lateral flow immunochromatographic platforms to improve band sensitivity and quantitative capabilities. This invention focuses on the synthesis and design of signal probes, comprehensively regulating the light scattering effect, immune capture efficiency, and immune reaction kinetics of nanomaterials. The designed labeled probe not only improves signal amplification but also exhibits excellent immune capture efficiency and chemical reaction kinetics. The synthesis process is simple and controllable, showing broad prospects for industrial production. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the synthesis route of the STQS tubular assembly in an embodiment of the present invention;

[0033] Figure 2 This is a comparative diagram of the immunoassay principle of the hollow tubular probe assembly of the present invention and the existing solid spherical assembled probe;

[0034] Figure 3 These are TEM images of the products at each stage of the preparation process of the STQS tubular assembly in Example 1 of the present invention; (a) solid silicon tube template SNTs@Ni-N2H4, (b) hollow silicon tube template SNTs, (c) three-layer oil phase assembly SNTs / QD3, (d) STQS tubular assembly structure.

[0035] Figure 4The results show the performance test results of assemblies prepared under different quantum dot feed ratios; a) Relationship between quantum dot assembly rate and feed ratio (the inset shows daylight photographs of the supernatant (top) and the assemblies dispersed in toluene solution (bottom) after quantum dot assembly at each stage); b) Relationship between the fluorescence intensity of the quantum dot assemblies and the quantum dot assembly rate.

[0036] Figure 5 The results are the fluorescence performance test results of different quantum dot materials in Example 3 of this invention; a) fluorescence spectra of the STQS assembly structure of hydrophobic quantum dots and their derivatives with red (R), green (G) and blue (B); b) fluorescence spectra of STQS nanomaterials at different stages (the percentage content on the spectrum is defined as follows: the first curve is the fluorescence intensity of 100% loaded free quantum dots, and the percentage content of the remaining stages is the fluorescence intensity relative to the first curve).

[0037] Figure 6 This is a shape light scattering effect verification, showing the fluorescence intensity relationship between hollow tubular assembly structures and solid spherical assembly structures with different particle numbers (in Example 4 of this invention, a single material uniformly assembles the same number of quantum dots); Figure 6 The ratio of the slopes of the fitted curves represents the signal amplification factor. The insets above the fitted curves show: upper left: sunlight image of the hollow tubular assembly solution; lower left: fluorescence image of the solution; upper right: sunlight image of the solid spherical assembly solution; lower right: fluorescence image of the solution. The inset in the middle of the fitted curves illustrates the experimental principle.

[0038] Figure 7 This is a form effect solution immunokinetics verification, showing the relationship between the fluorescence intensity of hollow tubular assembly structures and solid spherical assembly structures with the same number of particles (in Example 4 of this invention, a single material is uniformly assembled with the same number of quantum dots) and the fluorescence intensity of streptavidin-FITC fluorescent dye in the reaction supernatant (the middle of the fitting curve is a schematic diagram of the experimental principle).

[0039] Figure 8 This describes the relationship between the R-value signal intensity and the number of particles on the detection line for hollow tubular or solid spherical assembled probes (in Example 4 of this invention, a single material uniformly assembles the same number of quantum dots) in the biotin-streptavidin system; a) Test results of hollow tubular assembled probes; b) Test results of solid spherical assembled probes. The upper left inset shows the fluorescence pattern of the detection line of the two corresponding materials under ultraviolet light irradiation, and the lower inset shows a schematic diagram of the detection principle.

[0040] Figure 9 These are fluorescence images of the fluorescent lateral flow immunochromatographic test strips within the SARS-CoV-2 NP antigen concentration range of 0-500 ng / mL.

[0041] Figure 10The linear and nonlinear fitting curves of the T-line R value and different concentrations of SARS-CoV-2 NP antigen were read using the ColorPicker color reading software on a smartphone.

[0042] Figure 11 The results show the linear and nonlinear fitting curves of the T-line R signal intensity and different concentrations of SARS-CoV-2 NP antigen measured using a commercial reading machine. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0044] In this embodiment of the invention, the concentration of ammonia water is 25-28%.

[0045] See the schematic diagram of the synthesis route of the STQS tubular assembly in this embodiment of the invention. Figure 1 .

[0046] A comparative schematic diagram of the immunoassay principle of the hollow tubular probe of this invention and the existing solid spherical probe is provided. Figure 2 , Figure 2 The probe is first coupled with a labeled antibody to the material to obtain a probe. Then, the probe and the antigen are specifically bound together. The corresponding capture antibodies are laid on the C and T sections of the test strip to form a double-antibody sandwich immunoassay structure for detection.

[0047] Example 1:

[0048] 1. Synthesis of Hollow Silica Nanotube (SNT) Templates

[0049] Step 1) Synthesis of nickel-hydrazine@silica nanotubes: 8.5 g of Brij58 was dissolved in 15 mL of cyclohexane and stirred at 50 °C. 1.5 mL of NiCl2·6H2O aqueous solution (concentration 0.8 mol / L) was added, followed by dropwise addition of 0.45 mL of hydrazine hydrate (hydrazine concentration 64%). The mixture was stirred for 3 hours, and then 1 mL of diethylamine and 2 mL of tetraethyl silicate (TEOS) were added to the system. The reaction was continued by stirring, and the silica coating process was carried out for 2 hours. The product was centrifuged and washed with isopropanol to obtain nickel-hydrazine@silica nanotubes, which were labeled as solid silica tube templates SNTs@Ni-N2H4.

[0050] Step 2) Etching of nickel-hydrazine@silica nanotubes: Solid silica nanotube templates SNTs@Ni-N2H4 were dispersed in 300 mL of HCl (1 mol / L) aqueous solution. The dispersion was stirred at room temperature for 1 hour, and the product was collected by centrifugation. The product was washed with water until the pH was neutral to obtain hollow silica nanotubes SNTs (i.e., hollow silica nanotube templates SNTs), which were stored in anhydrous ethanol (dispersion concentration of approximately 10 mg / mL).

[0051] TEM images of the solid silicon tube template SNTs@Ni-N2H4 and the hollow silicon tube template SNTs are shown below. Figure 3 Subplots a and b.

[0052] 2. Synthesis of Thiolized Hollow Silica Nanotubes (SNTs-SH) Templates

[0053] Take 18 mL of the ethanol dispersion containing 180 mg SNTs, add 182 mL of ethanol, 2 mL of ammonia, and 200 μL of (3-mercaptopropyl)trimethoxysilane (MPS), and stir at 520 rpm for 12 h at room temperature. Finally, centrifuge to collect the precipitate, wash it three times with ethanol to obtain thiol-modified hollow silica nanotubes (SNTs-SH), and disperse them in ethanol (dispersion concentration approximately 10 mg / mL).

[0054] 3. Multi-layer coordination-driven affinity assembly

[0055] Take 1 mL of the SNTs-SH ethanol solution obtained in step 2, centrifuge to remove the supernatant to obtain a precipitate (approximately 10 mg SNTs-SH), and allow it to air dry slightly. Then add 1 mL (10 mg / mL) of toluene solution containing cadmium selenide / cadmium sulfide / zinc sulfide red quantum dots (QDs), and sonicate for 7 min to obtain a uniformly dispersed solution. Centrifuge to remove the supernatant, and wash with toluene to remove unbound QDs, obtaining an oil-soluble assembly (SNTs / QD1) with one layer of QDs. Add 2 mL of an ethanol solution (3 mg / mL) of dendritic polyamide (PAMA-NH2) to the above SNTs / QD1 assembly precipitate, sonicate for 10 min to obtain a uniformly dispersed solution, centrifuge to remove the supernatant, and wash with ethanol to remove excess PAMA-NH2, obtaining a nanocomposite of SNTs-SH / QD1 / PAMA-NH2 with the outermost layer coated by PAMA-NH2 (SNTs / QD1 / P). Subsequently, to assemble the second layer of QDs, 1 mL of a toluene solution (10 mg / mL) of red QDs was added to the SNTs / QD1 / P precipitate. After sonication for 7 min, a uniformly dispersed solution was obtained. The supernatant was removed by centrifugation, and excess QDs were removed by washing with toluene, yielding an oil-soluble assembly of two layers of QDs (SNTs / QD2). The above operation was repeated, with 2 mL of an ethanol solution (3 mg / mL) of dendritic polyamide (PAMA-NH2) added to the wet precipitate of the SNTs / QD2 assembly. After sonication for 10 min, a uniformly dispersed solution was obtained. The supernatant was removed by centrifugation, and excess PAMA-NH2 polymer was removed by washing with ethanol, yielding a nanocomposite of SNTs-SH / QD2 / PAMA-NH2 (SNTs / QD2 / P). Finally, to assemble the third layer of QDs, 1 mL of a toluene solution of red QDs (10 mg / mL) was added to the SNTs / QD2 / P precipitate. After sonication for 7 min, a uniformly dispersed solution was obtained. The supernatant was removed by centrifugation, and excess unbound QDs were removed by washing with toluene to obtain the oil-soluble assembly of the three-layer QDs (SNTs / QD3).

[0056] TEM images of the oil-soluble assembly SNTs / QD3 of the above-mentioned three-layer QDs are shown below. Figure 3 The subplot c.

[0057] 4. Phase transition of the assembly

[0058] 200 μL of octyltrimethoxysilane (OTMS) was added to the oil-soluble SNTs / QD3 precipitate and sonicated for several minutes until it dissolved into a homogeneous solution. Then, a mixture of 30 mL methanol and 0.75 mL ammonia was added, and sonication was continued for 30 min. The precipitate was then collected by centrifugation, and unbound OTMS was washed with methanol. The product was then dispersed in a mixture of 33 mL water and 66 μL sodium silicate and stirred at 400 rpm for 18 h at room temperature to form an organosilicon shell, yielding a silane-modified SNTs / QD3-OTMS complex.

[0059] 5. Preparation of STQS tubular assemblies

[0060] pass Silane growth was performed on the surface of SNTs / QD3-OTMS using a specific method. The SNTs / QD3-OTMS were centrifuged to remove the supernatant, and then ultrasonically dispersed in 20 mL of ethanol. Subsequently, 5 mL of water and 0.625 mL of ammonia were added, along with 25 μL of tetraethyl orthosilicate (TEOS). The reaction was carried out with medium-speed stirring. TEOS was added again after a 1-hour interval (25 μL each time), and the reaction was continued for a total of 2 hours. After the reaction was complete, the product was collected by centrifugation and then washed three times with ethanol to remove excess TEOS, yielding STQS tubular assemblies coated with silica.

[0061] TEM images of the above STQS tubular assembly are shown below. Figure 3 The subplot d.

[0062] Figure 3 The results lead to the following conclusions: 1) Comparison of sub-images a and b proves that the solid silicon tube template was transformed into a hollow silicon tube template through the etching process; 2) Comparison of sub-images b and c proves that quantum dots were assembled on the hollow silicon tube template; 3) Comparison of sub-images c and d proves that the tubular assembly was coated with silicon dioxide.

[0063] The fluorescence spectra of the products at different stages of the preparation of the above-mentioned STQS tubular assemblies are shown in [reference]. Figure 5 b. Definition of percentage content on the spectrum: The first curve represents the fluorescence intensity of 100% free quantum dots, and the percentage content of the remaining stages represents the fluorescence intensity relative to the first curve. Figure 5 In section b, QDs are bare quantum dots, SNTs / QD1 is a single-layer quantum dot assembly, SNTs / QD2 is a two-layer quantum dot assembly, SNTs / QD3 is a three-layer quantum dot assembly, SNTs / QD3-OTMS is a silane-modified complex, and STQS is a tubular assembly coated with silica. The ability of the SNTs-SH template to assemble multilayer quantum dots was investigated by measuring the fluorescence intensity at different assembly stages. Figure 5As shown in b, we define 100% as the fluorescence intensity value of the oil-phase quantum dots with the same amount as the first layer of unassembled quantum dots. The fluorescence intensity of SNTs / QD1 with one layer of quantum dots is slightly lower than that of CdSe / CdS / ZnS QDs themselves. This may be due to aggregation-induced quenching caused by the dense assembly of quantum dots. However, the relative fluorescence intensity increases after the second layer of quantum dots is successfully assembled, and the relative fluorescence intensity of the assembly continues to increase after the introduction of the third layer of quantum dots. Thus, the fluorescence intensity is significantly improved due to the assembly of more quantum dots. When phase transfer and silica coating strategies are used, the fluorescence intensity of the material decreases slightly, possibly due to the loss of assembly material during washing, centrifugation, and other operations, as well as the scattering effect caused by the silica coating material.

[0064] 6. Preparation of carboxylated STQS-COOH tubular assemblies

[0065] The above-mentioned STQS was dissolved in 40 mL of ethanol, followed by the addition of 1 mL of ammonia and 40 μL of 3-aminopropyltriethoxysilane (APTES). The mixture was stirred at room temperature for 12 h to obtain amino-modified STQS-NH2. Excess APTES was then removed by washing with ethanol, and the product was dispersed in 20 mL of N,N-dimethylformamide (DMF). 100 mg of succinic anhydride was added, and the mixture was stirred for another 4 h to obtain carboxyl-modified STQS microtubes (STQS-COOH). The product was washed several times with ethanol and water.

[0066] 7. STQS-COOH tubular assembly conjugate antibody

[0067] The monoclonal antibody against SARS-CoV-2NP (AB0046-2, Nanjing Baikang Biotechnology) was conjugated to STQS-COOH using a typical carbodiimide-assisted conjugation method. The specific steps are as follows:

[0068] First, 2 mg of STQS-COOH, 2 mg of N-hydroxythiosuccinimide sodium salt (Sulfo-NHS), and 1 mg of carbodiimide hydrochloride (EDC) were dissolved and dispersed in 1 mL of PB (pH = 6.0, 0.01 mol / L) buffer solution. The mixture was incubated on a shaker for 30 min at room temperature, followed by centrifugation at 10,000 rpm for 5 min. The supernatant was discarded, and the precipitate was redispersed in 1 mL of PB (pH = 7.4, 0.01 mol / L) buffer solution. 0.04 mg of SARS-CoV-2NP antibody (AB0046-2, Nanjing Baikang Biotechnology) was added, and the mixture was sonicated for 30 s. The mixture was then incubated on a shaker for 2.5 h at room temperature. Subsequently, the mixture was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the precipitate was redispersed in 1 mL of PB (pH = 7.4, 0.01 mol / L, containing 1% BSA) blocking solution. The mixture was blocked on a shaker for 2 h at room temperature. Finally, after the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm and washed twice with PB (pH=7.4, 0.01 mol / L) buffer solution. Finally, it was dispersed in 1 mL of PB (pH=7.4, 0.01 mol / L) buffer solution to obtain the iSTQS signal probe, which was stored at 4 °C for subsequent use.

[0069] 8. Preparation of Immunochromatographic Test Strips

[0070] First, the sample pad was treated with sample pad treatment solution (pure water, containing 1.2% Tris, 0.12% EDTA, 0.04% sodium caseinate, 1% trehalose, 0.5% Tween-20, pH=7.4) and dried overnight at 37℃. SARS-CoV-2 NP monoclonal antibody (AB0046-1, Nanjing Baikang Biotechnology) (2 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) were immobilized on the T and C lines of the NC membrane using a gold sputtering apparatus at a rate of 0.75 μL / cm, respectively, and then dried overnight at 37℃. Finally, the absorbent pad, NC membrane, and sample pad were assembled sequentially, cut into 3 mm wide strips, and sealed for storage under dry conditions.

[0071] 9. Immunochromatographic detection of SARS-CoV-2 NP antigen

[0072] The SARS-CoV-2 NP antigen was diluted to a specific concentration using 10 mM PBS buffer (pH 7.4) containing 0.1% sodium caseinate, 0.2% BSA, 0.4% Tween, and 0.2% PVP360. The antigen concentration range was 0-500 ng / mL. 80 μL of sample solutions containing different concentrations of SARS-CoV-2 NP antigen and 2 μg of iSTQS signal probe were thoroughly mixed and dropped onto the sample pad. The antigen flowed through the NC membrane to the absorption pad via capillary action. As the antigen concentration increased, the T line gradually changed from colorless to red, while the C line remained red. After 10 minutes, the sample was photographed using a smartphone under excitation with any type of small UV counterfeit money detector lamp (365 nm excitation light). The fluorescence images of the fluorescent lateral flow immunochromatographic test strip within the 0-500 ng / mL SARS-CoV-2 NP antigen concentration range are shown below. Figure 9 As shown.

[0073] 10. Quantitative analysis via mobile app on immunochromatographic platform

[0074] Subsequently, based on the test results of step 9, the R value of the T-line (the R value channel of the T-line photograph image) was read using the Color Picker software on a smartphone. A standard curve was plotted with the SARS-CoV-2 NP antigen concentration on the x-axis and the R value on the y-axis. The results are shown below. Figure 10 .from Figure 10 The standard curve shows that the nonlinear curve fitting range for the concentration of the SARS-CoV-2 N protein is 0-500 ng / mL, corresponding to R0. 2 The value was 0.989; the linear range was 0-2 ng / mL, corresponding to R0. 2 The linear range was 0.982, indicating a good linearity, and a detection limit of 0.02 ng / mL (uncorrected visual detection limit) and a quantitation limit of 0.019 ng / mL were obtained. Therefore, the signal readout model can achieve good linearity and detection sensitivity.

[0075] 11. Quantitative analysis at the instrument end of the immunochromatographic platform

[0076] Based on the test results of step 9, the R signal intensity of the T line was measured using a commercially available reading machine (the commercial reading machine was provided by the Shenzhen Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, model TND09-MJ). A standard curve was plotted with the SARS-CoV-2 NP antigen concentration on the x-axis and the R signal intensity of the T line measured using the commercial reading machine on the y-axis. The results are shown in [Figure 1]. Figure 11 .from Figure 11 The standard curve shows that the nonlinear curve fitting range for the concentration of the SARS-CoV-2 N protein is 0-500 ng / mL, corresponding to R0. 2 The value was 0.996; the linear range was 0-0.2 ng / mL, corresponding to R0. 2The linear range was 0.962, indicating a good linear range, and a detection limit as low as 0.081 ng / mL was obtained.

[0077] Example 2:

[0078] Repeat steps 1-3 of Example 1, except that the following operating conditions are changed in step 3:

[0079] 1) In the operation steps of one-layer assembly of quantum dots (QDs), the volume of QDs toluene solution (10mg / mL) is 0.2mL-1mL, the total QDs feed ratio is 20% to 100%, and other operating conditions remain unchanged. Finally, a series of one-layer assemblies SNTs / QD1 under different QDs feed ratio conditions are obtained.

[0080] 2) The SNTs / QD1 layer assembly prepared under the condition of 100% QDs feed ratio was further subjected to the second layer assembly of QDs. The difference was that the volume of the QDs toluene solution (10 mg / mL) was 0.2 mL to 1 mL, so that the total QDs feed ratio ranged from 120% to 200%. Other operating conditions remained unchanged, and finally a series of second layer assemblies SNTs / QD2 under different quantum dot QDs feed ratio conditions were obtained.

[0081] 3) The two-layer assembly SNTs / QD1 prepared under the condition of 200% QDs feed ratio was further subjected to the three-layer assembly of QDs. The difference was that the volume of QDs toluene solution (10 mg / mL) was 0.2 mL to 1 mL, so that the total QDs feed ratio ranged from 220% to 300%. Other operating conditions remained unchanged, and finally a series of three-layer assemblies SNTs / QD3 under different quantum dot QDs feed ratio conditions were obtained.

[0082] In the above-mentioned first to third layer assembly process, the assemblies prepared under different QDs feed ratios were subjected to performance tests. The test methods and steps are as follows:

[0083] The relationship between the quantum dot assembly rate and the quantum dot feed ratio in the assembly is shown in the appendix. Figure 4 Figure a shows a daylight photograph of the supernatant (top) and the assemblies dispersed in toluene solution after each stage of quantum dot assembly (bottom). Figure 4 As shown in sub-figure a, for the assembly of each layer of quantum dots, the assembly rate reaches saturation with the increase of the feed ratio. Therefore, it is necessary to find the inflection point where the assembly rate reaches saturation to ensure that the material assembles as many quantum dots as possible to improve the optical performance of the material. Furthermore, the relationship between the fluorescence intensity of the quantum dot assembly and the quantum dot assembly rate is shown in the appendix. Figure 4 Sub-image b. Figure 4 The quantum dot assembly rate in sub-figure b, and Figure 4 The assembly rate corresponds to that in sub-plot a. Figure 4 The quantum dot assembly rate in sub-figure b is the result of assembling one, two, or three layers together.

[0084] Figure 4 The assembly of the second and third layers of quantum dots is carried out after the assembly of the first layer has reached saturation. The second layer continues to add quantum dots based on the 100% addition ratio of the first layer, and the third layer continues to add quantum dots based on the 100% addition ratio of the second layer. The quantum dot addition ratio is the mass ratio of oil-phase quantum dots to silicon template. The quantum dot assembly rate is quantitatively detected by a fluorescence spectrometer. The quantum dot assembly rate in the assembly is calculated as follows: (fluorescence intensity of the quantum dot stock solution - fluorescence intensity of the supernatant after centrifugation) / fluorescence intensity of the quantum dot stock solution * quantum dot addition ratio.

[0085] Furthermore, the assembly results from one to three layers demonstrate the importance of PAMA-NH2 polymer filling in improving the quantum dot assembly yield.

[0086] Example 3:

[0087] The preparation process of the STQS tubular assembly in Example 1 was repeated, except that the red QDs were replaced with an equal mass of red, blue, or green QDs, thus ultimately obtaining red STQS, blue STQS, or green STQS, respectively. The red, blue, and green QDs, as well as the red, blue, and green STQS, were subjected to fluorescence intensity tests, and the measured fluorescence intensities were normalized. The test results are shown in […]. Figure 5 As shown in a, Figure 5 The illustrations in a are fluorescent photographs of suspensions of red STQS, blue STQS, or green STQS, respectively. Figure 5 The experimental results of a show that for quantum dots of the same color, the fluorescence emission wavelength did not change before and after assembly, thus ensuring the material's performance.

[0088] Example 4:

[0089] A performance comparison was conducted between hollow silicon tube assembly materials and solid spherical assembly materials.

[0090] The hollow tubular template is the hollow silica nanotubes (SNTs) prepared in step 1 of Example 1.

[0091] The preparation process of the solid spherical template includes the following steps:

[0092] 1) Mix 40ml ethanol, 2.43ml deionized water and 1.8ml ammonia water and heat to 55℃ with vigorous stirring. Quickly mix 4ml ethanol and 1.55ml TEOS and add to the above solution. Stir at 55℃ for 5 hours.

[0093] 2) Take 10 ml of the above reaction solution, add 70 ml of ethanol to a 250 ml bottle, add 7.5 ml of ammonia water, stir at room temperature, and then add 1 ml of TEOS and 10 ml of ethanol dropwise. React for 5 hours. Then add another 1 ml of TEOS and 10 ml of ethanol to the above system and continue the reaction for 5 hours.

[0094] 3) Finally, add 266 μL of TEOS and 3 mL of ethanol to the system and react for 80 min. Wash with ethanol to obtain solid silicon template dsi, which is stored in ethanol (dispersion concentration is about 10 mg / mL).

[0095] To facilitate a comparison of the performance of hollow silicon tube assembly materials and solid spherical assembly materials, it is necessary to control the number of quantum dots in a single hollow silicon tube assembly and a single solid spherical assembly to be the same. Theoretical calculations are required to ensure that the two materials are assembled in an unsaturated manner. The theoretical calculation process is described below.

[0096] Based on electron microscopy images of the material and measurements using nanometer software, the diameter (2r) of the solid spherical template dSI is 145 nm; (V) 实心球 =(4 / 3)πr 3 m = ρ SiO2 V. The mass m of a single solid silicon sphere of this size can be calculated as: 3.52 × 10⁻⁶. -12 mg.

[0097] The major axis (2a1) of the hollow tubular template is 139 nm, and the minor axis (2b1) is 36 nm. The major axis (2a2) of the cavity is 121 nm, and the minor axis (2b2) ​​is 21 nm. (V) 空心管 =a1×b1 2 ×π-a²×b² 2 ×π, m=ρ SiO2 V; The mass m of a single hollow tubular template with such particle size can be calculated as: 2.2 × 10⁻⁶ -13 mg.

[0098] That is, for a single material, the mass of a single solid silicon sphere is 16 times the mass of a single hollow tubular template. If both materials are used in 10mg batches, the number of hollow tubular materials is 16 times the number of solid silicon spheres (N spheres). Therefore, to control the assembly of the same number of quantum dots on both types of materials, for the same mass of both materials, the assembly rate of hollow tubular material QDs should be 16 times that of solid spherical materials.

[0099] Obtained from Example 2 Figure 4 The results show that for hollow tubular materials, the assembly rate is 12% when the feed ratio is 20%. Based on the above calculations, the assembly rate for solid spherical materials of the same mass should be 0.75%. Looking at the supernatant from the assembly of solid spherical materials, when the feed ratio is 0.75%, the supernatant is colorless, and the fluorescence detector can barely detect any fluorescence, proving that the assembly rate is 0.75%. Through the above theoretical calculations, the number of QDs assembled from the two individual materials was controlled to be the same.

[0100] Hollow tubular assembly structures are prepared from hollow tubular template materials, which is to repeat the operation steps 2-6 of Example 1. The only difference is that the following operating conditions are changed in step 3: only one layer of QDs is assembled, the volume of QDs toluene solution (10mg / mL) is 0.2mL, the total QDs feed ratio is 20%, and other operating conditions remain unchanged.

[0101] The solid spherical assembly structure is prepared by repeating steps 2-6 of Example 1. The only difference is that the following operating conditions are changed in step 3: only one layer of quantum dot (QDs) is assembled, the volume of QDs toluene solution (10 mg / mL) is 0.075 mL, the total QDs feed ratio is 0.75%, and other operating conditions remain unchanged.

[0102] The effect of shape-induced light scattering intensity on fluorescence intensity ( Figure 6 Verification scheme: Two assembled structural materials with different particle numbers were dispersed in 1 mL of ethanol. The fluorescence intensity of the assembled materials was measured, and multiple points were plotted as linear slopes. The ratio of the slopes represents the amplification ratio of the fluorescence signal intensity. The test results are shown in [reference needed]. Figure 6 .

[0103] Shape effect solution ( Figure 7 Immunokinetics validation protocol:

[0104] (1) FITC fluorescent dye labeling of streptavidin (SA)

[0105] 1. Dissolve the FITC fluorescent dye in deionized water to a concentration of 10 mg / mL, and mix thoroughly to completely dissolve the FITC fluorescent dye;

[0106] 2. Dissolve 1 mg of SA in 0.5 mL of 50 mM borate buffer (pH = 8.5), add excess FITC fluorescent dye (the molar amount of FITC fluorescent dye is 18 times the molar amount of streptavidin (SA), and mix the reaction mixture immediately;

[0107] 3. Incubate in the dark at room temperature for 1 hour;

[0108] 4. Ultrafiltration removes excess FITC fluorescent dye.

[0109] (2) Two carboxylation materials were coupled with biotin to prepare a probe: 2 mg of hollow tubular assembly or solid spherical assembly was dissolved and dispersed with 2 mg of N-hydroxythiosuccinimide sodium salt (Sulfo-NHS) and 1 mg of carbodiimide hydrochloride (EDC) in 1 mL of PB (pH=6.0, 0.01 mol / L) buffer solution. The mixture was incubated on a shaker at room temperature for 30 min, then centrifuged at 10000 rpm for 5 min. The supernatant was discarded, and the precipitate was redispersed in 1 mL of PB (pH=7.4, 0.01 mol / L) buffer solution. 0.04 mg of biotin was added, and the mixture was sonicated for 30 s and then incubated on a shaker at room temperature for 2.5 h.

[0110] (3) Take two materials with the same number of particles and add the same concentration of FITC-labeled SA (nBiotin:nSA = 1:8) to 10mM PB buffer (pH = 7.4). React at room temperature for 15 minutes. Centrifuge the solution after reaction and measure the fluorescence intensity of SA-FITC fluorescence in the supernatant. Repeat the above experiment for each material with multiple different particle numbers to obtain... Figure 7 Results: The lower the fluorescence intensity of the supernatant, the better the solution immunokinetics of the material.

[0111] Figures 6-7 The particle number of a material refers to the number of particles dispersed in a solvent, which can be measured using instruments.

[0112] Shape effect test strips ( Figure 8 Immunokinetics validation protocol:

[0113] 1. Preparation of SA-Biotin system test strips: First, treat the sample pad with sample pad treatment solution (pure water, containing 1.2% Tris, 0.12% EDTA, 0.04% sodium caseinate, 1% trehalose, 0.5% Tween-20, pH=7.4) and dry overnight at 37℃. Fix SA at 1 mg / mL onto the T-line of the NC membrane using a gold sputtering apparatus at a rate of 0.75 μL / cm, and then dry overnight at 37℃. Finally, assemble the absorbent pad, NC membrane, and sample pad sequentially, cut them into 3 mm wide strips using a strip cutter, and seal and store under dry conditions.

[0114] 2. Immunochromatographic assay using the SA-Biotin system: Two probes (conjugated with biotin) with different particle numbers were added to 80 μL of 10 mM PBS buffer (pH = 7.4) (containing 0.1% sodium caseinate, 0.2% BSA, 0.4% Tween, and 0.2% PVP360). After thorough mixing, the mixture was dropped onto the sample pad and allowed to flow through the NC membrane to the absorption pad via capillary action. As the number of probe particles increased, the T-line gradually changed from colorless to red. The R-value of the T-line was read using a color picker, and the number of particles at the first signal inflection point was compared to determine the kinetic effect of the two probe assembly structures. The fewer particles at the signal inflection point, the better the kinetic effect of the probe, which can improve the sensitivity of the chromatography platform.

[0115] The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the scope of protection of this invention should not be regarded as limited to the specific forms described in the embodiments.

Claims

1. A method for preparing an immunochromatographic probe with a quantum dot tubular assembly structure, characterized in that... Ni salts were reacted with hydrazine hydrate in the presence of a surfactant to form nickel-hydrazine complex nanorods. These nanorods were then coated with silica using a sol-gel process and selectively etched with hydrochloric acid to obtain one-dimensional hollow tubular silica nanotubes (SNTs). The SNTs were then thiolized to obtain SNTs-SH. Using SNTs-SH as a carrier and CdSe / CdS / ZnS red quantum dots (QDs) as signal tags, oil-soluble tubular assemblies dSi / tQDs were constructed on the SNTs-SH carrier through multilayer assembly of quantum dots. A hydrolytic condensation reaction was performed on the surface of the tubular assembly using n-octyltrimethoxysilane (OTMS) to achieve phase transfer from the organic phase to the aqueous phase. Then, a thin layer of silica was deposited by hydrolysis of tetraethyl orthosilicate (TEOS) using the Stöber method to improve the stability of the internally assembled quantum dots. Next, the assembly was modified with amino groups by 3-aminopropyltriethoxysilane and carboxyl groups by succinic anhydride to obtain STQS-COOH. The carboxyl-modified assembly STQS-COOH was coupled with a labeled antibody and then blocked with pH=7.4 PB buffer containing 0.5-2% BSA for 1.5-3 h to finally obtain the probe. The preparation method of the one-dimensional hollow tubular silica nanotubes (SNTs) specifically includes the following steps: S1: Dissolve the surfactant Brij58 in cyclohexane, stir at 45-55℃, add Ni salt, then add hydrazine hydrate dropwise, stir for 2.8-3.2h to react Ni salt with hydrazine hydrate to form nickel-hydrazine complex nanorods; S2: Then add diethylamine and TEOS to the system in step S1, and continue stirring the reaction for 1.5-2.5h to allow silica deposition to occur. After the reaction is complete, centrifuge and wash. S3: The product obtained in step S2 is dispersed in a 0.8-1.2M hydrochloric acid solution and selectively etched by stirring at room temperature for 0.5-2 hours. The product is then collected by centrifugation and washed with water until the pH is neutral to obtain one-dimensional hollow tubular silica nanotubes (SNTs), which are stored in anhydrous ethanol. The process of multilayer assembly of quantum dots on template SNTs-SH is as follows: 1) Assembly of the first layer of quantum dots: Take a toluene solution of CdSe / CdS / ZnS quantum dots, add the template SNTs-SH, sonicate for 7-10 min to obtain a homogeneous solution, then centrifuge to collect the precipitate. After the residual toluene solvent in the precipitate evaporates, the assembly of the first layer of quantum dots, SNTs / QD1, is obtained; the mass ratio of the template SNTs-SH to CdSe / CdS / ZnS quantum dots is 1:0.8~1.

0. 2) Coating with aminated dendritic polymer: The assembled product obtained in step 1) is added to an ethanol solution containing aminated dendritic polyamide PAMA-NH2, sonicated for 7-10 min, and the supernatant is removed by centrifugation to obtain the nanocomposite SNTs / QD-PAMA with the outermost layer coated with amino groups. Then, the amino groups on its surface can coordinate with quantum dots to continue the assembly of quantum dots. 3) Repeat steps 1)-2) above to achieve the alternating assembly of organic phase quantum dots and aminated dendritic polymer polyamide PAMA-NH2 in multiple layers, assembling a total of 2-4 layers of quantum dots, and finally obtaining the constructed oil-soluble assembly SNTs / tQD product.

2. The method for preparing a quantum dot tubular assembled structure immunochromatographic probe as described in claim 1, characterized in that... In step S1, the surfactant is Brij58, and its concentration in cyclohexane is 0.5-0.6 g / mL; the molar ratio of hydrazine hydrate to Ni salt is 4-10:1, and the concentration of Ni salt in cyclohexane is 50-100 mmol / L. In step S2, the volume ratio of diethylamine to TEOS is 0.4-0.6:1, and the volume ratio of TEOS in step S2 to the molar ratio of Ni salt in step S1 is 1:0.5-0.

8. The volume unit is mL, and the molar unit is mmol.

3. The method for preparing a quantum dot tubular assembled structure immunochromatographic probe as described in claim 1, characterized in that... In the process of multilayer assembly of quantum dots on template SNTs-SH, three layers of quantum dots are assembled.

4. The method for preparing a quantum dot tubular assembled structure immunochromatographic probe as described in claim 1, characterized in that... The process of conjugating carboxyl-modified assemblies with labeled antibodies is as follows: The carboxyl-modified assemblies are added to pH 6.0 PB buffer, along with N-hydroxythiosuccinimide sodium salt (Sulfo-NHS) and carbodiimide hydrochloride (EDC), and incubated in a shaker for 20–40 min. The product is then collected by centrifugation. The product is resuspended in pH 7.4 PB buffer, labeled antibody is added, and the mixture is reacted in a shaker for 2–3 h. After blocking, the tubular probes are collected by centrifugation, washed with pH 7.4 PB buffer, and stored in pH 7.4 PB buffer under cold conditions.

5. A quantum dot tubular assembly structure immunochromatographic probe prepared by the method described in any one of claims 1-4.

6. The application of the quantum dot tubular assembly structure immunochromatographic probe as described in claim 5, characterized in that... The probe is used to detect the corresponding antigen. This detection is not for the purpose of disease detection or treatment, and includes the following steps: S1 Immunochromatographic test strip construction: The immunochromatographic test strip includes a sample pad, an NC membrane and an absorbent pad assembled sequentially on a plastic base plate. The T line and C line of the NC membrane are coated with capture antibody and goat anti-mouse IgG antibody, respectively. S2 Antigen Concentration Detection: The antigen was diluted with a pH 7.4 PBS buffer containing 0.05-0.2% sodium caseinate, 0.1-1% BSA, 0.1-1% Tween 20, and 0.1-0.5% PVP360 to prepare a series of standard solutions with different antigen concentrations. The probe was added to the standard solutions and mixed thoroughly. The solutions were then dropped onto the sample pad of the test strip. Under capillary action, the probe flowed through the NC membrane to the absorption pad. After 5-20 min, the test strip was photographed using a smartphone under 365 nm UV excitation light to obtain images of the T-line and C-line. The R-value of the T-line image was read using Color Picker software on the smartphone. A standard curve was plotted with the R-value on the ordinate and the antigen concentration on the abscissa.

7. The application as described in claim 6, characterized in that... The labeled antibody is SARS-CoV-2 NP monoclonal antibody 2, the capture antibody is SARS-CoV-2 NP monoclonal antibody 1, and the antigen is SARS-CoV-2 NP antigen.

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