A shell-coated magnetic fluorescent composite probe, a kit thereof and application thereof

Abstract

The application takes Fe3O4 magnetic nanoparticles as a core, loads multilayer quantum dots through surface polyethylene imine modification, and further coats a SiO2 protective shell layer, to construct a multifunctional magnetic fluorescence nanoprobe (FeDQD@Si) with high fluorescence intensity, excellent stability and good dispersity. The probe integrates magnetic separation and enrichment, signal amplification and anti-matrix interference, can efficiently capture target viruses in complex fecal samples and inhibit background interference, realizes pg / mL level ultra-high sensitivity detection, the sensitivity is 50-100 times higher than that of a traditional colloidal gold immunochromatography method, and quantitative analysis can be realized through a portable fluorescence equipment. Meanwhile, the detection system does not need complex pretreatment, and can complete detection within 25 minutes, and is suitable for various application scenes such as on-site, family and primary medical treatment.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection technology for foodborne pathogens, specifically relating to a shell-coated magnetic fluorescent complex probe, its kit, and its applications. Background Technology

[0002] Foodborne viral acute gastroenteritis has long posed a significant threat to global public health, infecting over 600 million people and causing hundreds of thousands of deaths annually. Among the many related pathogens, norovirus (NoV) is the most common foodborne viral infection globally, accounting for the vast majority of foodborne viral diseases and being the leading viral pathogen causing acute gastroenteritis outbreaks. Adenovirus (AdV) is another foodborne enterovirus of concern, often co-infecting humans with norovirus. These two types of viruses share the following characteristics: i) strong environmental resistance, surviving in the natural environment for 10 days to 4 weeks; ii) extremely low infectious doses, requiring only a few dozen viral particles (approximately pg / mL) to cause disease; iii) initial symptoms such as fever, diarrhea, and vomiting are highly similar to bacterial foodborne illnesses, often leading to misdiagnosis and more insidious transmission. Therefore, early, simple, and rapid viral diagnosis is crucial for blocking transmission and guiding clinical treatment. However, while existing detection technologies such as qPCR and gene sequencing offer high accuracy, they lack timeliness and place stringent demands on instrument precision, operator skills, and experimental environments, making them unsuitable for on-the-spot testing scenarios such as border quarantine, community healthcare, and emergency response to outbreaks. Currently, there is a lack of reliable technologies that combine high on-site detection sensitivity with the ability to simultaneously detect multiple foodborne viruses, failing to address pressing real-world needs.

[0003] Lateral chromatographic immunoassay (LFA), based on the antigen-antibody specific recognition mechanism, enables rapid on-site detection of biochemical molecules. It boasts advantages such as ease of operation, low cost, and immediate results visualization, making it a common method for pathogen screening in resource-constrained scenarios. However, this technology remains limited by two major bottlenecks in foodborne virus detection: First, the colorimetric sensitivity of traditional colloidal gold labeling is insufficient, with detection limits typically in the ng / mL range, making it difficult to meet the accurate identification requirements of samples with low viral loads (such as early-stage infections or environmental swabs). Second, clinical samples such as feces and vomit have complex matrices, rich in intestinal flora, food residues, and interfering components such as proteases, which can easily cause problems such as test strip pore blockage, non-specific adsorption, and fluorescence quenching, leading to signal distortion or even complete ineffectiveness of conventional nanolabels in untreated raw samples. Although novel labeling strategies such as fluorescence, SERS, nanozymes, and photothermal materials have been introduced to improve the sensitivity of fluorescence-based fecal imaging (LFA), the quenching effect of components such as bile salts, humic acids, and proteases in the fecal matrix on fluorophores makes it difficult to directly apply existing fluorescent LFA methods for the on-site diagnosis of gastrointestinal infections. Therefore, overcoming the interference and suppression of fluorescence signals by complex biological matrices and establishing a highly sensitive LFA method adapted to raw fecal samples remains a critical technical challenge in this field.

[0004] Magnetic fluorescent nanoprobes integrate magnetic responsiveness and fluorescence signals, demonstrating great potential in simultaneously improving detection sensitivity and anti-interference capabilities. Fe3O4 is widely used as a magnetic core material due to its simple preparation, strong magnetic responsiveness, and good biocompatibility. However, traditional magnetic fluorescent probes still suffer from key drawbacks: poor fluorescence stability under matrix interference, easy adsorption of biomolecules on the surface, and insufficient chromatographic flowability. To address these issues, silica (SiO2) shell coating has become a crucial surface engineering strategy. The SiO2 shell exhibits high chemical inertness and good biocompatibility, physically isolating internal functional materials, effectively blocking interference from bile salts, humic acids, etc., maintaining fluorescence stability, reducing non-specific adsorption, and enabling direct detection of complex samples.

[0005] Existing magnetic fluorescent probe technologies are all designed for bacterial detection scenarios. Their core principle is to rely on the electrostatic adsorption effect generated by the positively charged silicon shell to achieve broad-spectrum capture of bacteria. They are mainly suitable for the enrichment and analysis of pathogenic bacteria in food or environmental samples. The detection objects, recognition mechanisms, and applicable matrices are all fundamentally different from those of this invention.

[0006] First, the detection targets are fundamentally different: existing methods target bacteria, which have large particle sizes and significant surface charge characteristics; this invention targets norovirus and enteric adenovirus, whose particle sizes are much smaller than bacteria, whose antigenic epitopes are easily masked, and whose infectivity is extremely low, making specific identification through electrostatic adsorption impossible. Second, the recognition mechanisms are different: existing technologies rely on broad-spectrum electrostatic adsorption between positively charged silicon shells and negatively charged bacteria, lacking virus-specific recognition capabilities; this invention employs antigen-antibody specific immune recognition, generating a binding response only for the target virus, and is not a simple structural application or scenario replacement. Third, the applicable matrices are fundamentally different: existing technologies are only suitable for simple matrices such as food and the environment, and cannot withstand the strong fluorescence quenching effect of fecal samples; while this invention targets strong quenching substances in feces such as bile salts, humic acids, and proteases, precisely constructing a 40 nm SiO2 shell through the co-condensation of TEOS and APTMS, specifically solving the quantum dot fluorescence quenching problem from a structural design perspective. This is a technical effect that existing bacterial probes completely lack and cannot anticipate. Finally, there is a fundamental difference between signal amplification and shell function: existing technologies mostly use single-layer quantum dot labeling and do not design signal amplification for the low abundance characteristics of viruses; this invention uses PEI-mediated double-layer quantum dot assembly to achieve a 1.98-fold amplification of fluorescence signal, and simultaneously achieves anti-quenching, improved fluidity, and reduced background signal through a precisely thick SiO2 shell. The overall technical concept is substantially different from existing bacterial detection probes.

[0007] In summary, foodborne viruses and bacteria differ significantly: viruses have smaller particle sizes, their antigenic epitopes are more easily obscured by the matrix, and components such as bile salts, proteases, and humic acids in feces can easily cause fluorescence quenching. Therefore, existing bacterial detection probes cannot be directly applied to the specific, highly sensitive, and highly stable detection of norovirus and enteric adenovirus in feces. Structural reconstruction and methodological innovation are still needed to address the specific needs of virus detection.

[0008] In view of this, the present invention is hereby proposed. Summary of the Invention

[0009] To address the shortcomings of existing technologies, one objective of this invention is to provide a method for preparing and applying a SiO2-shell-coated magnetic fluorescent composite probe (FeDQD@Si). This invention addresses the pain points of traditional magnetic fluorescent probes in complex biological samples (such as feces), including susceptibility to interference, signal quenching, cumbersome pretreatment, and limited sensitivity. It proposes a three-layer "core-sandwich-shell" structure design: a core of ~200nm Fe3O4 magnetic microspheres provides strong superparamagnetic response, enabling efficient capture and rapid enrichment of target viruses from complex samples; efficient electrostatic assembly of bilayer CdSe / ZnS quantum dots is achieved through the cationic polymer polyethyleneimine (PEI), utilizing the fluorescence superposition effect of multiple quantum dots to significantly amplify the signal; finally, a SiO2 shell is coated on the outer layer, prepared through a TEOS and APTMS co-condensation reaction, with the shell thickness precisely controllable to 40 nm. nm not only physically isolates the internal quantum dots from interfering components such as bile salts and humic acid in the fecal matrix, effectively protecting the luminescence performance of the probe in complex matrices, but also significantly improves the mobility of the probe during chromatography and reduces background signal interference, thus constructing a high-performance magnetic fluorescent composite probe that combines enrichment ability, amplification effect and anti-interference protection.

[0010] The second objective of this invention is to provide a magnetic enrichment-fluorescence detection module based on the aforementioned FeDQD@Si probe. This invention innovatively integrates a magnetic enrichment module with a fluorescence detection module, utilizing an external magnetic field to achieve rapid separation and concentration of the target virus from complex samples, simultaneously removing matrix interference. Furthermore, the fluorescence superposition effect of the double-layer quantum dots provides a highly sensitive quantitative signal output, solving the problems of signal distortion and insufficient sensitivity in complex samples inherent in traditional LFA technology, thus achieving accurate identification of low-concentration virus samples.

[0011] The third objective of this invention is to provide a high-performance foodborne virus detection strategy. This invention deeply integrates the aforementioned SiO2-shell-coated magnetic fluorescent composite probe with lateral chromatography immunoassay technology, coupled with a portable fluorescence reader, to establish an ultra-high sensitivity detection method that can be completed within 25 minutes with a detection limit as low as pg / mL. This strategy not only achieves rapid, simultaneous, and highly sensitive detection of norovirus and enteric adenovirus, but is also suitable for rapid on-site screening under UV light visual observation mode, and enables accurate diagnosis in primary healthcare institutions using portable instruments. This invention integrates magnetic enrichment, fluorescence amplification, and SiO2 anti-interference functions through a single probe, overcoming the challenge of traditional lateral chromatography's inability to directly detect complex fecal samples. It is suitable for immediate detection at border crossings, primary healthcare facilities, and epidemic outbreak sites, and has significant potential for widespread application.

[0012] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a SiO2-shell-coated magnetic fluorescent composite probe comprising nanomaterials and a specific antibody.

[0013] In some embodiments of the present invention, the nanomaterial has a core-shell structure, wherein the core of the core-shell structure is an Fe3O4 nanoparticle, and a fluorescent interlayer and a protective shell are sequentially coated from the outer surface of the core outward.

[0014] In some embodiments of the present invention, the fluorescent interlayer comprises PEI and CdSe / ZnS quantum dots.

[0015] In some embodiments of the present invention, the fluorescent interlayer is a double layer.

[0016] In some embodiments of the present invention, the protective shell is a SiO2 shell with a thickness of 20-60 nm.

[0017] In some embodiments of the present invention, the SiO2 shell is coupled with a specific antibody.

[0018] In some embodiments of the present invention, the specific antibody is a norovirus antibody and an enteroadenovirus antibody.

[0019] In some embodiments of the present invention, the Fe3O4 nanoparticles have a uniform spherical morphology and superparamagnetic properties, which are used to provide magnetic response capability and a core for loading fluorescent quantum dots, and the particle size is 100-300 nm.

[0020] In some embodiments of the present invention, the CdSe / ZnS quantum dots have excellent fluorescence properties and a diameter of 5-20 nm.

[0021] In some embodiments of the present invention, the thickness of the SiO2 shell is 20-60 nm.

[0022] A second aspect of the present invention provides a method for preparing a SiO2-shell-coated magnetic fluorescent composite probe according to the first aspect of the present invention, comprising the following steps: 1) Fe3O4 nanoparticles were mixed with PEI aqueous solution and subjected to ultrasonic treatment to obtain Fe3O4-PEI nanospheres; then the Fe3O4-PEI nanospheres were mixed with CdSe / ZnS quantum dot solution and subjected to ultrasonic treatment to obtain FeQD; 2) The FeQD nanoparticles obtained in step 1) are mixed with PEI aqueous solution again and subjected to ultrasonic treatment, then mixed with CdSe / ZnS quantum dot solution and subjected to ultrasonic treatment, and the second layer of CdSe / ZnS quantum dots is repeatedly adsorbed to obtain FeDQD; 3) The FeDQD nanoparticles obtained in step 2) are dispersed in anhydrous ethanol, and ammonia, TEOS and APTMS are added in sequence to carry out a co-condensation reaction to obtain FeDQD@Si nanoprobes; 4) After surface amination and carboxylation of the FeDQD@Si nanoprobe obtained in step 3), it is activated by EDC / NHS and incubated with anti-norovirus antibody and anti-enteric adenovirus antibody to obtain SiO2 shell-coated magnetic fluorescent composite probe.

[0023] In some embodiments of the present invention, in step 1), the molecular weight of the PEI is 600-25000.

[0024] In some embodiments of the present invention, the concentration of the PEI aqueous solution is 0.5-5 mg / mL.

[0025] In some embodiments of the present invention, the mass ratio of the Fe3O4 nanoparticles to PEI is 1:(2-20).

[0026] In some embodiments of the present invention, the concentration of the CdSe / ZnS quantum dot solution is 10-50 mg / mL.

[0027] In some embodiments of the present invention, in step 2), the molecular weight of the PEI is 600-25000.

[0028] In some embodiments of the present invention, the concentration of the PEI aqueous solution is 0.5-5 mg / mL.

[0029] In some embodiments of the present invention, the mass ratio of FeQD to PEI is 1:(2-20).

[0030] In some embodiments of the present invention, the concentration of the CdSe / ZnS quantum dot solution is 10-50 mg / mL.

[0031] In some embodiments of the present invention, in step 3), the SiO2 shell thickness is precisely controlled to ~40 nm through the co-condensation reaction of TEOS and APTMS in an alkaline environment; the ultrasonic modification time is 30-120 min, and the reaction system contains anhydrous ethanol, ammonia, TEOS and APTMS.

[0032] In some embodiments of the present invention, in step 4), the amination conditions are as follows: FeDQD@Si is dispersed in a 1% (v / v) 3-aminopropyltriethoxysilane (APTES) ethanol solution and reacted at room temperature; the carboxylation conditions are as follows: 10 mg / mL DMSO solution of succinic anhydride is added and reacted at 4 °C to convert the surface amino groups into carboxyl groups; the EDC / NHS activation conditions are as follows: 100 mM EDC and 100 mM NHS are used in a ratio of 1:2 and activated at room temperature for 15 min; the antibody covalent coupling conditions are as follows: anti-norovirus antibody and anti-adenovirus antibody (6 μg / mg probe each) are added and incubated at room temperature for 2 h, followed by blocking unreacted sites with 10% bovine serum albumin (BSA) for 60 min.

[0033] A third aspect of the present invention provides the application of the SiO2-shell-coated magnetic fluorescent composite probe of the first aspect and the preparation method of the second aspect in the preparation of products for detecting foodborne viruses.

[0034] In some embodiments of the present invention, the test samples for the product include fecal samples.

[0035] In some embodiments of the present invention, the foodborne viruses include norovirus and enteric adenovirus.

[0036] A fourth aspect of the present invention provides a kit for the simultaneous detection of norovirus and enteric adenovirus, the kit comprising the SiO2-shell-coated magnetic fluorescent composite probe described in the first aspect of the present invention.

[0037] In some embodiments of the present invention, the kit further includes an immunochromatographic test strip comprising a sample pad, a chromatography membrane, and an absorbent pad connected in sequence.

[0038] In some embodiments of the present invention, the chromatography membrane is provided with two detection lines and one control line, wherein the two detection lines are respectively loaded with anti-norovirus antibody and anti-enteric adenovirus antibody.

[0039] In some embodiments of the present invention, the control line is loaded with IgG.

[0040] This kit is versatile and can be used in various testing scenarios. The fluorescence signal observed under UV light is suitable for field or home self-testing, enabling rapid qualitative screening; the quantitative mode of the portable fluorescence reader is suitable for professional, highly sensitive quantitative testing in hospitals or third-party testing institutions, helping clinicians determine the degree of disease progression and guide medication.

[0041] The beneficial effects of this invention are: The SiO2-shell-coated magnetic fluorescent composite probe proposed in this application has three integrated functions: magnetic enrichment for sample pretreatment to magnetically capture antigens from feces and eliminate interference from complex matrices; fluorescence output for signal amplification; and SiO2 shell isolation to protect the probe from fluorescence quenching in fecal samples, improve chromatographic fluidity, and suppress background signals from complex samples. The magnetic enrichment-immunochromatography technique using the SiO2-shell-coated magnetic fluorescent composite probe can be used in various testing scenarios, including field testing, home self-testing, hospitals, and third-party testing institutions, for qualitative or quantitative detection of norovirus and adenovirus in clinical fecal samples.

[0042] In summary, this invention proposes a novel magnetic enrichment-fluorescence detection technology. It achieves efficient antigen capture from feces through magnetic enrichment while simultaneously eliminating interference from complex matrices. Building upon this, magnetic separation and enrichment, fluorescence signal amplification, and SiO2 shell anti-interference protection are integrated into a single nanolabel. Combined with bilayer quantum dots, fluorescence signal amplification is achieved, enabling immunochromatographic detection sensitivity to reach the pg / mL level. Simultaneously, the 40 nm thick SiO2 shell forms a dense physical barrier, completely isolating quenching substances such as bile salts, humic acids, and proteases in feces, preventing quantum dot fluorescence quenching, enhancing fluidity during chromatography, and effectively suppressing background signal interference from complex samples. This technology combines the sensitive fluorescence signal for rapid on-site screening with anti-interference capabilities for direct detection of complex samples. We designed and synthesized a magnetic fluorescent probe (FeDQD@Si) with a three-layer structure of "core-intermediate layer-shell". A ~200 nm Fe3O4 magnetic core provides a strong superparamagnetic response for rapid separation and enrichment of target viruses. The intermediate layer, densely packed with bilayer CdSe / ZnS quantum dots, exhibits excellent fluorescence performance, providing a fluorescence signal superposition amplification effect and significantly improving detection sensitivity. The ~40 nm SiO2 shell, precisely constructed through the co-condensation of TEOS and APTMS, provides strong physical isolation and protection, significantly improving the fluorescence stability and chromatographic mobility of the probe in fecal matrix. This magnetic enrichment-immunochromatography platform is a high-performance, highly flexible, and user-friendly immunoassay tool capable of completing detection within 25 minutes, with detection limits reaching the pg / mL level. It is suitable for on-the-fly detection needs at border crossings, primary healthcare institutions, and outbreak sites, while also meeting the precision diagnostic requirements of clinical laboratories, providing reliable technical support for food safety monitoring and public health emergency response. Attached Figure Description

[0043] Figure 1 This is a schematic diagram illustrating the preparation method for synthesizing the SiO2-shell-coated magnetic fluorescent composite probe of Example 1 of the present invention.

[0044] Figure 2Electron microscopy characterization of the structure of the SiO2-shell-coated magnetic fluorescent composite probe of Example 1 of this invention. Wherein: a is a TEM image of the Fe3O4 magnetic core; b and e are TEM images of FeQD loaded with monolayer quantum dots; c and f are TEM images of FeDQD loaded with bilayer quantum dots; d and g are TEM images of FeDQD@Si; h is a magnified TEM image of the silica shell region in the FeDQD@Si probe; i and j are EDS elemental line scan results of FeDQD@Si; k is the elemental distribution map of FeDQD@Si, showing the layered distribution of Fe, O, S, Zn, Si, and Cd.

[0045] Figure 3 The image shows a comparison of the fluorescence emission spectra of the SiO2-shell-coated magnetic fluorescent composite probe and the uncoated SiO2-shell-coated bilayer magnetic fluorescent probe in fecal matrix. The inset image shows a comparison of the fluorescence quenching of the corresponding probes in fecal matrix.

[0046] Figure 4 This is a schematic diagram illustrating the immunochromatographic technique of the SiO2-shell-coated magnetic fluorescent composite probe used in Example 2 of the present invention for the detection of norovirus and enteric adenovirus.

[0047] Figure 5 This is the result of the membrane concentration optimization for the SiO2-shell-coated magnetic fluorescent composite probe in the immunochromatographic technique of Example 2 of the present invention.

[0048] Figure 6 This is the result of optimizing the incubation time of the immunochromatographic technique for the SiO2-shell-coated magnetic fluorescent composite probe of Example 2 of the present invention.

[0049] Figure 7 This paper presents the principle and results of the fluorescence signal difference between the uncoated SiO2-shelled double-layer magnetic fluorescent probe and the SiO2-shell-coated magnetic fluorescent composite probe in fecal samples according to Example 3 of the present invention. In this paper, a is a schematic diagram of the detection principle; b is the detection results of probes without silica shell (i) and with silica shell coating (ii).

[0050] Figure 8 The results show the flowability comparison of the uncoated SiO2-shelled bilayer magnetic fluorescent probe (a) and the SiO2-shell-coated magnetic fluorescent composite probe (b) in the buffer system in Example 3 of the present invention.

[0051] Figure 9The images show the detection results of norovirus and enteric adenovirus using the SiO2-shell-coated magnetic fluorescent composite probe in Immunochromatography according to Example 3 of this invention. In the images: a) is the fluorescence image of the norovirus and adenovirus test strips at various concentrations; b) is the corresponding thermogram; c) and e) are the fluorescence intensity bar graphs and calibration curves of the norovirus detection line at various concentrations, respectively; d) and f) are the fluorescence intensity bar graphs and calibration curves of the adenovirus detection line at various concentrations, respectively.

[0052] Figure 10 The image shows the detection results of norovirus (a) and enteric adenovirus (b) using the colloidal gold method in Example 3 of this invention.

[0053] Figure 11 This is an immunochromatographic verification of the specificity of the SiO2-shell-coated magnetic fluorescent composite probe of Example 3 of the present invention, wherein: a is the fluorescence image result of the specific verification against 11 common gastrointestinal and respiratory pathogens, and b is the corresponding heat map result.

[0054] Figure 12 For the repeatability verification of the immunochromatographic technique of the SiO2 shell-coated magnetic fluorescent composite probe of Example 3 of the present invention, where: a and b are T-line fluorescence images (i) and fluorescence intensity bar graphs (ii) of six repeated detections of high-concentration and low-concentration virus samples, respectively.

[0055] Figure 13 The results of immunochromatographic analysis of the SiO2-shell-coated magnetic fluorescent composite probe of Example 3 of the present invention in actual samples are shown below: a) is the fluorescence image (i) and quantitative fluorescence intensity result (ii) of the spiked recovery experiment of norovirus and adenovirus in lake water sample; b) is the fluorescence image (i) and quantitative fluorescence intensity result (ii) of the spiked recovery experiment of norovirus and adenovirus in fecal sample.

[0056] Figure 14 The results of immunochromatographic analysis of the SiO2-shell-coated magnetic fluorescent composite probe of Example 3 of this invention in clinical fecal samples are shown below. Specifically: a and b are heatmap comparisons of FeDQD@Si-LFA and qPCR detection results, respectively; c and d are violin plots showing the distribution of FeDQD@Si-LFA and qPCR detection values, respectively; e and f are ROC curve analysis diagrams for norovirus clinical sample detection and adenovirus clinical sample detection, respectively. Detailed Implementation

[0057] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0058] Example 1 A method for preparing the SiO2-shell-coated magnetic fluorescent composite probe of the above embodiment, such as... Figure 1 As shown, it includes the following steps: (1) Preparation of Fe3O4 magnetic nanoparticles by solvothermal method. 1.35 g of ferric chloride hexahydrate (FeCl3·6H2O) was dissolved in 40 mL of ethylene glycol, and 3.6 g of anhydrous sodium acetate and 1.0 g of polyethylene glycol (PEG-6000) were added. After stirring vigorously for 30 min, the mixture was transferred to a 50 mL polytetrafluoroethylene-lined stainless steel high-pressure reactor and reacted at 200 ℃ for 10 h. After the reaction was completed, the mixture was cooled to room temperature, and the product was collected by magnetic separation. The product was washed three times each with ethanol and deionized water and dispersed in 40 mL of deionized water for later use.

[0059] (2) The Fe3O4 surface prepared in step (1) was modified to be positively charged by the cationic polymer PEI. Subsequently, a large number of negatively charged CdSe / ZnS quantum dots were electrostatically adsorbed on the Fe3O4 surface to obtain Fe3O4 / PEI / QDs (FeQD) nanoparticles. Fe3O4 nanoparticles (10 mg / mL) were mixed with PEI aqueous solution (1.7 mg / mL), sonicated for 30 min, and then magnetically separated and washed to obtain Fe3O4 / PEI. Fe3O4 / PEI was dispersed in 50 mL of carboxylated CdSe / ZnS quantum dot solution (20 mg / mL, diameter ~12 nm), sonicated for 30 min to achieve electrostatic adsorption, magnetically separated to remove unbound quantum dots, and washed to obtain FeQD.

[0060] (3) The surface charge of FeQD prepared in step (2) was modified to positive charge using PEI, and the second layer of CdSe / ZnS quantum dots was repeatedly adsorbed to obtain Fe3O4 / PEI / QDs / PEI / QDs (FeDQD). FeQD was redispersed in fresh PEI solution (1.7 mg / mL), sonicated for 30 min, and then dispersed again in 50 mL of CdSe / ZnS quantum dot solution and sonicated for 30 min. After magnetic separation and washing, FeDQD was obtained.

[0061] (4) A SiO2 shell was coated onto the surface of FeDQD via a co-condensation reaction of TEOS and APTMS to obtain FeDQD@Si nanoprobes. FeDQD (10 mg / mL) was dispersed in 36 mL of anhydrous ethanol and sonicated for 20 min to form a homogeneous suspension. 2.4 mL of ammonia, 10 μL of TEOS, and 500 μL of APTMS were added sequentially, and the mixture was sonicated for 60 min. The product was washed with deionized water, collected by magnetic separation, and dispersed in 20 mL of anhydrous ethanol to obtain a SiO2-shell-coated magnetic fluorescent composite probe (FeDQD@Si).

[0062] (5) Preparation of FeDQD@Si antibody probe. FeDQD@Si was dispersed in 1% APTES ethanol solution and reacted at room temperature to introduce surface amino groups; succinic anhydride DMSO solution (10 mg / mL) was added and reacted at 4 °C to convert amino groups to carboxyl groups; it was activated with EDC (100 mM) and NHS (100 mM) for 15 min; antibodies (6 μg anti-norovirus antibody and 6 μg anti-adenovirus antibody per mg probe) were added and incubated at room temperature for 2 h; unreacted sites were blocked with 10% BSA for 60 min; after magnetic separation and washing, it was dispersed in 200 mL PBS buffer (10 mM, pH 7.4) and stored at 4 °C for later use.

[0063] Figure 1 This is a schematic diagram illustrating the preparation method for synthesizing a SiO2-shell-coated magnetic fluorescent composite probe. Figure 2 Electron microscopy characterization of the structure of the SiO2-shell-coated magnetic fluorescent composite probe. First, the prepared FeDQD@Si nanostructure was comprehensively characterized using transmission electron microscopy (TEM). Figure 2 Figure a shows that the synthesized Fe3O4 magnetic nanoparticles have excellent dispersibility and uniform particle size (~200 nm). Figure 2 Figures b and e show that a large number of CdSe / ZnS-COOH quantum dots (~12 nm) were effectively adsorbed onto the Fe3O4 / PEI surface through ultrasonic treatment, forming FeQDs with a rough surface. After repeating the PEI modification and quantum dot coating steps, the quantum dot density on the FeQD surface increased significantly, forming a dense bilayer quantum dot coated structure. Figure 2 c, f). Figure 2 As shown in d and g, FeDQD@Si prepared by the co-condensation reaction of TEOS and APTMS possesses a complete SiO2 shell and excellent dispersibility. High-resolution TEM (HRTEM) characterization further revealed that the outer layer of FeDQD@Si is a SiO2 shell with a thickness of ~40 nm. Figure 2EDS line scan results show that Fe3O4 has a double-layer quantum dot structure and a SiO2 shell; elemental mapping images show that the Fe signal is concentrated in the core, Cd, S, and Zn are distributed in the middle layer, and Si is concentrated in the outer shell region. Figure 2 (ij), confirming the expected core-sandwich-shell structure. Figure 3 This is a comparison of the fluorescence emission spectra of FeDQD and FeDQD@Si in a fecal matrix. The fluorescence signal of FeDQD without SiO2 coating was significantly quenched in the fecal matrix, with a substantial decrease in emission peak intensity; while FeDQD@Si maintained strong fluorescence emission under the same conditions, indicating that the SiO2 shell effectively isolates quenching substances in the feces, protecting the luminescent properties of the quantum dots.

[0064] Example 2 This embodiment describes the construction of an immunochromatographic probe with a SiO2 shell-coated magnetic fluorescent composite probe, which involves two steps: (1) To construct a dual-target lateral chromatography detection system for norovirus (NoV) and adenovirus (AdV), a polyvinyl chloride (PVC) backing board was used as the supporting substrate, and a sample pad, a nitrocellulose (NC) membrane, and an absorbent pad were stacked sequentially to form the chromatography structure. The NC membrane had two detection lines (T1 / T2) and one control line (C line) pre-installed: the T1 line was coated with anti-AdV monoclonal antibody, the T2 line with anti-NoV monoclonal antibody, and the C line with goat anti-mouse IgG polyclonal antibody, with mass concentrations of 0.9 mg / mL, 0.9 mg / mL, and 0.8 mg / mL, respectively, and was sprayed onto the membrane at a linear velocity of 0.8 μL / cm. After spraying, the NC membrane was placed in a 34℃ hot air circulating oven for dehydration and curing for 120 min, and then laminated with the PVC backing board, sample pad, and absorbent pad with an overlap width of 0.1 mm. Cut the assembled large board into individual test strips with a width of 3.0 mm, vacuum seal them in aluminum foil bags, and store them at 4°C away from light.

[0065] (2) Optimize the operating conditions of the immunochromatographic technique of SiO2 shell-coated magnetic fluorescent composite probe, including antibody streaking concentration and incubation time, to achieve the best detection performance of the platform.

[0066] Figure 4 This is a flowchart illustrating the immunochromatographic technique using SiO2-shell-coated magnetic fluorescent composite probes for the detection of norovirus and enteric adenovirus. This embodiment optimizes the operating conditions of the SiO2-shell-coated magnetic fluorescent composite probe immunochromatographic technique to achieve the platform's best detection performance. Figure 5To optimize the concentration of online capture antibodies, FeDQD@Si-LFA showed the highest signal-to-noise ratio for both norovirus and enteric adenovirus when the concentrations of both online anti-norovirus and anti-adenovirus antibodies were 0.9 mg / mL. Figure 6 The optimized incubation time for the SiO2-shell-coated magnetic fluorescent composite probe resulted in strong fluorescence signals and the highest signal-to-noise ratio in the immunochromatographic detection of norovirus and enteric adenovirus after an incubation time of 10 min. This optimization ensures smooth flow of the FeDQD@Si probe and the immune complex formed after virus capture on the NC membrane, guaranteeing the stable operation of the immunochromatographic detection system using this magnetic fluorescent probe.

[0067] Example 3 To achieve stable and ultra-sensitive detection of foodborne viruses in fecal samples, this embodiment constructs an immunochromatographic detection platform based on FeDQD@Si probes (FeDQD@Si-LFA). The core design of this platform lies in achieving efficient pre-concentration of target analytes through a magnetic enrichment strategy, while simultaneously leveraging the SiO2 shell to impart excellent matrix tolerance to the probe, thereby overcoming the technical bottleneck of accurate and ultra-sensitive detection of viruses in complex fecal samples. Subsequently, this embodiment systematically investigates the impact of the SiO2 shell on the detection performance of FeDQD@Si-LFA.

[0068] Figure 7 Figure a shows a schematic diagram comparing the fluorescence signals of uncoated FeDQD probes and SiO2-coated magnetic fluorescent composite probes in fecal samples. In fecal samples, the uncoated FeDQD probes are exposed, causing the quantum dots (QDs) to directly contact interfering substances such as bile salts, humic acid, and proteases, leading to rapid fluorescence signal decay. In contrast, the FeDQD@Si probes, thanks to the physical isolation provided by the SiO2 shell, effectively shield against matrix interference and maintain stable fluorescence signals. This core advantage is further confirmed by immunochromatographic test strips. Figure 7 (b) After incubating and magnetically enriching fecal samples containing norovirus with both probes, the samples were loaded onto the substrate. Visual observation revealed weak fluorescence signals in the FeDQD group's detection line (T-line) and a scattered signal distribution in the thermal image; while the FeDQD@Si group showed bright fluorescence signals in the T-line and a concentrated, sharp signal in the thermal image. Quantitative analysis showed that the limit of detection (LOD) of the FeDQD group was approximately one order of magnitude higher than that of the FeDQD@Si group, confirming that the SiO2 shell is the key guarantee for the ultra-sensitive detection of foodborne viruses in fecal samples using FeDQD@Si-LFA.

[0069] Furthermore, the FeDQD probe exposed on the surface is prone to non-specific adsorption on the nitrocellulose (NC) membrane, resulting in obvious red fluorescence in the background area of ​​the test strip, and cluttered background signals and blurred T-line edges in the thermal image. Figure 8 (a); Conversely, the FeDQD@Si probe benefits from the steric hindrance effect and charge modulation of the SiO2 shell, resulting in a clean background area with no stray signals on the test strip, and a clear T-line outline and concentrated signal in the thermal image. Figure 8 (b) This difference indicates that the SiO2 shell significantly improves the dispersibility and chromatographic flowability of the probe, reduces non-specific adsorption, and provides a reliable basis for quantitative detection.

[0070] Figure 9 Immunochromatographic images and thermograms of different concentrations of norovirus and enteric adenovirus were obtained using an immunochromatographic probe with a SiO2-coated magnetic fluorescent composite probe. When both norovirus and adenovirus concentrations decreased to 0.005 ng / mL, the fluorescence signal of the T-line was still observable under UV light. In the FeDQD@Si-mediated fluorescence detection mode, the limit of detection (vLOD) of FeDQD@Si-LFA was 0.005 ng / mL. Subsequently, we evaluated the virus quantification capability of FeDQD@Si-LFA in fluorescence mode. Calibration curves of the T-line fluorescence signal intensity versus norovirus and enteric adenovirus concentrations showed a good positive correlation between the two viruses in the range of 0.005–10 ng / mL, with R² values ​​of 0.999 (norovirus) and 0.959 (adenovirus), respectively. According to the IUPAC definition (LOD = mean blank signal + 3 × blank standard deviation), the detection limits of FeDQD@Si-LFA for norovirus and adenovirus are 0.725 pg / mL and 1.28 pg / mL, respectively.

[0071] Compared with traditional colloidal gold immunochromatography (AuNP-LFA), FeDQD@Si-LFA has a sensitivity that is 50-100 times higher. Figure 10 This result demonstrates that the immunochromatographic method using SiO2-shell-coated magnetic fluorescent composite probes exhibits significant advantages in rapid on-site screening: its detection time is comparable to that of conventional colloidal gold test strips, while its sensitivity is significantly improved, providing a more competitive technical option for the efficient identification of viral infections.

[0072] Meanwhile, we selected a variety of common enteric pathogens, including rotavirus, Escherichia coli O157:H7, Shigella, Clostridium difficile, Vibrio cholerae, Campylobacter jejuni, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pharyngitis, and Streptococcus pyogenes (10). 5 Using cells / mL as an interfering agent, the specificity of the immunochromatographic assay for the SiO2-shell-coated magnetic fluorescent composite probe was tested. Figure 11 The fluorescence images and thermograms of the pilot-scale test strips clearly demonstrated that the immunochromatographic platform using the SiO2-shell-coated magnetic fluorescent composite probe exhibited good selectivity for the target virus and no cross-reactivity with non-target pathogens. Therefore, the specific viral antibodies applied to the detection line of the SiO2-shell-coated magnetic fluorescent composite probe immunochromatographic platform are sufficient to ensure the high specificity of the detection platform. The reproducibility of the SiO2-shell-coated magnetic fluorescent composite probe immunochromatographic platform was verified by calculating the relative standard deviation (RSD) after six consecutive tests on high-concentration (5 ng / mL) and low-concentration (0.05 ng / mL) norovirus and adenovirus samples. Figure 12 The results of the repeatability test of the immunochromatographic platform of SiO2-shell-coated magnetic fluorescent composite probe are shown. The RSD values ​​of each test group are less than 4.71%, indicating that the established immunochromatographic platform of SiO2-shell-coated magnetic fluorescent composite probe has good repeatability.

[0073] This embodiment examines the performance of SiO2-shell-coated magnetic fluorescent composite probes in immunochromatography for the direct detection of foodborne viruses in complex real-world samples. Firstly, as... Figure 13 As shown, high concentrations (10 ng / mL), medium concentrations (0.05 ng / mL), and low concentrations (0.005 ng / mL) of norovirus and adenovirus were added to lake water samples and healthy human fecal samples, respectively, to simulate real, complex samples containing viruses. The detection results were consistent with those of the PBS buffer system, with average recoveries ranging from 85.24% to 111.15% and RSD values ​​all below 11.1%. These results confirm the adaptability of this detection method to complex matrix samples, demonstrating a good proportional relationship between target analyte concentration and T-line fluorescence intensity, indicating stable and reliable methodological performance.

[0074] Furthermore, by comparing with quantitative real-time PCR, the diagnostic potential of the SiO2-shell-coated magnetic fluorescent composite probe immunochromatographic technique for clinical viral foodborne infections was evaluated. Figure 14A total of 75 clinical stool samples were tested, including 18 norovirus-positive, 12 adenovirus-positive, and 45 healthy control samples. FeDQD@Si-LFA effectively distinguished between virus-positive and negative samples (***p<0.001 compared to negative controls), showing high consistency with qPCR results. Compared to quantitative real-time PCR, the total detection time of FeDQD@Si-LFA is approximately 25 min (including 10 min of incubation and magnetic enrichment, 10 min of chromatography, and 5 min of quantitative fluorescence signal analysis), significantly shorter than the 2–3 hours required for quantitative real-time PCR. The operation process is also simplified, eliminating the need for nucleic acid extraction, amplification, and clean laboratory conditions, thus meeting both qualitative screening and quantitative analysis requirements. The addition of the magnetic enrichment step allows the platform to directly process raw stool matrix, eliminating the cumbersome pretreatment steps in traditional detection methods. Therefore, FeDQD@Si-LFA technology demonstrates good adaptability in scenarios such as epidemic prevention and control, food safety monitoring, and rapid clinical diagnosis, meeting the application requirements under different testing conditions and possessing broad prospects for promotion.

[0075] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A SiO2-shell-coated magnetic fluorescent composite probe, characterized in that, The probe comprises nanomaterials and specific antibodies; The nanomaterial has a core-shell structure, the core of which is Fe3O4 nanoparticles, and fluorescent interlayers and protective shells are sequentially wrapped from the outer surface of the core outwards. The fluorescent interlayer comprises PEI and CdSe / ZnS quantum dots; The fluorescent interlayer is double-layered; The protective shell is a SiO2 shell, and the thickness of the SiO2 shell is 20-60 nm; The SiO2 shell is coupled with a specific antibody; The specific antibodies are norovirus antibodies and enteroadenovirus antibodies.

2. The SiO2-shell-coated magnetic fluorescent composite probe according to claim 1, characterized in that: The Fe3O4 nanoparticles have a particle size of 100-300 nm; The diameter of the CdSe / ZnS quantum dots is 5-20 nm; The thickness of the SiO2 shell is 20-60 nm.

3. The preparation method of the SiO2 shell-coated magnetic fluorescent composite probe according to claim 1 or 2, comprising the following steps: 1) Fe3O4 nanoparticles were mixed with PEI aqueous solution and subjected to ultrasonic treatment to obtain Fe3O4-PEI nanospheres; then the Fe3O4-PEI nanospheres were mixed with CdSe / ZnS quantum dot solution and subjected to ultrasonic treatment to obtain FeQD; 2) The FeQD nanoparticles obtained in step 1) are mixed with PEI aqueous solution again and subjected to ultrasonic treatment, then mixed with CdSe / ZnS quantum dot solution and subjected to ultrasonic treatment, and the second layer of CdSe / ZnS quantum dots is repeatedly adsorbed to obtain FeDQD; 3) The FeDQD nanoparticles obtained in step 2) are dispersed in anhydrous ethanol, and ammonia, TEOS and APTMS are added in sequence to carry out a co-condensation reaction to obtain FeDQD@Si nanoprobes; 4) After surface amination and carboxylation of the FeDQD@Si nanoprobe obtained in step 3), it is activated by EDC / NHS and incubated with anti-norovirus antibody and anti-enteric adenovirus antibody to obtain SiO2 shell-coated magnetic fluorescent composite probe.

4. The preparation method according to claim 3, characterized in that: In step 1), The molecular weight of the PEI is 600-25000; The concentration of the PEI aqueous solution is 0.5-5 mg / mL; The mass ratio of Fe3O4 nanoparticles to PEI is 1:(2-20). The concentration of the CdSe / ZnS quantum dot solution is 10-50 mg / mL.

5. The preparation method according to claim 3, characterized in that: In step 2), The molecular weight of the PEI is 600-25000; The concentration of the PEI aqueous solution is 0.5-5 mg / mL; The mass ratio of FeQD to PEI is 1:(2-20). The concentration of the CdSe / ZnS quantum dot solution is 10-50 mg / mL.

6. The preparation method according to claim 3, characterized in that: In step 3), The co-condensation reaction conditions of the FeDQD nanoparticles are as follows: the SiO2 shell thickness is precisely controlled to ~40 nm through the co-condensation reaction of TEOS and APTMS in an alkaline environment; the ultrasonic modification time is 30-120 min; and the reaction system contains anhydrous ethanol, ammonia, TEOS and APTMS.

7. The preparation method according to claim 3, characterized in that: In step 4), The amination conditions are as follows: FeDQD@Si is dispersed in 0.5~2% (v / v) 3-aminopropyltriethoxysilane (APTES) ethanol solution and reacted at room temperature; The carboxylation conditions are as follows: add 5~15 mg / mL DMSO solution of succinic anhydride, react at 4 °C to convert surface amino groups into carboxyl groups; The activation conditions for the EDC / NHS are as follows: 100 mM EDC and 100 mM NHS in a volume ratio of 1:(1~3), activated at room temperature for 10~30 min; The conditions for antibody covalent conjugation are as follows: add anti-norovirus antibody and anti-adenovirus antibody, incubate at room temperature for 2 h, and then block unreacted sites with bovine serum albumin for 30-90 min.

8. The application of the SiO2 shell-coated magnetic fluorescent composite probe according to claim 1 or 2, and the preparation method according to any one of claims 3 to 7, in the preparation of products for detecting foodborne viruses; The test samples for the product include fecal samples.

9. A kit for simultaneously detecting norovirus and enteric adenovirus, characterized in that: The kit includes the SiO2-shell-coated magnetic fluorescent composite probe as described in claim 1 or 2.

10. The reagent kit according to claim 9, characterized in that: The kit also includes immunochromatographic test strips.

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