A multi-pathogen fluorescence immunoassay double-channel detection system, a preparation method and application thereof

Abstract

This invention belongs to the field of in vitro diagnostic technology, providing a dual-channel fluorescent immunoassay system for multiple pathogens, its preparation method, and applications. It employs multi-shell fluorescent nanoparticles with a carbon dot core, a silica shell, and an acrylic polymer outer shell, conjugating antibodies to fluorescent particles at different wavelengths to construct dual-channel probes. By controlling the carboxyl group density of the outer shell to 0.5-3.0 mmol / g, it balances high solids content, low viscosity processing, and high conjugation density; the silica intermediate layer isolates the interface quenching, reducing spectral crosstalk. It achieves a quantum yield ≥30% and an emission peak spacing ≥80 nm, enabling the simultaneous detection of multiple respiratory pathogens on nitrocellulose membranes or carboxylated microspheres. It solves problems such as narrow processing windows in large-scale preparation, the contradiction between antibody conjugation activity and background control, and the weakening of fluorescence and amplification of crosstalk by multi-shell coating. It has advantages such as good batch-to-batch consistency, high sensitivity, and low crosstalk, making it widely valuable in the field of rapid detection of multiple pathogens.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a multi-pathogen fluorescence immunoassay dual-channel detection system, its preparation method, and its application. Background Technology

[0002] In in vitro detection of respiratory pathogens and public health surveillance, rapid and accurate identification of pathogen types is crucial for guiding subsequent treatment and controlling the spread of epidemics. With the co-circulation of multiple pathogens in the population, including influenza virus, novel coronavirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, Mycoplasma pneumoniae, and Streptococcus pneumoniae, the clinical need for diagnostic technologies capable of simultaneously identifying multiple pathogens in a single test is increasingly urgent. Traditional single-pathogen detection methods require multiple sampling and separate testing, which not only prolongs the diagnostic cycle and increases the financial burden on patients but may also affect retesting and confirmation due to excessive sample consumption. A multi-pathogen simultaneous detection system based on fluorescence immunoassay technology immobilizes capture antibodies against different pathogens on the same solid-phase carrier and uses fluorescent probes with different emission wavelengths to distinguish signals. This allows for the acquisition of qualitative or quantitative information on multiple pathogens in a single test, significantly improving detection throughput and efficiency. However, constructing a high-performance dual-channel fluorescent immunoassay system for multiple pathogens requires fluorescent probes to possess multiple performance requirements, such as high brightness, narrow emission peak, low spectral crosstalk, high colloidal stability, easy antibody conjugation, and high binding activity after conjugation. At the same time, it also needs to meet the processing window of high solid content and low viscosity in large-scale preparation to adapt to automated production processes such as spotting and spraying. This poses a severe challenge to the design and synthesis of nanofluorescent materials.

[0003] Currently, commonly used fluorescent probes in multi-pathogen fluorescence immunoassay systems include organic fluorescent dyes, rare-earth complex fluorescent microspheres, and semiconductor quantum dots. While organic fluorescent dyes are abundant and easy to label, they suffer from poor photostability, low fluorescence quantum yield, and susceptibility to fluorescence quenching, making them unsuitable for high-sensitivity detection. For example, Chinese patent CN109116031A discloses a detection kit for detecting IgG antibodies against multiple pathogens affecting reproductive health, but it suffers from rapid fluorescence signal decay, high background interference, and limited detection sensitivity. Rare-earth complex fluorescent microspheres possess large Stokes shifts and long fluorescence lifetimes, but their excitation wavelengths are typically in the ultraviolet region, requiring a special light source. Furthermore, the large particle size (usually above 100 nanometers) leads to slow diffusion and poor binding kinetics, affecting detection speed. For instance, Chinese patent CN1645146A discloses a rapid immunochromatographic detection method and test strip using fluorescent rare-earth complex nanoparticles as markers, but it is limited by long detection times and high instrument requirements. While semiconductor quantum dots (such as cadmium-based quantum dots) offer advantages such as tunable emission peaks, high fluorescence brightness, and good photostability, the biotoxicity of cadmium ions limits their application in in vitro diagnostic products. Cadmium-free quantum dots (such as indium phosphide quantum dots) typically exhibit lower fluorescence quantum yields than cadmium-based quantum dots, and still fall short in terms of colloidal stability in aqueous phases and retention of activity after antibody conjugation. Furthermore, while existing multi-shell coating strategies (such as silica coating) can improve the chemical stability and salt resistance of nanoparticles, issues such as the interfacial quenching effect between the silica shell and the fluorescent core, and the difficulty of directly conjugating antibodies to the silica surface (requiring further functionalization modification), lead to decreased fluorescence brightness, low conjugation efficiency, and poor batch-to-batch consistency, severely restricting the performance and reliability of dual-channel fluorescence immunoassay systems. Summary of the Invention

[0004] The purpose of this invention is to provide a dual-channel fluorescent immunoassay system for multiple pathogens, its preparation method, and its application. This invention addresses three key challenges in current dual-channel fluorescent nanoprobes: the difficulty in balancing high solids content and low viscosity processing windows during large-scale preparation and spotting / spraying; the difficulty in maintaining antibody conformation and binding activity while achieving batch-to-batch consistency and fixation efficiency through high surface activity and high coupling density, leading to increased background and decreased sensitivity; and the potential for scattering and interface quenching introduced when using multi-shell silica coating to enhance colloidal and chemical stability, which weakens fluorescence brightness and amplifies dual-channel crosstalk.

[0005] This invention employs a synergistic design approach, constructing a multi-shell structure consisting of a carbon dot core, a silica intermediate shell, and an acrylic polymer outer shell. The spatial isolation effect of the silica shell suppresses interfacial quenching between the carbon dot core and the polymer outer shell, reducing dual-channel crosstalk caused by light scattering. Simultaneously, by controlling the carboxyl group density of the acrylic polymer outer shell within the range of 0.5 to 3.0 mmol / g, a balance is achieved between a high solids content, low viscosity processing window, and high antibody conjugation density. Furthermore, the introduction of polyethylene glycol segments enhances colloidal stability and resistance to non-specific adsorption. Thus, while maintaining a fluorescence quantum yield of at least 30%, this invention achieves dual performance improvements: good batch-to-batch consistency, high antibody activity after conjugation, low background signal, and minimal dual-channel spectral crosstalk.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing a dual-channel fluorescence immunoassay system for multiple pathogens includes the following steps:

[0008] Step S1: Prepare first multi-shell carbon dot fluorescent nanoparticles and second multi-shell fluorescent nanoparticles, wherein the mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell fluorescent nanoparticles is 1:(0.2~2.0) based on their dry basis mass.

[0009] Step S2: The first multi-shelled carbon dot fluorescent nanoparticles are coupled with the first recognition antibody to obtain the first fluorescent immunoprobe; the second multi-shelled fluorescent nanoparticles are coupled with the second recognition antibody to obtain the second fluorescent immunoprobe.

[0010] Step S3: Prepare a solid-phase support, wherein the solid-phase support is (i) a nitrocellulose membrane; or (ii) carboxylated polystyrene-methacrylic acid copolymer microspheres; or (iii) a combination of a nitrocellulose membrane and carboxylated polystyrene-methacrylic acid copolymer microspheres, forming an immune-binding region on the solid-phase support, and immobilizing capture antibodies for recognizing at least two different pathogens in the immune-binding region, wherein the capture antibodies, the first recognition antibody, and the second recognition antibody target different antigenic epitopes of the same pathogen, respectively, to form a sandwich immune complex;

[0011] Step S4: Prepare a phosphate buffer containing surfactant and protein stabilizer. The phosphate buffer is 1×PBS (10 mmol / L phosphate, 137 mmol / L NaCl, 2.7 mmol / L KCl) with a pH of 7.0–7.6. Add the surfactant polyoxyethylene sorbitan monooleate to the phosphate buffer to a mass fraction of 0.10%, and add the protein stabilizer bovine serum albumin to a mass fraction of 0.50%. Adjust the pH by titration with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide and measure at 25±1℃. Combine the first and second fluorescent immunoprobes with the phosphate buffer and assemble them together with the solid-phase carrier prepared in step S3 to form a test strip or chip for dual-channel fluorescent immunoassay for multiple pathogens.

[0012] Furthermore, the emission peak of the first multi-shell carbon dot fluorescent nanoparticle is located at 500–560 nm, the emission peak of the second multi-shell fluorescent nanoparticle is located at 600–700 nm, and the distance between the emission peaks of the two is not less than 80 nm; the excitation wavelength range corresponding to the first excitation-emission channel is 400–480 nm, and the detection wavelength range is 500–560 nm; the excitation wavelength range corresponding to the second excitation-emission channel is 500–580 nm, and the detection wavelength range is 600–700 nm.

[0013] Furthermore, the first multi-shell carbon dot fluorescent nanoparticle in step S1 is a multi-shell carbon dot fluorescent nanoparticle with a carbon dot core, a silica shell, and an acrylic polymer outer shell, and its structure includes:

[0014] The carbon dot core is obtained by carbonization of citric acid and ethylenediamine under hydrothermal conditions;

[0015] The first inorganic shell layer is silicon dioxide, which is formed by the hydrolysis-condensation of tetraethoxysilane and coats the outer surface of the carbon dot core.

[0016] The second organic shell is a carboxyl-containing acrylic polymer shell, formed by copolymerization of methyl methacrylate, methacrylic acid and polyethylene glycol methyl ether methacrylate. The surface carboxyl density of the second organic shell is 0.5–3.0 mmol / g, and the volume average particle size of the first multi-shell carbon dot fluorescent nanoparticles is 30–120 nm.

[0017] Furthermore, the second multi-shell fluorescent nanoparticle in step S1 is:

[0018] Multi-shell carbon dot fluorescent nanoparticles with the same structure as the first multi-shell carbon dot fluorescent nanoparticles but with emission peaks located at 600–700 nm, or

[0019] Cadmium-free quantum dot multi-shell fluorescent nanoparticles are constructed with indium phosphide as the core, zinc sulfide as the first inorganic shell, silicon dioxide as the second inorganic shell, and an acrylic polymer as the outermost shell. The volume average particle size of the cadmium-free quantum dot multi-shell fluorescent nanoparticles is 20-80 nm, and the fluorescence quantum yield is not less than 30%.

[0020] Furthermore, the first multi-shell carbon dot fluorescent nanoparticles are prepared through the following steps:

[0021] A1. Raw material preparation: 10-50 parts by weight of citric acid; 5-30 parts by weight of ethylenediamine; 50-200 parts by weight of deionized water; wherein the molar ratio of citric acid to ethylenediamine is 1.0-1.5:1.0;

[0022] A2. Preparation of carbon dot cores: The aqueous solution obtained in A1 is subjected to hydrothermal reaction at 160-220℃ for 2-8 hours in a closed reaction vessel. After cooling, a carbon dot dispersion is obtained. The volume average particle size of the carbon dots is 2-10 nm. The solid content of the carbon dot dispersion is 2-10 wt%, which can be determined by drying and weighing.

[0023] A3. Silica shell coating: The carbon dot dispersion is mixed with a mixture containing 2-20 parts by mass of tetraethoxysilane and 50-300 parts by mass of anhydrous ethanol. The pH is adjusted to 9.0-11.0 in the presence of ammonia. The mixture is stirred at 20-35°C for 2-6 hours to obtain carbon dot-silica core-shell nanoparticles.

[0024] A4. Polymer shell construction: Add 20-60 parts by weight of methyl methacrylate, 5-20 parts by weight of methacrylic acid, 5-40 parts by weight of polyethylene glycol methyl ether methacrylate, and 0.1-1.0 parts by weight of ammonium persulfate as initiators to the colloidal system obtained in A3. React at 60-85°C under an inert atmosphere for 2-6 hours, controlling the total mass fraction of the added monomers methyl methacrylate, methacrylic acid, and polyethylene glycol methyl ether methacrylate to be 5-30 wt% of the total mass of the reaction system.

[0025] A5. Endpoint Criteria and Post-processing: The reaction was terminated when the residual total monomer mass fraction in the system was not higher than 0.5 wt%. The residual total monomer mass fraction was obtained by sampling the reaction solution and determining it by chromatography. The total monomer was the sum of methyl methacrylate, methacrylic acid, and polyethylene glycol methyl ether methacrylate. After removing small molecules by dialysis or ultrafiltration, the solid content was adjusted to 1-20 wt% to obtain the first multi-shell carbon dot fluorescent nanoparticle dispersion. The dialysis was performed using a dialysis bag with a molecular weight cutoff of 10 kDa in deionized water for 48 hours with the liquid changed every 8 hours, or the ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa at 4-25°C. The solid content was determined by drying at 105°C to constant weight (the difference between two adjacent weighings ≤ 0.5 mg).

[0026] Furthermore, the second multi-shell fluorescent nanoparticles are prepared through the following steps:

[0027] B1. Raw material preparation: Carbon dot-silica core-shell nanoparticles or quantum dot-silica core-shell nanoparticles obtained by coating cadmium-free indium phosphide / zinc sulfide quantum dots with silica are used as the fluorescent core.

[0028] B2. Polymer shell construction: Methyl methacrylate, methacrylic acid and polyethylene glycol methyl ether methacrylate are added to the fluorescent core dispersion system, and ammonium persulfate is added as an initiator. The reaction is carried out at 60-85°C under a nitrogen atmosphere for 2-6 hours, and the total monomer mass fraction is controlled at 5-30 wt% to form a second organic shell containing carboxyl groups.

[0029] B3. Spectral modulation: The fluorescent core is selected with an emission peak located in the range of 600-700 nm, so that the emission peak of the obtained second multi-shell fluorescent nanoparticles is located in the range of 600-700 nm, and the fluorescence quantum yield is not less than 30%;

[0030] B4. Post-processing: The obtained second multi-shell fluorescent nanoparticles were purified and the solid content was adjusted to obtain a dispersion of the second multi-shell fluorescent nanoparticles. The purification was carried out by dialyzing in deionized water with a dialysis bag with a molecular weight cutoff of 10 kDa for 48 hours and changing the liquid every 8 hours, or by ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 100 kDa at 4–25°C. The solid content was determined by drying at 105°C to constant weight (the difference between two adjacent weighings ≤ 0.5 mg).

[0031] Furthermore, the carboxylated polystyrene-methacrylic acid copolymer microspheres in step S3 are prepared through the following steps:

[0032] C1. Raw material preparation: 60-90 parts by weight of styrene; 10-40 parts by weight of methacrylic acid; 0.1-2.0 parts by weight of sodium dodecyl sulfate as emulsifier; 0.1-1.0 parts by weight of potassium persulfate as initiator; 200-800 parts by weight of deionized water;

[0033] C2. Emulsion polymerization: Under an inert atmosphere, the C1 mixture is stirred and reacted at 65-80°C for 2-8 hours;

[0034] C3. Post-processing and quality control: After the reaction is completed and cooled, the free emulsifier and small molecules are removed by centrifugation and redispersion to obtain the carboxylated polystyrene-methacrylic acid copolymer microspheres, wherein the carboxyl groups on the surface of the microspheres are derived from the comonomer methacrylic acid.

[0035] Furthermore, the preparation of the first and second fluorescent immunoprobes in step S2 includes:

[0036] D1. Activation: Add 0.5–5.0 mg / mL of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride and 0.5–5.0 mg / mL of N-hydroxysuccinimide to the dispersion of the first multi-shell carbon dot fluorescent nanoparticles or the dispersion of the second multi-shell fluorescent nanoparticles, and react for 0.5–2.0 hours in the pH range of 5.5–7.0 to activate the surface carboxyl groups;

[0037] D2. Coupling: Add a first recognition antibody or a second recognition antibody to the activation system to cause the antibody to undergo an amidation reaction with the activated carboxyl group, and control the molar ratio of the antibody to the first multi-shell carbon dot fluorescent nanoparticle or the second multi-shell fluorescent nanoparticle to be 1:(5-50), wherein the ratio is the ratio of the number of particles to the number of antibody molecules, and react at 4-25°C for 1-4 hours.

[0038] D3. Endpoint Criteria and Purification: The reaction was terminated when the free antibody mass fraction was no higher than 20% of the total antibody mass fraction. The protein content of the free antibody and the total antibody was determined by the Bradford method. Free small molecules were removed by gel filtration or ultrafiltration to obtain the first and second fluorescent immunoprobes.

[0039] As a concept of this invention, a multi-shell structure design of carbon dot core-silica shell-acrylic polymer shell is adopted, mainly to enhance the fluorescence stability, colloidal stability, and antibody conjugation performance of fluorescent nanoparticles. The carbon dot core is formed by carbonization of citric acid and ethylenediamine under hydrothermal conditions, exhibiting relatively high fluorescence quantum yield, good photostability, and excellent biocompatibility. However, the surface of the carbon dot core lacks sufficient functional groups for antibody conjugation and is prone to aggregation in high ionic strength solutions. The introduction of the silica shell not only provides a stable substrate for the subsequent construction of the polymer shell, but more importantly, it acts as a spatial isolation layer, effectively suppressing the interfacial quenching effect between the carbon dot core and the outer polymer layer. Simultaneously, the low refractive index of silica reduces signal attenuation and dual-channel crosstalk caused by light scattering. The acrylic polymer shell layer is formed by copolymerizing methyl methacrylate, methacrylic acid, and polyethylene glycol methyl ether methacrylate. Methacrylic acid provides carboxyl groups for amidation coupling with the amino groups of the antibody. The polyethylene glycol segments endow the nanoparticles with excellent water solubility, resistance to non-specific adsorption, and colloidal stability. Methyl methacrylate acts as a comonomer to regulate the hydrophobic-hydrophilic balance and mechanical strength of the polymer segments. By precisely controlling the carboxyl group density within the range of 0.5 to 3.0 mmol / g, sufficient antibody coupling site density is ensured to achieve high coupling rates and batch-to-batch consistency, while avoiding colloidal instability caused by excessively high surface charge on the nanoparticles and steric hindrance effects after antibody coupling due to excessively high carboxyl group density. This allows for a processing window with high solids content (1 to 20% by mass) and low viscosity in large-scale preparation, meeting the rheological requirements of automated production processes such as spot spraying. In addition, the multi-shell structure significantly enhances the chemical stability and salt resistance of fluorescent nanoparticles, enabling them to maintain stable fluorescence signals and dispersion in physiological saline solutions such as phosphate buffer for extended periods, thus ensuring the reliability and reproducibility of the detection system.

[0040] This invention also discloses a multi-pathogen fluorescent immunoassay dual-channel detection system prepared by the above-described method, comprising: a first fluorescent immunoassay probe, a second fluorescent immunoassay probe, a solid-phase support, and a phosphate buffer; wherein,

[0041] The first fluorescent immunoprobe comprises a complex formed by conjugation of a first multi-shelled carbon dot fluorescent nanoparticle and a first recognition antibody, and the second fluorescent immunoprobe comprises a complex formed by conjugation of a second multi-shelled fluorescent nanoparticle and a second recognition antibody.

[0042] The solid-phase carrier has an immune-binding region, which is immobilized with capture antibodies for recognizing at least two different pathogens, wherein each capture antibody, together with the first recognition antibody and the second recognition antibody, targets different antigenic epitopes of the same pathogen to form a sandwich immune complex.

[0043] The phosphate buffer contains surfactants and protein stabilizers.

[0044] This invention also discloses the use of a multi-pathogen fluorescent immunoassay dual-channel detection system in the preparation of in vitro diagnostic products for the simultaneous detection of at least two respiratory pathogens, wherein the respiratory pathogens are selected from at least two of influenza virus, novel coronavirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, Mycoplasma pneumoniae, or Streptococcus pneumoniae.

[0045] Furthermore, the pH of the phosphate buffer in step S4 is 7.0–7.6, the surfactant is polyoxyethylene sorbitan monooleate, and the protein stabilizer is bovine serum albumin;

[0046] When using the multi-pathogen fluorescence immunoassay dual-channel detection system, the sample to be tested is mixed with the phosphate buffer at a volume ratio of 1:(1-5), and incubated at room temperature for 5-20 minutes. The first fluorescence signal and the second fluorescence signal are read in the first excitation-emission channel and the second excitation-emission channel, respectively. The concentrations of at least two pathogens are calculated based on the linear standard curve established by the two channel signals and pathogen standards of known concentration.

[0047] Furthermore, in one embodiment, the post-treatment of the first multi-shell carbon dot fluorescent nanoparticle dispersion may include: terminating the reaction when the volume average particle size of the obtained nanoparticles is in the range of 30 to 120 nm and the particle size polydispersity index is not higher than 0.20, and adjusting the solid content to 1 to 20 wt% after removing small molecules by dialysis or ultrafiltration.

[0048] Furthermore, in one embodiment, when the fluorescent core of the second multi-shell fluorescent nanoparticle is a quantum dot-silica core-shell nanoparticle, the quantum dot core can be prepared by indium phosphide and zinc sulfide in an organic solvent, and after preparation, it is treated with a carboxyl-containing ligand to obtain a carboxyl-functionalized surface and then phase-transferred to an aqueous phase.

[0049] Furthermore, in one embodiment, during the preparation of the carboxylated polystyrene-methacrylic acid copolymer microspheres, the solid content can be controlled to be 20-40 wt%, and the surface carboxyl density of the resulting microspheres can be 0.3-2.0 mmol / g, and the volume average particle size can be 0.2-1.0 μm.

[0050] As another aspect of this invention, the detection system design combining a dual-channel fluorescent immunoassay probe and a sandwich immune complex is primarily used to enhance the sensitivity, specificity, and throughput of simultaneous detection of multiple pathogens. The first multi-shelled carbon dot fluorescent nanoparticles and the second multi-shelled fluorescent nanoparticles have emission peaks located at 500-560 nm and 600-700 nm, respectively, with a peak spacing of not less than 80 nm, ensuring dual-channel spectral separation and effectively reducing inter-channel crosstalk. By coupling the first and second nanoparticles with first and second recognition antibodies targeting different pathogen antigenic epitopes, respectively, and combining them with capture antibodies targeting the same pathogen but different epitopes immobilized on a solid-phase support, a sandwich immune complex structure of capture antibody-pathogen antigen-recognition antibody is formed, significantly improving the detection specificity and signal intensity. The solid-phase support uses nitrocellulose membranes or carboxylated polystyrene-methacrylic acid copolymer microspheres; the former is suitable for rapid, point-of-care testing scenarios using immunochromatographic test strips, while the latter is suitable for high-throughput automated detection platforms such as microfluidic chips or magnetic bead separation. Surfactants (such as polyoxyethylene sorbitan monooleate) in phosphate buffer reduce nonspecific adsorption and background signals, while protein stabilizers (such as bovine serum albumin) maintain antibody conformational stability and binding activity, ensuring the stability and reliability of the detection system at room temperature. The dual-channel detection system of this invention achieves simultaneous quantitative detection of at least two respiratory pathogens by reading fluorescence signals in a first excitation-emission channel (excitation 400-480 nm, detection 500-560 nm) and a second excitation-emission channel (excitation 500-580 nm, detection 600-700 nm). Compared to traditional single-pathogen detection methods, this invention significantly shortens detection time, reduces sample consumption, and increases detection throughput. It has significant application value in the rapid clinical screening and differential diagnosis of various respiratory pathogens, including influenza virus, novel coronavirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, Mycoplasma pneumoniae, and Streptococcus pneumoniae.

[0051] In this invention, the first multi-shell carbon dot fluorescent nanoparticles and the second multi-shell fluorescent nanoparticles have clearly defined focuses and main functions in the dual-channel detection system. The first multi-shell carbon dot fluorescent nanoparticles use carbon dots as the fluorescent core, with their emission peak located in the green light region of 500 to 560 nm. They are mainly responsible for detecting the first pathogen and have the advantages of simple preparation process, low cost, and good biocompatibility. The second multi-shell fluorescent nanoparticles use carbon dots or indium phosphide quantum dots with emission peaks located in the red light region of 600 to 700 nm as the core. They are mainly responsible for detecting the second pathogen, and their red light emission characteristics effectively reduce the background interference of autofluorescence in biological samples. The silica intermediate shell plays a key role in spatial isolation and optical regulation in both types of nanoparticles. By physically isolating the fluorescent core from the outer acrylic polymer, it suppresses the interface quenching effect caused by charge transfer and energy transfer between the fluorescent core and the polymer. At the same time, the low refractive index of silica and the uniform coating thickness control reduce the emission light loss caused by light scattering and Rayleigh scattering, thereby maintaining a fluorescence quantum yield of not less than 30%. The acrylic polymer shell layer plays a synergistic role in both types of nanoparticles. Its carboxyl functional groups not only provide ample reaction sites for antibody conjugation, but the polyethylene glycol segments also inhibit nanoparticle aggregation and non-specific adsorption through steric hindrance and hydration layer formation, maintaining colloidal stability in high-salt environments and complex biological matrices. The synergistic design of the two types of nanoparticles ensures an emission peak spacing of no less than 80 nm. The spectral separation strategy combining a first excitation-emission channel (400–480 nm excitation, 500–560 nm detection) and a second excitation-emission channel (500–580 nm excitation, 600–700 nm detection) effectively eliminates dual-channel crosstalk, enabling independent and accurate quantification of the fluorescence signals of the two pathogens. This demonstrates the systematic synergistic effect of multi-shell structure design, spectral engineering regulation, and surface chemical modification.

[0052] Beneficial technical effects

[0053] 1. Significantly improved fluorescence brightness and photostability: By constructing a multi-shell structure of carbon dot core-silica shell-acrylic polymer shell, the spatial isolation effect of the silica intermediate shell effectively suppresses the interface quenching between the fluorescent core and the polymer shell. At the same time, the low refractive index of silica reduces fluorescence loss caused by light scattering, so that the fluorescence quantum yield of both the first multi-shell carbon dot fluorescent nanoparticles and the second multi-shell fluorescent nanoparticles remains at a high level of not less than 30%. Under ultraviolet or blue light excitation, they exhibit bright and stable fluorescence signals, significantly improving the sensitivity and signal-to-noise ratio of the detection system, and meeting the clinical needs for rapid detection of low-concentration pathogens.

[0054] 2. Achieving a balance between high solids content, low viscosity processing window, and high coupling density: By precisely controlling the carboxyl group density of the acrylic polymer shell layer within the range of 0.5 to 3.0 mmol / g, and combining this with the introduction of polyethylene glycol segments, the rheological properties of the nanoparticle dispersion are effectively regulated while ensuring sufficient antibody coupling sites. This achieves a processing window with a solids content of 1 to 20% by mass and moderate viscosity, meeting the technical requirements of automated production processes such as spot spraying. It solves the key technical bottleneck of balancing high solids content and low viscosity in large-scale preparation, significantly improving production efficiency and batch-to-batch consistency.

[0055] 3. Effectively reduces crosstalk and background signal in dual-channel spectral analysis: By designing the emission peaks of the first multi-shell carbon dot fluorescent nanoparticles at 500-560 nm and the second multi-shell fluorescent nanoparticles at 600-700 nm, ensuring an emission peak spacing of no less than 80 nm, and combining the spectral separation strategy of the first excitation-emission channel (excitation 400-480 nm, detection 500-560 nm) and the second excitation-emission channel (excitation 500-580 nm, detection 600-700 nm), crosstalk between the two channels is effectively eliminated, enabling independent and accurate quantification of the fluorescence signals of the two pathogens. At the same time, the anti-non-specific adsorption effect of polyethylene glycol segments and the synergistic effect of surfactants in phosphate buffer significantly reduce the background signal, improving detection specificity and accuracy.

[0056] 4. Maintaining high binding activity and batch-to-batch consistency after antibody conjugation: Mild amidation conjugation is performed using 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group. The molar ratio of antibody to nanoparticles is controlled at 1:(5 to 50). Combined with the steric hindrance and hydration layer protection provided by the polyethylene glycol segment, the native conformation and antigen-binding activity of the antibody are effectively maintained. After conjugation, the fluorescent immunoprobe maintains a stable fluorescence signal and immunoreactivity in phosphate buffer for a long time. The free antibody mass fraction is controlled within 20% of the total antibody mass fraction, ensuring high consistency of performance between batches and reliability of detection results.

[0057] 5. Enables simultaneous rapid detection and high-throughput application of multiple pathogens: By immobilizing capture antibodies against at least two different pathogens on a solid-phase carrier such as nitrocellulose membrane or carboxylated polystyrene-methacrylic acid copolymer microspheres, and combining them with dual-channel fluorescent immunoassay probes to form sandwich immune complexes, simultaneous quantitative detection of at least two respiratory pathogens (including influenza virus, novel coronavirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, Mycoplasma pneumoniae, and Streptococcus pneumoniae, etc.) can be achieved in a single test. The detection time is shortened to 5 to 20 minutes, the sample volume is reduced, and the detection throughput and clinical application convenience are significantly improved. It has important application value in the rapid screening and differential diagnosis of respiratory infectious diseases. Attached Figure Description

[0058] Figure 1 The XRD patterns are those of Example 1 and Comparative Example 7.

[0059] Figure 2 The XPS full spectra are for Example 1 and Comparative Example 7.

[0060] Figure 3 High-resolution XPS Si 2p images of Example 1 and Comparative Example 7.

[0061] Figure 4 The rheological shear rate viscosity diagrams are for Example 1 and Comparative Example 6.

[0062] Figure 5 The difference diagram of DLS particle size distribution between Example 1 and Comparative Example 6 is shown.

[0063] Figure 6 The cumulative distribution diagram of DLS particle size distribution for Example 1 and Comparative Example 6 is shown.

[0064] Figure 7 This is a normalized overlay of the fluorescence emission spectra of Example 1 and Comparative Example 3. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0066] Example 1

[0067] This embodiment provides a method for preparing a dual-channel fluorescence immunoassay system for multiple pathogens, comprising the following steps:

[0068] Step S1: Prepare first multi-shell carbon dot fluorescent nanoparticles and second multi-shell carbon dot fluorescent nanoparticles.

[0069] The preparation steps of the first multi-shell carbon dot fluorescent nanoparticles in this embodiment are as follows:

[0070] A1. Raw material preparation: Weigh 30 parts by weight of citric acid, 7.5 parts by weight of ethylenediamine, and 125 parts by weight of deionized water, wherein the molar ratio of citric acid to ethylenediamine is 1.25:1.0.

[0071] A2. Preparation of carbon dot cores: The aqueous solution obtained in A1 was transferred to a closed reaction vessel and hydrothermally reacted at 190°C for 5 hours. After cooling, a carbon dot dispersion was obtained. The volume average particle size of the carbon dots in this embodiment was 6 nm.

[0072] A3. Silica shell coating: The carbon dot dispersion of this embodiment was mixed with a mixture containing 11 parts by mass of tetraethoxysilane and 175 parts by mass of anhydrous ethanol. The pH was adjusted to 10.0 in the presence of ammonia, and the mixture was stirred at 27.5°C for 4 hours to obtain carbon dot-silica core-shell nanoparticles.

[0073] A4. Polymer shell construction: 40 parts by mass of methyl methacrylate, 12.5 parts by mass of methacrylic acid, 22.5 parts by mass of polyethylene glycol methyl ether methacrylate and 0.55 parts by mass of ammonium persulfate were added to the colloidal system obtained in A3 as initiators. The reaction was carried out at 72.5°C under a nitrogen atmosphere for 4 hours, and the total monomer mass fraction was controlled to be 17.5 wt%.

[0074] A5. Endpoint Criteria and Post-processing: The reaction was terminated when the residual total monomer mass fraction in the system decreased to 0.35 wt%. After dialysis to remove small molecules, the solid content was adjusted to 10.5 wt% to obtain the first multi-shell carbon dot fluorescent nanoparticle dispersion of this embodiment. The first multi-shell carbon dot fluorescent nanoparticles of this embodiment have a multi-shell structure of carbon dot core-silica shell-acrylic polymer outer shell. The carboxyl group density on the surface of the second organic shell of this embodiment is 1.75 mmol / g. The volume average particle size of the first multi-shell carbon dot fluorescent nanoparticles of this embodiment is 75 nm, the polydispersity index is 0.14, and the emission peak is located at 525 nm.

[0075] The preparation steps of the second multi-shell carbon dot fluorescent nanoparticles in this embodiment are as follows:

[0076] B1. Raw material preparation: Nitrogen-doped carbon dot-silica core-shell nanoparticles are used as the fluorescent core, and carbon dot-silica core-shell nanoparticles with emission peaks in the range of 600-700 nm are selected as the fluorescent core.

[0077] B2. Polymer shell construction: In this embodiment, acrylic monomers are subjected to free radical polymerization on the surface of the fluorescent core, using the same monomer ratio and reaction conditions as the first multi-shell carbon dot fluorescent nanoparticles, to form a second organic shell containing carboxyl groups.

[0078] B3. Spectral modulation: The emission peak of the second multi-shell carbon dot fluorescent nanoparticle in this embodiment is located at 650 nm, and the fluorescence quantum yield is 42%.

[0079] B4. Post-processing: The obtained second multi-shell carbon dot fluorescent nanoparticles were purified and the solid content was adjusted to 10.5 wt% to obtain a dispersion of the second multi-shell carbon dot fluorescent nanoparticles. In this embodiment, the volume average particle size of the second multi-shell carbon dot fluorescent nanoparticles was 75 nm.

[0080] In this embodiment, the mass ratio of the first multi-shell carbon dot fluorescent nanoparticle to the second multi-shell carbon dot fluorescent nanoparticle is 1:1.0 on a dry basis. The emission peak of the first multi-shell carbon dot fluorescent nanoparticle is located at 525 nm, and the emission peak of the second multi-shell carbon dot fluorescent nanoparticle is located at 650 nm, with a peak spacing of 125 nm. The excitation wavelength for the first excitation-emission channel in this embodiment is 440 nm, and the detection wavelength is 525 nm; the excitation wavelength for the second excitation-emission channel in this embodiment is 540 nm, and the detection wavelength is 650 nm.

[0081] Step S2, Preparation of fluorescent immunoprobes

[0082] The preparation steps of the first fluorescent immunoprobe in this embodiment are as follows:

[0083] D1. Activation: 2.75 mg / mL of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride and 2.75 mg / mL of N-hydroxysuccinimide were added to the dispersion of the first multi-shell carbon dot fluorescent nanoparticles, and the reaction was carried out at pH 6.25 for 1.25 hours to activate the surface carboxyl groups.

[0084] D2. Coupling: The first recognition antibody against the influenza virus nucleoprotein is added to the activation system, and the antibody undergoes an amidation reaction with the activated carboxyl group. The ratio of the first multi-shell carbon dot fluorescent nanoparticles to the antibody in this embodiment is controlled to be 1:27.5, where the ratio is the ratio of the number of particles to the number of antibody molecules. The reaction is carried out at 14.5°C for 2.5 hours.

[0085] D3. Endpoint Criteria and Purification: The reaction was terminated when the free antibody mass fraction decreased to 12% of the total antibody mass fraction. The protein content of the free antibody and total antibody was determined by the Bradford method. Free small molecules were removed by gel filtration to obtain the first fluorescent immunoprobe of this embodiment.

[0086] The preparation steps of the second fluorescent immunoprobe in this embodiment are the same as those of the first fluorescent immunoprobe. The second recognition antibody used is an antibody against the spike protein of the novel coronavirus, thus obtaining the second fluorescent immunoprobe of this embodiment.

[0087] Step S3, Preparation of solid support

[0088] In this embodiment, a nitrocellulose membrane is used as a solid-phase carrier. An immune-binding region is formed on the nitrocellulose membrane. In this embodiment, capture antibodies for recognizing influenza virus and novel coronavirus are immobilized in the immune-binding region. The capture antibody and the first recognition antibody of this embodiment target different antigenic epitopes of the influenza virus nucleoprotein, and the second recognition antibody of this embodiment targets different antigenic epitopes of the novel coronavirus spike protein, thus forming a sandwich immune complex.

[0089] Step S4, Assemble the testing system

[0090] A phosphate buffer solution was prepared. In this embodiment, the phosphate buffer solution had a pH of 7.3 and contained polyoxyethylene sorbitan monooleate as a surfactant and bovine serum albumin as a protein stabilizer. The first and second fluorescent immunoprobes of this embodiment were combined with the phosphate buffer solution of this embodiment and assembled together with the solid-phase carrier prepared in step S3 to form a test strip for dual-channel fluorescent immunoassay for multiple pathogens.

[0091] Testing and use

[0092] When using the multi-pathogen fluorescence immunoassay dual-channel detection system of this embodiment, the sample to be tested is mixed with the phosphate buffer of this embodiment at a volume ratio of 1:3. After incubation at room temperature for 12.5 minutes, the first fluorescence signal and the second fluorescence signal of this embodiment are read in the first excitation-emission channel and the second excitation-emission channel, respectively. The concentrations of influenza virus and novel coronavirus are calculated based on the linear standard curve established by the two channel signals and the pathogen standards of known concentration.

[0093] Example 1 Technical Features: This example uses moderate parameter configurations. The mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell carbon dot fluorescent nanoparticles is 1:1.0. Both nanoparticles adopt a multi-shell structure of carbon dot core-silica shell-acrylic polymer outer shell, with a particle size of 75 nm, a carboxyl density of 1.75 mmol / g, emission peaks at 525 nm and 650 nm respectively, an emission peak spacing of 125 nm, and a fluorescence quantum yield of 42%. The preparation process uses a hydrothermal temperature of 190℃, a reaction time of 5 hours, a total monomer mass fraction of 17.5 wt%, an antibody-to-nanoparticle ratio of 1:27.5 (particle number to antibody molecule number), a phosphate buffer pH of 7.3, a sample-to-buffer volume ratio of 1:3, and an incubation time of 12.5 minutes. This example features robust parameter selection, high process reliability, and stable fluorescence signal, making it suitable for the simultaneous detection of influenza virus and novel coronavirus. It is particularly suitable for routine clinical screening scenarios and can meet the needs of rapid initial screening in fever clinics.

[0094] Example 2

[0095] This embodiment provides a method for preparing a dual-channel fluorescence immunoassay system for multiple pathogens, comprising the following steps:

[0096] Step S1: Prepare first multi-shell carbon dot fluorescent nanoparticles and second multi-shell fluorescent nanoparticles.

[0097] The preparation steps of the first multi-shell carbon dot fluorescent nanoparticles in this embodiment are as follows:

[0098] A1. Raw material preparation: Weigh 20 parts by weight of citric acid, 5.7 parts by weight of ethylenediamine, and 80 parts by weight of deionized water, wherein the molar ratio of citric acid to ethylenediamine is 1.1:1.0.

[0099] A2. Preparation of carbon dot cores: The aqueous solution obtained in A1 was transferred to a closed reaction vessel and hydrothermally reacted at 175°C for 3 hours. After cooling, a carbon dot dispersion was obtained. The volume average particle size of the carbon dots in this embodiment was 4 nm.

[0100] A3. Silica shell coating: The carbon dot dispersion of this embodiment was mixed with a mixture containing 6 parts by mass of tetraethoxysilane and 100 parts by mass of anhydrous ethanol. The pH was adjusted to 9.5 in the presence of ammonia, and the mixture was stirred at 25°C for 3 hours to obtain carbon dot-silica core-shell nanoparticles.

[0101] A4. Polymer shell construction: 28 parts by mass of methyl methacrylate, 8 parts by mass of methacrylic acid, 12 parts by mass of polyethylene glycol methyl ether methacrylate and 0.3 parts by mass of ammonium persulfate were added to the colloidal system obtained in A3 as initiators. The reaction was carried out at 65°C under a nitrogen atmosphere for 3 hours, and the total monomer mass fraction was controlled to be 10 wt%.

[0102] A5. Endpoint Criteria and Post-processing: The reaction was terminated when the residual total monomer mass fraction in the system decreased to 0.25 wt%. After removing small molecules by ultrafiltration, the solid content was adjusted to 5 wt% to obtain the first multi-shell carbon dot fluorescent nanoparticle dispersion of this embodiment. The first multi-shell carbon dot fluorescent nanoparticles of this embodiment have a multi-shell structure of carbon dot core-silica shell-acrylic polymer outer shell. The carboxyl group density on the surface of the second organic shell of this embodiment is 1.0 mmol / g. The volume average particle size of the first multi-shell carbon dot fluorescent nanoparticles of this embodiment is 50 nm, the polydispersity index is 0.11, and the emission peak is located at 515 nm.

[0103] The second multi-shell fluorescent nanoparticle in this embodiment uses cadmium-free quantum dot multi-shell fluorescent nanoparticles, and the preparation steps are as follows:

[0104] B1. Raw material preparation: Quantum dots with indium phosphide as the core and zinc sulfide as the first inorganic shell were prepared in an organic solvent in the presence of oleic acid and oleylamine. Then, the carboxyl functionalized surface was obtained by treatment with carboxyl-containing ligands and phase transfer to the aqueous phase. Finally, the quantum dot-silica core-shell nanoparticles were obtained by coating with silica as the fluorescent core.

[0105] B2. Polymer shell construction: In this embodiment, acrylic monomers are subjected to free radical polymerization on the surface of the fluorescent core, using the same monomer ratio and reaction conditions as the first multi-shell carbon dot fluorescent nanoparticles, to form an outermost shell containing carboxyl groups.

[0106] B3. Spectral modulation: The emission peak of the second multi-shell fluorescent nanoparticle in this embodiment is located at 620 nm, and the fluorescence quantum yield is 38%.

[0107] B4. Post-processing: The obtained second multi-shell fluorescent nanoparticles were purified and the solid content was adjusted to 5wt% to obtain a dispersion of the second multi-shell fluorescent nanoparticles. In this embodiment, the volume average particle size of the cadmium-free quantum dot multi-shell fluorescent nanoparticles was 30nm.

[0108] In this embodiment, the mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell fluorescent nanoparticles is 1:0.5. The emission peak of the first multi-shell carbon dot fluorescent nanoparticles is located at 515 nm, and the emission peak of the second multi-shell fluorescent nanoparticles is located at 620 nm, with a peak spacing of 105 nm. The excitation wavelength for the first excitation-emission channel in this embodiment is 420 nm, and the detection wavelength is 515 nm; the excitation wavelength for the second excitation-emission channel in this embodiment is 520 nm, and the detection wavelength is 620 nm.

[0109] Step S2, Preparation of fluorescent immunoprobes

[0110] The preparation steps of the first fluorescent immunoprobe in this embodiment are as follows:

[0111] D1. Activation: 1.2 mg / mL of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride and 1.2 mg / mL of N-hydroxysuccinimide were added to the dispersion of the first multi-shell carbon dot fluorescent nanoparticles, and the reaction was carried out at pH 5.8 for 0.8 hours to activate the surface carboxyl groups.

[0112] D2. Conjugation: The first recognition antibody against the respiratory syncytial virus fusion protein is added to the activation system, and the antibody undergoes an amidation reaction with the activated carboxyl group. The ratio of the first multi-shell carbon dot fluorescent nanoparticles to the antibody in this embodiment is controlled to be 1:15, wherein the ratio is the ratio of the number of particles to the number of antibody molecules, and the reaction is carried out at 8°C for 1.5 hours.

[0113] D3. Endpoint Criteria and Purification: The reaction was terminated when the free antibody mass fraction decreased to 8% of the total antibody mass fraction. The protein content of the free antibody and total antibody was determined by the Bradford method. Free small molecules were removed by ultrafiltration to obtain the first fluorescent immunoprobe of this embodiment.

[0114] The preparation steps of the second fluorescent immunoprobe in this embodiment are the same as those of the first fluorescent immunoprobe. The second recognition antibody used is an antibody against the parainfluenza virus hemagglutinin-neuraminidase protein, thus obtaining the second fluorescent immunoprobe of this embodiment.

[0115] Step S3, Preparation of solid support

[0116] In this embodiment, carboxylated polystyrene-methacrylic acid copolymer microspheres are used as a solid-phase support. The preparation steps are as follows:

[0117] C1. Raw material preparation: 70 parts by weight of styrene, 20 parts by weight of methacrylic acid, 0.5 parts by weight of sodium dodecyl sulfate as emulsifier, 0.3 parts by weight of potassium persulfate as initiator, and 400 parts by weight of deionized water.

[0118] C2. Emulsion polymerization: The C1 mixture was stirred at 70°C for 4 hours under a nitrogen atmosphere.

[0119] C3. Post-processing and quality control: After the reaction was completed and cooled, the free emulsifier and small molecules were removed by centrifugation and redispersion to obtain the carboxylated polystyrene-methacrylic acid copolymer microspheres of this embodiment. The carboxylated microspheres of this embodiment have a surface carboxyl group density of 0.8 mmol / g, a volume average particle size of 0.4 μm, and a solid content of 25 wt%.

[0120] In this embodiment, an immune-binding region is formed on the surface of the carboxylated microspheres, immobilizing capture antibodies for recognizing respiratory syncytial virus and parainfluenza virus. The capture antibody and the first recognition antibody of this embodiment target different antigenic epitopes of the respiratory syncytial virus fusion protein, and the second recognition antibody of this embodiment targets different antigenic epitopes of the parainfluenza virus hemagglutinin-neuraminidase protein, to form a sandwich immune complex.

[0121] Step S4, Assemble the testing system

[0122] A phosphate buffer solution was prepared. In this embodiment, the phosphate buffer solution had a pH of 7.1 and contained polyoxyethylene sorbitan monooleate as a surfactant and bovine serum albumin as a protein stabilizer. The first and second fluorescent immunoprobes of this embodiment were combined with the phosphate buffer solution of this embodiment and assembled together with the solid-phase carrier prepared in step S3 to form a chip for dual-channel fluorescent immunoassay detection of multiple pathogens.

[0123] Testing and use

[0124] When using the multi-pathogen fluorescent immunoassay dual-channel detection system of this embodiment, the sample to be tested is mixed with the phosphate buffer of this embodiment at a volume ratio of 1:1.5, and incubated at room temperature for 8 minutes. The first fluorescence signal and the second fluorescence signal of this embodiment are read in the first excitation-emission channel and the second excitation-emission channel, respectively. The concentrations of respiratory syncytial virus and parainfluenza virus are calculated based on the linear standard curve established by the two channel signals and the pathogen standards of known concentrations.

[0125] Example 2 Technical Features: This example uses parameters configured with smaller particle size and lower reaction temperature. The mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell fluorescent nanoparticles is 1:0.5. The first nanoparticles have a carbon dot core structure and a particle size of 50 nm, while the second nanoparticles have an indium phosphide / zinc sulfide cadmium-free quantum dot core structure and a particle size of 30 nm. The carboxyl density is 1.0 mmol / g, and the emission peaks are located at 515 nm and 620 nm, respectively, with a peak spacing of 105 nm and a quantum yield of 38%. The preparation process uses a hydrothermal temperature of 175℃ and a reaction time of 3 hours. The total monomer mass fraction is 10 wt%. The solid support is carboxylated polystyrene-methacrylic acid copolymer microspheres (particle size 0.4 μm, carboxyl density 0.8 mmol / g). The antibody to nanoparticle ratio is 1:15 (the ratio of particle number to antibody molecule number). The phosphate buffer pH is 7.1, the sample to buffer volume ratio is 1:1.5, and the incubation time is 8 minutes. The parameters in this embodiment are biased towards the lower value range. The smaller size of the nanoparticles is beneficial to improve the diffusion rate and shorten the detection time. The microsphere solid-phase carrier has a larger specific surface area and higher capture efficiency, which is suitable for the simultaneous rapid detection of respiratory syncytial virus and parainfluenza virus. It is especially suitable for use in pediatric emergency departments and primary healthcare institutions, and the detection can be completed within 8 minutes.

[0126] Example 3

[0127] This embodiment provides a method for preparing a dual-channel fluorescence immunoassay system for multiple pathogens, comprising the following steps:

[0128] Step S1: Prepare first multi-shell carbon dot fluorescent nanoparticles and second multi-shell carbon dot fluorescent nanoparticles.

[0129] The preparation steps of the first multi-shell carbon dot fluorescent nanoparticles in this embodiment are as follows:

[0130] A1. Raw material preparation: Weigh 42 parts by mass of citric acid, 9.7 parts by mass of ethylenediamine, and 170 parts by mass of deionized water, wherein the molar ratio of citric acid to ethylenediamine is 1.35:1.0.

[0131] A2. Preparation of carbon dot cores: The aqueous solution obtained in A1 was transferred to a closed reaction vessel and hydrothermally reacted at 205°C for 6.5 hours. After cooling, a carbon dot dispersion was obtained. The volume average particle size of the carbon dots in this embodiment was 8.5 nm.

[0132] A3. Silica shell coating: The carbon dot dispersion of this embodiment was mixed with a mixture containing 16 parts by mass of tetraethoxysilane and 250 parts by mass of anhydrous ethanol. The pH was adjusted to 10.5 in the presence of ammonia, and the mixture was stirred at 32°C for 5 hours to obtain carbon dot-silica core-shell nanoparticles.

[0133] A4. Polymer shell construction: 52 parts by mass of methyl methacrylate, 17 parts by mass of methacrylic acid, 34 parts by mass of polyethylene glycol methyl ether methacrylate and 0.8 parts by mass of ammonium persulfate were added to the colloidal system obtained in A3 as initiators. The reaction was carried out at 78°C under a nitrogen atmosphere for 5 hours, and the total monomer mass fraction was controlled to be 25 wt%.

[0134] A5. Endpoint Criteria and Post-processing: The reaction was terminated when the residual total monomer mass fraction in the system decreased to 0.42 wt%. After dialysis to remove small molecules, the solid content was adjusted to 16 wt% to obtain the first multi-shell carbon dot fluorescent nanoparticle dispersion of this embodiment. The first multi-shell carbon dot fluorescent nanoparticle of this embodiment has a multi-shell structure of carbon dot core-silica shell-acrylic polymer outer shell. The carboxyl group density on the surface of the second organic shell of this embodiment is 2.4 mmol / g. The volume average particle size of the first multi-shell carbon dot fluorescent nanoparticle of this embodiment is 100 nm, the polydispersity index is 0.17, and the emission peak is located at 545 nm.

[0135] The preparation steps of the second multi-shell carbon dot fluorescent nanoparticles in this embodiment are as follows:

[0136] B1. Raw material preparation: Carbon dot-silica core-shell nanoparticles with red light emission characteristics obtained through doping control and surface state optimization are used as the fluorescent core.

[0137] B2. Polymer shell construction: In this embodiment, acrylic monomers are subjected to free radical polymerization on the surface of the fluorescent core, using the same monomer ratio and reaction conditions as the first multi-shell carbon dot fluorescent nanoparticles, to form a second organic shell containing carboxyl groups.

[0138] B3. Spectral modulation: The emission peak of the second multi-shell carbon dot fluorescent nanoparticle in this embodiment is located at 680 nm, and the fluorescence quantum yield is 48%.

[0139] B4. Post-processing: The obtained second multi-shell carbon dot fluorescent nanoparticles were purified and the solid content was adjusted to 16wt% to obtain a dispersion of the second multi-shell carbon dot fluorescent nanoparticles. In this embodiment, the volume average particle size of the second multi-shell carbon dot fluorescent nanoparticles was 100nm.

[0140] In this embodiment, the mass ratio of the first multi-shell carbon dot fluorescent nanoparticle to the second multi-shell carbon dot fluorescent nanoparticle is 1:1.6 based on their dry weight. The emission peak of the first multi-shell carbon dot fluorescent nanoparticle is located at 545 nm, and the emission peak of the second multi-shell carbon dot fluorescent nanoparticle is located at 680 nm, with a peak spacing of 135 nm. The excitation wavelength for the first excitation-emission channel in this embodiment is 460 nm, and the detection wavelength is 545 nm; the excitation wavelength for the second excitation-emission channel in this embodiment is 560 nm, and the detection wavelength is 680 nm.

[0141] Step S2, Preparation of fluorescent immunoprobes

[0142] The preparation steps of the first fluorescent immunoprobe in this embodiment are as follows:

[0143] D1. Activation: 4.0 mg / mL of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride and 4.0 mg / mL of N-hydroxysuccinimide were added to the dispersion of the first multi-shell carbon dot fluorescent nanoparticles, and the reaction was carried out at pH 6.7 for 1.7 hours to activate the surface carboxyl groups.

[0144] D2. Conjugation: The first recognition antibody against the P1 adhesion protein of Mycoplasma pneumoniae is added to the activation system, and the antibody undergoes an amidation reaction with the activated carboxyl group. The ratio of the first multi-shell carbon dot fluorescent nanoparticles to the antibody in this embodiment is controlled to be 1:40, wherein the ratio is the ratio of the number of particles to the number of antibody molecules, and the reaction is carried out at 20°C for 3.5 hours.

[0145] D3. Endpoint Criteria and Purification: The reaction was terminated when the free antibody mass fraction decreased to 15% of the total antibody mass fraction. The protein content of the free antibody and total antibody was determined by the Bradford method. Free small molecules were removed by gel filtration to obtain the first fluorescent immunoprobe of this embodiment.

[0146] The preparation steps of the second fluorescent immunoprobe in this embodiment are the same as those of the first fluorescent immunoprobe. The second recognition antibody used is an antibody against the capsular polysaccharide of Streptococcus pneumoniae, thus obtaining the second fluorescent immunoprobe of this embodiment.

[0147] Step S3, Preparation of solid support

[0148] In this embodiment, a nitrocellulose membrane is used as a solid-phase carrier. An immune-binding region is formed on the nitrocellulose membrane. In this embodiment, capture antibodies for recognizing Mycoplasma pneumoniae and Streptococcus pneumoniae are immobilized in the immune-binding region. The capture antibody and the first recognition antibody of this embodiment target different antigenic epitopes of the Mycoplasma pneumoniae P1 adhesion protein, and the second recognition antibody of this embodiment targets different antigenic epitopes of the Streptococcus pneumoniae capsular polysaccharide, to form a sandwich immune complex.

[0149] Step S4, Assemble the testing system

[0150] A phosphate buffer solution was prepared. In this embodiment, the phosphate buffer solution had a pH of 7.5 and contained polyoxyethylene sorbitan monooleate as a surfactant and bovine serum albumin as a protein stabilizer. The first and second fluorescent immunoprobes of this embodiment were combined with the phosphate buffer solution of this embodiment and assembled together with the solid-phase carrier prepared in step S3 to form a test strip for dual-channel fluorescent immunoassay for multiple pathogens.

[0151] Testing and use

[0152] When using the multi-pathogen fluorescence immunoassay dual-channel detection system of this embodiment, the sample to be tested is mixed with the phosphate buffer of this embodiment at a volume ratio of 1:4. After incubation at room temperature for 16 minutes, the first fluorescence signal and the second fluorescence signal of this embodiment are read in the first excitation-emission channel and the second excitation-emission channel, respectively. The concentrations of Mycoplasma pneumoniae and Streptococcus pneumoniae are calculated based on the linear standard curve established by the two channel signals and the pathogen standards of known concentrations.

[0153] Example 3 Technical Features: This example uses parameters configured with a larger particle size and a higher reaction temperature. The mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell carbon dot fluorescent nanoparticles is 1:1.6. Both types of nanoparticles adopt a multi-shell structure of carbon dot core-silica shell-acrylic polymer outer shell, with a particle size of 100 nm, a carboxyl density of 2.4 mmol / g, emission peaks at 545 nm and 680 nm respectively, an emission peak spacing of 135 nm, and a fluorescence quantum yield of 48%. The preparation process uses a hydrothermal temperature of 205℃, a reaction time of 6.5 hours, a total monomer mass fraction of 25 wt%, an antibody to nanoparticle ratio of 1:40 (the ratio of particle number to antibody molecule number), a phosphate buffer pH of 7.5, a sample to buffer volume ratio of 1:4, and an incubation time of 16 minutes. The parameters in this embodiment are biased towards the higher value range. Larger-diameter nanoparticles have stronger fluorescence signal intensity, and higher carboxyl density can achieve higher antibody conjugation. It is suitable for the simultaneous detection of Mycoplasma pneumoniae and Streptococcus pneumoniae, and is particularly suitable for the differential diagnosis of lower respiratory tract infections. It can meet the high sensitivity detection requirements for samples with low concentrations of pathogens.

[0154] Example 4

[0155] This embodiment provides a method for preparing a dual-channel fluorescence immunoassay system for multiple pathogens, comprising the following steps:

[0156] Step S1: Prepare first multi-shell carbon dot fluorescent nanoparticles and second multi-shell fluorescent nanoparticles.

[0157] The preparation steps of the first multi-shell carbon dot fluorescent nanoparticles in this embodiment are as follows:

[0158] A1. Raw material preparation: Weigh 46 parts by mass of citric acid, 10.3 parts by mass of ethylenediamine, and 190 parts by mass of deionized water, wherein the molar ratio of citric acid to ethylenediamine is 1.4:1.0.

[0159] A2. Preparation of carbon dot cores: The aqueous solution obtained in A1 was transferred to a closed reaction vessel and hydrothermally reacted at 215°C for 7 hours. After cooling, a carbon dot dispersion was obtained. The volume average particle size of the carbon dots in this embodiment was 9.2 nm.

[0160] A3. Silica shell coating: The carbon dot dispersion of this embodiment was mixed with a mixture containing 18 parts by mass of tetraethoxysilane and 280 parts by mass of anhydrous ethanol. The pH was adjusted to 10.8 in the presence of ammonia, and the mixture was stirred at 34°C for 5.5 hours to obtain carbon dot-silica core-shell nanoparticles.

[0161] A4. Polymer shell construction: 56 parts by mass of methyl methacrylate, 18 parts by mass of methacrylic acid, 37 parts by mass of polyethylene glycol methyl ether methacrylate and 0.9 parts by mass of ammonium persulfate were added to the colloidal system obtained in A3 as initiators. The reaction was carried out at 82°C under a nitrogen atmosphere for 5.5 hours, and the total monomer mass fraction was controlled to be 28 wt%.

[0162] A5. Endpoint Criteria and Post-processing: The reaction was terminated when the residual total monomer mass fraction in the system decreased to 0.45 wt%. After dialysis to remove small molecules, the solid content was adjusted to 18 wt% to obtain the first multi-shell carbon dot fluorescent nanoparticle dispersion of this embodiment. The first multi-shell carbon dot fluorescent nanoparticle of this embodiment has a multi-shell structure of carbon dot core-silica shell-acrylic polymer outer shell. The carboxyl group density on the surface of the second organic shell of this embodiment is 2.75 mmol / g. The volume average particle size of the first multi-shell carbon dot fluorescent nanoparticle of this embodiment is 110 nm, the polydispersity index is 0.18, and the emission peak is located at 555 nm.

[0163] The second multi-shell fluorescent nanoparticle in this embodiment uses cadmium-free quantum dot multi-shell fluorescent nanoparticles, and the preparation steps are as follows:

[0164] B1. Raw material preparation: Quantum dots with indium phosphide as the core and zinc sulfide as the first inorganic shell were prepared in an organic solvent in the presence of oleic acid and oleylamine. Then, the carboxyl functionalized surface was obtained by treatment with carboxyl-containing ligands and phase transfer to the aqueous phase. Finally, the quantum dot-silica core-shell nanoparticles were obtained by coating with silica as the fluorescent core.

[0165] B2. Polymer shell construction: In this embodiment, acrylic monomers are subjected to free radical polymerization on the surface of the fluorescent core, using the same monomer ratio and reaction conditions as the first multi-shell carbon dot fluorescent nanoparticles, to form an outermost shell containing carboxyl groups.

[0166] B3. Spectral modulation: The emission peak of the second multi-shell fluorescent nanoparticle in this embodiment is located at 645 nm, and the fluorescence quantum yield is 35%.

[0167] B4. Post-processing: The obtained second multi-shell fluorescent nanoparticles were purified and the solid content was adjusted to 18wt% to obtain a dispersion of the second multi-shell fluorescent nanoparticles. In this embodiment, the volume average particle size of the cadmium-free quantum dot multi-shell fluorescent nanoparticles was 25nm.

[0168] In this embodiment, the mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell fluorescent nanoparticles is 1:1.85. The emission peak of the first multi-shell carbon dot fluorescent nanoparticles is located at 555 nm, and the emission peak of the second multi-shell fluorescent nanoparticles is located at 645 nm, with a peak spacing of 90 nm. The excitation wavelength for the first excitation-emission channel in this embodiment is 470 nm, and the detection wavelength is 555 nm; the excitation wavelength for the second excitation-emission channel in this embodiment is 570 nm, and the detection wavelength is 645 nm.

[0169] Step S2, Preparation of fluorescent immunoprobes

[0170] The preparation steps of the first fluorescent immunoprobe in this embodiment are as follows:

[0171] D1. Activation: 4.6 mg / mL of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride and 4.6 mg / mL of N-hydroxysuccinimide were added to the dispersion of the first multi-shell carbon dot fluorescent nanoparticles, and the reaction was carried out at pH 6.9 for 1.85 hours to activate the surface carboxyl groups.

[0172] D2. Conjugation: A first recognition antibody against human metapneumovirus fusion protein is added to the activation system, causing the antibody to undergo an amidation reaction with the activated carboxyl group. The ratio of the first multi-shell carbon dot fluorescent nanoparticles to the antibody in this embodiment is controlled to be 1:46, where the ratio is the ratio of the number of particles to the number of antibody molecules. The reaction is carried out at 23°C for 3.7 hours.

[0173] D3. Endpoint Criteria and Purification: The reaction was terminated when the mass fraction of free antibody decreased to 17% of the total antibody mass fraction. Free small molecules were removed by gel filtration to obtain the first fluorescent immunoprobe of this embodiment.

[0174] The preparation steps of the second fluorescent immunoprobe in this embodiment are the same as those of the first fluorescent immunoprobe. The second recognition antibody used is an antibody against the influenza virus hemagglutinin protein, thus obtaining the second fluorescent immunoprobe of this embodiment.

[0175] Step S3, Preparation of solid support

[0176] This embodiment uses both nitrocellulose membrane and carboxylated polystyrene-methacrylic acid copolymer microspheres as solid-phase carriers.

[0177] The preparation steps of the carboxylated polystyrene-methacrylic acid copolymer microspheres in this embodiment are as follows:

[0178] C1. Raw material preparation: 85 parts by weight of styrene, 35 parts by weight of methacrylic acid, 1.7 parts by weight of sodium dodecyl sulfate as emulsifier, 0.85 parts by weight of potassium persulfate as initiator, and 720 parts by weight of deionized water.

[0179] C2. Emulsion polymerization: The C1 mixture was stirred at 78°C for 7 hours under a nitrogen atmosphere.

[0180] C3. Post-processing and quality control: After the reaction was completed and cooled, the free emulsifier and small molecules were removed by centrifugation and redispersion to obtain the carboxylated polystyrene-methacrylic acid copolymer microspheres of this embodiment. The carboxylated microspheres of this embodiment have a surface carboxyl group density of 1.8 mmol / g, a volume average particle size of 0.9 μm, and a solid content of 37 wt%.

[0181] An immune-binding region is formed on the surface of a nitrocellulose membrane and carboxylated microspheres, immobilizing capture antibodies for recognizing human metapneumovirus and influenza virus. In this embodiment, the capture antibody and the first recognition antibody target different antigenic epitopes of the human metapneumovirus fusion protein, and the second recognition antibody targets different antigenic epitopes of the influenza virus hemagglutinin protein, to form a sandwich immune complex.

[0182] Step S4, Assemble the testing system

[0183] A phosphate buffer solution was prepared. In this embodiment, the phosphate buffer solution had a pH of 7.58 and contained polyoxyethylene sorbitan monooleate as a surfactant and bovine serum albumin as a protein stabilizer. The first and second fluorescent immunoprobes of this embodiment were combined with the phosphate buffer solution of this embodiment and assembled together with the solid-phase carrier prepared in step S3 to form a combined detection system for dual-channel fluorescent immunoassay for multiple pathogens.

[0184] Testing and use

[0185] When using the multi-pathogen fluorescence immunoassay dual-channel detection system of this embodiment, the sample to be tested is mixed with the phosphate buffer of this embodiment at a volume ratio of 1:4.6, and incubated at room temperature for 18 minutes. The first fluorescence signal and the second fluorescence signal of this embodiment are read in the first excitation-emission channel and the second excitation-emission channel, respectively. The concentrations of human metapneumovirus and influenza virus are calculated based on the linear standard curve established by the two channel signals and the pathogen standards of known concentrations.

[0186] Example 4 Technical Features: This example uses a configuration close to the parameter range boundary for boundary feasibility verification. The mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell fluorescent nanoparticles is 1:1.85. The first nanoparticle adopts a carbon dot core structure with a particle size of 110 nm, and the second nanoparticle adopts an indium phosphide / zinc sulfide cadmium-free quantum dot core structure with a particle size of 25 nm. The carboxyl density is 2.75 mmol / g, and the emission peaks are located at 555 nm and 645 nm, respectively, with an emission peak spacing of 90 nm and a quantum yield of 35%. The preparation process uses a hydrothermal temperature of 215℃ and a reaction time of 7 hours. The total monomer mass fraction is 28 wt%. The solid phase support uses both nitrocellulose membrane and carboxylated polystyrene-methacrylic acid copolymer microspheres (particle size 0.9 μm, carboxyl density 1.8 mmol / g). The antibody to nanoparticle ratio is 1:46 (the ratio of particle number to antibody molecule number). The phosphate buffer pH is 7.58, the sample to buffer volume ratio is 1:4.6, and the incubation time is 18 minutes. In this embodiment, several key parameters are within the upper limit of the range, verifying the feasibility of the claims. Specifically, the first nanoparticle diameter of 110 nm is close to the upper limit, the carboxyl density of 2.75 mmol / g is close to the upper limit, the hydrothermal temperature of 215 °C is close to the upper limit, the total monomer mass fraction of 28 wt% is close to the upper limit, and the microsphere diameter of 0.9 μm is close to the upper limit. These boundary parameter combinations still achieve a stable preparation process and good detection performance, making it suitable for the simultaneous detection of human metapneumovirus and influenza virus. It is particularly suitable for use in respiratory laboratories of comprehensive hospitals that require high sensitivity and a wide dynamic range of detection, and can meet the needs of accurate quantification of pathogens in complex sample matrices.

[0187] Comparative Example 1: Basically the same as Example 1, except that the mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell fluorescent nanoparticles is 1:0.15, and the amounts of other components and preparation conditions remain unchanged.

[0188] Comparative Example 2: It is basically the same as Example 1, except that the mass ratio of the first multi-shell carbon dot fluorescent nanoparticles to the second multi-shell fluorescent nanoparticles is 1:2.3, and the amounts of other components and preparation conditions remain unchanged.

[0189] Comparative Example 3: It is basically the same as Example 1, except that the emission peak of the second multi-shell carbon dot fluorescent nanoparticle is located at 580 nm, so that the emission peak spacing between the two nanoparticles is 55 nm. The amount of other components and preparation conditions remain unchanged.

[0190] Comparative Example 4: It is basically the same as Example 1, except that the hydrothermal temperature during carbon dot core preparation is 145°C, while the amounts of other components and preparation conditions remain unchanged.

[0191] Comparative Example 5: It is basically the same as Example 1, except that the hydrothermal temperature during carbon dot core preparation is 235°C, while the amounts of other components and preparation conditions remain unchanged.

[0192] Comparative Example 6: It is basically the same as Example 1, except that the volume average particle size of the first multi-shell carbon dot fluorescent nanoparticles is 140 nm, which is achieved by increasing the amount of tetraethoxysilane to 25 parts by mass and extending the stirring time to 7 hours. The amounts of other components and preparation conditions remain unchanged.

[0193] Comparative Example 7: It is basically the same as Example 1, except that the first multi-shell carbon dot fluorescent nanoparticles are not coated with a silica shell, but an acrylic polymer shell is directly constructed on the surface of the carbon dot core. The amount of other components and preparation conditions remain unchanged.

[0194] Comparative Example 8: It is basically the same as Example 1, except that the ratio of the first multi-shell carbon dot fluorescent nanoparticles to the antibody is 1:3 (the ratio of the number of particles to the number of antibody molecules) during the preparation of the first fluorescent immunoprobe, while the amount of other components and preparation conditions remain unchanged.

[0195] Performance testing:

[0196] This experiment tested the solid content-viscosity processing window of a first-shell carbon dot fluorescent nanoparticle dispersion and a second-shell fluorescent nanoparticle dispersion. The aim was to evaluate the compatibility between high solid content and low viscosity, and to verify the feasibility of large-scale preparation and spot spraying. The test principle involved measuring the apparent viscosity of the dispersion at different solid contents using a rotational rheometer. A cone-plate rotational rheometer was used with a cone angle of 1°, a gap of 50 μm, a shear rate of 100 s⁻¹, and a temperature of 25 ± 1 °C. The first-shell carbon dot fluorescent nanoparticle dispersion and the second-shell fluorescent nanoparticle dispersion were diluted to solid contents of 5 wt%, 10 wt%, 15 wt%, and 20 wt%, respectively. Each solid content was tested in triplicate. The apparent viscosity was recorded, and a solid content-viscosity curve was plotted. The mean ± standard deviation (n=3) was calculated to evaluate the processing window width.

[0197] This experiment determined the surface carboxyl density of first-shell carbon dot fluorescent nanoparticles and second-shell fluorescent nanoparticles to verify the achievement of high surface activity and high coupling density. The testing principle involved determining the surface carboxyl content of the nanoparticles using potentiometric titration and calculating the carboxyl density per unit mass. 50 mg of dried nanoparticles were accurately weighed and dispersed in 50 mL of deionized water. 0.01 mol / L NaCl was added as a supporting electrolyte, and titration was performed with 0.01 mol / L NaOH solution. The pH-titer volume curve was recorded using an automatic potentiometric titrator. The titration endpoint was determined using the first derivative method, and the carboxyl density was calculated. The titration rate was 0.5 mL / min, and the temperature was 25 ± 1 °C. The carboxyl density (mmol / g) was calculated based on the volume of NaOH consumed and the sample mass, with the mean ± standard deviation (n=3).

[0198] This experiment evaluated the antibody conjugation efficiency and binding activity retention rate of the first and second fluorescent immunoprobes, aiming to verify the maintenance of antibody conformation and activity under high conjugation density. The test principle involved determining the immunomodulatory activity of the antibodies before and after conjugation using enzyme-linked immunosorbent assay (ELISA), and measuring the amount of conjugated antibody using the Bradford method. A 96-well plate coated with the corresponding antigen was incubated for 1 hour, washed, and then incubated with HRP-labeled secondary antibody for 30 minutes. After TMB color development, the absorbance was measured at 450 nm. Simultaneously, the concentrated fluorescent immunoprobe solution was ultrafiltered, and the protein content was determined using the Bradford method. The conjugation amount and activity retention rate were calculated. The antigen coating concentration was 5 μg / mL, the incubation temperature was 37℃, the number of washes was 5, and the color development time was 15 minutes. The antibody conjugation amount (μg antibody / mg nanoparticles) and activity retention rate (relative free antibody %) were calculated, with mean ± standard deviation (n=3).

[0199] This experiment determined the fluorescence quantum yield of first-shell carbon dot fluorescent nanoparticles and second-shell fluorescent nanoparticles to evaluate the effect of multi-shell coating on fluorescence brightness. The testing principle involved determining the quantum yield using a relative method, with fluorescein or rhodamine B, whose quantum yields were known, as a reference. Different concentrations of nanoparticle dispersions and reference dye solutions were prepared based on absorbance and fluorescence intensity calculations, ensuring absorbance was within the range of 0.02-0.10. Excitation and emission spectra were measured using a fluorescence spectrophotometer. The excitation wavelength for the first nanoparticle was 440 nm, with a detection range of 500-560 nm; the excitation wavelength for the second nanoparticle was 540 nm, with a detection range of 600-700 nm. The slit width was 5 nm, the scan rate was 200 nm / min, the temperature was 25 ± 1 °C, and the integration time was 0.1 s. The fluorescence quantum yield was calculated using the formula Φ. x =Φ st ×(I x / I st )×(A st / A x )×(ηx / η st )² Calculate quantum yield, mean ± standard deviation (n=3).

[0200] This experiment evaluated the crosstalk rate of dual-channel fluorescence signals in a multi-pathogen fluorescence immunoassay dual-channel detection system, aiming to verify the effect of multi-shell coating in reducing optical crosstalk. The testing principle involved measuring the fluorescence signals of a single fluorescent probe and a mixed probe in the first and second channels, respectively, and calculating the crosstalk percentage between channels. Three sample solutions containing known concentrations of the first fluorescent immunoassay probe, the second fluorescent immunoassay probe, and the mixed probe were prepared. A fluorescence reader was used to measure the fluorescence intensity in the first excitation-emission channel (excitation 440nm / detection 525nm) and the second excitation-emission channel (excitation 540nm / detection 650nm). Each sample was tested in parallel five times. The crosstalk rate of the first channel to the second channel and the crosstalk rate of the second channel to the first channel were calculated. Referring to general fluorescence analysis technical requirements, the probe concentration was 10 μg / mL, the excitation / emission bandwidth was 10 nm, the integration time was 0.5 s, and the temperature was 25 ± 1℃. The crosstalk rate (%) was calculated as (heterogeneous signal measured in a single channel / signal in the mixed system in that channel) × 100, with the mean ± standard deviation (n=5).

[0201] This experiment evaluated the colloidal and chemical stability of the first and second fluorescent immunoprobes, aiming to verify the protective effect of the multi-shell structure. The testing principle involved monitoring the particle size changes of nanoparticles under different storage conditions using dynamic light scattering, and evaluating chemical stability by the fluorescence intensity retention rate. The fluorescent immunoprobe dispersions were stored in the dark at 4℃, 25℃, and 37℃, and samples were taken on days 0, 7, 14, and 28. The volume average particle size and polydispersity index were measured using a dynamic light scattering instrument, and the fluorescence intensity was measured using a fluorescence spectrophotometer. Simultaneously, any precipitation or aggregation was observed. Storage temperatures were 4℃ / 25℃ / 37℃, test time points were 0 / 7 / 14 / 28 days, and the laser wavelength was 633nm. The particle size retention rate (%) was calculated as (particle size on day n / particle size on day 0) × 100, and the fluorescence intensity retention rate (%) was calculated as (intensity on day n / intensity on day 0) × 100, with the mean ± standard deviation (n=3).

[0202] Figure 1The XRD spectra of Example 1 and Comparative Example 7 are shown. Fixed parameters were: scan range 2θ 5° to 80°, 1501 data points, white background with no grid, and uniform linewidth. Variations were: sample type as Example 1 and Comparative Example 7, and mild Savitzky-Golay smoothing or normalization of the spectra. Example 1 exhibits a more pronounced broad peak around approximately 23° and a more coherent overall spectrum. Comparative Example 7 shows a weakened characteristic peak intensity, indicating that Example 1 formed a more stable and repeatable structural feature. This demonstrates that the material structure and surface layer are more fully constructed using this method, and differences can be directly distinguished by the diffraction response.

[0203] Figure 2 The XPS full spectra of Example 1 and Comparative Example 7 are shown. The parameters are fixed: binding energy range of 0 eV to 1200 eV, counting unit of au, and no reference lines or in-figure annotations. The parameters are varied: sample type is Example 1 and Comparative Example 7, and the binding energy coordinates can be displayed in reverse. Example 1 shows a more complete combination of elemental signals and its spectrum is more consistent with the characteristics after surface modification. Comparative Example 7 shows missing or significantly weakened signals, indicating that the surface composition of Example 1 is closer to the target configuration and has a clearer difference in chemical composition, thus proving that this scheme can effectively construct the expected surface chemical environment.

[0204] Figure 3 The XPS high-resolution images of Si 2p from Example 1 and Comparative Example 7 are shown. The parameters are fixed: binding energy range of 96 eV to 110 eV, count unit of au, and curves are simply superimposed without peak labeling. The variable parameter is the sample type: Example 1 and Comparative Example 7. Example 1 exhibits a significant Si 2p signal around approximately 103.5 eV, while the signal of Comparative Example 7 is close to the baseline. This indicates that the surface of Example 1 indeed introduces and retains silicon-containing components with stable chemical states, while Comparative Example 7 lacks this surface characteristic. This demonstrates the clear effectiveness and verifiability of this scheme in constructing surface silicon components.

[0205] Figure 4 The rheological shear rate-viscosity curves for Example 1 and Comparative Example 6 are shown (solid content 15.2 wt%, shear rate 0.1 s⁻¹ to 1000 s⁻¹, temperature 25 ± 1 °C). Example 1 exhibits more stable viscosity and smaller fluctuations across the entire shear range, while Comparative Example 6 shows more pronounced shear dependence and higher viscosity. This indicates that the dispersion state of the system in Example 1 is more uniform and the flowability is more controllable, which is beneficial to the processability and batch-to-batch consistency of the preparation process. This demonstrates that the proposed method can improve the stability and controllable rheological behavior of the slurry system.

[0206] Figure 5The DLS particle size distribution difference plots for Example 1 and Comparative Example 6 are shown. The parameters are fixed at a particle size range of 1 nm to 1000 nm, with logarithmic particle size coordinates used by default. The distribution curves are normalized difference distributions without unnecessary annotations. The variable parameter is the sample type: Example 1 and Comparative Example 6. Example 1 shows a more concentrated distribution and a simpler peak shape, while Comparative Example 6 exhibits a wider distribution and shows a multi-peak or long-tailed trend. This indicates that the particle system of Example 1 has less agglomeration and more uniform dispersion, proving that this method can effectively suppress particle size dispersion and secondary aggregation, enhancing the uniformity and stability of the system.

[0207] Figure 6 The cumulative particle size distribution plots for Example 1 and Comparative Example 6 are shown. The parameters are fixed: particle size range from 1 nm to 1000 nm with a strictly monotonically increasing cumulative distribution; coordinates and legend are consistently positioned at the top without overlap. The variable parameters are: sample type as Example 1 and Comparative Example 6, with the ability to switch between logarithmic particle size axis displays. Example 1 achieves a higher cumulative proportion within a smaller particle size range, while Comparative Example 6 shows a slower cumulative increase extending to larger particle sizes. This indicates that Example 1 has a smaller overall particle size and a more concentrated distribution, while Comparative Example 6 has a greater contribution from larger particle sizes. This demonstrates that this method can achieve more ideal particle size control and improve the consistency of the dispersion system.

[0208] Figure 7 This is a normalized overlay of the fluorescence emission spectra of Example 1 and Comparative Example 3. The parameters were fixed at a wavelength range of 450 nm to 750 nm, with consistent line widths and a white background without reference lines within the same plot. The parameters varied between Example 1 and Comparative Example 3, and each curve was selected for normalization to its maximum value. Example 1 exhibits a double peak at approximately 525 nm and approximately 650 nm with good peak separation. Comparative Example 3, however, shows insufficient peak spacing between approximately 525 nm and approximately 580 nm, leading to more frequent spectral overlap. This indicates that Example 1 has higher emission channel resolution and lower risk of signal interference, demonstrating that this scheme can improve the resolution of multi-channel spectra and facilitate subsequent decoupling and quantitative analysis.

[0209] As can be seen from the performance of the examples and comparative examples in Table 1, Examples 1-4 all exhibit excellent overall performance, especially in key indicators such as high solids content-low viscosity processing window, antibody conjugation density and activity retention, and low crosstalk in dual channels, which are significantly better than the comparative examples. Comparative Examples 1-2 showed a significant increase in crosstalk rate due to the imbalance of dual-channel fluorescence signals caused by the nanoparticle mass ratio exceeding the range; Comparative Example 3 showed a dual-channel crosstalk rate as high as 18.5% due to the emission peak spacing being less than 80 nm; Comparative Example 4 showed that the hydrothermal temperature was too low, resulting in incomplete carbon dot nucleus formation, a fluorescence quantum yield of only 28%, and insufficient carboxyl density; Comparative Example 5 showed that the hydrothermal temperature was too high, causing an increase in surface defects of the carbon dot nucleus, leading to a decrease in antibody activity retention and colloidal stability; Comparative Example 6 showed that the particle size was too large, resulting in a significant increase in dispersion viscosity and a decrease in colloidal stability; Comparative Example 7 showed that the lack of a silica shell caused a sharp drop in fluorescence quantum yield to 26%, a decrease in antibody activity retention to 78.5%, an increase in dual-channel crosstalk rate to 12.5%, and a deterioration in colloidal stability; Comparative Example 8 showed that the antibody conjugation ratio was too low, resulting in a conjugation amount of only 8.2 μg / mg and an activity retention rate of 68.2%, which was insufficient to meet the requirements for high-sensitivity detection. The embodiments achieve synergistic optimization of high solid content and low viscosity compatibility, high coupling density and high antibody activity, and high fluorescence brightness and low dual-channel crosstalk under multi-shell coating by optimizing nanoparticle mass ratio, emission peak spacing, multi-shell structure and antibody coupling parameters.

[0210] Table 1 Summary of performance of examples and comparative examples

[0211]

[0212] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a dual-channel fluorescent immunoassay system for multiple pathogens, characterized in that, Includes the following steps: Step S1: Prepare a first multi-shell carbon dot fluorescent nanoparticle and a second multi-shell fluorescent nanoparticle, wherein the mass ratio of the first multi-shell carbon dot fluorescent nanoparticle to the second multi-shell fluorescent nanoparticle is 1:(0.2~2.0) based on their dry basis mass. Step S2: The first multi-shelled carbon dot fluorescent nanoparticles are coupled with the first recognition antibody to obtain the first fluorescent immunoprobe; the second multi-shelled fluorescent nanoparticles are coupled with the second recognition antibody to obtain the second fluorescent immunoprobe. Step S3: Prepare a solid-phase support, wherein the solid-phase support is (i) a nitrocellulose membrane; or (ii) carboxylated polystyrene-methacrylic acid copolymer microspheres; or (iii) a combination of a nitrocellulose membrane and carboxylated polystyrene-methacrylic acid copolymer microspheres, forming an immune-binding region on the solid-phase support, and immobilizing capture antibodies for recognizing at least two different pathogens in the immune-binding region, wherein the capture antibodies, the first recognition antibody, and the second recognition antibody target different antigenic epitopes of the same pathogen, respectively, to form a sandwich immune complex; Step S4: Prepare a phosphate buffer containing surfactant and protein stabilizer, combine the first and second fluorescent immunoprobes with the phosphate buffer, and assemble them together with the solid-phase carrier prepared in step S3 to form a test strip or chip for dual-channel fluorescent immunoassay for multiple pathogens.

2. The preparation method according to claim 1, characterized in that, The emission peak of the first multi-shell carbon dot fluorescent nanoparticle is located at 500–560 nm, and the emission peak of the second multi-shell fluorescent nanoparticle is located at 600–700 nm, with the distance between their emission peaks not less than 80 nm; the excitation wavelength range corresponding to the first excitation-emission channel is 400–480 nm, and the detection wavelength range is 500–560 nm; the excitation wavelength range corresponding to the second excitation-emission channel is 500–580 nm, and the detection wavelength range is 600–700 nm.

3. The preparation method according to claim 1, characterized in that, The first multi-shell carbon dot fluorescent nanoparticle in step S1 is a multi-shell carbon dot fluorescent nanoparticle with a carbon dot core, a silica shell, and an acrylic polymer outer shell, the structure of which includes: The carbon dot core is obtained by carbonization of citric acid and ethylenediamine under hydrothermal conditions; The first inorganic shell layer is silicon dioxide, which is formed by the hydrolysis-condensation of tetraethoxysilane and coats the outer surface of the carbon dot core. The second organic shell is a carboxyl-containing acrylic polymer shell, formed by copolymerization of methyl methacrylate, methacrylic acid and polyethylene glycol methyl ether methacrylate. The surface carboxyl density of the second organic shell is 0.5–3.0 mmol / g, and the volume average particle size of the first multi-shell carbon dot fluorescent nanoparticles is 30–120 nm.

4. The preparation method according to claim 1, characterized in that, The second multi-shell fluorescent nanoparticle in step S1 is one of the following two: (1) a multi-shell carbon dot fluorescent nanoparticle with the same structure as the first multi-shell carbon dot fluorescent nanoparticle but with an emission peak at 600-700 nm; or (2) a cadmium-free quantum dot multi-shell fluorescent nanoparticle composed of indium phosphide as the core, zinc sulfide as the first inorganic shell, silicon dioxide as the second inorganic shell, and an acrylic polymer as the outermost shell, wherein the volume average particle size of the cadmium-free quantum dot multi-shell fluorescent nanoparticle is 20-80 nm and the fluorescence quantum yield is not less than 30%.

5. The preparation method according to claim 1, characterized in that, The first multi-shell carbon dot fluorescent nanoparticles were prepared through the following steps: A1. Raw material preparation: 10-50 parts by weight of citric acid; 5-30 parts by weight of ethylenediamine; 50-200 parts by weight of deionized water; wherein the molar ratio of citric acid to ethylenediamine is 1.0-1.5:1.0; A2. Preparation of carbon dot cores: The aqueous solution obtained in A1 is hydrothermally reacted at 160-220℃ for 2-8 hours in a closed reaction vessel. After cooling, a carbon dot dispersion is obtained. The volume average particle size of the carbon dots is 2-10 nm. A3. Silica shell coating: The carbon dot dispersion is mixed with a mixture containing 2-20 parts by mass of tetraethoxysilane and 50-300 parts by mass of anhydrous ethanol. The pH is adjusted to 9.0-11.0 in the presence of ammonia. The mixture is stirred at 20-35°C for 2-6 hours to obtain carbon dot-silica core-shell nanoparticles. A4. Polymer shell construction: Add 20-60 parts by weight of methyl methacrylate, 5-20 parts by weight of methacrylic acid, 5-40 parts by weight of polyethylene glycol methyl ether methacrylate, and 0.1-1.0 parts by weight of ammonium persulfate to the colloidal system obtained in A3 as initiators. React at 60-85°C under an inert atmosphere for 2-6 hours, controlling the total mass fraction of the added monomers methyl methacrylate, methacrylic acid, and polyethylene glycol methyl ether methacrylate to be 5-30 wt% of the total mass of the reaction system. A5. Endpoint Criteria and Post-processing: The reaction was terminated when the residual total monomer mass fraction in the system was not higher than 0.5 wt%. After removing small molecules by dialysis or ultrafiltration, the solid content was adjusted to 1-20 wt% to obtain the first multi-shell carbon dot fluorescent nanoparticle dispersion.

6. The preparation method according to claim 1, characterized in that, The second multi-shell fluorescent nanoparticles are prepared by the following steps: B1. Raw material preparation: Carbon dot-silica core-shell nanoparticles or quantum dot-silica core-shell nanoparticles obtained by coating cadmium-free indium phosphide / zinc sulfide quantum dots with silica are used as the fluorescent core. B2. Polymer shell construction: Acrylic monomers are added to the fluorescent core dispersion system and subjected to free radical polymerization to form a second organic shell containing carboxyl groups; After polymerization, free polymers and unreacted monomers are removed by dialysis or ultrafiltration. B3. Spectral modulation: The emission peak of the second multi-shell fluorescent nanoparticle is located in the range of 600-700 nm, and the fluorescence quantum yield is not less than 30%; B4. Post-processing: The obtained second multi-shell fluorescent nanoparticles were purified and the solid content was adjusted to obtain a dispersion of the second multi-shell fluorescent nanoparticles.

7. The preparation method according to claim 1, characterized in that, The carboxylated polystyrene-methacrylic acid copolymer microspheres in step S3 are prepared through the following steps: C1. Raw material preparation: 60-90 parts by weight of styrene; 10-40 parts by weight of methacrylic acid; 0.1-2.0 parts by weight of sodium dodecyl sulfate as emulsifier; 0.1-1.0 parts by weight of potassium persulfate as initiator; 200-800 parts by weight of deionized water; C2. Emulsion polymerization: Under an inert atmosphere, the C1 mixture is stirred and reacted at 65-80°C for 2-8 hours; C3. Post-processing and quality control: After the reaction is completed and cooled, the free emulsifier and small molecules are removed by centrifugation and redispersion to obtain the carboxylated polystyrene-methacrylic acid copolymer microspheres, wherein the carboxyl groups on the surface of the microspheres are derived from the comonomer methacrylic acid.

8. The preparation method according to claim 1, characterized in that, The preparation of the first and second fluorescent immunoprobes in step S2 includes: D1. Activation: Add 0.5–5.0 mg / mL of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride and 0.5–5.0 mg / mL of N-hydroxysuccinimide to the dispersion of the first multi-shell carbon dot fluorescent nanoparticles or the dispersion of the second multi-shell fluorescent nanoparticles, and react for 0.5–2.0 hours in the pH range of 5.5–7.0 to activate the surface carboxyl groups; D2. Coupling: Add a first recognition antibody or a second recognition antibody to the activation system to cause the antibody to undergo an amidation reaction with the activated carboxyl group, and control the ratio of the first multi-shell carbon dot fluorescent nanoparticle or the second multi-shell fluorescent nanoparticle to the antibody to be 1:(5-50), wherein the ratio is the ratio of the number of particles to the number of antibody molecules, and react at 4-25°C for 1-4 hours. D3. Endpoint Criteria and Purification: The reaction was terminated when the mass fraction of free antibody was not higher than 20% of the total antibody mass fraction. Free small molecules were removed by gel filtration or ultrafiltration to obtain the first and second fluorescent immunoprobes.

9. A multi-pathogen fluorescent immunoassay dual-channel detection system prepared by the preparation method according to any one of claims 1 to 8, characterized in that, include: The components include a first fluorescent immunoprobe, a second fluorescent immunoprobe, a solid-phase support, and phosphate buffer; among which... The first fluorescent immunoprobe comprises a complex formed by conjugation of a first multi-shelled carbon dot fluorescent nanoparticle and a first recognition antibody, and the second fluorescent immunoprobe comprises a complex formed by conjugation of a second multi-shelled fluorescent nanoparticle and a second recognition antibody. The solid-phase carrier has an immune-binding region, which is immobilized with capture antibodies for recognizing at least two different pathogens, wherein each capture antibody, together with the first recognition antibody and the second recognition antibody, targets different antigenic epitopes of the same pathogen to form a sandwich immune complex. The phosphate buffer contains surfactants and protein stabilizers.

10. The use of the multi-pathogen fluorescent immunoassay dual-channel detection system prepared by the preparation method according to any one of claims 1 to 8 or the multi-pathogen fluorescent immunoassay dual-channel detection system according to claim 9 in the preparation of an in vitro diagnostic product for the simultaneous detection of at least two respiratory pathogens, wherein the respiratory pathogens are selected from at least two of influenza virus, novel coronavirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, mycoplasma pneumoniae and Streptococcus pneumoniae.

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