A respiratory pathogen detection kit based on mask sampling and an in vitro detection method thereof

Abstract

The application relates to a respiratory pathogen detection kit based on mask sampling and an in-vitro detection method thereof, and relates to the technical field of detection kits. The kit comprises a disposable sampling mask, a reaction box inner cover, a reaction box outer cover, a reaction bottom box and a reaction solution; the disposable sampling mask is provided with a separable to-be-detected area to capture exhaled aerosols, the reaction box inner cover is provided with a microcavity preloaded with a detection compound, the compound is released to the to-be-detected area through wetting and pressing, and a color development signal is generated by using specific binding of antigens and antibodies to cause nanometer gold particles to gather. The respiratory pathogen detection kit based on mask sampling and the in-vitro detection method thereof provided by the application can realize the integration of non-invasive sampling and in-situ detection, and improve the sampling compliance of groups such as children and the reliability of detection results.

Description

Technical Field

[0001] This application belongs to the field of detection kit technology, and in particular relates to a respiratory pathogen detection kit based on mask sampling and its in vitro detection method. Background Technology

[0002] Community-acquired pneumonia is a common cause of hospitalization in children. Early identification of the pathogen is crucial for targeted treatment and appropriate medication in children with respiratory infections. Current respiratory pathogen testing typically relies on samples such as nasal swabs, throat swabs, sputum, or blood. However, young children are less cooperative during sampling, and nasal and throat swabs can cause discomfort, leading to insufficient sample collection or false negative results. Furthermore, during peak respiratory infection seasons, children visiting medical facilities for testing may increase the risk of cross-infection.

[0003] Gold nanoparticles possess excellent visual colorimetric properties. When gold nanoparticles transition from a dispersed to an aggregated state, their local surface plasmon resonance absorption peaks change, resulting in a color change. Based on this property, a pathogen detection system that can be visually interpreted without complex instruments can be constructed.

[0004] However, existing testing protocols typically still require steps such as individual sampling, sample transfer, and reagent mixing, which are not user-friendly for young children and home settings. Summary of the Invention

[0005] In view of this, this application provides a respiratory pathogen detection kit based on mask sampling and its in vitro detection method to solve the technical problem that existing sampling and detection schemes cannot simultaneously achieve non-invasive sampling and timely detection.

[0006] In a first aspect, this application provides a respiratory pathogen detection kit based on mask sampling, comprising: A disposable sampling mask, the disposable sampling mask including a separable test area for capturing the wearer's exhaled aerosol, the test area being provided with multiple detection zones and positioning marks corresponding one-to-one with the multiple detection zones; The reaction box inner cover includes a carrier layer and a plurality of microcavities disposed on the carrier layer. The plurality of microcavities are disposed corresponding to a plurality of detection areas, and each microcavity is pre-filled with a detection complex. The outer cover of the reaction chamber is used to apply pressure to the inner cover of the reaction chamber when the inner cover of the reaction chamber is in contact with the area to be tested. The reaction chamber is used to support the area to be tested and contain the reaction solution. The reaction solution includes at least a first reaction solution for wetting the test area and a second reaction solution for rinsing the test area; The detection complex comprises gold nanoparticles and recognition molecules attached to the surface of the gold nanoparticles. The recognition molecules can specifically bind to the target respiratory pathogen antigen. When the test area is wetted by the first reaction solution, and the microcavity of the inner cover of the reaction box is in contact with and pressurized against the detection area of ​​the test area, the detection complex is released from the microcavity to the corresponding detection area. If the target respiratory pathogen antigen is present in the detection area, the detection complex specifically binds to the target respiratory pathogen antigen and causes the gold nanoparticles to aggregate, resulting in a visual color signal in the corresponding detection area.

[0007] In some embodiments, the disposable sampling mask includes: The outer layer is used to prevent external liquids from seeping into the inside of the mask; The inner layer is used to absorb the moisture exhaled by the wearer; The intermediate layer is sandwiched between the outer layer and the inner layer. The intermediate layer is a meltblown fabric layer. The area to be tested is located in the middle of the meltblown fabric layer and corresponds to the wearer's mouth and nose area when worn.

[0008] In some embodiments, the area to be tested is a circular area, and the outer periphery of the circular area is provided with scratches, dotted lines or easy-tear lines to separate it from the meltblown fabric layer; at least two detection zones are provided within the circular area.

[0009] In some embodiments, the diameter of the circular region is 3-8 cm, preferably 5 cm. The circular area is provided with 4 circular detection zones, which are evenly distributed within the circular area and are respectively provided with positioning marks numbered 1 to 4.

[0010] In some embodiments, the inner cover of the reaction box includes a hydrophilic porous carrier layer and hydrophobic barrier layers disposed on both sides of the carrier layer, wherein a plurality of through holes are provided on one side of the hydrophobic barrier layer, and the through holes together with the carrier layer and the hydrophobic barrier layer on the other side form the microcavity.

[0011] In some embodiments, the carrier layer is a filter paper layer, and the hydrophobic barrier layer is a wax paper layer; the plurality of through holes correspond one-to-one with the plurality of detection areas on the area to be tested.

[0012] In some embodiments, polystyrene sulfonate is pre-placed in the microcavity, the detection complex is dried and fixed in the microcavity, and can be released to the test area under the action of the first reaction solution.

[0013] In some embodiments, the recognition molecule is an antibody, and the detection complex is an antibody-gold nanocomplex; The antibody-gold nanocomplexes in different microcavities correspond to different target respiratory pathogen antigens, and each gold nanoparticle is linked to only one specific antibody.

[0014] In some embodiments, the target respiratory pathogen antigen includes one or more of SARS-CoV-2 NP (Severe Acute Respiratory Syndrome Coronavirus 2 Nucleocapsid Protein), H1N1HA, and RSV FP; At least one of the multiple microcavities is pre-filled with an anti-BSA (anti-Bovine Serum Albumin)-gold nanocomplex, which serves as a quality control complex.

[0015] In some embodiments, the surface of the gold nanoparticles in the antibody-gold nanocomplex is connected to HS-PEG-COOH (Thiol-poly(ethylene glycol)-carboxyl), and after activation by EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (hydrochloride), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (hydrochloride)) / sulfo-NHS (N-Hydroxysulfosuccinimide (sodium salt), N-hydroxysulfosuccinimide (sodium salt)), they are linked to a secondary antibody, and after blocking treatment, they bind to a specific monoclonal antibody corresponding to the target pathogen antigen.

[0016] In some embodiments, the first reaction solution is a PBS (Phosphate Buffered Saline) solution containing BSA (Bovine Serum Albumin), preferably a PBS solution with 0.5% BSA, and the second reaction solution is a PBST (Phosphate Buffered Saline with Tween-20) solution.

[0017] In some embodiments, the outer cover of the reaction chamber includes a circular pressure plate and a handle disposed on the circular pressure plate, the circular pressure plate being used to press the inner cover of the reaction chamber and the area to be tested; and / or, The reaction chamber is a circular box, and the kit contains at least two reaction chambers, which are used for wetting reaction and rinsing treatment, respectively.

[0018] Secondly, this application provides a method for in vitro detection of respiratory pathogens based on mask sampling as described in the first aspect, comprising the following steps: Provide a disposable sampling mask that has captured exhaled aerosols, and separate the area to be tested from the disposable sampling mask; The area to be tested is placed in the reaction box, and the first reaction solution is added to wet the area to be tested; Place the microcavity surface of the inner cover of the reaction box facing the area to be tested, and align the microcavity with the detection area; The outer cover of the reaction box is placed above the inner cover of the reaction box and pressure is applied to release the detection complex in the microcavity and guide it to the corresponding detection area; After the reaction is complete, remove the outer cover and the inner cover of the reaction box, and rinse the area to be tested with the second reaction solution; Observe the color development of each detection area in the area to be tested to determine whether the mask-captured sample contains the corresponding target respiratory pathogen antigen; The method described herein is an in vitro detection method for isolated samples and does not include steps for diagnosing or treating diseases in humans or animals.

[0019] In some embodiments, the disposable sampling mask that has captured exhaled aerosols is obtained by the subject wearing it for 0.5 to 1 hour while awake, and the test area is separated from the meltblown fabric layer of the disposable sampling mask.

[0020] In some embodiments, the reaction time is 1 to 10 minutes, and the rinsing time is 10 to 60 seconds.

[0021] In some embodiments, when a colorimetric signal is generated in the quality control detection area, the in vitro detection process is deemed valid. Provided that this in vitro detection process is effective, if a colorimetric signal is generated in the detection area corresponding to any target pathogen, it is determined that the target pathogen antigen is present in the sample captured by the mask.

[0022] This application provides a respiratory pathogen detection kit and in vitro detection method based on mask sampling. The method utilizes the separable test area of ​​a disposable sampling mask as a sample carrier to capture target respiratory pathogen antigens in the wearer's exhaled aerosol. A closed-loop detection system is constructed by combining a reaction chamber inner cover pre-filled with a detection complex, a pressurized reaction chamber outer cover, a reaction base carrying the test area, and a reaction solution containing wetting and rinsing functions. The test area is wetted by the first reaction solution, and the detection complex within the microcavity is released and guided to the corresponding detection area under the pressure of the outer cover. The specific binding of the recognition molecule to the target antigen triggers the aggregation of gold nanoparticles, thereby generating a visible color signal in the detection area.

[0023] This process achieves a high degree of spatial integration between sample collection and testing, avoiding the complex sample transfer steps of traditional methods. It effectively solves the long-standing contradiction in existing respiratory pathogen testing practices between low sampling compliance and difficulty in achieving testing accuracy. In particular, for young children, the elderly, or people sensitive to pain, invasive or semi-invasive sampling processes often cause strong discomfort, fear, and even physiological rejection, resulting in extremely low sampling compliance. This leads to unstable sampling quality, insufficient sample volume, false negatives, and missed detections, and makes it difficult to meet the technical needs of rapid screening for large populations and convenient home testing. Therefore, it avoids sampling failures or distorted test results due to non-cooperation of subjects, significantly improving the comfort, convenience, consistency, and reliability of the testing process, and is suitable for rapid screening in multiple scenarios. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the structure of the detection kit provided in the embodiments of this application; Figure 2 This is a schematic cross-sectional view of the sampling mask provided in the embodiments of this application; Figure 3 This is a schematic diagram of the planar structure of the sampling mask provided in the embodiments of this application; Figure 4 This is a schematic diagram of the structure of the testing area of ​​the sampling mask provided in this application embodiment; Figure 5 This is an exploded structural diagram of the reaction chamber of the test kit provided in the embodiments of this application; Figure 6 This is an exploded structural diagram of the inner cover of the reaction box of the test kit provided in this application embodiment; Figure 7 This is a schematic diagram of the planar structure of the inner cover of the reaction box of the test kit provided in the embodiments of this application; Figure 8 This is a schematic diagram of the structure of the first and second bottom boxes of the reaction box of the detection kit provided in the embodiments of this application.

[0026] The attached icon numbers are as follows: 10. Disposable sampling mask; 11. Outer layer; 12. Middle layer; 120. Area to be tested; 121. Quality control area; 122. Testing area; 13. Inner layer; 20. Reactor body; 21. First bottom box; 22. Second bottom box; 23. Inner cover of the reaction box; 230. Microcavity; 231. First hydrophobic barrier layer; 2310. Through hole; 232. Carrier layer; 233. Second hydrophobic barrier layer; 24. Outer cover of the reaction box; 241. Circular pressure plate; 242. Handle. Detailed Implementation

[0027] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that the embodiments of this application may also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods are omitted so as not to obscure the description of the embodiments of this application with unnecessary detail.

[0028] It should also be understood that the term "and / or" as used in the specification of embodiments of this application and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0029] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as being "connected to" another component, it can be directly connected to or indirectly connected to that other component.

[0030] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0031] Furthermore, in the description of the embodiments and the appended claims of this application, the terms "first," "second," "third," etc., are used only for distinguishing descriptions and should not be construed as indicating or implying relative importance.

[0032] In this specification, references to "one embodiment" or "some embodiments" mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in still other embodiments" appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized. "A plurality" refers to two or more.

[0033] In the detection of pathogens causing respiratory diseases in children, existing invasive sampling methods present operational challenges due to insufficient cooperation from young children. Nasal or pharyngeal swabs, in particular, easily induce crying and physical resistance in children, increasing the difficulty of specimen collection and reducing specimen quality due to contamination by saliva or secretions. Consequently, false negatives occur, the accuracy of pathogen identification decreases, and the timeliness of clinical treatment decisions is affected.

[0034] If the aforementioned sampling mismatch problem is not resolved, the reliability of respiratory pathogen detection cannot be maintained, clinical treatment decisions will be made based on erroneous information, the risk of antibiotic abuse will increase, the possibility of drug-resistant strains will rise, the effective treatment window will be delayed, the risk of rapid progression of the child's condition to severe pneumonia will increase, the complexity of medical intervention will be increased, and the health risks of the child will be exacerbated.

[0035] like Figures 1 to 4As shown, this embodiment provides a respiratory pathogen detection kit based on mask sampling. The kit includes a disposable sampling mask 10 and a reaction box body 20. The reaction box body 20 includes an inner reaction box cover 23, an outer reaction box cover 24, a reaction bottom box, and a reaction solution. The disposable sampling mask 10 includes a separable test area 120 for capturing the wearer's exhaled aerosols. The test area 120 is provided with multiple detection zones 122 and positioning markers corresponding to each detection zone 122. One of the detection zones 122 is used for quality inspection, serving as a quality control zone 121. Specifically, the disposable sampling mask 10 serves as the direct carrier for sample collection. Its core feature is the integrated, separable test area 120. This area is typically located on the inner side of the mask, opposite the wearer's mouth and nose respiratory tract, allowing for efficient interception and enrichment of pathogen particles in exhaled aerosols during breathing. The test area 120 is designed as a separable structure, meaning that after sampling, this part can be completely removed from the mask body through tearing, cutting, or peeling, and directly used as a sample carrier for subsequent testing into the reaction chamber. This avoids the traditional process of transferring samples from a swab to the eluent and then to the test card, significantly reducing the risk of sample loss and cross-contamination. Multiple detection zones 122 on the test area 120 are specific sites for the occurrence of specific immune responses. The location markers corresponding to each detection zone 122 can be printed numbers, letters, symbols, or color blocks, indicating the type of pathogen (such as influenza virus, respiratory syncytial virus, etc.) corresponding to different detection zones 122. This helps users or testing personnel quickly and accurately locate and identify the meaning of each detection zone 122 during result interpretation, preventing confusion. The form of the positioning mark is not limited. For example, it can be an ink printing layer or an embossed mark formed by indentation, as long as it can clearly indicate the position and attributes of the detection area 122.

[0036] The inner cover 23 of the reaction chamber includes a carrier layer 232 and multiple microcavities 230 disposed on the carrier layer 232. The multiple microcavities 230 correspond to multiple detection areas 122, and each microcavity 230 contains a pre-placed detection complex. In this structure, the inner cover 23 is a key component carrying the core detection reagent. Its carrier layer 232 mainly serves as physical support and structural shaping, and is typically made of a polymer material with a certain degree of hardness and toughness (such as polystyrene, polycarbonate, polypropylene, etc.) to ensure shape stability under pressure without excessive deformation. The multiple microcavities 230 disposed on the carrier layer 232 are arranged in an array, their spatial positions strictly corresponding to the multiple detection areas 122 on the test area 120, ensuring that each microcavity 230 accurately covers one detection area 122 when the cover is closed. The shape of the microcavity 230 can be cylindrical, conical, or hemispherical, etc., and its volume is precisely designed to accommodate the required volume of detection reagent. The detection complex pre-placed in each microcavity 230 is a reagent in a dry or lyophilized state, and its distribution form in the microcavity 230 can be powder, film, or gel. Through the physical isolation of the microcavities 230, the mutual diffusion or interference of reagents between different detection zones 122 is effectively prevented, realizing the parallelization and independence of multi-target detection.

[0037] The outer cover 24 of the reaction chamber is used to apply pressure to the inner cover 23 of the reaction chamber when it is in contact with the test area 120. Specifically, the outer cover 24 is usually wrapped around or fastened to the outside of the reaction chamber, and its function is to provide a uniform, controllable and easy-to-operate mechanical pressure source. After the inner cover 23 of the reaction chamber is placed on the test area 120, by closing or tightening the outer cover 24, the inner wall of the outer cover will directly or indirectly press against the carrier layer 232 of the inner cover 23. This pressure transmission mechanism allows the microcavity 230 to fit tightly against the detection area 122 below, which not only ensures that the reagent released by the microcavity 230 can act vertically on the corresponding detection area 122 and prevent lateral diffusion, but also maintains a good contact interface during the reaction and promotes the capillary flow of liquid from the microcavity 230 to the detection area 122. The outer cover 24 of the reaction box can be designed using conventional fastening methods in the field, such as snap-fit ​​connection, threaded tightening or magnetic adsorption, as long as sufficient pressure can be applied to the inner cover to make the microcavity 230 fit tightly against the detection area 122.

[0038] The reaction chamber is used to hold the test area 120 and contain the reaction solution; it constitutes the bottom container and reaction pool of the kit. Internally, it features a recessed cavity whose shape is adapted to the size and shape of the test area 120, used to lay flat, fix, and hold the test area 120 separated from the mask during testing. The cavity of the reaction chamber also functions to contain the reaction solution; its depth and volume are designed to ensure that the test area 120 is completely immersed or fully wetted after the reaction solution is added, while allowing sufficient space for liquid expansion to prevent overflow. The reaction chamber is typically made of chemically resistant and low-permeability materials such as polyethylene or polypropylene.

[0039] The reaction solution includes at least a first reaction solution for wetting the test area 120 and a second reaction solution for rinsing the test area 120. In this embodiment, the reaction solution is a chemical medium system that drives the immune reaction and washes away the background. The first reaction solution is mainly used to wet the test area 120. Its function is to activate the sample antigen on the test area 120 and to act as a solvent to dissolve and elute the dried detection complex in the microcavity 230 to the detection area 122. The first reaction solution can be phosphate buffer (PBS), Tris-HCl buffer, or a compound buffer containing a surfactant (such as Tween-20). Its pH and ionic strength are optimized to maintain the activity of the antigen and antibody while promoting the release and flow of the complex. The second reaction solution is mainly used to rinse the test area 120. Its function is to remove unbound free detection complexes and other non-specifically adsorbed impurities on the detection area 122 after the specific binding reaction is completed, thereby reducing background noise and improving the signal-to-noise ratio and accuracy of the detection. The second reaction solution is usually a buffer solution containing washing aids (such as Tween, Triton X-100, etc.), which has a higher elution strength than the first reaction solution. It can effectively remove non-specific conjugates without destroying the specific immune complexes that have been formed.

[0040] The detection complex comprises gold nanoparticles and recognition molecules attached to the surface of the gold nanoparticles. These recognition molecules specifically bind to antigens of target respiratory pathogens; specifically, the detection complex is the core probe for signal visualization. Gold nanoparticles (AuNPs), as signal markers, possess unique localized surface plasmon resonance characteristics, and their optical properties are closely related to the particle aggregation state. The recognition molecules attached to the surface of the gold nanoparticles are typically biomolecules with specific recognition capabilities, such as monoclonal antibodies, polyclonal antibodies, or aptamers. These molecules are immobilized on the surface of the gold nanoparticles through physical adsorption or chemical cross-linking. When gold nanoparticles are dispersed, their solution is typically red; however, when the gold nanoparticles aggregate, due to the plasmon coupling effect between the particles, their absorption spectrum undergoes a redshift, causing the solution or spot color to change from red to blue or from red to purple, resulting in a color change that is visible to the naked eye.

[0041] When the test area 120 is wetted by the first reaction solution, and the microcavity 230 of the inner cover 23 of the reaction chamber is in contact with and pressurized against the detection area 122 of the test area 120, the detection complex is released from the microcavity 230 into the corresponding detection area 122. In this step, the test area 120 is first pretreated with the first reaction solution to fully hydrate the aerosol sample on the test area 120, exposing potential antigen epitopes. Subsequently, the inner cover 23 of the reaction chamber is closed onto the test area 120, and pressure is applied by the outer cover 24 of the reaction chamber to create a sealed contact environment between the microcavity 230 and the detection area 122. At this time, the first reaction solution enters the microcavity 230 through capillary action or pressure drive, redissolving the pre-placed dried detection complex. Driven by the concentration gradient and pressure difference, the dissolved detection complex flows out from the outlet of the microcavity 230, diffuses vertically, and infiltrates the detection area 122 below. This process enables on-demand release of reagents and in-situ reaction, ensuring that the detection complex can act precisely on the target detection area 122.

[0042] If the target respiratory pathogen antigen is present in the detection area 122, the detection complex specifically binds to the target respiratory pathogen antigen, causing the gold nanoparticles to aggregate and generating a visual color signal in the corresponding detection area 122. Specifically, when the target respiratory pathogen antigen is captured on the detection area 122 of the test area 120, the recognition molecules in the detection complex released from the microcavity 230 will specifically bind to the antigen. Since antigens typically have multiple epitopes, and the surface of the gold nanoparticles is modified with multiple recognition molecules, one antigen molecule can simultaneously connect to multiple gold nanoparticles, or one gold nanoparticle can connect to multiple antigen molecules. This multivalent cross-linking leads to the physical aggregation of gold nanoparticles in a three-dimensional network in the detection area 122. As the degree of aggregation increases, the plasmon resonance peak of the gold nanoparticles shifts, and the wavelength of its reflected or transmitted light changes, resulting in a significant color change in the detection area 122 (e.g., from red to blue / gray). This color signal is direct evidence of the presence of the target antigen and can be interpreted qualitatively or semi-quantitatively by visual observation without the need for complex instruments.

[0043] The core innovation of this application lies in the construction of a respiratory pathogen screening system that integrates non-invasive sampling and in-situ detection. By combining a separable test area 120 with aerosol capture function with an integrated microcavity reaction inner cover 230, and using a biphasic reaction solution to drive the immune reaction and washing, a fully closed-loop operation from sample collection to result output is realized, which effectively solves the technical bottlenecks of existing swab sampling methods in terms of compliance, operational complexity and risk of cross-infection.

[0044] The working process and principle of this application are as follows: the wearer breathes normally through a disposable sampling mask 10, and pathogens in the exhaled aerosol are intercepted and enriched in the test area 120 of the mask; after sampling, the test area 120 is separated from the mask and laid flat in the reaction box; the test area 120 is moistened with the first reaction solution, and then the inner cover 23 of the reaction box with the pre-placed detection complex is fastened, and the outer cover 24 of the reaction box is used to apply pressure to make the microcavity 230 and the detection area 122 fit tightly; under the action of the first reaction solution, the nano-gold labeled complex in the microcavity 230 dissolves and is released into the detection area 122. If the detection area 122 contains the corresponding antigen, the nano-gold particles aggregate and develop color through antigen-antibody specific binding; finally, the device is rinsed with the second reaction solution to remove the unbound complex, and the detection result is determined based on the color change of the detection area 122.

[0045] like Figure 2 and Figure 3As shown, this embodiment provides a disposable sampling mask 10. The disposable sampling mask 10 includes an outer layer 11, an inner layer 13, and a middle layer 12. The outer layer 11 is used to prevent external liquids from seeping into the mask, the inner layer 13 is used to absorb the moisture exhaled by the wearer, and the middle layer 12 is sandwiched between the outer layer 11 and the inner layer 13. The middle layer 12 is a meltblown fabric layer, and the separable test area 120 is disposed in the middle of the meltblown fabric layer, corresponding to the wearer's mouth and nose area when worn.

[0046] The outer layer 11, as the outermost protective structure of the mask, mainly serves as a physical barrier to prevent liquid droplets, splashes, or other contaminants from the external environment from penetrating into the mask, thereby protecting the filtration performance of the middle layer 12 from damage by external liquids. In specific implementations, the outer layer is usually made of a non-woven fabric material with hydrophobic properties, such as polypropylene spunbond non-woven fabric. This application embodiment does not specifically limit the specific material of the outer layer, as long as it can meet the function of preventing liquid penetration. The inner layer 13 is located on the inside of the mask and directly contacts the wearer's facial skin. Its main function is to absorb the moisture exhaled by the wearer, thereby improving wearing comfort and preventing the feeling of dampness caused by the condensation of exhaled air. Exemplarily, the inner layer 13 can be made of a non-woven fabric material with good hydrophilicity, such as hot-air non-woven fabric or spunbond non-woven fabric that has been treated with hydrophilicity. This application embodiment does not specifically limit this. The middle layer 12 is sandwiched between the outer layer 11 and the inner layer 13, constituting the core filtration layer of the mask. In this embodiment, the middle layer 12 is a meltblown fabric layer. The meltblown fabric layer is a nonwoven fabric made by stretching a fine stream of polymer melt sprayed from a die head using a high-speed hot air stream, forming ultrafine fibers, which are then collected on a condensation screen to form a fiber web, and reinforced by self-bonding. Due to its high porosity, small pore size, and high filtration efficiency, the meltblown fabric layer can effectively capture airborne particulate matter and aerosols. A separable testing area 120 is located in the middle of the meltblown fabric layer and corresponds to the wearer's mouth and nose area when worn. This means that when the user wears the mask, the testing area 120 is directly facing the wearer's mouth and nose, thereby maximizing the capture of exhaled aerosols. The meltblown fabric layer typically utilizes electrostatic adsorption to enhance its ability to capture small particles; its fibers carry electrostatic charges, enabling them to adsorb charged or neutral particles in the air. It should be understood that the specific shape of the separable test area 120 can be set according to actual needs, such as a circle, square or polygon. The separation method can be achieved by pre-setting marks, dot breaks or easy-tear lines on the meltblown fabric layer so that it can be completely removed from the mask during the testing process.

[0047] In one optional embodiment, the test area 120 is a circular area, and the outer periphery of the circular area is provided with scoring, dotted lines, or easy-tear lines to facilitate separation from the meltblown fabric layer. At least two detection zones 122 are provided within the circular area.

[0048] The detection area 122 is a specific functional area within the test area 120 used to hold the sample and undergo a colorimetric reaction. Setting at least two detection areas 122 is to meet the need for parallel detection of multiple targets, enabling the same test sample to be screened for multiple different respiratory pathogen antigens simultaneously. For example, in a specific implementation scenario, the detection area 122 may include specific reaction sites for different viral or bacterial antigens. Integrating multiple detection areas 122 within a circular area not only improves detection efficiency but also saves sample volume and reagent costs. Furthermore, the layout of the multiple detection areas 122 within the circular area can be designed according to actual needs, such as an array or a ring distribution, to ensure that the detection areas 122 do not interfere with each other and can achieve a precise one-to-one correspondence with the microcavity 230 structure on the inner cover 23 of the reaction chamber, thereby ensuring the accuracy and repeatability of the test results.

[0049] In some embodiments, the diameter of the circular region is 3-8 cm. This size range is determined based on a comprehensive consideration of aerosol capture efficiency and the convenience of subsequent reaction operations. If the diameter is less than 3 cm, the amount of aerosol sample captured may be insufficient, thus affecting the detection sensitivity; if the diameter is greater than 8 cm, it will increase the consumption of reaction solution and may increase the risk of non-uniform reagent diffusion due to the excessively long flow path of the microcavity 230. Preferably, the diameter of the circular region can be set to 5 cm to maintain good hydrodynamic characteristics while ensuring the amount of sample captured.

[0050] like Figure 4 As shown, the circular area contains four circular detection zones 122, which are evenly distributed within the circular area and each is marked with a positioning label numbered 1 to 4. The four detection zones 122 meet the needs of multi-indicator joint screening for common respiratory pathogens; for example, two or three viral antigens can be detected simultaneously, with a quality control zone 121 included. This evenly distributed layout helps ensure consistent wetting of each detection zone 122 during the reaction process, avoiding uneven color development due to edge effects. The specific arrangement of the circular detection zones 122 can be determined according to actual design requirements, such as a rectangular array or a cross-shaped arrangement.

[0051] The positioning markers 1 to 4 mentioned above are used to ensure precise alignment between the microcavity 230 of the inner cover 23 of the reaction chamber and the detection area 122 of the test area 120 during the testing process. Through these positioning markers, operators can quickly identify the detection target corresponding to each detection area 122, thereby accurately interpreting the test results. The positioning markers can be printed, embossed, or affixed to the perimeter of the detection area 122, and can be in the form of numbers, color codes, or symbols.

[0052] like Figure 5 and Figure 6As shown, this embodiment provides a reaction chamber inner cover 23, which includes a hydrophilic porous carrier layer 232 and hydrophobic barrier layers disposed on both sides of the carrier layer 232. The hydrophobic barrier layers include a first hydrophobic barrier layer 231 and a second hydrophobic barrier layer 233. The hydrophilic porous carrier layer 232, as the core functional layer of the reaction chamber inner cover 23, is mainly used to support and pre-place the detection complex, and during the detection process, it adsorbs the reaction liquid through capillary action, thereby wetting and releasing the dry detection complex. The hydrophilic porous carrier layer 232 can be made of porous materials such as filter paper, nitrocellulose membrane, or non-woven fabric. Its pore size and porosity can be selected according to the flow rate of the reaction liquid and the particle size of the detection complex. For example, a filter paper layer with a pore size in the range of 0.1 to 10 micrometers can be selected. The hydrophobic barrier layers are disposed on both sides of the carrier layer 232, mainly to prevent lateral diffusion of liquid, limit the reaction area, and provide mechanical support. The hydrophobic barrier layer can be made of waxed paper, polyethylene film or hydrophobically treated polymer sheet, which is tightly bonded to the carrier layer 232 by hot pressing, bonding or lamination process to ensure that the carrier layer 232 is completely sealed except for the area of ​​through hole 2310.

[0053] like Figure 6 and Figure 7 As shown, a plurality of through-holes 2310 are provided on one side of the hydrophobic barrier layer. The through-holes 2310, together with the carrier layer 232 and the hydrophobic barrier layer on the other side, form a microcavity 230. The through-holes 2310 are formed on one side of the hydrophobic barrier layer, and their positions correspond one-to-one with the multiple detection areas 122 on the area to be tested 120. The shape and size of the through-holes 2310 can be designed according to the shape and size of the detection areas 122, for example, they can be circular, square or strip-shaped. In the vertical direction, the through-holes 2310 penetrate the hydrophobic barrier layer on that side, exposing the underlying hydrophilic porous carrier layer 232; while in the horizontal direction, the sidewalls of the through-holes 2310 are composed of a hydrophobic barrier layer. When the inner cover 23 of the reaction chamber is in contact with the test area 120, the through hole 2310, the pores of the carrier layer 232 located below the through hole 2310, and the hydrophobic barrier layer located on the other side of the carrier layer 232 together form a relatively closed three-dimensional space, namely the microcavity 230. This sandwich-like layered structure design, utilizing the strong hydrophobicity of the hydrophobic barrier layer, can effectively restrict the reaction liquid to flow only in the vertical direction defined by the through hole 2310, preventing the liquid from spreading laterally or leaking within the plane of the carrier layer 232. This ensures that the detection complex pre-placed in the microcavity 230 can be accurately guided to the corresponding detection area 122 on the test area 120, avoiding cross-contamination between different detection areas 122.

[0054] When the inner cover 23 of the reaction chamber adheres to and is pressed against the test area 120, which has been moistened by the first reaction solution, the first reaction solution enters the interior of the microcavity 230 through the through-hole 2310, rapidly wetting the hydrophilic porous carrier layer 232. The carrier layer 232 swells after absorbing the liquid, and the detection complex pre-dried in its pores dissolves or disperses into the liquid. Driven by pressure and capillary action, the liquid containing the detection complex flows vertically downward through the through-hole 2310, directly covering the corresponding detection area 122 of the test area 120. If the detection area 122 contains a target respiratory pathogen antigen, the recognition molecule (such as an antibody) in the detection complex specifically binds to the antigen, thereby causing the aggregation of gold nanoparticles and generating a visible color signal.

[0055] Specifically, the carrier layer 232 is a filter paper layer, and the hydrophobic barrier layer is a wax paper layer; multiple through holes 2310 correspond one-to-one with multiple detection areas 122 on the test area 120. The carrier layer 232, as the core functional layer of the inner cover 23 of the reaction chamber, is made of filter paper. Utilizing the natural hydrophilicity and high porosity of the filter paper, it can effectively adsorb and store the liquid detection complex, and under pressure, use capillary force to rapidly and uniformly release the complex to the test area 120. The filter paper layer can be selected from commonly used laboratory qualitative or quantitative filter paper, typically made of cotton fiber or wood pulp fiber. Its thickness and pore size can be selected according to the loading and release rate of the detection complex; for example, the pore size range can be set between 5 and 20 micrometers. The hydrophobic barrier layer is made of waxed paper, which has excellent hydrophobic and barrier properties. It effectively prevents the reaction liquid from diffusing laterally in the non-microcavity 230 area, ensuring that the detection complex can only be vertically guided to the corresponding detection area 122 through the through-hole 2310, thereby avoiding signal crosstalk between different detection areas 122. The waxed paper layer can be prepared by impregnating or coating the base paper with wax. The wax component can be paraffin wax, microcrystalline wax, or a mixture thereof. The hydrophobic barrier effect and mechanical strength can be controlled by adjusting the wax layer thickness.

[0056] Through-holes 2310 are formed on one side of the wax paper layer, and their positional distribution strictly matches the spatial layout of the detection areas 122 within the test area 120. When the inner cover 23 of the reaction chamber covers the test area 120, the through-holes 2310 completely cover the corresponding detection areas 122 in the vertical direction, ensuring that the detection complex released from the microcavity 230 can accurately land within the detection area 122. The shape of the through-holes 2310 can be adapted to the shape of the detection area 122; for example, when the detection area 122 is circular, the through-holes 2310 are also designed to be circular. The size of the through-holes 2310 should be slightly smaller than or equal to the size of the detection area 122 to further limit the liquid diffusion range. This one-to-one spatial coding relationship ensures the accuracy of the detection results, allowing the operator to directly determine the pathogen type based on the color development location without the need for complex image recognition equipment.

[0057] In some embodiments, polystyrene sulfonate is also pre-placed in the microcavity 230, and the detection complex is dried and fixed in the microcavity 230 and can be released to the test area 120 under the action of the first reaction solution.

[0058] Polystyrene sulfonate, as a water-soluble polymer electrolyte, primarily functions as a stabilizer and release aid in this embodiment. During the drying and fixation of the detection complex in the microcavity 230, polystyrene sulfonate can inhibit the non-specific aggregation of gold nanoparticles in the dry state through the charge interaction or steric hindrance effect between its sulfonic acid groups and the surface of the gold nanoparticles. This maintains the dispersion and bioactivity of the detection complex, which helps extend the shelf life of the kit and ensures the stability of the reagent during storage. Furthermore, polystyrene sulfonate has strong hydrophilicity. When the first reaction solution contacts the microcavity 230, it can rapidly absorb water and swell, accelerating the wetting and penetration of the dried detection complex inside the microcavity 230. This rapid liquid wetting shortens the dissolution and release time of the detection complex, allowing it to be released more quickly and completely from the microcavity 230 and channeled to the corresponding detection area 122 of the test area 120, thereby improving the initiation speed of the detection reaction and the uniformity of the signal intensity.

[0059] In some embodiments, the recognition molecule is an antibody, specifically an immunoglobulin capable of specifically binding to a particular antigen. In specific implementations, the antibody can be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody, to ensure high specificity and low cross-reactivity of the detection. The detection complex is an antibody-gold nanoparticle complex, which forms a biological probe by chemically conjugating the antibody to the surface of gold nanoparticles. The antibody-gold nanoparticle complexes in different microcavities 230 correspond to different target respiratory pathogen antigens, and each gold nanoparticle is conjugated to only one specific antibody.

[0060] The aforementioned structural configuration enables the kit to detect multiple pathogens in parallel within the same reaction system. Utilizing the high affinity and specificity between antibodies and antigens, the antibody-gold nanoparticle complex precisely captures the target pathogen antigen in the test area 120. When the target antigen is present, the antibody, acting as a recognition molecule, binds to the antigen, inducing the aggregation of gold nanoparticles and resulting in a significant color change. Because different microcavities 230 are pre-contained with single antibody-gold nanoparticle complexes targeting different pathogens, and each gold nanoparticle is linked to only one antibody, this technology effectively eliminates the risk of false positives due to probe cross-reactivity, ensuring a clear and traceable source of the detection signal. This design allows the kit to accurately distinguish and identify multiple respiratory pathogen antigens in a single testing operation, providing a reliable multiplex detection method for clinical diagnosis or home screening.

[0061] In some embodiments, the target respiratory pathogen antigens include one or more of SARS-CoV-2NP, H1N1HA, and RSV FP. Specifically, SARS-CoV-2NP, or Severe Acute Respiratory Syndrome Coronavirus Type 2 nucleocapsid protein, is one of the main structural proteins of the novel coronavirus. It is highly expressed during viral infection, exhibits good immunogenicity, and is suitable as a target for high-sensitivity detection. H1N1HA is the hemagglutinin protein of the H1N1 subtype of influenza A virus. This protein is located on the surface of the viral envelope and mediates the adsorption process between the virus and host cells; it is a key antigen for distinguishing influenza virus subtypes and identifying circulating strains. RSV FP refers to the fusion protein of respiratory syncytial virus (RSV). This protein plays a crucial role in the viral invasion of host cells; its antigenicity is stable, and it is an important indicator for the clinical diagnosis of RSV infection. This embodiment, by limiting the above three specific antigens, can accurately cover the spectrum of common viral pathogens in community-acquired pneumonia in children, demonstrating strong targeting and facilitating rapid identification of the etiology.

[0062] At least one of the multiple microcavities 230 is pre-contained with an anti-BSA-gold nanocomplex as a quality control complex. The anti-BSA-gold nanocomplex can refer to gold nanoparticles with anti-bovine serum albumin antibody attached to their surface. This quality control complex is used for intrinsic quality control of the kit's detection process. In practical applications, since the first reaction solution in the kit typically contains BSA (Bovine Serum Albumin) as a blocking agent or stabilizer, when the detection process proceeds normally, the first reaction solution wets the test area 120 and releases the detection complex within the microcavity 230. At this time, the pre-contained anti-BSA-gold nanocomplex specifically binds to the BSA molecules in the first reaction solution. As the reaction proceeds, the gold nanoparticles aggregate under the bridging effect of BSA, thereby causing a change in the localized surface plasmon resonance (LSPR) absorption peak. This aggregation state causes a visible color change (e.g., from red to blue-purple) in the quality control detection area 122. If the expected colorimetric signal does not appear in the quality control detection area 122, it indicates that the reaction solution was not properly wetted, the release from the microcavity 230 was obstructed, or the reagent was ineffective, thus rendering the test result invalid. By setting up a quality control complex, false negative results caused by operational errors or reagent inactivation can be effectively eliminated, significantly improving the reliability and clinical applicability of the test results.

[0063] In some embodiments, the surface of the gold nanoparticles in the antibody-gold nanocomplex is linked to HS-PEG-COOH, which is activated by EDC / sulfo-NHS and then linked to a secondary antibody. After blocking, it binds to a specific monoclonal antibody corresponding to the target pathogen antigen. HS-PEG-COOH, or thiol-polyethylene glycol-carboxyl group, has a molecular structure with a thiol group at one end, a carboxyl group at the other end, and a long polyethylene glycol chain in the middle. During preparation, HS-PEG-COOH forms a strong Au-S bond with the gold atoms on the surface of the gold nanoparticles through its terminal thiol group, thus stably anchoring it to the gold nanoparticle surface. Simultaneously, the long polyethylene glycol chain acts as a hydrophilic spacer arm, increasing the dispersion stability of the gold nanoparticles in aqueous solution, reducing non-specific adsorption, and providing sufficient flexible space for subsequent antibody conjugation to alleviate steric hindrance and improve antibody binding activity. After HS-PEG-COOH linkage, it is activated by EDC / sulfo-NHS. EDC stands for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and sulfo-NHS stands for sodium N-hydroxysuccinimide sulfonate. This activation process aims to convert the carboxyl group at the end of HS-PEG-COOH into an active ester, thereby enhancing its reactivity with amino groups. In the specific chemical reaction, EDC first reacts with the carboxyl group to form an unstable O-acylisourea reactive intermediate, which is then attacked by sulfo-NHS to form a relatively stable NHS ester in water.

[0064] Subsequently, the activated gold nanoparticles are conjugated with a secondary antibody. The secondary antibody can be an antibody targeting immunoglobulins of a specific species (such as rabbits or mice), and its molecular structure contains multiple amino groups. These amino groups can nucleophilically attack the NHS ester groups on the surface of the gold nanoparticles, forming stable amide bonds, thereby covalently coupling the secondary antibody to the end of the PEG chain. By introducing the secondary antibody as a bridging molecule, it can further bind to the subsequently added specific monoclonal antibody. This indirect conjugation strategy can effectively control the spatial orientation of the specific antibody, allowing its antigen-binding site (Fab segment) to be more fully exposed, thus significantly improving the antigen capture efficiency and sensitivity of the detection complex. After the secondary antibody conjugation is complete, a blocking treatment is performed. The purpose of the blocking treatment is to use a blocking agent (such as bovine serum albumin BSA, casein, or glycine) to occupy the remaining unreacted active sites and non-specific adsorption sites on the surface of the gold nanoparticles. This step can block the non-specific binding of the detection complex to non-target components in the sample during subsequent detection, thereby reducing background noise and improving the signal-to-noise ratio and the accuracy of the detection results. After the blocking treatment, a specific monoclonal antibody corresponding to the target pathogen antigen is added. This specific monoclonal antibody can specifically bind to the Fc fragment of the secondary antibody, thereby being immobilized on the surface of the gold nanoparticles. The final constructed antibody-gold nanoparticle complex has a multi-level structure of gold nanoparticle core-PEG spacer arm-secondary antibody bridging-specific monoclonal antibody. This structure ensures both the colloidal stability of the gold nanoparticles and maximizes the antigen recognition ability of the specific antibody.

[0065] In some embodiments, the reaction solution components include a first reaction solution and a second reaction solution. The first reaction solution is a PBS solution containing BSA, and the second reaction solution is a PBST solution.

[0066] The first reaction solution is a PBS solution containing BSA, designed to provide a stable physiological environment for the antigen-antibody reaction. PBS (Phosphate Buffered Saline) is a commonly used balanced salt solution in biochemical experiments. Its main components typically include sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride, providing a pH value (usually 7.2–7.4) and ionic strength close to physiological conditions, which is beneficial for maintaining the biological activity and stability of the antibody-gold nanoparticle complex. The addition of BSA (Bovine Serum Albumin) plays a dual crucial role: On the one hand, as a common non-specific blocking agent, BSA can pre-occupy non-specific adsorption sites on the surface of the test area 120 (such as the meltblown fabric fiber of a mask), thereby reducing the physical adsorption of the detection complex in non-target areas during subsequent reactions, effectively reducing background interference, and improving the detection signal-to-noise ratio; on the other hand, combined with the quality control design of the above embodiment, BSA also acts as a target molecule for the quality control reaction. When the test area 120 is wetted by the first reaction solution, the released anti-BSA-gold nanocomplex can bind with BSA molecules in the liquid phase, thereby forming gold nanoparticles in the quality control area 121 and generating a color signal to verify the effectiveness of the reaction process.

[0067] The second reaction solution is a PBST solution, which is mainly used for the rinsing step after the reaction. The PBST solution can be a rinsing solution formulated by adding Tween-20 (polyoxyethylene dehydrated sorbitan monolaurate) nonionic surfactant to PBS buffer. Tween-20 has mild detergency; its hydrophilic-lipophilic balance allows it to effectively disrupt hydrophobic interactions, thereby eluting away excess antibody-gold nanoparticle complexes that are not specifically bound to the target antigen or are only retained on the surface of the test area 120 through nonspecific adsorption. By using the PBST solution for rinsing, non-specifically bound gold nanoparticles can be removed to the greatest extent, avoiding the generation of unexpected background color deposition in the detection area 122. This ensures that the finally observed colorimetric signal is caused by specific aggregation due to the binding of the target antigen and the specific antibody, thus significantly improving the accuracy and specificity of the detection results. The concentration of Tween-20 can be adjusted according to the actual rinsing intensity requirements, for example, it can be a volume concentration between 0.05% and 0.5%, which is not specifically limited in this embodiment.

[0068] like Figure 5As shown in the figure, this application embodiment provides a reaction box body 20 for a respiratory pathogen detection kit based on mask sampling. The structure mainly includes an outer reaction box cover 24 and a reaction bottom box. The outer reaction box cover 24 includes a circular pressure plate 241 and a handle 242 disposed on the circular pressure plate 241. The circular pressure plate 241 is used to press the inner reaction box cover 23 and the test area 120 tightly.

[0069] The outer cover 24 of the reaction chamber, as an operating component that applies mechanical pressure to the reaction system, has the core function of ensuring a tight fit between the inner cover 23 of the reaction chamber and the test area 120, thereby allowing the detection complex in the microcavity 230 to be smoothly released and uniformly guided to the detection area 122. The geometry of the circular pressure plate 241 is adapted to the circular reaction base and the test area 120. This coaxial symmetrical design ensures the uniformity of pressure distribution during pressing, effectively avoiding problems such as insufficient release of the detection complex or uneven color development in the detection area 122 due to uneven local force. The handle 242 is provided on the circular pressure plate 241, providing a grip for the operator to easily apply vertical downward pressure. The handle 242 can be integrally formed with the circular pressure plate 241, or it can be fixedly connected by snap-fit, threaded connection, or other methods. To improve the comfort and stability of operation, the shape of the handle 242 can be designed according to ergonomic principles, such as using a cylindrical, elongated shape, or a structure with anti-slip texture on the surface.

[0070] In some embodiments, such as Figure 5 and Figure 8 As shown, the reaction chamber is a circular box. The kit contains at least two reaction chambers, including a first chamber 21 and a second chamber 22, used for wetting and rinsing, respectively. The reaction chamber, serving as a container for the test area 120 and the reaction solution, has a circular outline that matches the shape of the test area 120, facilitating rapid positioning and placement. The reaction chamber can be made of chemically resistant plastic materials such as polyethylene or polypropylene to adapt to different reaction solution environments. By using at least two independent reaction chambers, the wetting and rinsing steps in the detection process are physically separated. One reaction chamber holds the first reaction solution, supporting the test area 120 for wetting and colorimetric reactions; the other reaction chamber holds the second reaction solution, supporting the test area 120 for rinsing. This dual-chamber configuration effectively avoids direct mixing of residual liquid after the wetting reaction with the rinsing solution in a single container, thus preventing cross-contamination, ensuring the cleanliness of rinsing, and improving the accuracy of the detection results. Meanwhile, the two reaction chambers can be used independently, which simplifies the operation process. Operators do not need to perform complex liquid replacement in the same chamber, reducing the risk of operational errors.

[0071] Secondly, this application provides an in vitro detection method using a respiratory pathogen detection kit based on mask sampling, the method comprising the following steps: Step S100: Provide a disposable sampling mask that has captured exhaled aerosols, and separate the area to be tested from the disposable sampling mask.

[0072] A disposable sampling mask that has captured exhaled aerosols refers to a mask sample where, after being worn by a test subject, the test area in the middle of the meltblown fabric layer has absorbed the wearer's exhaled aerosols. This test area, serving as the sample carrier for subsequent testing, needs to be completely separated from the mask structure. During separation, the edge of the disposable sampling mask is cut to expose the middle meltblown fabric layer located between the outer and inner layers; subsequently, along the pre-made notches, dotted lines, or easy-tear lines on the meltblown fabric layer, the circular test area is torn off the main body of the meltblown fabric layer. The separated test area retains the porous fibrous structure of the meltblown fabric, and aerosol particles containing respiratory pathogen antigens are trapped in the gaps between the fibers.

[0073] Step S200: Place the area to be tested in the reaction box and add the first reaction solution to wet the area to be tested.

[0074] The reaction chamber holds the isolated test area and serves as a container for wetting and reaction. After the test area is placed flat on the bottom of the reaction chamber, an appropriate amount of the first reaction solution is added. The first reaction solution is a phosphate-buffered saline (PBS) solution containing bovine serum albumin (BSA). The liquid wets the meltblown fabric fibers of the test area through capillary action. This wetting process activates the bioactivity of the antigen in the test area, enabling it to react with the subsequently added recognition molecules. Furthermore, the BSA in the first reaction solution non-specifically occupies some sites on the fiber surface of the test area, reducing non-specific adsorption of the detection complex in subsequent steps and thus lowering background interference.

[0075] Step S300: Place the microcavity surface of the inner cover of the reaction box facing the area to be tested, and align the microcavity with the detection area.

[0076] The microcavity surface of the reaction chamber inner cover refers to the surface on which a carrier layer and microcavity structure are located. During placement, ensure that the microcavities on the reaction chamber inner cover are spatially aligned with the pre-fabricated detection areas on the test area. This alignment process is achieved through the correspondence between positioning marks on the test area and the preset positions of the microcavities; for example, align the microcavity marked 1 with detection area 1 on the test area, and so on. The reaction chamber inner cover fits tightly against the top of the test area, with the openings of the microcavities directly facing the corresponding detection area surface, forming a closed reaction space. This ensures that the material pre-placed within the microcavities can be directionally transported to the target detection area.

[0077] Step S400: Place the outer cover of the reaction chamber above the inner cover of the reaction chamber and apply pressure to release the detection complex in the microcavity and guide it to the corresponding detection area.

[0078] The outer cover of the reaction chamber is placed on top of the inner cover, and a uniform downward pressure is applied to the inner cover using the circular pressure plate on the outer cover. This pressure squeezes out the air between the inner cover and the test area to eliminate air bubble interference and forces the dried detection complex in the microcavity to contact the fibers of the test area, which are wetted by the first reaction solution. The detection complex includes gold nanoparticles and recognition molecules (such as antibodies) attached to their surface. Under the dissolving effect of the first reaction solution, the detection complex, which was originally dried and fixed in the microcavity, rapidly hydrates and is released, flowing down to the corresponding detection area in the direction of liquid flow. After entering the detection area, the detection complex is uniformly distributed in the meltblown fabric fiber network under the diffusion effect of the liquid.

[0079] Step S500: After the reaction is complete, remove the outer cover and inner cover of the reaction chamber, and rinse the test area with the second reaction solution.

[0080] With the inner lid of the reaction chamber in contact with the test area for a preset time, the reaction chamber allows the recognition molecules in the detection complex to fully contact and specifically bind to the target respiratory pathogen antigens that may be present in the test area. After the reaction is complete, the outer lid and inner lid of the reaction chamber are removed sequentially to expose the test area. The test area is then rinsed with a second reaction solution, which is phosphate-buffered saline (PBST) containing Tween. The rinsing aims to remove free detection complexes that have not specifically bound to the antigen, as well as residual components of the first reaction solution, retaining only the detection complexes that are firmly bound to the test area through an immune reaction.

[0081] Step S600: Observe the color development of each detection area in the area to be tested to determine whether the target respiratory pathogen antigen is present in the sample captured by the mask.

[0082] After rinsing, the test area is observed for color development. The color development is based on the aggregation effect of the gold nanoparticles: if a target respiratory pathogen antigen is present in a detection area, the recognition molecule in the detection complex binds to the antigen, causing the gold nanoparticles to aggregate under antigen bridging. When the gold nanoparticles change from a dispersed state to an aggregated state, their local surface plasmon resonance (LSPR) absorption peak redshifts, resulting in a change in the macroscopic color of the detection area (e.g., from red to purplish-brown or gray). If the detection area does not contain the corresponding antigen, the gold nanoparticles remain dispersed, and the detection area does not change color or displays a background color. By observing whether each detection area produces a specific color change signal with the naked eye or an auxiliary colorimetric card, it can be determined whether the corresponding target respiratory pathogen antigen is present in the mask-captured sample.

[0083] The aforementioned technical processes work synergistically to achieve a complete transformation from mask samples to test results. The separation and wetting of the test area provides the necessary liquid-phase environment and biological activity basis for the antigen-antibody reaction; the fit between the inner and outer lids of the reaction chamber, driven by physical pressure, enables the precise release and directional transport of trace detection complexes, ensuring high reaction specificity; the rinsing step effectively reduces background noise by removing non-specific bindings, highlighting the colorimetric signal of antigen-mediated gold nanoparticle aggregation. Each step strictly corresponds to the physical structural characteristics of the reagent kit, simplifying the complex immunochemical reaction into a standardized mechanical operation procedure. This method is an in vitro detection method for ex vivo samples and does not include steps for disease diagnosis or treatment in humans or animals.

[0084] In some embodiments, a disposable sampling mask that has captured exhaled aerosols is obtained by the subject wearing it for 0.5 to 1 hour while awake, and the test area is separated from the meltblown fabric layer of the disposable sampling mask.

[0085] A conscious state refers to the process of wearing a mask when the subject is fully conscious and able to control their breathing rhythm, excluding non-natural physiological states such as sleep, anesthesia, or sedation. This state limitation aims to eliminate differences in aerosol emission dynamics caused by decreased respiratory rate, tidal volume changes, or special body positions during sleep, ensuring that the collected exhaled aerosols accurately reflect the level of respiratory pathogens carried by the subject during normal waking activities. For children, wearing the mask in a conscious state requires the child to complete the sampling under relatively quiet and cooperative conditions, avoiding interference from drastic fluctuations in breathing patterns caused by crying or strenuous exercise. In this state, the aerosol flow generated by the subject's natural breathing can stably penetrate the inner layer of the mask and be effectively intercepted by the intermediate layer.

[0086] The wearing time was set at 0.5–1 hour, a time window determined based on a balance between aerosol enrichment efficiency and detection sensitivity. If the wearing time is less than 0.5 hours, the number of pathogen particles captured by the meltblown fabric layer may fall below the lower limit of the detection method, increasing the risk of false negatives. If the wearing time is longer than 1 hour, although it increases the capture amount, it may lead to excessive humidity inside the mask, reducing wearing comfort and potentially altering the physical state of the sample or increasing non-specific adsorption. Therefore, 0.5–1 hour is an experimentally validated preferred range, achieving the best balance between sample detectability and operational compliance. During this time period, the subject continues to breathe, causing aerosols containing the target respiratory pathogen antigens to continuously deposit on the mask's filter material.

[0087] The test area is separated from the meltblown fabric layer of a disposable sampling mask. Disposable sampling masks typically employ a multi-layered composite structure. The outer layer is a hydrophobic barrier layer to block external liquid splashes; the inner layer is a hydrophilic soft layer to absorb moisture and provide wearing comfort; the meltblown fabric layer, as the middle layer, is a high-efficiency filter material made of ultrafine fibers such as polypropylene through high-temperature meltblowing, and is the core functional layer in the mask that actually captures aerosol particles. Limiting the test area to separation from the meltblown fabric layer means that during the sample acquisition step, the composite structure of the mask must be physically opened to precisely locate and cut or tear off a specific area of ​​the meltblown fabric layer, rather than directly using the entire mask or other layers. This operation ensures that the carrier contacted by the subsequent detection reaction is the filter medium actually enriched with pathogens, effectively avoiding interference from the outer or inner layer materials on the detection reaction, thus guaranteeing the accuracy of the test results. The separation process may include cutting along preset notches or break lines to obtain a standardized test sample piece.

[0088] In some embodiments, the reaction time is 1–10 minutes, and the rinsing time is 10–60 seconds. Limiting the reaction time to 1–10 minutes aims to cover the kinetic processes required for the specific binding of the antibody to the target respiratory pathogen antigen and the aggregation of the gold nanoparticles. Setting a lower limit of 1 minute ensures sufficient contact between the antibody-gold nanoparticle complex and the antigen to form a stable immune complex, thereby avoiding false negative results due to inadequate reaction. Setting an upper limit of 10 minutes helps prevent the accumulation of nonspecific adsorption over time, which reduces the signal-to-noise ratio, while meeting the timeliness requirements of rapid detection scenarios. In practical applications, the reaction time can be set according to the ambient temperature or a fixed value recommended in the kit instructions, such as 3 minutes, 5 minutes, or 8 minutes.

[0089] After the colorimetric reaction is complete, the second reaction solution is used to remove unbound free detection complexes and other non-specific adsorbed substances from the surface of the detection area. The duration of this process is called the rinsing time, which is limited to 10-60 seconds. The lower limit of 10 seconds ensures basic cleaning effect and effectively removes background interference; the upper limit of 60 seconds is used to prevent excessive rinsing from causing the bound gold nanoparticle complexes to detach, thereby ensuring the stability of the colorimetric signal. In actual operation, the operator can use a stopwatch or timer to precisely control the rinsing time, for example, setting it to 15 seconds, 30 seconds, or 45 seconds.

[0090] In some embodiments, the detection method further includes: determining that the in vitro detection process is valid when a colorimetric signal is generated in the quality control detection area; Provided that this in vitro detection process is effective, if a colorimetric signal is generated in the detection area corresponding to any target pathogen, it is determined that the antigen of that target pathogen is present in the mask-captured sample.

[0091] The quality control detection area serves as a crucial reference for verifying the completeness of the kit's functionality and the correctness of the operational procedures. Its internal microcavities are pre-filled with an anti-BSA-gold nanoparticle complex, while the first reaction solution used to wet the test area contains bovine serum albumin (BSA). When the inner lid of the reaction chamber is attached to and pressed against the test area, the first reaction solution dissolves and releases the anti-BSA-gold nanoparticle complex into the quality control detection area. The BSA in the solution rapidly undergoes a specific immune reaction with the anti-BSA-gold nanoparticle complex. Because the BSA protein acts as a bridge in the solution, it connects multiple antibody-labeled gold nanoparticles, leading to high-density aggregation of the gold nanoparticles. This aggregation causes a redshift in the local surface plasmon resonance absorption peak of the gold nanoparticles, resulting in a visually perceptible color change in the quality control detection area, such as a change from red to bluish-gray or purplish-brown.

[0092] The aforementioned color change demonstrates, in principle, that the series of physicochemical processes—wetting, release, diffusion, and immunobinding—have all occurred normally. This means the first reaction solution has successfully infiltrated the test area, the detection complex within the microcavity has been successfully released, and an effective antigen-antibody reaction and gold nanoparticle aggregation and color development have occurred, thus confirming the validity of the in vitro detection process. Conversely, if the control test area does not develop color, it indicates an abnormality in at least one of the aforementioned key steps, such as insufficient reaction solution, microcavity blockage, or complex inactivation. In this case, further pathogen identification should not be performed, and the test should be deemed invalid.

[0093] After confirming the color development of the quality control testing area to establish the effectiveness of the detection system, the corresponding testing areas for each target pathogen are interpreted. The target pathogen-corresponding testing area indicates whether the mask-captured sample contains a specific pathogen component. Its color development mechanism relies on specific immune binding: if a corresponding target respiratory pathogen antigen is captured on a specific testing area of ​​the mask, when an antibody-gold nanoparticle complex containing the corresponding specific antibody is released into that area, the antibody binds to the target antigen. Since the antigen on the testing area is usually encapsulated by proteins in aerosols or presents as a multivalent antigenic epitope, it can simultaneously bind to multiple antibody-labeled gold nanoparticles, thereby inducing cross-linking and aggregation of the gold nanoparticles, thus generating a visible color signal.

[0094] Based on the premise that the system has been proven to be functioning correctly in the quality control area, once a colorimetric signal is observed in the detection area corresponding to any target pathogen, it can be confirmed that the signal is a physical aggregation reaction caused by specific immune binding, rather than non-specific adsorption or interference from impurities. This confirms the presence of the target pathogen antigen in the mask-captured sample. Through this dual confirmation logic of first verifying the system's effectiveness and then performing qualitative analysis, the interference of false positive or false negative results is minimized, ensuring the reliability of the in vitro detection results.

[0095] In one specific embodiment, the detection method includes: When using the test kit, remove the disposable sampling mask from the test kit described in the first aspect. The person being tested should wear the mask for 0.5 to 1 hour while awake, ensuring that the circular test area in the meltblown fabric layer of the mask is directly facing the wearer's mouth and nose. After wearing, remove the mask and allow it to dry at room temperature.

[0096] Cut open the mask, remove the middle meltblown fabric layer, and tear off the circular test area along the outer perimeter of the test area using the scoring or dotted lines. Place the circular test area into the first base box, preferably with the inside of the mask facing up and the side with the printed number facing down. Add the first reaction solution A to the first base box to moisten the circular test area.

[0097] Remove the inner lid of the reaction chamber from the aluminum foil packaging. Place it face down over the circular test area, ensuring the microcavity number on the inner lid corresponds to the detection area number on the circular test area. Then, place the outer lid of the reaction chamber over the inner lid and apply downward pressure using the handle to ensure a tight seal between the inner lid and the test area on the mask. At this point, the antibody-gold nanocomposite in the microcavities is released under the action of the first reaction solution and flows to the corresponding detection area.

[0098] If the target pathogen antigen is present in the area of ​​the mask to be tested, the target pathogen antigen will specifically bind to the antibody-gold nanoparticle complex, forming a bridged aggregate structure of gold nanoparticles. After the gold nanoparticles aggregate, the absorption peak red-shifts, causing the corresponding detection area to appear blue-purple or other recognizable color changes.

[0099] After approximately 5 minutes of reaction, remove the outer cover of the reaction chamber and peel off the inner cover using tweezers. Pour the second reaction solution B into the second bottom box, use tweezers to pick up the circular test area, and rinse it in the second reaction solution B for approximately 30 seconds. After rinsing, place the circular test area flat on a dry surface and observe the color change in each test area.

[0100] In this embodiment, detection area 1 corresponds to SARS-CoV-2 NP, detection area 2 corresponds to H1N1 HA, detection area 3 corresponds to RSV FP, and detection area 4 is the quality control area.

[0101] Since the first reaction solution A contains BSA, and microcavity 4 is pre-filled with an anti-BSA-gold nanocomplex, a colorimetric signal should be generated in control zone 4 under correct operating conditions. If no colorimetric signal is generated in control zone 4, it indicates that there may be an operational error, reagent failure, or abnormal sample processing during the detection process, and the result of this test is invalid.

[0102] Under the premise that color development is achieved in control zone 4, observe whether color development signals are generated in detection zones 1, 2, and 3. If color development is observed in detection zone 1, it indicates the presence of SARS-CoV-2 NP corresponding antigen in the mask-captured sample; if color development is observed in detection zone 2, it indicates the presence of H1N1 HA corresponding antigen in the mask-captured sample; and if color development is observed in detection zone 3, it indicates the presence of RSV FP corresponding antigen in the mask-captured sample. If no color development is observed in detection zones 1, 2, and 3, but color development is observed in control zone 4, it indicates that the above-mentioned target pathogen antigens in the mask-captured sample were not detected in this in vitro test.

[0103] It should be noted that the SARS-CoV-2 NP, H1N1 HA, and RSV FP mentioned above are merely exemplary detection targets. Those skilled in the art can replace the pre-placed antibody-gold nanocomplex in the microcavity with specific recognition complexes corresponding to other respiratory pathogen antigens, based on actual detection needs, thereby achieving in vitro detection of biomarkers related to other viruses, bacteria, mycoplasma, chlamydia, or other pathogens.

[0104] The respiratory pathogen detection kit and its in vitro detection method based on mask sampling provided in this application have the following advantages compared with the prior art: 1. The embodiments of this application use masks to capture exhaled aerosols as test samples, eliminating the need to collect nasal or pharyngeal swabs, making them suitable for young children and people with poor sampling cooperation.

[0105] 2. This application combines the sampling carrier with the detection carrier, and the area to be tested comes directly from the meltblown fabric layer of the sampling mask, which reduces the sample transfer steps and lowers the complexity of operation.

[0106] 3. In this embodiment, different detection complexes are pre-placed in the microcavity array of the inner cover, which enables parallel detection of multiple respiratory pathogen antigens on the test area of ​​the same sampling mask.

[0107] 4. The embodiments of this application adopt the principle of nano-gold aggregation and color development. The detection results can be observed with the naked eye without the need for complicated detection instruments, making it suitable for home, primary healthcare or rapid screening scenarios.

[0108] 5. In the embodiments of this application, a quality control area is set up. For example, the BSA in the first reaction solution reacts with the anti-BSA-gold nanocomposite to form a quality control color development, which can determine whether the detection process is effective and improve the reliability of the results.

[0109] 6. The detection target of this application embodiment can be expanded by replacing the corresponding specific antibody, and is applicable to the in vitro detection of various viruses, bacteria or other respiratory pathogen antigens.

[0110] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0111] The above-described embodiments are only used to illustrate the technical solutions of the embodiments of this application, and are not intended to limit them. Although the embodiments of this application have been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of the embodiments of this application.

Claims

1. A respiratory pathogen detection kit based on mask sampling, characterized in that, include: A disposable sampling mask, the disposable sampling mask including a separable test area for capturing the wearer's exhaled aerosol, the test area being provided with multiple detection zones and positioning marks corresponding one-to-one with the multiple detection zones; The reaction box inner cover includes a carrier layer and a plurality of microcavities disposed on the carrier layer. The plurality of microcavities are disposed corresponding to a plurality of detection areas, and each microcavity is pre-filled with a detection complex. The outer cover of the reaction chamber is used to apply pressure to the inner cover of the reaction chamber when the inner cover of the reaction chamber is in contact with the area to be tested. The reaction chamber is used to support the area to be tested and contain the reaction solution. The reaction solution includes at least a first reaction solution for wetting the test area and a second reaction solution for rinsing the test area; The detection complex comprises gold nanoparticles and recognition molecules attached to the surface of the gold nanoparticles. The recognition molecules can specifically bind to the target respiratory pathogen antigen. When the test area is wetted by the first reaction solution, and the microcavity of the inner cover of the reaction box is in contact with and pressurized against the detection area of ​​the test area, the detection complex is released from the microcavity to the corresponding detection area. If the target respiratory pathogen antigen is present in the detection area, the detection complex specifically binds to the target respiratory pathogen antigen and causes the gold nanoparticles to aggregate, resulting in a visual color signal in the corresponding detection area.

2. The respiratory pathogen detection kit based on mask sampling as described in claim 1, characterized in that, The disposable sampling masks include: The outer layer is used to prevent external liquids from seeping into the inside of the mask; The inner layer is used to absorb the moisture exhaled by the wearer; The intermediate layer is sandwiched between the outer layer and the inner layer. The intermediate layer is a meltblown fabric layer. The area to be tested is located in the middle of the meltblown fabric layer and corresponds to the wearer's mouth and nose area when worn.

3. The respiratory pathogen detection kit based on mask sampling as described in claim 2, characterized in that, The area to be tested is a circular area, and the outer periphery of the circular area is provided with scratches, dotted lines or easy-tear lines to facilitate separation from the meltblown fabric layer; at least two detection zones are provided within the circular area.

4. The respiratory pathogen detection kit based on mask sampling as described in claim 3, characterized in that, The diameter of the circular region is 3-8 cm; The circular area is provided with 4 circular detection zones, which are evenly distributed within the circular area and are respectively provided with positioning marks numbered 1 to 4.

5. The respiratory pathogen detection kit based on mask sampling as described in claim 1, characterized in that, The inner cover of the reaction chamber includes a hydrophilic porous carrier layer and hydrophobic barrier layers disposed on both sides of the carrier layer. One side of the hydrophobic barrier layer has multiple through-holes, which, together with the carrier layer and the hydrophobic barrier layer on the other side, form the microcavity; and / or, The outer cover of the reaction chamber includes a circular pressure plate and a handle disposed on the circular pressure plate. The circular pressure plate is used to press the inner cover of the reaction chamber and the area to be tested; and / or The reaction chamber is a circular box, and the kit contains at least two reaction chambers, which are used for wetting reaction and rinsing treatment, respectively.

6. The respiratory pathogen detection kit based on mask sampling as described in claim 1, characterized in that, The microcavity is also pre-filled with polystyrene sulfonate. The detection complex is dried and fixed within the microcavity and can be released into the test area under the action of the first reaction solution; and / or, The recognition molecule is an antibody, and the detection complex is an antibody-gold nanocomplex. The antibody-gold nanocomplexes in the different microcavities correspond to different target respiratory pathogen antigens, and each gold nanoparticle is linked to only one specific antibody; and / or, The target respiratory pathogen antigens include one or more of SARS-CoV-2 NP, H1N1HA, and RSVFP; At least one of the plurality of microcavities is pre-filled with an anti-BSA-gold nanocomposite as a quality control composite; and / or, The first reaction solution is a PBS solution containing BSA, and the second reaction solution is a PBST solution.

7. The respiratory pathogen detection kit based on mask sampling as described in claim 6, characterized in that, The surface of the gold nanoparticles in the antibody-gold nanocomplex is connected to HS-PEG-COOH, and after activation by EDC / sulfo-NHS, they are linked to the secondary antibody, and after blocking treatment, they bind to the specific monoclonal antibody corresponding to the target respiratory pathogen antigen.

8. A method for in vitro detection using the respiratory pathogen detection kit based on mask sampling according to any one of claims 1 to 7, characterized in that, Includes the following steps: Provide a disposable sampling mask that has captured exhaled aerosols, and separate the area to be tested from the disposable sampling mask; The area to be tested is placed in the reaction box, and the first reaction solution is added to wet the area to be tested; Place the microcavity surface of the inner cover of the reaction box facing the area to be tested, and align the microcavity with the detection area; The outer cover of the reaction box is placed above the inner cover of the reaction box and pressure is applied to release the detection complex in the microcavity and guide it to the corresponding detection area; After the reaction is complete, remove the outer cover and the inner cover of the reaction box, and rinse the area to be tested with the second reaction solution; Observe the color development of each detection area in the area to be tested to determine whether the mask-captured sample contains the corresponding target respiratory pathogen antigen; The method described herein is an in vitro detection method for isolated samples and does not include steps for diagnosing or treating diseases in humans or animals.

9. The method as described in claim 8, characterized in that, The disposable sampling mask that has captured exhaled aerosols is obtained by the subject wearing it for 0.5 to 1 hour while awake, and the test area is separated from the disposable sampling mask; and / or, The reaction time is 1 to 10 minutes, and the rinsing time is 10 to 60 seconds.

10. The method as described in claim 8, characterized in that, When a colorimetric signal is generated in the quality control area, the in vitro testing process is deemed valid. Provided that the in vitro detection process is effective, if a colorimetric signal is generated in the detection area corresponding to any target pathogen, it is determined that the target respiratory pathogen antigen is present in the sample captured by the mask.

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