Bicolor quantum dot visual quantitative monkey pox antigen detection kit and application thereof

Abstract

The invention discloses a bicolor quantum dot visual quantitative type monkey pox antigen detection kit and application thereof, the bicolor quantum dot visual quantitative type monkey pox antigen detection kit comprises a chromatography test strip, and the chromatography test strip is composed of a sample pad, a combination pad, an NC membrane and a water absorption pad which are sequentially overlapped and fixed on a plastic backboard; the combination pad is sprayed with an iPQDS immunochromatography probe and an rQD-BSA-biotin reference probe solution to form an immunoreaction hue recognition system; a T line coated with MPXV A29L mAb2 and SA and a C line coated with a goat anti-mouse IgG antibody are sprayed on the NC membrane. Dendritic silicon dioxide with central-radial orientation and a macroporous structure is used as a template, so that efficient loading of green fluorescent quantum dots is realized; pEI is used as a connecting layer, so that layer-by-layer assembly of the QD is realized, and the stacking density of the quantum dots is remarkably improved; a green QD assembly probe-red QD reference probe co-chromatography color recognition mode is adopted, so that signal amplification and accurate visual interpretation and quantification are realized.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a two-color quantum dot visualization quantitative monkeypox antigen detection kit and its application. Background Technology

[0003] Lateral flow immunochromatography (LFIA) is a proven point-of-care diagnostic technique that offers an easy-to-use, cost-effective, portable, and rapid on-site testing solution. However, commercially available immunochromatographic reagents using colloidal gold nanoparticles (AuNPs) have limitations such as low sensitivity, inaccurate quantification, and inability to detect multiple targets. Color recognition analysis is a visualization and image-based detection method based on spectral composition analysis, suitable for naked-eye qualitative interpretation and quantitative analysis of smartphone images. This method, based on the principles of three primary color mixing and color decomposition decoding, enables the simultaneous detection of multiple targets. The real-time diagnostic technology employing the RGB three-primary-color mode has the following core advantages: First, this mode highly aligns with the physiological basis of human visual perception—red, green, and blue cone cells in the human retina respond to specific wavelengths, respectively. By using spectrally matched RGB luminescent probes, high-precision and high-reliability naked-eye identification can be achieved. Second, this mode is highly compatible with the color perception mechanism of machine vision. RGB signal input can directly match the color separation and reconstruction process of the Bayer array color filter system in a mobile phone CMOS sensor (generating raw RGB data through color filters and converting it into a standard three-primary-color image through image processing), thereby effectively simplifying the image analysis process and improving the signal-to-noise ratio. Based on the consistency between human vision and machine vision in color perception, selecting red, green, and blue three-primary-color luminescent materials with narrow emission characteristics as signal probes not only significantly enhances visual discrimination but also minimizes color information loss during image acquisition and reconstruction, thereby comprehensively improving the analytical performance and diagnostic reliability of immunochromatographic images.

[0004] Quantum dots, as an emerging class of fluorescent labeling materials, possess extremely high fluorescence quantum efficiency (close to 100%), zero bleaching, and high color purity (half-width at half maximum < 30 nm), giving them unique advantages in color recognition analysis. However, the small size of individual quantum dots (5-10 nm) makes them impossible to separate, and they are unstable to chemical modification, resulting in limited luminescence intensity of the prepared probes. Therefore, developing quantum dot assembly strategies, such as encapsulating multiple quantum dots in polymer or silica bundles to prepare multi-level quantum dot structures, provides high luminescence and stability. Porous silicon templates, with their large internal space and high optical transparency, are ideal templates for encapsulating large numbers of quantum dot units as aqueous dispersion components. Currently used quantum dot assembly strategies, including evaporation-induced self-assembly, solid template surface assembly, and near-surface embedding in latex swelling, suffer from problems such as uncontrolled size dispersion, inability to enter the internal space, and limited / non-uniform quantum dot embedding, respectively. Therefore, a well-designed template-based assembly strategy with uniform and compact quantum dot encapsulation is beneficial for achieving the high brightness and good color purity required for LFIA applications.

[0005] This invention proposes a three-dimensional layer-by-layer sequential assembly and modification method for quantum dot building blocks within a central-radial channel template. Utilizing a silane bonding layer and a polymer intermediate layer, a metal-ligand affinity assembly driving force is constructed, enabling the three-dimensional assembly of oil-phase quantum dots within the template. This results in a tightly packed / efficient "brick-and-mortar" structure with coordination bonds. The luminescence properties of the quantum dots are maintained through a low-damage modification method using a hydrophobic layer / composite silanization layer, thereby enhancing the brightness sensitivity of the tag material. A novel color recognition immunochromatographic mode based on narrow-emission dual-color quantum dots is established, replacing the colloidal gold light-dark gradient with a green-red gradient. This improves the visual recognition of the results and the color information acquisition and analysis performance of smartphone terminals, providing accurate and highly sensitive detection results for MPXV infection in a home-based point-of-care diagnostic solution. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit and its applications.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit, comprising a chromatographic test strip; the chromatographic test strip consists of a sample pad, a conjugate pad, an NC membrane, and an absorbent pad sequentially overlapped and fixed to a plastic backing plate; the conjugate pad is immersed in a processing buffer and sprayed with iPQDS immunochromatographic probe and rQD-BSA-biotin reference probe solution to form an immunochromatographic recognition system; the NC membrane is sprayed with a T line coated with MPXV A29L mAb2 and streptavidin and a C line coated with goat anti-mouse IgG antibody.

[0008] Further, the treatment buffer comprises 40-75 mM PB solution, 0.8-1.5 wt% BSA, 0.8-1.5 wt% Tween 20 and 0.04-0.075 wt% sodium caseinate, with a pH of 7.3-7.5.

[0009] Furthermore, the iPQDS immunochromatographic probe solution comprises 0.008~0.015M PBS solution and 3.5~6 mg / mL... -1 iPQDS immunoassay probe, 0.08–0.15 wt% BSA and 0.008–0.015 wt% Tween 20.

[0010] Furthermore, the iPQDS immunoassay probe is prepared through the following steps: (a.1) Add 55-100g of triethanolamine to ultrapure water, heat and stir, then add 300-570mg of cetyltrimethylammonium bromide and 135-250g of sodium salicylate and react for 1h; add 3.5-6ml of tetraethyl orthosilicate and react for 2h, centrifuge and wash with ethanol to obtain a precipitate; react the precipitate in a mixture of hydrochloric acid / methanol at 60℃ for 6h, then wash with ethanol to obtain SiO2 microspheres; (a.2) SiO2 microspheres were assembled layer by layer using green fluorescent QD chloroform dispersion and PEI ethanol solution to obtain a first-layer assembly, a second-layer assembly and a third-layer assembly; (a.3) The first, second, or third layer assembly was dried under a nitrogen gas flow and then dissolved in OTMS under sonication. Methanol and ammonia were then added, followed by sonication and centrifugation to obtain the product. The product was washed with methanol, and the precipitate was dispersed in water and ammonia with stirring to form an assembly precipitate. The assembly precipitate was dispersed in a mixture of ethanol, water, and ammonia, and TEOS was added and stirred once to obtain PQDS. After centrifugation and washing, the PQDS was dispersed again in ethanol and ammonia, and APTMS was added and stirred once. After centrifugation and washing, the precipitate was dispersed and stirred in DMF containing succinic anhydride to obtain PQDS-COOH precipitate, which was then centrifuged, washed, and dispersed in a solution with a concentration of 4 mg / mL. -1 PQDS-COOH solution was obtained from PB solution; (a.4) Take 50 μL of a solution with a concentration of 10 mg / mL -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added to 1 mL of PQDS-COOH solution, shaken and centrifuged, washed with PB solution and resuspended in 1 mL of PBS solution. Then, 8.46 μL of a 9.47 mg / mL solution was added. -1MPXV A29L mAb1 was shaken for 2.5 h; then shaken with ethanolamine to obtain the iPQDS immunoassay probe.

[0011] Furthermore, step (a.2) specifically includes the following sub-steps: (a.2.1) Add 0.25-1.5 mL of green fluorescent QD chloroform dispersion to 8-15 mg SiO2 microspheres, sonicate for 7 min and centrifuge, then remove free QD with chloroform to obtain the first layer assembly; (a.2.2) The first layer assembly was prepared at a concentration of 8-15 mg / mL in 0.8-1.5 mL. -1 After sonication in PEI ethanol solution, the supernatant was removed by centrifugation, and the precipitate was washed with ethanol to obtain SQ1P. (a.2.3) Repeat step (a.2.1) with SQ1P to obtain the second layer assembly; (a.2.4) Repeat steps (a.2.2) and (a.2.1) on the second-layer assembly to obtain the third-layer assembly.

[0012] Further, the rQD-BSA-biotin reference probe solution is prepared through the following steps: (b.1) Dissolve 90–160 μL of MPA in 20 mL of water and adjust the pH to 8.5–9.5 with NaOH solution; then add 20 mL of a solution with a concentration of 0.8–15 mg / mL. -1 A chloroform solution of red luminescent QD was stirred at 60°C for 1 h to obtain a crude solution, which was then ultrafiltered using an MWCO~3000 membrane. Subsequently, it was ultrafiltered with PB solution to obtain rQD-MPA. (b.2) Dissolve 8-15 mg BSA in 5 mL of PB solution to obtain BSA solution. Dissolve 0.8-1.5 mg EZ-link-sulfo-NHS-LC-biotin in 180 μL of PB solution and transfer it to BSA solution. Stir for 1 h. Then use MWCO~3000 membrane for ultrafiltration and wash with PB solution to obtain BSA-biotin solution. (b.3) Disperse 1 mg rQD-MPA in PB solution and add 50 μL of a solution with a concentration of 10 mg / mL. -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added and stirred for 25 min; then ultrafiltration was performed using an MWCO~3000 membrane, and the sample was washed with PB solution. The sample was dispersed in 1 mL of PB solution, and then 100 μL of BSA-Biotin solution was added and stirred for 2.5 h to obtain the rQD-BSA-Biotin reference probe solution.

[0013] Furthermore, the conjugate pad is prepared by the following method: Immerse the conjugate pad in the treatment buffer, remove and dry; then prepare a solution with a concentration of 3.5–6 mg / mL. -1 The iPQDS immunochromatographic probe solution has a concentration of 0.16~0.3 mg / mL. -1 rQD-BSA-Biotin reference probe solution at 5 μL cm -1 The solution is sprayed evenly onto the bonding pad at a rate of [missing information], and dried at 37°C for at least 4 hours to obtain the bonding pad.

[0014] Furthermore, the NC film is prepared by the following method: The concentration was 1.2~2.2 mg / mL. -1 MPXV A29L mAb2 and concentrations of 0.4~0.75 mg / mL -1 A mixed solution of streptavidin was prepared at 1.0 μL cm⁻¹. -1 The rate was plotted as a T-line on the NC membrane, and concentrations of 1.2–2.2 mg / mL were measured. -1 Goat anti-mouse IgG antibody was administered at 1.0 μL cm -1 The rate was drawn as a C line on the NC film, and the NC film was obtained after drying at 37°C.

[0015] Secondly, this invention provides an application of a two-color quantum dot visualization quantitative monkeypox antigen detection kit in the detection of monkeypox virus.

[0016] Furthermore, the application specifically includes the following steps: (c.1) Sample collection: Using sterile collection tools, monkeypox virus herpes fluid is directly aspirated or dipped from the herpes lesions within 1 minute; (c.2) The collected monkeypox virus herpes fluid was then lysed in running buffer for 1 min and then dropped onto the sample pad in the two-color quantum dot visualization quantitative monkeypox antigen detection kit; (c.3) Result Interpretation: Result interpretation begins 10 minutes after the immunochromatographic reaction: When the C line on the NC membrane is green and the T line is pure red, the monkeypox virus test result is negative; when the C line on the NC membrane is green and the T line is orange-red, orange, yellow, yellow-green, or pure green, the monkeypox virus test result is positive, and the viral load gradually increases in the order of orange-red-orange-yellow-yellow-green-pure green; when the C line on the NC membrane does not show color, the test result is invalid, and the kit should be replaced and the test repeated.

[0017] The beneficial effects of this invention are: 1) Using center-radial oriented, macroporous dendritic silica as a template, efficient loading of fluorescent quantum dots is achieved; 2) By using PEI as a connecting layer, layer-by-layer assembly of QDs was achieved, which significantly improved the packing density of quantum dots and enhanced the fluorescence signal intensity; 3) A red-green QD dual-color probe collaborative recognition mode was designed, which breaks through the limitations of traditional single-color signals; 4) Utilizing the high affinity between immune capture and streptavidin-biotin, the signal probe and reference probe are ensured to be uniformly mixed; 5) The target protein selected for detection in this invention is the newly emerging monkeypox virus A29L protein, and there are currently no mature related products on the market. Attached Figure Description

[0018] Figure 1 This document presents a flowchart of the preparation process for quantum dot-filled three-dimensional fluorescent microspheres (PQDS), along with a cross-sectional structure and a schematic diagram of the molecular structure of the PQDS. Figure 1 In the diagram, 'a' represents the fabrication process of quantum dot-filled three-dimensional fluorescent microspheres (PQDS). Figure 1 b in the figure is a schematic diagram of the cross-sectional structure and molecular structure of PQDS; Figure 2 Transmission, scanning, and cross-sectional transmission electron microscopy images of SiO2 microspheres, the first-layer assembly, the second-layer assembly, the third-layer assembly, and PQDS, among which, Figure 2 a1 in the image is a transmission electron microscope image of SiO2 microspheres; Figure 2 a2 in the image is a transmission electron microscope image of the first layer assembly; Figure 2 a3 in the image is a transmission electron microscope image of the second-layer assembly; Figure 2 a4 in the image is a transmission electron microscope image of the third-layer assembly; Figure 2 a5 in the image is a transmission electron microscope image from PQDS; Figure 2 b1 in the image is a scanning electron microscope image of SiO2 microspheres; Figure 2 b2 in the image is a scanning electron microscope image of the first layer assembly; Figure 2 b3 in the image is a scanning electron microscope image of the second-layer assembly; Figure 2 b4 in the image is a scanning electron microscope image of the third-layer assembly; Figure 2 b5 in the image is a scanning electron microscope image from PQDS; Figure 2 c1 in the image is a cross-sectional transmission electron microscope image of SiO2 microspheres; Figure 2 c2 in the image is a cross-sectional transmission electron microscope image of the first layer assembly; Figure 2 c3 in the image is a cross-sectional transmission electron microscope image of the second-layer assembly; Figure 2 c4 in the image is a cross-sectional transmission electron microscope image of the third-layer assembly; Figure 2c5 in the image is a cross-sectional transmission electron microscope image of the PQDS. Figure 3 This is a graph showing the change in loading ratio as a function of feed ratio during quantum dot assembly. Figure 4 The fluorescence intensity varies with wavelength and is plotted in the visible light spectrum. Figure 5 The correlation between fluorescence intensity and probe particle number for a single quantum dot modified with mercaptopropionic acid; Figure 6 The correlation between the fluorescence intensity of PQDS-COOH precipitate and the number of probe particles is shown in the figure. Figure 7 Correlation between the G channel number and the number of probe particles for a single quantum dot-biotin conjugate modified with mercaptopropionic acid. Figure 8 Correlation plot of G channel values ​​and probe particle number for PQDS-COOH precipitate-biotin conjugate; Figure 9 This document presents a schematic diagram of the detection process, detection principle, and detection results for a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. Figure 9 In the diagram, 'a' represents the detection flowchart of a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. Figure 9 In the diagram, b is a schematic representation of the detection principle of the dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. Figure 9 In the diagram, 'c' represents the detection results obtained using a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. Figure 10 This image shows the results of a dual-color quantum dot-based visualization and quantitative monkeypox antigen detection kit for monkeypox virus antigen detection and visual interpretation. Figure 11 The results of detection and visual interpretation of MPXV A29L using a single-color bright-dark type-LFIA based on colloidal gold. Figure 12 This image shows the detection results of a dual-color quantum dot-based visualization quantitative monkeypox antigen detection kit used on herpes fluid samples from monkeypox virus-infected individuals. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0020] This invention discloses a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit and its applications, and prepares an iPQDS immunoassay probe with high brightness and high color purity quantum dots. First, a centrally-radially oriented, macroporous dendritic silica (SiO2 microspheres) is selected as a template and modified with high-density alkyl thiols. Through metal-ligand coordination in the organic phase, green hydrophobic quantum dots are densely immobilized onto the template pore surface, forming an assembled structure. Then, using an amino-affinity polymer (polyethyleneimine, PEI) as an intermediate connecting polymer layer, the layer-by-layer assembly of green fluorescent quantum dots is achieved, significantly improving template utilization and the spatial integration of green quantum dots. A stepwise, low-damage modification method based on alkyl chain intercalation, hydrophobic silane micellization coating, and dense silica shell deposition is proposed. A color-recognition immunochromatographic system was established. Biotinylated red quantum dot reference probes and green quantum dot microsphere signal probes were mixed and chromatographically analyzed, producing a color band at the T-line on the NC membrane. As the concentration of MPXV A29L increased, the T-line changed from red (negative) to green (positive). The T-line co-captured the reference and signal probes through antibody-antigen immunoreaction and streptavidin-biotin affinity, effectively avoiding the uneven color mixing problem of pre-laid reference probe modes. This ensured accurate and vivid (antigen concentration-dependent) color gradation, suitable for precise and highly sensitive human visual interpretation and detailed quantitative analysis of mobile images.

[0021] Preparation of iPQDS immunoassay probes: (a.1) Preparation of center-radial macroporous SiO2 microspheres (dSiO2): 55-100 g of triethanolamine was added to ultrapure water and stirred in an 80 °C water bath for 30 min; 300-570 mg of cetyltrimethylammonium bromide and 135-250 g of sodium salicylate were added, and the reaction was continued for 1 h to obtain a reaction solution; then 3.5-6 mL of tetraethyl orthosilicate was added to the reaction solution, and the reaction was continued for 2 h to obtain a product; the product was centrifuged and washed three times with ethanol to obtain a precipitate; the precipitate was reacted in a mixture of hydrochloric acid and methanol at 60 °C for 6 h and repeated 2-3 times to obtain a final product, which was used to remove the residual organic template in the pores; after washing the final product with ethanol, the final product was dispersed in 50 mL of ethanol to obtain center-radial macroporous SiO2 microspheres.

[0022] (a.2) SiO2 microspheres were assembled layer by layer using green fluorescent QD chloroform dispersion and PEI ethanol solution to obtain a first-layer assembly, a second-layer assembly and a third-layer assembly.

[0023] Step (a.2) specifically includes the following sub-steps: (a.2.1) Add 0.25-1.5 mL of a 10 mg / mL solution to 8-15 mg SiO2 microspheres. -1The green fluorescent QD chloroform dispersion was sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the first layer assembly (SQ1).

[0024] (a.2.2) The first layer assembly was prepared at a concentration of 8-15 mg / mL in 0.8-1.5 mL. -1 Sonicate in PEI (MW~1800) ethanol solution for 10 minutes, centrifuge to remove supernatant, wash precipitate with ethanol to obtain SQ1P.

[0025] (a.2.3) and add 0.25~1.5 mL of a 10 mg / mL solution to SQ1P. -1 The green fluorescent QD chloroform dispersion was obtained and sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the second-layer assembly (SQ2).

[0026] (a.2.4) Then the second layer assembly was prepared at a concentration of 8-15 mg / mL in 0.8-1.5 mL. -1 The sample was sonicated in PEI (MW~1800) ethanol solution for 10 minutes, centrifuged to remove the supernatant, and the precipitate was washed with ethanol to obtain SQ2P. Then, 0.25~1.5 mL of a 10 mg / mL solution was added to the SQ2P. -1 The green fluorescent QD chloroform dispersion was obtained and sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the third-layer assembly (SQ3).

[0027] (a.3) To transfer the hydrophobic phase to the aqueous phase, the first-layer assembly (SQ1), the second-layer assembly (SQ2), or the third-layer assembly (SQ3) was slightly dried under a nitrogen gas flow and then dissolved in 260 μL of OTMS under sonication. Subsequently, 40 mL of methanol and 1 mL of ammonia were added, and the mixture was sonicated for 30 minutes. The product was then centrifuged and washed twice with methanol. The precipitate was dispersed in 40 mL of water and 80 μL of ammonia and stirred for 24 hours to form the assembly precipitate. A silica shell was generated using the Stöber method: the assembly precipitate was dispersed in a mixture containing 40 mL of ethanol, 10 mL of water, and 1.25 mL of ammonia. 25 μL of TEOS was added, and the mixture was stirred for 2 hours. This step was repeated once to obtain quantum dot-filled fluorescent microspheres (PQDS). The obtained PQDS was centrifuged and washed twice with ethanol, and then redispersed in 40 mL of ethanol and 0.5 mL of ammonia to obtain a dispersion. Add 20 μL APTMS to the dispersion and stir for 3 hours, then repeat this process once. Centrifuge, wash thoroughly with ethanol, and redisperse in 20 mL DMF containing 100 mg succinic anhydride, stirring for 12 hours to obtain PQDS-COOH precipitate. Centrifuge the PQDS-COOH precipitate, wash thoroughly with ethanol and water, and then redisperse in a 4 mg / mL solution.-1 PQDS-COOH solution was obtained by adding PB solution (phosphate buffer, 0.02M, pH=6.0).

[0028] (a.4) Take 50 μL of a solution with a concentration of 10 mg / mL -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added to 1 mL of iPQDS-COOH solution, vortexed for 30 min and centrifuged. After washing with PB solution, the solution was resuspended in 1 mL of PBS solution (phosphate-buffered saline, 0.02 M, pH = 7.4). Then, 8.46 μL of a 9.47 mg / mL solution was added. -1 MPXV A29L mAb1 was added and shaken for 2.5 h; then a concentration of 3 mg / mL was used. -1 The surface was blocked by shaking with ethanolamine for 2 hours to obtain the iPQDS immunoassay probe.

[0029] The prepared iPQDS immunoassay probe was then washed twice with PBS solution and dispersed in PBS solution with a concentration of 0.008-0.015 M containing 0.08-0.15 wt% BSA and 0.008-0.015 wt% Tween 20 to obtain the iPQDS immunochromatographic probe solution, which was stored at 4 °C.

[0030] Preparation of rQD-BSA-Biotin reference probe solution: (b.1) Dissolve 90–160 μL of MPA in 20 mL of water and adjust the pH to 8.5–9.5 with 1 M NaOH solution; then add 20 mL of a solution with a concentration of 0.8–15 mg / mL. -1 A two-phase solution was formed in chloroform of red luminescent quantum dots (rQDs). The solution was stirred at 60°C for 1 h to obtain a crude solution, thus transferring the hydrophobic rQD phase to the aqueous phase. The crude solution was then ultrafiltered using an MWCO~3000 membrane, followed by three ultrafiltrations with PBS solution to obtain water-soluble red luminescent quantum dots rQD-MPA.

[0031] (b.2) Dissolve 8-15 mg BSA in 5 mL of PB solution (0.02 M, pH=7.4) to obtain a BSA solution. Dissolve 0.8-1.5 mg EZ-link-sulfo-NHS-LC-biotin in 180 μL of PB solution (0.02 M, pH=7.4) and transfer it to the BSA solution. Stir at room temperature for 1 h. Then, perform ultrafiltration using a MWCO~3000 membrane and wash three times with PB solution (0.02 M, pH=7.4) to obtain a BSA-biotin solution, which is stored at 4 °C.

[0032] (b.3) Disperse 1 mg rQD-MPA in PB solution (0.02 M, pH=6.0) and add 50 μL of a solution with a concentration of 10 mg / mL. -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was stirred for 25 min; then ultrafiltration was performed using an MWCO~3000 membrane, followed by washing with PB solution (0.02 M, pH=6.0), dispersion in 1 mL PB solution (0.02 M, pH=7.4), and then 100 μL BSA-Biotin solution was added and stirred for 2.5 h to obtain the rQD-BSA-Biotin reference probe solution, which was stored at 4 °C.

[0033] This invention also provides a dual-color quantum dot visualization quantitative monkeypox antigen detection kit, comprising a chromatographic test strip; the chromatographic test strip consists of a sample pad, a conjugate pad, an NC membrane, and an absorbent pad sequentially overlapped and fixed to a plastic backing plate; the conjugate pad is immersed in a processing buffer and sprayed with iPQDS immunochromatographic probe and rQD-BSA-biotin reference probe solution to form an immunochromatographic recognition system; the NC membrane is sprayed with a T line coated with MPXV A29L mAb2 and streptavidin and a C line coated with goat anti-mouse IgG antibody (goat anti-mouse IgG antibody).

[0034] The conjugate pad is prepared by the following method: The conjugate pad was immersed in the treatment buffer for 12 hours, then removed and dried; subsequently, a concentration of 3.5–6 mg / mL was prepared. -1 The iPQDS immunochromatographic probe solution has a concentration of 0.16~0.3 mg / mL. -1 rQD-BSA-Biotin reference probe solution at 5 μL cm -1 The solution is sprayed evenly onto the bonding pad at a rate of [missing information], and dried at 37°C for at least 4 hours to obtain the bonding pad.

[0035] The treatment buffer solution comprises 40-75 mM PB solution, 0.8-1.5 wt% BSA, 0.8-1.5 wt% Tween 20, and 0.04-0.075 wt% sodium caseinate, with a pH of 7.3-7.5.

[0036] The NC membrane is prepared by the following method: The concentration was 1.2~2.2 mg / mL. -1 MPXV A29L mAb2 and concentrations of 0.4~0.75 mg / mL -1 A mixed solution of streptavidin (SA) was prepared at 1.0 μL cm⁻¹. -1 The rate was plotted as a T-line on the NC membrane, and concentrations of 1.2–2.2 mg / mL were measured. -1Goat anti-mouse IgG antibody was administered at 1.0 μL cm -1 The rate was drawn as a C line on the NC film, and the NC film was obtained after drying at 37°C.

[0037] The plastic backing plate, which is assembled from the sample pad, conjugate pad and absorbent pad, was cut into strips 3.80 mm wide using an automatic strip cutter (ZQ2002, Shanghai Jinbiao Biotechnology Co., Ltd.).

[0038] This invention also provides an application of a two-color quantum dot-based visualized quantitative monkeypox antigen detection kit in the detection of monkeypox virus, specifically including the following steps: (c.1) Sample collection: Using sterile collection tools (such as syringes or cotton swabs), directly aspirate or dip into the herpes lesions to collect monkeypox virus herpes fluid (MPXV herpes fluid) within 1 minute.

[0039] (c.2) The collected monkeypox virus herpes fluid was then lysed in running buffer for 1 min and then dropped onto the sample pad in the two-color quantum dot visualization quantitative monkeypox antigen detection kit.

[0040] The running buffer consists of 0.01M PBS solution, 0.2wt% BSA, and 0.4wt% Tween 20.

[0041] (c.3) Result Interpretation: Result interpretation begins 10 minutes after the immunochromatographic reaction: When the C line on the NC membrane is green and the T line is pure red, the monkeypox virus test result is negative; when the C line on the NC membrane is green and the T line is orange-red, orange, yellow, yellow-green, or pure green, the monkeypox virus test result is positive, and the viral load gradually increases in the order of orange-red-orange-yellow-yellow-green-pure green; when the C line on the NC membrane does not show color, the test result is invalid, and the kit should be replaced and the test repeated.

[0042] Detection principle: Collected monkeypox virus herpes fluid is dropped onto a sample pad, which then flows to a conjugate pad coated with an iPQDS immunoassay probe and an rQD-BSA-biotin reference probe. MPXVA29L mAb1 on the iPQDS immunoassay probe specifically binds to the MPXV A29L antigen in the monkeypox virus herpes fluid, forming an antigen-antibody-quantum dot complex. Subsequently, the antigen-antibody-quantum dot complex and the rQD-BSA-biotin reference probe flow to the NC membrane. Then, MPXV A29L mAb2 on the T line of the NC membrane captures the antigen-antibody-quantum dot complex, forming a double antibody sandwich structure, and the quantum dots aggregate and show color (such as green fluorescence). Streptavidin on the T line of the NC membrane captures the rQD-BSA-biotin reference probe, forming a visible color signal (and showing a gradient change from pure red to orange-red to orange to yellow to yellow-green to pure green as the concentration of MPXV A29L antigen increases). Unbound iPQDS immunoassay probes continue to move to the C line, where they are captured by goat anti-mouse IgG antibody, and the resulting color development serves as a marker of effective detection.

[0043] When the MPXV A29L antigen is not present in the monkeypox virus herpes fluid, MPXV A29L mAb1 does not bind specifically and flows directly to the T line, while the rQD-BSA-biotin reference probe is captured by streptavidin. Because the rQD-BSA-biotin reference probe has red quantum dots (rQD), the T line appears red. When the MPXV A29L antigen in monkeypox virus herpes fluid is at a low viral load, a small amount of MPXV A29L antigen specifically binds to MPXV A29L mAb1 to form an antigen-antibody-quantum dot complex. When this complex flows through the T line, it is captured by MPXV A29L mAb2. However, due to the small amount of antigen, the complex aggregation is low, and the green quantum dots (gQDs) are more dispersed, resulting in color mixing (red and green superimposed to form orange). Therefore, the T line appears orange. When the MPXV A29L antigen in monkeypox virus herpes fluid is at a moderate viral load, the distribution of green quantum dots in the T line is more concentrated, resulting in color mixing (red and green superimposed to form yellow), and the T line appears yellow. When the MPXV A29L antigen in monkeypox virus herpes fluid is at a high viral load, the distribution of green quantum dots in the T line is dense, causing the green to completely cover the red, and the T line will show green.

[0044] Key points and areas to be protected in this invention: 1.1) Dendritic silica template and directional assembly technology, characterized in that: a centrally-radially oriented, macroporous dendritic silica is used as a template, combined with the hydrophobic-metal coordination of the organic phase, to achieve efficient integration of green fluorescent quantum dots (QDs), construct an ordered multilayer structure, and improve the stability and signal transmission efficiency of the material.

[0045] 1.2) PEI Intermediate Layer Enhances Quantum Dot Packing Density: By using PEI (polyethyleneimine) as an intermediate connecting layer, the layer-by-layer assembly of green QDs is achieved, which significantly improves the quantum dot packing density, enhances signal intensity, and maintains structural uniformity, laying the foundation for subsequent color gradient formation.

[0046] 1.3) Red-Green QD Dual-Color Probe Collaborative Recognition System: A hybrid system of a red QD reference probe modified with biotin and a green QD signal probe is designed. The T-line color changes from red to green (as the MPXV A29L concentration changes) to achieve visualized quantitative detection, breaking through the limitations of traditional single-color signals.

[0047] 1.4) Streptavidin-Biotin synergistic mixing mechanism: Utilizing the high affinity between immune capture and streptavidin-biotin, the signal probe and reference probe are uniformly mixed, avoiding component separation and improving the accuracy and repeatability of detection results.

[0048] 1.5) Signal amplification and color gradient visualization technology: Through the cascade amplification effect of QD assembled signal probes, distinguishable color gradients are generated. Combined with visual observation and image quantification analysis, high-sensitivity and real-time detection can be achieved without complex equipment, taking into account both qualitative and quantitative needs.

[0049] Example 1: Preparation of iPQDS immunoassay probe.

[0050] (a.1) Preparation of center-radial macroporous SiO2 microspheres (dSi): 68 mg of triethanolamine was added to 25 mL of ultrapure water and stirred in an 80 °C water bath for 30 min. Then, 380 mg of hexadecyltrimethylammonium bromide and 168 mg of sodium salicylate were added, and the reaction was continued for 1 h to obtain a reaction solution. Subsequently, 4 mL of tetraethyl orthosilicate was added to the reaction solution, and the reaction was continued for 2 h to obtain the product. The product was centrifuged and washed three times with ethanol to obtain a precipitate. The precipitate was reacted in a hydrochloric acid / methanol mixture at 60 °C for 6 h and repeated twice to obtain the final product. After washing the final product with ethanol, the final product was dispersed in 50 mL of ethanol to obtain center-radial macroporous SiO2 microspheres.

[0051] (a.2) Preparation of the first layer assembly: Add different volumes (0.25~1.5 mL) of 10 mg / mL SiO2 microspheres to 10 mg SiO2 microspheres. -1The green fluorescent QD chloroform dispersion was prepared and sonicated for 7 min. After centrifugation, free QD was removed with chloroform to obtain the first layer assembly (SQ1). The relationship between the amount of green fluorescent QD chloroform dispersion and the loading ratio of the assembly was investigated. As the amount of green fluorescent QD chloroform dispersion added increased, the loading ratio also gradually increased. When the amount of green fluorescent QD chloroform dispersion increased from 25% (total amount of green fluorescent QD chloroform dispersion was 0.25 mL) to 150% (total amount of green fluorescent QD chloroform dispersion was 1.5 mL), the loading ratio increased from 25% to 119%.

[0052] (a.3) To transfer the hydrophobic phase to the aqueous phase, the first-layer assembly (SQ1) was slightly dried under a nitrogen gas flow and then dissolved in 260 μL of OTMS under ultrasonication. Subsequently, 40 mL of methanol and 1 mL of ammonia were added, and the mixture was ultrasonicated for 30 minutes. The product was then centrifuged and washed twice with methanol. The precipitate was dispersed in 40 mL of water and 80 μL of ammonia and stirred for 24 hours to form the assembly precipitate SQ1-OTMS. A silica shell was generated using the Stöber method: the assembly precipitate SQ1-OTMS was dispersed in a mixture containing 40 mL of ethanol, 10 mL of water, and 1.25 mL of ammonia. 25 μL of TEOS was added, and the mixture was stirred for 2 hours. This step was repeated once to obtain quantum dot-filled fluorescent microspheres PQDS. The obtained PQDS was centrifuged and washed twice with ethanol, and then redispersed in 40 mL of ethanol and 0.5 mL of ammonia to obtain a dispersion. Add 20 μL of APTMS to the dispersion and stir for 3 hours, then repeat this process once. Centrifuge, wash thoroughly with ethanol, and redisperse in 20 mL of DMF containing 100 mg succinic anhydride, stirring for 12 hours to obtain PQDS-COOH precipitate. Centrifuge the PQDS-COOH precipitate, wash thoroughly with ethanol and water, and then redisperse in a 4 mg / mL solution. -1 PQDS-COOH solution was obtained by adding PB solution (0.02M, pH=6.0).

[0053] Figure 1 The diagram in Figure 'a' is a flowchart of the preparation process of quantum dot three-dimensional filled fluorescent microspheres (PQDS). It shows the preparation process of pomegranate seed filled fluorescent microspheres (PQDS) by using central-radial macroporous SiO2 microspheres as templates and achieving three-dimensional high-density assembly of green hydrophobic quantum dots within the template through metal-ligand coordination in the organic phase and coordination mediated by affinity polymers.

[0054] Figure 1b in the diagram is a cross-sectional structure and molecular structure diagram of PQDS, showing the coordination-hydrophobic forces and chemical bonds and their layer-by-layer intercalation / encapsulation structure between the silicon template modified layer, quantum dot layer, affinity polymer layer, organosilicon layer, and dense silica layer. The prepared quantum dot-filled three-dimensional fluorescent microspheres (PQDS) use silica as a template, with affinity ligands (such as hydrophobic ligands or alkyl thiols) modified within the channels. Through coordination interactions between the ligands and quantum dots (such as metal-thiol bonds), the quantum dots are guided to oriented assemblies on the template surface, forming an ordered structure. Coordination assembly technology is used to achieve layer-by-layer stacking of quantum dots (green spheres) on the template. Single-layer structures: a single layer of quantum dots covers the template surface; double / triple-layer structures: through a "brick-and-mortar ordered stacking" strategy (alternating quantum dot layers and organic layers), multiple dense arrangements are formed, improving structural stability and functional density. Polyethyleneimine (PEI) acts as a connecting layer or charge regulator, promoting the ordered adsorption of quantum dots. Hydrophobic quantum dots are fixed to the template surface through hydrophobic interactions or coordination bonds. The quantum dot-filled three-dimensional fluorescent microspheres (PQDS) possess a dense silica shell: providing chemical stability and biocompatibility, and an organosilicon coating layer: the shell surface is modified by covalent bonds, introducing active groups (such as hydroxyl, amino, and thiol groups).

[0055] (a.4) Take 50 μL of a solution with a concentration of 10 mg / mL -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added to 1 mL of iPQDS-COOH solution, shaken for 30 min and centrifuged. After washing with PB solution, the solution was resuspended in 1 mL of PBS solution (0.02 M, pH 7.4), and 8.46 μL of a 9.47 mg / mL solution was added. -1 MPXV A29L mAb1 was added and shaken for 2.5 h; then a concentration of 3 mg / mL was used. -1 The surface was blocked by shaking with ethanolamine for 2 hours to obtain the iPQDS immunoassay probe.

[0056] The prepared iPQDS immunoassay probe was then washed twice with PBS solution and dispersed in 0.01M PBS solution containing 0.15wt% BSA and 0.01wt% Tween 20 to obtain the iPQDS immunochromatographic probe solution, which was stored at 4°C.

[0057] Preparation of rQD-BSA-Biotin reference probe solution: (b.1) Dissolve 113 μL of MPA in 20 mL of water and adjust the pH to 9.0 with 1 M NaOH solution; then add 20 mL of a solution with a concentration of 1 mg / mL. -1A two-phase solution was formed from a chloroform solution of the red luminescent quantum dots (rQDs). The solution was stirred at 60°C for 1 hour to obtain a crude solution. The crude solution was then ultrafiltered using an MWCO 3000 membrane, followed by three ultrafiltrations with PBS solution to obtain water-soluble red luminescent quantum dots (rQD-MPA).

[0058] (b.2) Dissolve 10 mg BSA in 5 mL of PB solution (0.02 M, pH=7.4) to obtain a BSA solution. Dissolve 1 mg EZ-link-sulfo-NHS-LC-biotin in 180 μL of PB solution (0.02 M, pH=7.4) and transfer it to the BSA solution. Stir at room temperature for 1 h. Then, perform ultrafiltration using a MWCO~3000 membrane and wash three times with PB solution (0.02 M, pH=7.4) to obtain a BSA-biotin solution, which is stored at 4 °C.

[0059] (b.3) Disperse 1 mg rQD-MPA in PB solution (0.02 M, pH=6.0) and add 50 μL of a solution with a concentration of 10 mg / mL. -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was stirred for 25 min; then ultrafiltration was performed using an MWCO~3000 membrane, followed by washing with PB solution (0.02 M, pH=6.0), dispersion in 1 mL PB solution (0.02 M, pH=7.4), and then 100 μL BSA-Biotin solution was added and stirred for 2.5 h to obtain the rQD-BSA-Biotin reference probe solution, which was stored at 4 °C.

[0060] Preparation of the binding pad: The conjugate pad was immersed in the treatment buffer for 12 hours, then removed and dried; subsequently, a concentration of 4 mg / mL was added. -1 The iPQDS immunochromatographic probe solution and its concentration were 0.2 mg / mL. -1 rQD-BSA-Biotin reference probe solution at 5 μL cm -1 The solution is sprayed evenly onto the bonding pad at a rate of [missing information], and dried at 37°C for 5 hours to obtain the bonding pad.

[0061] The treatment buffer solution comprises 50 mM PB solution, 1 wt% BSA, 1 wt% Tween 20 and 0.05 wt% sodium caseinate, pH=7.4.

[0062] Preparation of NC membrane: A concentration of 1.5 mg / mL -1 MPXV A29L mAb2 and a concentration of 0.5 mg / mL -1 A mixed solution of streptavidin was prepared at 1.0 μL cm⁻¹. -1The rate was plotted as a T-line on the NC membrane, and the concentration was 1.5 mg / mL. -1 Goat anti-mouse IgG antibody at 1.0 μL cm -1 The rate was drawn as a C line on the NC film, and the NC film was obtained after drying at 37°C.

[0063] Example 2: Preparation of iPQDS immunoassay probe.

[0064] (a.1) Preparation of center-radial macroporous SiO2 microspheres (dSi): 68 mg of triethanolamine was added to 25 mL of ultrapure water and stirred in an 80 °C water bath for 30 min. Then, 380 mg of hexadecyltrimethylammonium bromide and 168 mg of sodium salicylate were added, and the reaction was continued for 1 h to obtain a reaction solution. Subsequently, 4 mL of tetraethyl orthosilicate was added to the reaction solution, and the reaction was continued for 2 h to obtain the product. The product was centrifuged and washed three times with ethanol to obtain a precipitate. The precipitate was reacted in a hydrochloric acid / methanol mixture at 60 °C for 6 h and repeated three times to obtain the final product. After washing the final product with ethanol, the final product was dispersed in 50 mL of ethanol to obtain center-radial macroporous SiO2 microspheres.

[0065] (a.2) Preparation of the second-layer assembly: Add 1.5 mL of a 10 mg / mL solution to 10 mg SiO2 microspheres. -1 The green fluorescent QD chloroform dispersion was sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the first layer assembly (SQ1) with the maximum loading ratio.

[0066] Based on the maximum loading ratio of 119% for the first-layer assembly SQ1, the relationship between the dosage and loading ratio was further explored. The first-layer assembly SQ1 was added at a concentration of 10 mg / mL per 1 mL. -1 SQ1P was obtained by sonication in PEI (MW~1800) ethanol solution for 10 minutes, centrifugation to remove the supernatant, and washing the precipitate with ethanol. Subsequently, different volumes (0.25~1.5 mL) of 10 mg / mL solution were added to the SQ1P. -1 The green fluorescent QD chloroform dispersion was prepared and sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the second-layer assembly (SQ2). The amount added was increased from 175% (total amount of green fluorescent QD chloroform dispersion was 1.75 mL) to 300% (total amount of green fluorescent QD chloroform dispersion was 3 mL), and the loading ratio was increased from 145% to 220%.

[0067] (a.3) To transfer the hydrophobic phase to the aqueous phase, the second-layer assembly (SQ2) was slightly dried under a nitrogen gas flow and then dissolved in 260 μL of OTMS under sonication. Subsequently, 40 mL of methanol and 1 mL of ammonia were added, and the mixture was sonicated for 30 minutes. The product was then centrifuged and washed twice with methanol. The precipitate was dispersed in 40 mL of water and 80 μL of ammonia and stirred for 24 hours to form the assembly precipitate SQ2-OTMS. A silica shell was generated using the Stöber method: the assembly precipitate SQ2-OTMS was dispersed in a mixture containing 40 mL of ethanol, 10 mL of water, and 1.25 mL of ammonia. 25 μL of TEOS was added, and the mixture was stirred for 2 hours. This step was repeated once to obtain PQDS. The obtained PQDS was centrifuged and washed twice with ethanol, and then redispersed in 40 mL of ethanol and 0.5 mL of ammonia to obtain a dispersion. 20 μL of APTMS was added to the dispersion and stirred for 3 hours. This process was then repeated once. Centrifuge, wash thoroughly with ethanol, and redisperse in 20 mL of DMF containing 100 mg succinic anhydride. Stir for 12 h to obtain PQDS-COOH precipitate. Centrifuge the PQDS-COOH precipitate, wash thoroughly with ethanol and water, and then redisperse in a 4 mg / mL solution. -1 PQDS-COOH solution was obtained by adding PB solution (0.02M, pH=6.0).

[0068] (a.4) Take 50 μL of a solution with a concentration of 10 mg / mL -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added to 1 mL of iPQDS-COOH solution, shaken for 30 min and centrifuged. After washing with PB solution, the solution was resuspended in 1 mL of PBS solution (0.02 M, pH 7.4), and 8.46 μL of a 9.47 mg / mL solution was added. -1 MPXV A29L mAb1 was added and shaken for 2.5 h; then a concentration of 3 mg / mL was used. -1 The surface was blocked by shaking with ethanolamine for 2 hours to obtain the iPQDS immunoassay probe.

[0069] The prepared iPQDS immunoassay probe was then washed twice with PBS solution and dispersed in 0.01M PBS solution containing 0.15wt% BSA and 0.01wt% Tween 20 to obtain the iPQDS immunochromatographic probe solution, which was stored at 4°C.

[0070] Preparation of rQD-BSA-Biotin reference probe solution: (b.1) Dissolve 113 μL of MPA in 20 mL of water and adjust the pH to 9.0 with 1 M NaOH solution; then add 20 mL of a solution with a concentration of 1 mg / mL. -1 A two-phase solution was formed from a chloroform solution of the red luminescent quantum dots (rQDs). The solution was stirred at 60°C for 1 hour to obtain a crude solution. The crude solution was then ultrafiltered using an MWCO 3000 membrane, followed by three ultrafiltrations with PBS solution to obtain water-soluble red luminescent quantum dots (rQD-MPA).

[0071] (b.2) Dissolve 10 mg BSA in 5 mL of PB solution (0.02 M, pH=7.4) to obtain a BSA solution. Dissolve 1 mg EZ-link-sulfo-NHS-LC-biotin in 180 μL of PB solution (0.02 M, pH=7.4) and transfer it to the BSA solution. Stir at room temperature for 1 h. Then, perform ultrafiltration using a MWCO~3000 membrane and wash three times with PB solution (0.02 M, pH=7.4) to obtain a BSA-biotin solution, which is stored at 4 °C.

[0072] (b.3) Disperse 1 mg rQD-MPA in PB solution (0.02 M, pH=6.0) and add 50 μL of a solution with a concentration of 10 mg / mL. -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was stirred for 25 min; then ultrafiltration was performed using an MWCO~3000 membrane, followed by washing with PB solution (0.02 M, pH=6.0), dispersion in 1 mL PB solution (0.02 M, pH=7.4), and then 100 μL BSA-Biotin solution was added and stirred for 2.5 h to obtain the rQD-BSA-Biotin reference probe solution, which was stored at 4 °C.

[0073] Preparation of the binding pad: The conjugate pad was immersed in the treatment buffer for 12 hours, then removed and dried; subsequently, a concentration of 4 mg / mL was added. -1 The iPQDS immunochromatographic probe solution and its concentration were 0.2 mg / mL. -1 rQD-BSA-Biotin reference probe solution at 5 μL cm -1 The solution is sprayed evenly onto the bonding pad at a rate of [missing information], and dried at 37°C for 5 hours to obtain the bonding pad.

[0074] The treatment buffer solution comprises 50 mM PB solution, 1 wt% BSA, 1 wt% Tween 20 and 0.05 wt% sodium caseinate, pH=7.4.

[0075] Preparation of NC membrane: A concentration of 1.5 mg / mL -1MPXV A29L mAb2 and a concentration of 0.5 mg / mL -1 A mixed solution of streptavidin was prepared at 1.0 μL cm⁻¹. -1 The rate was plotted as a T-line on the NC membrane, and the concentration was 1.5 mg / mL. -1 Goat anti-mouse IgG antibody at 1.0 μL cm -1 The rate was drawn as a C line on the NC film, and the NC film was obtained after drying at 37°C.

[0076] Example 3: Preparation of iPQDS immunoassay probe.

[0077] (a.1) Preparation of center-radial macroporous SiO2 microspheres (dSi): 68 mg of triethanolamine was added to 25 mL of ultrapure water and stirred in an 80 °C water bath for 30 min. Then, 380 mg of hexadecyltrimethylammonium bromide and 168 mg of sodium salicylate were added, and the reaction was continued for 1 h to obtain a reaction solution. Subsequently, 4 mL of tetraethyl orthosilicate was added to the reaction solution, and the reaction was continued for 2 h to obtain the product. The product was centrifuged and washed three times with ethanol to obtain a precipitate. The precipitate was reacted in a hydrochloric acid / methanol mixture at 60 °C for 6 h and repeated three times to obtain the final product. After washing the final product with ethanol, the final product was dispersed in 50 mL of ethanol to obtain center-radial macroporous SiO2 microspheres.

[0078] (a.2) Preparation of the third-layer assembly: Add 1.5 mL of a 10 mg / mL solution to 10 mg SiO2 microspheres. -1 The green fluorescent QD chloroform dispersion was sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the first layer assembly (SQ1) with the maximum loading ratio.

[0079] The first-layer assembly SQ1 was prepared at a concentration of 10 mg / mL in 1 mL. -1 The sample was sonicated in a PEI (MW~1800) ethanol solution for 10 minutes, centrifuged to remove the supernatant, and the precipitate was washed with ethanol to obtain SQ1P. Then, 1.5 mL of a 10 mg / mL solution was added to the SQ1P. -1 The green fluorescent QD chloroform dispersion was obtained and sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the second-layer assembly (SQ2).

[0080] Building upon the highest loading ratio of 220% achieved in the first two layers, a third layer assembly was attempted to further explore the relationship between the dosage and loading ratio. The second layer assembly, SQ2, was prepared at a concentration of 10 mg / mL in 1 mL. -1SQ2P was obtained by sonication in PEI (MW~1800) ethanol solution for 10 minutes, centrifugation to remove the supernatant, and washing the precipitate with ethanol. Subsequently, different volumes (0.25~1.5 mL) of 10 mg / mL solution were added to the SQ2P. -1 The green fluorescent QD chloroform dispersion was prepared and sonicated for 7 min; after centrifugation, free QD was removed with chloroform to obtain the third-layer assembly (SQ3). The increase in loading ratio gradually slowed down with increasing input amount, eventually ceasing. The maximum loading ratio of the third-layer assembly SQ3 was 279%.

[0081] (a.3) To transfer the hydrophobic phase to the aqueous phase, the third-layer assembly (SQ3) was slightly dried under a nitrogen gas flow and then dissolved in 260 μL of OTMS under sonication. Subsequently, 40 mL of methanol and 1 mL of ammonia were added, and the mixture was sonicated for 30 minutes. The product was then centrifuged and washed twice with methanol. The precipitate was dispersed in 40 mL of water and 80 μL of ammonia and stirred for 24 hours to form the assembly precipitate SQ3-OTMS. The silica shell was generated using the Stöber method: the assembly precipitate SQ3-OTMS was dispersed in a mixture containing 40 mL of ethanol, 10 mL of water, and 1.25 mL of ammonia. 25 μL of TEOS was added, and the mixture was stirred for 2 hours. This step was repeated once to obtain PQDS. The obtained PQDS was centrifuged and washed twice with ethanol, and then redispersed in 40 mL of ethanol and 0.5 mL of ammonia to obtain a dispersion. 20 μL of APTMS was added to the dispersion and stirred for 3 hours. This process was then repeated once. Centrifuge, wash thoroughly with ethanol, and redisperse in 20 mL of DMF containing 100 mg succinic anhydride. Stir for 12 h to obtain PQDS-COOH precipitate. Centrifuge the PQDS-COOH precipitate, wash thoroughly with ethanol and water, and then redisperse in a 4 mg / mL solution. -1 PQDS-COOH solution was obtained by adding PB solution (0.02M, pH=6.0).

[0082] (a.4) Take 50 μL of a solution with a concentration of 10 mg / mL -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added to 1 mL of iPQDS-COOH solution, shaken for 30 min and centrifuged. After washing with PB solution, the solution was resuspended in 1 mL of PBS solution (0.02 M, pH 7.4), and 8.46 μL of a 9.47 mg / mL solution was added. -1 MPXV A29L mAb1 was added and shaken for 2.5 h; then a concentration of 3 mg / mL was used. -1 The surface was blocked by shaking with ethanolamine for 2 hours to obtain the iPQDS immunoassay probe.

[0083] The prepared iPQDS immunoassay probe was then washed twice with PBS solution and dispersed in 0.01M PBS solution containing 0.15wt% BSA and 0.01wt% Tween 20 to obtain the iPQDS immunochromatographic probe solution, which was stored at 4°C.

[0084] Preparation of rQD-BSA-Biotin reference probe solution: (b.1) Dissolve 113 μL of MPA in 20 mL of water and adjust the pH to 9.0 with 1 M NaOH solution; then add 20 mL of a solution with a concentration of 1 mg / mL. -1 A two-phase solution was formed from a chloroform solution of the red luminescent quantum dots (rQDs). The solution was stirred at 60°C for 1 hour to obtain a crude solution. The crude solution was then ultrafiltered using an MWCO 3000 membrane, followed by three ultrafiltrations with PBS solution to obtain water-soluble red luminescent quantum dots (rQD-MPA).

[0085] (b.2) Dissolve 10 mg BSA in 5 mL of PB solution (0.02 M, pH=7.4) to obtain a BSA solution. Dissolve 1 mg EZ-link-sulfo-NHS-LC-biotin in 180 μL of PB solution (0.02 M, pH=7.4) and transfer it to the BSA solution. Stir at room temperature for 1 h. Then, perform ultrafiltration using a MWCO~3000 membrane and wash three times with PB solution (0.02 M, pH=7.4) to obtain a BSA-biotin solution, which is stored at 4 °C.

[0086] (b.3) Disperse 1 mg rQD-MPA in PB solution (0.02 M, pH=6.0) and add 50 μL of a solution with a concentration of 10 mg / mL. -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was stirred for 25 min; then ultrafiltration was performed using an MWCO~3000 membrane, followed by washing with PB solution (0.02 M, pH=6.0), dispersion in 1 mL PB solution (0.02 M, pH=7.4), and then 100 μL BSA-Biotin solution was added and stirred for 2.5 h to obtain the rQD-BSA-Biotin reference probe solution, which was stored at 4 °C.

[0087] Preparation of the binding pad: The conjugate pad was immersed in the treatment buffer for 12 hours, then removed and dried; subsequently, a concentration of 4 mg / mL was added. -1 The iPQDS immunochromatographic probe solution and its concentration were 0.2 mg / mL. -1 rQD-BSA-Biotin reference probe solution at 5 μL cm -1The solution is sprayed evenly onto the bonding pad at a rate of [missing information], and dried at 37°C for 5 hours to obtain the bonding pad.

[0088] The treatment buffer solution comprises 50 mM PB solution, 1 wt% BSA, 1 wt% Tween 20 and 0.05 wt% sodium caseinate, pH=7.4.

[0089] Preparation of NC membrane: A concentration of 1.5 mg / mL -1 MPXV A29L mAb2 and a concentration of 0.5 mg / mL -1 A mixed solution of streptavidin was prepared at 1.0 μL cm⁻¹. -1 The rate was plotted as a T-line on the NC membrane, and the concentration was 1.5 mg / mL. -1 Goat anti-mouse IgG antibody at 1.0 μL cm -1 The rate was drawn as a C line on the NC film, and the NC film was obtained after drying at 37°C.

[0090] Figure 2 Transmission, scanning, and cross-sectional transmission electron microscopy images of SiO2 microspheres, the first-layer assembly, the second-layer assembly, the third-layer assembly, and PQDS, among which, Figure 2 a1 in the image is a transmission electron microscope image of SiO2 microspheres, which confirms that the central-radial macroporous SiO2 microspheres have an open central-radial pore structure. Figure 2 b1 in the image is a scanning electron microscope image of SiO2 microspheres, showing that the average pore opening size of the SiO2 microspheres is 40 nm, which provides ample space for filling the quantum dots. Figure 2 a2 in the image is a transmission electron microscope image of the first-layer assembly SQ1. Figure 2 b2 in the image is a scanning electron microscope image of the first-layer assembly SQ1, showing that hydrophobic quantum dots are directly and efficiently integrated into the thiol-modified SiO2 microspheres through organic phase thiol-metal coordination. Figure 2 a3 in the image is a transmission electron microscope image of the second-layer assembly. Figure 2 b3 in the image is a scanning electron microscope image of the second-layer assembly, which shows that the originally saturated assembly can be assembled into two layers after being bridged by PEI, further improving the quantum dot filling degree. Figure 2 a4 in the image is a transmission electron microscope image of the third-layer assembly. Figure 2 b4 in the image is a scanning electron microscope image of the third-layer assembly, showing that after the three-layer assembly, the quantum dot packing density is maximized, the interior is filled and the monodispersity of the microspheres is maintained; Figure 2 a5 in the image is a transmission electron microscope image from PQDS. Figure 2b5 in the image is a scanning electron microscope image from PQDS, showing that a complete SiO2 coating layer was formed through composite modification with organosilicon and dense silica. Figure 2 c1 in the image is a transmission electron microscope image of the cross-section of SiO2 microspheres. Figure 2 c2 in the image is a cross-sectional transmission electron microscope image of the first-layer assembly. Figure 2 c3 in the image is a cross-sectional transmission electron microscope image of the second-layer assembly. Figure 2 c4 in the image is a cross-sectional transmission electron microscope image of the third-layer assembly. Figure 2 c5 in the image is a cross-sectional transmission electron microscope image of PQDS. The cross-sectional transmission electron microscope image confirms that the interior of the quantum dot three-dimensional filled fluorescent microsphere PQDS has a tree-like macroporous structure. The quantum dot filling depth reaches 130 nm and extends to the central region. The layer-by-layer assembly method significantly improves the filling density of quantum dots and ultimately forms a complete garnet-shaped structure.

[0091] Figure 3 The graph shows the change in loading ratio as a function of feed ratio during quantum dot assembly, demonstrating that the loading ratio of quantum dots (the mass ratio of fixed quantum dots to SiO2 microspheres) gradually increases with the increase of feed ratio (the mass ratio of added quantum dots to template). The staged, layer-by-layer assembly method mediated by affinity polymers effectively suppresses the saturation effect of each layer, increasing the loading to 279%. The inset shows that the color of the assembly dispersion gradually deepens with increasing loading.

[0092] Figure 4 The fluorescence intensity varies with wavelength and is plotted in the visible light spectrum. The fluorescence peak (PL peak) of QDs (quantum dots with hydrophobic surfaces) is 526 nm, the full width at half maximum (FWHM) is 22.1 nm, and the photoluminescence quantum yield (PLQY) is 83%. The first-layer assembly (SQ1) has a PL peak of 527 nm, an FWHM of 22.9 nm, and a PLQY of 69%. The second-layer assembly (SQ2) has a PL peak of 528 nm, an FWHM of 22.9 nm, and a PLQY of 58%. The third-layer assembly (SQ3) has a PL peak of 528 nm, an FWHM of 23.2 nm, and a PLQY of 53%. The quantum dot-filled fluorescent microspheres (PQDS) have a PL peak of 528 nm, an FWHM of 23.3 nm, and a PLQY of 41%. The PQDS-COOH precipitate (PQDS-COOH) has a PL peak of 526 nm, an FWHM of 22.1 nm, and a PLQY of 41%. The peak value was 528 nm, the FWHM value was 23.3 nm, and the PLQY value was 41%; according to Figure 4 It can be seen that the fluorescence emission peak shape, wavelength, full width at half maximum (FWHM), and apparent state of the solution of QDs, SQ1, SQ2, SQ3, PQDS, and PQDS-COOH remain basically stable.

[0093] Figure 5 Correlation between fluorescence intensity and probe particle number for single quantum dots (QD-MPA) modified with mercaptopropionic acid; Figure 6 The correlation between the fluorescence intensity of PQDS-COOH precipitate and the number of probe particles is shown in the figure. Figure 5 and Figure 6 The fluorescence intensity of PQDS-COOH precipitate and mercaptopropionic acid-modified single quantum dots showed a good linear relationship with the number of probe particles. The slope of PQDS-COOH precipitate was 6770 times larger than that of mercaptopropionic acid-modified single quantum dots, which is due to the synergistic effect of a large number of quantum dots embedded in SiO2 microspheres.

[0094] Figure 7 Correlation between the G channel number and the number of probe particles for a single quantum dot-biotin conjugate modified with mercaptopropionic acid. Figure 8 The correlation between the G channel value and the probe particle number of the PQDS-COOH precipitate-biotin conjugate is shown in the figure. Figure 7 and Figure 8 It can be seen that the imaging brightness of the mercaptopropionic acid-modified single quantum dot-biotin conjugate and the PQDS-COOH precipitate-biotin conjugate on a nitrocellulose membrane coated with streptavidin is positively correlated with the number of probe particles. The G channel value refers to the signal intensity detected by the fluorescence detection system in the green band. The imaging brightness increases with the increase of the number of probe particles, confirming that the probe is effectively captured by the immobilized SA. The lowest visible particle concentration of the mercaptopropionic acid-modified single quantum dot-biotin conjugate is 4.5 × 10⁻⁶. 9 The minimum visible particle concentration of the PQDS-COOH precipitate-biotin conjugate is 4.4 × 10⁻⁶. 6 This means that the signal was amplified by 1023 times.

[0095] Figure 9 This document presents a schematic diagram of the detection process, detection principle, and detection results for a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. Figure 9 In the diagram, 'a' is the detection flowchart of the dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. It shows that the detection process includes sample collection, virus lysis, immunochromatography, and visual / terminal reading, which takes a total of 15 minutes. The whole process is suitable for home point-of-care diagnostic scenarios. Figure 9 b in the diagram is a schematic diagram of the detection principle of the dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. It shows that the MPXV A29L antigen is bound by the iPQDS probe and the immobilized capture antibody sandwich as it flows through the sample pad, conjugate pad and NC membrane, as well as the working principle of the red quantum dot biotin-SA independent reference system. Figure 9In the diagram, c represents the detection results of a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. It shows the correspondence between different mixing ratios of green and red probes on the T line and the color results, i.e., the antigen concentration-dependent visible color changes and their risk levels.

[0096] Figure 10 The image shows the detection and visual interpretation results of MPXV A29L using a dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit. Figure 10 The concentration of MPXV A29L increased (0~500 ng / mL), showing the effect of concentration. -1 The T-line imaging result shows a gradient change from pure red to orange-red to orange to yellow to yellow-green to pure green, meeting the high recognition requirements of the human eye. Based on the visual colorimetric chart used by 30 independent / untrained users, five concentrations (0, 0.5, 5, 50, 500 ng / mL) were compared. -1 The band classification results show that only 2 people were at 0.5 and 1 ng / mL. -1 The results were incorrectly interpreted, with an average accuracy rate as high as 98.8%.

[0097] Figure 11 The results of detection and visual interpretation of MPXV A29L using a single-color bright-dark type-LFIA based on colloidal gold. Figure 11 The results showed that the concentration of MPXV A29L increased with increasing concentration (0~500 ng / mL). -1 The T-line imaging results show a gradual gradient from colorless to light red to dark red, which is relatively difficult for the human eye to discern precisely. Based on the visual colorimetric chart used by 30 independent / untrained users, five concentrations (0, 0.5, 5, 50, 500 ng / mL) were compared. -1 The band classification results are shown at concentrations of 0.5, 5, 50, and 500 ng / mL. -1 On the strip, a significant number of users made incorrect attribution judgments, with an average accuracy rate of 63.3%.

[0098] Figure 12 The image shows the detection results of a dual-color quantum dot-based visualization quantitative monkeypox antigen detection kit for herpes fluid samples from monkeypox virus-infected individuals. Figure 12This study demonstrates that as the viral load in monkeypox virus herpes fluid increases (Ct value for nucleic acid detection: 31.1~14.7), the T-line imaging results show a gradual gradient from pure red to orange-red to orange to yellow to yellow-green to pure green, meeting the high visual recognition requirements of the human eye. After loading the band image into the user's mobile application, the system can automatically perform color selection, G / R value extraction and calculation, standard curve concentration recalculation, risk level classification, and result output with a single click. The T-line color blocks, T-line G / R values, and infection risk levels of real samples are consistent with their nucleic acid detection Ct values, confirming that the dual-color quantum dot-based visualized quantitative monkeypox antigen detection kit provided by this invention can be used for home-based point-of-care diagnosis of MPXV.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A two-color quantum dot-based visualized quantitative monkeypox antigen detection kit, characterized in that, The test strip includes a chromatography strip; the chromatography strip consists of a sample pad, a conjugate pad, an NC membrane, and an absorbent pad, which are sequentially overlapped and fixed to a plastic backing plate; the conjugate pad is immersed in a processing buffer and sprayed with iPQDS immunochromatographic probe and rQD-BSA-biotin reference probe solution to form an immunochromatographic recognition system; the NC membrane is sprayed with a T line coated with MPXV A29L mAb2 and streptavidin and a C line coated with goat anti-mouse IgG antibody.

2. The dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to claim 1, characterized in that, The treatment buffer solution comprises 40-75 mM PB solution, 0.8-1.5 wt% BSA, 0.8-1.5 wt% Tween 20, and 0.04-0.075 wt% sodium caseinate, with a pH of 7.3-7.

5.

3. The dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to claim 1, characterized in that, The iPQDS immunochromatographic probe solution comprises 0.008~0.015M PBS solution and 3.5~6mg mL -1 iPQDS immunoassay probe, 0.08–0.15 wt% BSA and 0.008–0.015 wt% Tween 20.

4. The dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to claim 1, characterized in that, The iPQDS immunoassay probe is prepared through the following steps: (a.1) Add 55-100g of triethanolamine to ultrapure water, heat and stir, then add 300-570mg of cetyltrimethylammonium bromide and 135-250g of sodium salicylate and react for 1h; add 3.5-6ml of tetraethyl orthosilicate and react for 2h, centrifuge and wash with ethanol to obtain a precipitate; react the precipitate in a mixture of hydrochloric acid / methanol at 60℃ for 6h, then wash with ethanol to obtain SiO2 microspheres; (a.2) SiO2 microspheres were assembled layer by layer using green fluorescent QD chloroform dispersion and PEI ethanol solution to obtain a first-layer assembly, a second-layer assembly and a third-layer assembly; (a.3) The first, second, or third layer assembly was dried under a nitrogen gas flow and then dissolved in OTMS under sonication. Methanol and ammonia were then added, followed by sonication and centrifugation to obtain the product. The product was washed with methanol, and the precipitate was dispersed in water and ammonia with stirring to form an assembly precipitate. The assembly precipitate was dispersed in a mixture of ethanol, water, and ammonia, and TEOS was added and stirred once to obtain PQDS. After centrifugation and washing, the PQDS was dispersed again in ethanol and ammonia, and APTMS was added and stirred once. After centrifugation and washing, the precipitate was dispersed and stirred in DMF containing succinic anhydride to obtain PQDS-COOH precipitate, which was then centrifuged, washed, and dispersed in a solution with a concentration of 4 mg / mL. -1 PQDS-COOH solution was obtained from PB solution; (a.4) Take 50 μL of a solution with a concentration of 10 mg / mL -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added to 1 mL of LPQDS-COOH solution, shaken and centrifuged, washed with PB solution and resuspended in 1 mL of PBS solution. Then, 8.46 μL of a 9.47 mg / mL solution was added. -1 MPXV A29L mAb1 was shaken for 2.5 h; then shaken with ethanolamine to obtain the iPQDS immunoassay probe.

5. The dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to claim 1, characterized in that, Step (a.2) specifically includes the following sub-steps: (a.2.1) Add 0.25-1.5 mL of green fluorescent QD chloroform dispersion to 8-15 mg SiO2 microspheres, sonicate for 7 min and centrifuge, then remove free QD with chloroform to obtain the first layer assembly; (a.2.2) The first layer assembly was prepared at a concentration of 8-15 mg / mL in 0.8-1.5 mL. -1 After sonication in PEI ethanol solution, the supernatant was removed by centrifugation, and the precipitate was washed with ethanol to obtain SQ1P. (a.2.3) Repeat step (a.2.1) with SQ1P to obtain the second layer assembly; (a.2.4) Repeat steps (a.2.2) and (a.2.1) on the second-layer assembly to obtain the third-layer assembly.

6. The dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to claim 1, characterized in that, The rQD-BSA-biotin reference probe solution was prepared through the following steps: (b.1) Dissolve 90–160 μL of MPA in 20 mL of water and adjust the pH to 8.5–9.5 with NaOH solution; then add 20 mL of a solution with a concentration of 0.8–15 mg / mL. -1 A chloroform solution of red luminescent QD was stirred at 60°C for 1 h to obtain a crude solution, which was then ultrafiltered using an MWCO~3000 membrane. Subsequently, it was ultrafiltered with PB solution to obtain rQD-MPA. (b.2) Dissolve 8-15 mg BSA in 5 mL of PB solution to obtain BSA solution. Dissolve 0.8-1.5 mg EZ-link-sulfo-NHS-LC-biotin in 180 μL of PB solution and transfer it to BSA solution. Stir for 1 h. Then use MWCO~3000 membrane for ultrafiltration and wash with PB solution to obtain BSA-biotin solution. (b.3) Disperse 1 mg rQD-MPA in PB solution and add 50 μL of a solution with a concentration of 10 mg / mL. -1 EDC and a concentration of 10 mg / mL in 100 μL -1 The sulfonyl-NHS was added and stirred for 25 min; then ultrafiltration was performed using an MWCO~3000 membrane, and the sample was washed with PB solution, dispersed in 1 mL of PB solution, and then 100 μL of BSA-Biotin solution was added and stirred for 2.5 h to obtain the rQD-BSA-Biotin reference probe solution.

7. The dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to claim 1, characterized in that, The conjugate pad is prepared by the following method: Immerse the conjugate pad in the treatment buffer, remove and dry; then prepare a solution with a concentration of 3.5–6 mg / mL. -1 The iPQDS immunochromatographic probe solution has a concentration of 0.16~0.3 mg / mL. -1 rQD-BSA-Biotin reference probe solution at 5 μL cm -1 The solution is sprayed evenly onto the bonding pad at a rate of [missing information], and dried at 37°C for at least 4 hours to obtain the bonding pad.

8. The dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to claim 1, characterized in that, The NC membrane is prepared by the following method: The concentration was 1.2~2.2 mg / mL. -1 MPXV A29L mAb2 and concentrations of 0.4~0.75 mg / mL -1 A mixed solution of streptavidin was prepared at 1.0 μL cm⁻¹. -1 The rate was plotted as a T-line on the NC membrane, and concentrations of 1.2–2.2 mg / mL were measured. -1 Goat anti-mouse IgG antibody was administered at 1.0 μL cm -1 The rate was drawn as a C line on the NC film, and the NC film was obtained after drying at 37°C.

9. The use of the dual-color quantum dot visualization quantitative monkeypox antigen detection kit according to any one of claims 1-8 in the detection of monkeypox virus.

10. The application according to claim 9, characterized in that, The application specifically includes the following steps: (c.1) Sample collection: Using sterile collection tools, monkeypox virus herpes fluid is directly aspirated or dipped from the herpes lesions within 1 minute; (c.2) The collected monkeypox virus herpes fluid was then lysed in running buffer for 1 min and then dropped onto the sample pad in the two-color quantum dot visualization quantitative monkeypox antigen detection kit; (c.3) Result Interpretation: Result interpretation begins 10 minutes after the immunochromatographic reaction: When the C line on the NC membrane is green and the T line is pure red, the monkeypox virus test result is negative; when the C line on the NC membrane is green and the T line is orange-red, orange, yellow, yellow-green, or pure green, the monkeypox virus test result is positive, and the viral load gradually increases in the order of orange-red-orange-yellow-yellow-green-pure green; when the C line on the NC membrane does not show color, the test result is invalid, and the kit should be replaced and the test repeated.

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