Magnetic fluorescent nano-film probe as well as preparation method and application thereof

Abstract

The invention relates to a magnetic fluorescent nano-film probe as well as a preparation method and application thereof. The probe comprises a magnetic substrate film layer and a quantum dot shell layer which are sequentially arranged in the stacking direction. The quantum dot shell layer is of a multi-layer laminated structure formed by alternating quantum dot layers and third cationic polymer interlayers, and the first layer and the last layer in the laminating direction of the laminated structure are the quantum dot layers independently; and aminophenylboronic acid is coupled to the surface of one side, opposite to the graphene oxide nanosheet layer, of the last quantum dot layer. The magnetic fluorescent nano-film probe has strong fluorescence performance, has the capability of capturing various virus glycoproteins, and can quickly and accurately detect various viruses.

Description

[0001] CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to Chinese Patent Application No. 202410771353.5 filed on June 14, 2024, the entire contents of which are incorporated herein by these references for all purposes. Technical Field

[0002] The present disclosure relates to the field of novel nanomaterials and immunochromatographic detection technology, and in particular, to a magnetic fluorescent nanofilm probe and a preparation method and application thereof. Background Art

[0003] At present, some mature virus detection technologies have been widely used in laboratory and hospital examinations, such as virus isolation, real-time reverse transcription polymerase chain reaction (RT-PCR), genome sequencing, and enzyme-linked immunosorbent assay (ELISA). However, these methods require clean rooms, complex pre-treatment, and large output instruments. Therefore, they are not suitable for use in areas with limited conditions or scarce resources.

[0004] Immunochromatography (ICA) technology is one of the most mature and popular POCT detection methods, with obvious advantages such as rapidity, low cost and portability. ICA has a wide range of applications in the field of personal health self-examination. The principle of ICA technology currently used for virus detection is generally based on a double antibody sandwich format, directly detecting viral antigens (virions or viral proteins), and usually requires a pair of antibodies (a detection antibody and a capture antibody) to modify the nanoprobes and test lines of the ICA strip. The type and performance of antibodies have a great influence on the detection performance of ICA. The preparation of specific antibodies for emerging viruses takes a long time (generally 3-6 months) and is costly. In addition, the antibodies modified on the surface of nanoprobes are easy to change and have poor reproducibility. Detecting multiple viral antigens on ICA requires multiple pairs of specific antibodies, which further increases the difficulty of virus detection. In addition, spherical nanoprobes such as the current mainstream nanoprobes (such as colloidal AuNPs, latex microspheres and fluorescent microspheres) can only produce general signal intensity and weak stability in complex clinical samples, and lack sensitivity. Therefore, there is an urgent need to develop non-antibody-modified capture probes to establish a universal ICA technology to monitor multiple viruses simultaneously and sensitively. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present disclosure is to provide a magnetic fluorescent nanomembrane probe, which has strong fluorescence properties and the ability to capture a variety of viral glycoproteins, and can achieve rapid and accurate detection of a variety of viruses.

[0006] In order to achieve the above-mentioned object, the present disclosure provides a magnetic fluorescent nanofilm probe in a first aspect, the probe comprising a magnetic substrate film layer and a quantum dot shell layer sequentially arranged along a stacking direction; The quantum dot shell layer is a multilayer stacked structure formed by alternating quantum dot layers and third cationic polymer interlayers, the first layer and the last layer along the stacking direction of the stacked structure are each independently the quantum dot layer, and aminophenylboronic acid is coupled to the surface of the last quantum dot layer on the side facing away from the graphene oxide nanosheet layer.

[0007] Optionally, the stacked structure has three layers; the quantum dots in the quantum dot layer are CdSe / ZnS-MPA quantum dots.

[0008] Optionally, the magnetic base film layer includes a graphene oxide nanosheet layer, a first cationic polymer interlayer, a Fe3O4 magnetic nanoparticle layer and a second cationic polymer interlayer arranged in sequence along the stacking direction; wherein, in the graphene oxide nanosheet layer, the average diameter of the graphene oxide nanosheets is 100-2000nm, preferably 500-800nm; in the Fe3O4 magnetic nanoparticle layer, the average particle size of the Fe3O4 magnetic nanoparticles is 20-200nm, preferably 50-100nm.

[0009] Optionally, the first cationic polymer interlayer, the second cationic polymer interlayer and the third cationic polymer interlayer are each independently polyethyleneimine.

[0010] The second aspect of the present disclosure provides a method for preparing the magnetic fluorescent nanofilm probe according to the first aspect, the method comprising the following steps: S1, mixing an aqueous dispersion of graphene oxide nanosheets with an aqueous solution of a first cationic polymer and performing a first ultrasonic treatment to obtain GO@PEI nanosheets; S2, mixing the aqueous dispersion of the GO@PEI nanosheets with the aqueous dispersion of Fe3O4 magnetic nanoparticles and performing a second ultrasonic treatment to obtain a GOFe magnetic nanofilm; S3, mixing the aqueous dispersion of the GOFe magnetic nanofilm with the aqueous solution of the second cationic polymer and performing a third ultrasonic treatment to obtain a GOFe@PEI magnetic substrate film; S4, adding CdSe / ZnS-MPA quantum dots to the aqueous dispersion of the GOFe@PEI magnetic substrate film and performing a fourth ultrasonic treatment to obtain a GOFe@QD magnetic nanofilm; The aqueous dispersion of the GOFe@QD magnetic nanofilm is mixed with an aqueous solution of a third cationic polymer and subjected to a fifth ultrasonic treatment to obtain a GOFe@QD@PEI magnetic nanofilm; Adding CdSe / ZnS-MPA quantum dots to the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm and performing a sixth ultrasonic treatment to obtain a GOFe@DQD magnetic nanofilm; S5. Mix the GOFe@DQD magnetic nanofilm with an aqueous solution of aminophenylboronic acid for incubation.

[0011] Optionally, in step S1, the average diameter of the graphene oxide nanosheets is 100-2000 nm, preferably 500-800 nm; the mass concentration of the aqueous dispersion of the graphene oxide nanosheets is 0.1-5 mg / mL; the mass concentration of the aqueous solution of the first cationic polymer is 0.1-5 mg / mL; the volume ratio of the aqueous dispersion of the graphene oxide nanosheets to the aqueous solution of the first cationic polymer is 0.5-5:1; In step S2, the average particle size of the Fe3O4 magnetic nanoparticles is 20-200 nm, preferably 50-100 nm; the mass concentration of the aqueous dispersion of the Fe3O4 magnetic nanoparticles is 5-20 mg / mL; the mass concentration of the aqueous dispersion of the GO@PEI nanosheets is 0.1-20 mg / mL; the volume ratio of the aqueous dispersion of the GO@PEI nanosheets to the aqueous dispersion of the Fe3O4 magnetic nanoparticles is 500-1000:1; In step S3, the mass concentration of the aqueous dispersion of the GOFe magnetic nanofilm is 0.1-20 mg / mL; the mass concentration of the aqueous solution of the second cationic polymer is 0.1-5 mg / mL; the volume ratio of the aqueous dispersion of the GOFe magnetic nanofilm to the aqueous solution of the second cationic polymer is 1-5:1; In step S4, the mass concentration of the aqueous dispersion of the GOFe@PEI magnetic substrate membrane is 0.1-20 mg / mL; the amount of the CdSe / ZnS-MPA quantum dots added is 0.01-0.1 mg per 100 mL of the aqueous dispersion of the GOFe@PEI magnetic substrate membrane; the mass concentration of the aqueous solution of the third cationic polymer is 0.1-5 mg / mL; the mass concentration of the aqueous dispersion of the GOFe@QD magnetic nanofilm is 0.1-20 mg / mL; the volume ratio of the aqueous dispersion of the GOFe@QD magnetic nanofilm to the aqueous solution of the third cationic polymer is 1-5:1; the mass concentration of the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm is 0.1-20 mg / mL; the amount of the CdSe / ZnS-MPA quantum dots added is 0.01-0.1 mg per 100 mL of the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm; In step S5, the mass concentration of the aqueous solution of aminophenylboronic acid is 0.01-0.5 mg / mL; the added amount of the GOFe@DQD magnetic nanofilm is 0.01-0.1 mg per 100 mL of the aqueous solution of aminophenylboronic acid.

[0012] The third aspect of the present disclosure provides the use of the magnetic fluorescent nanomembrane probe described in the first aspect or the magnetic fluorescent nanomembrane probe prepared by the method described in the second aspect in detecting viral glycoproteins by immunochromatography for non-disease diagnosis and / or treatment purposes, or in preparing products for detecting viral glycoproteins.

[0013] A fourth aspect of the present disclosure provides a kit for detecting viral glycoproteins, the kit comprising the magnetic fluorescent nanomembrane probe described in the first aspect or the magnetic fluorescent nanomembrane probe prepared by the method described in the second aspect.

[0014] Optionally, the kit also includes an immunochromatographic test strip, which includes a sample pad, a chromatographic membrane and an absorption pad connected in sequence, and a plurality of detection lines and quality control lines are arranged on the chromatographic membrane; each of the detection lines is loaded with antiviral glycoprotein antibodies; and the quality control line is loaded with streptavidin.

[0015] Optionally, there are three test lines, which are respectively loaded with anti-SARS-CoV-2 antigen spike protein antibodies, anti-MPXVA29 envelope protein antibodies and anti-EBOV glycoprotein antibodies.

[0016] Through the above technical scheme, the present disclosure provides a magnetic fluorescent nanofilm probe and its preparation method and application. The magnetic fluorescent nanofilm probe has strong fluorescence performance, strong magnetic responsiveness and a large reaction interface, has the ability to capture multiple viral glycoproteins, and can achieve rapid and accurate detection of multiple viruses. The kit prepared by the magnetic fluorescent nanofilm probe disclosed in the present disclosure has high sensitivity, short detection time and wide detection range, and has good application prospects.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 It is a schematic diagram of the preparation method for synthesizing the magnetic fluorescent nanofilm probe and the quality control line probe disclosed in the present invention.

[0019] Figure 2The electron microscope characterization, Zeta potential and fluorescence intensity of the products in each stage of the magnetic fluorescent nanomembrane probe structure disclosed in the present invention.

[0020] Figure 3 This is to verify the ability of the magnetic fluorescent nanomembrane probe disclosed in the present invention to capture viral glycoproteins.

[0021] Figure 4 This is a diagram showing the working principle of the immunochromatography of the magnetic fluorescent nanomembrane probe disclosed in the present invention for SARS-CoV-2 antigen spike protein, MPXVA29 envelope protein and EBOV glycoprotein.

[0022] Figure 5 This is the feasibility verification result of the immunochromatography of the magnetic fluorescent nanomembrane probe disclosed in the present invention.

[0023] Figure 6 The optimized results of the membrane concentration for immunochromatography of the magnetic fluorescent nanomembrane probe disclosed in the present invention.

[0024] Figure 7 The results of optimizing the composition of the running buffer for immunochromatography of the magnetic fluorescent nanomembrane probe disclosed in the present invention.

[0025] Figure 8 The results of the optimization of the chromatographic reaction time of the immunochromatographic method using the magnetic fluorescent nanomembrane probe disclosed in the present invention.

[0026] Fig. 9 This is a graph showing the results of the immunochromatography of the magnetic fluorescent nanomembrane probe disclosed herein for simultaneously detecting SARS-CoV-2 antigen spike protein, MPXVA29 envelope protein and EBOV glycoprotein.

[0027] Fig.10 The reproducible results of immunochromatography of the magnetic fluorescent nanomembrane probe disclosed in the present invention.

[0028] Fig.11 The immunochromatographic analysis results of the magnetic fluorescent nanomembrane probe disclosed in the present invention on three viruses in simulated clinical samples.

[0029] Fig.12 The present invention provides an analysis of the detection results of throat swab samples of clinical respiratory tract infection patients detected by immunochromatography using the magnetic fluorescent nanomembrane probe disclosed herein and its correlation with the ELISA method. DETAILED DESCRIPTION

[0030] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0031] like Figure 1As shown in a, the first aspect of the present disclosure provides a magnetic fluorescent nanofilm probe, the probe comprising a magnetic substrate film layer and a quantum dot shell layer sequentially arranged along a stacking direction; The quantum dot shell layer is a multilayer stacked structure formed by alternating quantum dot layers and third cationic polymer interlayers, the first layer and the last layer along the stacking direction of the stacked structure are each independently the quantum dot layer, and aminophenylboronic acid is coupled to the surface of the last quantum dot layer on the side facing away from the graphene oxide nanosheet layer.

[0032] The magnetic fluorescent nanoprobe disclosed in the present invention is based on the electrostatic interaction of ionic polymer interlayers, and a layer of Fe3O4 magnetic nanoparticles and multiple quantum dot layers are adsorbed on the graphene oxide nanosheet layer, so that the magnetic fluorescent nanoprobe can show excellent dispersibility, stability, magnetism and fluorescence properties in different samples to be detected; one end of aminophenylboronic acid (APBA) is a boronic acid group, which can effectively capture and quickly enrich a variety of viral glycoproteins from the samples to be detected, which is conducive to the rapid quantitative detection of viruses.

[0033] In a preferred embodiment of the present disclosure, the aminophenylboronic acid is 4-aminophenylboronic acid.

[0034] In this disclosure, “stacking direction” refers to the direction from the graphene oxide nanosheet layer to the last quantum dot layer, e.g. Figure 1 In the bottom-up direction.

[0035] In one embodiment, the stacked structure has three layers. The first layer and the last layer are each independently a quantum dot layer, such as Figure 2 As shown in d and 2e, the second cationic polymer interlayer and the third cationic polymer interlayer enable each quantum dot layer to be adsorbed on the GOFe@PEI magnetic substrate film, and the density of the quantum dots is controllable. Specifically, each quantum dot layer contains 100-2000 quantum dots; the distance between the centers of two adjacent quantum dots is 12-20nm, such as Figure 2 As shown in j, the distance between the centers of two adjacent quantum dots is preferably 12 nm.

[0036] According to the present disclosure, the quantum dots in the quantum dot layer have negative charges. In a specific embodiment, the quantum dots in the quantum dot layer are CdSe / ZnS-MPA quantum dots. In the present disclosure, Figure 2As shown in Figure 1, the magnetic fluorescent nanomembrane probe with a double-layer quantum dot structure formed by hydrophilic carboxylated quantum dots and polyethyleneimine can not only improve the luminescence of the magnetic fluorescent nanomembrane probe, but also ensure that the magnetic fluorescent nanomembrane probe has excellent dispersibility in the virus solution to be tested. The fluorescence signal of the magnetic fluorescent nanomembrane probe containing a double-layer quantum dot layer is enhanced by 1.82 times compared with the fluorescence signal of the magnetic fluorescent nanomembrane probe containing a single-layer quantum dot layer. The carboxyl groups on the surface of CdSe / ZnS-MPA quantum dots can couple APBA with quantum dots through a condensation reaction with the amino groups of APBA, thereby making the magnetic fluorescent nanomembrane probe have excellent ability to capture viral glycoproteins.

[0037] In one embodiment, the magnetic substrate film layer includes a graphene oxide nanosheet layer, a first cationic polymer interlayer, a Fe3O4 magnetic nanoparticle layer, and a second cationic polymer interlayer, which are sequentially arranged along a stacking direction.

[0038] In this disclosure, Figure 2 As shown in Figure 1, the zeta potential of the graphene oxide nanosheet layer increases after PEI modification and decreases sharply after Fe3O4 magnetic nanoparticles and quantum dots are coated, indicating that the interaction between each layer of the magnetic fluorescent nanofilm disclosed in the present invention is driven by electrostatic interaction. The flexible structure of the graphene oxide nanosheet layer and its large surface interface ensure that the graphene oxide nanosheet layer has a large ability to load Fe3O4 magnetic nanoparticles and quantum dots. Figure 2 c, the Fe3O4 magnetic nanoparticles of the Fe3O4 magnetic nanoparticle layer are adsorbed on the graphene oxide nanosheet layer through the action of the first cationic polymer interlayer, and the density of the Fe3O4 magnetic nanoparticles is controllable. Specifically, the Fe3O4 magnetic nanoparticle layer contains 10-100 Fe3O4 magnetic nanoparticles, and the distance between the centers of two adjacent Fe3O4 magnetic nanoparticles is 90-500nm, as shown in FIG. Figure 2 As shown in g, the distance between the centers of two adjacent Fe3O4 magnetic nanoparticles is preferably 200 nm, which can give the magnetic fluorescent nanofilm probe better dispersibility. Since the graphene oxide nanosheet layer adsorbs more Fe3O4 magnetic nanoparticles, the magnetic fluorescent nanofilm probe has a higher magnetic responsiveness.

[0039] In one embodiment, in the graphene oxide nanosheet layer, the average diameter of the graphene oxide nanosheets is 100-2000 nm, preferably 500-800 nm.

[0040] According to the present disclosure, the Fe3O4 magnetic nanoparticles carry a negative charge. In the Fe3O4 magnetic nanoparticle layer, the average particle size of the Fe3O4 magnetic nanoparticles is 20-200 nm, preferably 50-100 nm.

[0041] According to the present disclosure, the first cationic polymer interlayer, the second cationic polymer interlayer and the third cationic polymer interlayer are each independently polyethyleneimine (PEI). The molecular weight of polyethyleneimine is preferably 5000-80000Da, preferably 6000-70000Da.

[0042] In the present disclosure, the polyethyleneimine interlayer can effectively enhance the binding force between the graphene oxide nanosheet layer and the Fe3O4 magnetic nanoparticles and quantum dots, thereby ensuring the fluorescence stability of the magnetic fluorescent nanofilm probe, and at the same time ensuring that the magnetic fluorescent nanofilm probe still has strong stability under long-term storage. Generally, the magnetic fluorescent nanofilm probe of the present disclosure is stored under freeze-dried conditions, specifically, the temperature is (-80)-(-60)°C.

[0043] The second aspect of the present disclosure provides a method for preparing the magnetic fluorescent nanofilm probe according to the first aspect, the method comprising the following steps: S1, mixing an aqueous dispersion of graphene oxide nanosheets with an aqueous solution of a first cationic polymer and performing a first ultrasonic treatment to obtain GO@PEI nanosheets; S2, mixing the aqueous dispersion of the GO@PEI nanosheets with the aqueous dispersion of Fe3O4 magnetic nanoparticles and performing a second ultrasonic treatment to obtain a GOFe magnetic nanofilm; S3, mixing the aqueous dispersion of the GOFe magnetic nanofilm with the aqueous solution of the second cationic polymer and performing a third ultrasonic treatment to obtain a GOFe@PEI magnetic substrate film; S4, adding CdSe / ZnS-MPA quantum dots to the aqueous dispersion of the GOFe@PEI magnetic substrate film and performing a fourth ultrasonic treatment to obtain a GOFe@QD magnetic nanofilm; The aqueous dispersion of the GOFe@QD magnetic nanofilm is mixed with an aqueous solution of a third cationic polymer and subjected to a fifth ultrasonic treatment to obtain a GOFe@QD@PEI magnetic nanofilm; Adding CdSe / ZnS-MPA quantum dots to the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm and performing a sixth ultrasonic treatment to obtain a GOFe@DQD magnetic nanofilm; S5. Mix the GOFe@QD magnetic nanofilm with an aqueous solution of aminophenylboronic acid for incubation.

[0044] In the present disclosure, ultrasonic treatment can be performed using an ultrasonic instrument commonly used by those skilled in the art, which will not be described in detail herein. The preparation method provided in the present disclosure is based on a cationic polymer-mediated electrostatic self-assembly method, and the preparation process of the method is simple, efficient, repeatable, and suitable for large-scale production.

[0045] In one embodiment, in step S1, the average diameter of the graphene oxide nanosheets is 100-2000nm, preferably 500-800nm; the mass concentration of the aqueous dispersion of the graphene oxide nanosheets is 0.1-5mg / mL; the mass concentration of the aqueous solution of the first cationic polymer is 0.1-5mg / mL; the volume ratio of the aqueous dispersion of the graphene oxide nanosheets to the aqueous solution of the first cationic polymer is 0.5-5:1. The conditions of the first ultrasonic treatment include: temperature of 10-30°C, frequency of 100-800 Hz, and time of 10-50min.

[0046] In one embodiment, in step S2, the average particle size of the Fe3O4 magnetic nanoparticles is 20-200nm, preferably 50-100nm. The content of Fe3O4 nanoparticles and GO@PEI nanosheets can vary in a large range. In a specific embodiment, the mass concentration of the aqueous dispersion of Fe3O4 magnetic nanoparticles is 5-20mg / mL; the mass concentration of the aqueous dispersion of GO@PEI nanosheets is 0.1-20mg / mL; the volume ratio of the aqueous dispersion of GO@PEI nanosheets to the aqueous dispersion of Fe3O4 magnetic nanoparticles is 500-1000:1. The conditions of the second ultrasonic treatment include: temperature of 10-30°C, frequency of 100-800Hz, and time of 10-50min.

[0047] In the present disclosure, by controlling the dosage of Fe3O4 magnetic nanoparticles, a suitable number of Fe3O4 nanoparticles are loaded on GO@PEI nanosheets, which not only enables the prepared magnetic fluorescent nanofilm probe to have higher magnetism and ensures the dispersibility of the magnetic fluorescent nanofilm probe, but also avoids the reduction of the dispersibility of the magnetic fluorescent nanofilm probe caused by excessive Fe3O4 magnetic nanoparticles adsorbed on graphene oxide nanosheets.

[0048] In one embodiment, in step S3, the mass concentration of the aqueous dispersion of the GOFe magnetic nanomembrane is 0.1-20 mg / mL; the mass concentration of the aqueous solution of the second cationic polymer is 0.1-5 mg / mL; the volume ratio of the aqueous dispersion of the GOFe magnetic nanomembrane to the aqueous solution of the second cationic polymer per 100 mL of the aqueous solution of the second cationic polymer is 1-5:1; the conditions of the third ultrasonic treatment include: temperature of 10-30°C, frequency of 100-800 Hz, and time of 10-50 min.

[0049] In one embodiment, in step S4, the mass concentration of the aqueous dispersion of the GOFe@PEI magnetic substrate film is 0.1-20 mg / mL; the amount of the CdSe / ZnS-MPA quantum dots added is 0.01-0.1 mg relative to every 100 mL of the aqueous dispersion of the GOFe@PEI magnetic substrate film; the conditions for the fourth ultrasonic treatment include: temperature of 10-30°C, frequency of 100-800 Hz, and time of 10-50 min. The mass concentration of the aqueous solution of the third cationic polymer is 0.1-5 mg / mL; the mass concentration of the aqueous dispersion of the GOFe@QD magnetic nanofilm is 0.1-20 mg / mL; the volume ratio of the aqueous dispersion of the GOFe@QD magnetic nanofilm to the aqueous solution of the third cationic polymer is 1-5:1; the conditions for the fifth ultrasonic treatment include: temperature of 10-30°C, frequency of 100-800 Hz, and time of 10-50 min. The mass concentration of the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm is 0.1-20 mg / mL; the amount of CdSe / ZnS-MPA quantum dots added is 0.01-0.1 mg per 100 mL of the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm; the conditions of the sixth ultrasonic treatment include: temperature of 10-30°C, frequency of 100-800 Hz, and time of 10-50 min.

[0050] Within the scope of the above implementation, the amount of CdSe / ZnS-MPA quantum dots added can be controlled so that the magnetic fluorescent nanofilm probe has a suitable number of quantum dots, thereby further ensuring the fluorescence of the magnetic fluorescent nanofilm probe.

[0051] In step S5, the mass concentration of the aqueous solution of aminophenylboronic acid is 0.01-0.5 mg / mL, preferably 0.01-0.05 mg / mL; the amount of the GOFe@DQD magnetic nanofilm added is 0.01-0.1 mg per 100 mL of the aqueous solution of aminophenylboronic acid. The incubation conditions include: a temperature of 10-30°C and a time of 2-5 hours. Furthermore, this step may also include blocking unreacted carboxyl groups with bovine serum albumin to obtain the magnetic fluorescent nanofilm probe.

[0052] The third aspect of the present disclosure provides the use of the magnetic fluorescent nanomembrane probe described in the first aspect or the magnetic fluorescent nanomembrane probe prepared by the method described in the second aspect in detecting viral glycoproteins by immunochromatography for non-disease diagnosis and / or treatment purposes, or in preparing products for detecting viral glycoproteins.

[0053] A fourth aspect of the present disclosure provides a kit for detecting viral glycoproteins, the kit comprising the magnetic fluorescent nanomembrane probe described in the first aspect or the magnetic fluorescent nanomembrane probe prepared by the method described in the second aspect.

[0054] The kit disclosed herein utilizes aminophenylboronic acid coupled in a magnetic fluorescent nanomembrane probe to effectively capture and rapidly enrich a variety of viral glycoproteins from a sample to be detected. The magnetic fluorescent nanomembrane probe disclosed herein avoids the complicated antibody screening and modification process and broadens the application scope of immunochromatography.

[0055] In one embodiment, the kit further comprises an immunochromatographic test strip (ICA), which comprises a sample pad, a chromatographic membrane and an absorption pad connected in sequence, and a plurality of detection lines and quality control lines are arranged on the chromatographic membrane; each of the detection lines is respectively loaded with antiviral glycoprotein antibodies; and the quality control line is loaded with streptavidin.

[0056] In a preferred embodiment, there are three test lines, and the three test lines are respectively loaded with anti-SARS-CoV-2 antigen spike protein antibodies, anti-MPXVA29 envelope protein antibodies, and anti-EBOV glycoprotein antibodies. The present disclosure can realize the simultaneous analysis of three highly pathogenic viral glycoproteins, thereby improving the detection throughput and detection sensitivity.

[0057] In the present disclosure, the sample pad is used to load the sample solution to be detected. The absorbent pad is used to provide capillary force. The material of the bottom plate is preferably PVC, and the bottom plate serves as the backing card of the dual-signal immunochromatographic test paper. The chromatographic membrane is a nitrocellulose membrane. Anti-SARS-CoV-2 antigen spike protein antibodies, anti-MPXVA29 envelope protein antibodies, anti-EBOV glycoprotein antibodies and streptavidin are sprayed on the surface of the nitrocellulose (NC) membrane, respectively, and then the modified NC membrane is placed in a constant temperature drying oven (37°C) to obtain the nitrocellulose membrane in the immunochromatographic test strip.

[0058] In one embodiment, the concentration of the anti-SARS-CoV-2 antigen spike protein antibody is preferably 0.1-2.5 mg / mL, preferably 1 mg / mL; the concentration of the anti-MPXVA29 envelope protein antibody is preferably 0.1-2.5 mg / mL, preferably 1.2 mg / mL; the concentration of the anti-EBOV glycoprotein antibody is preferably 0.1-2.5 mg / mL, preferably 1 mg / mL. The mass concentration of streptavidin is preferably 0.1-2.5 mg / mL, preferably 1 mg / mL.

[0059] In the present disclosure, the kit also includes a running buffer, a magnetic fluorescent nanofilm probe and a quality control line probe. The running buffer includes Tween, fetal bovine serum (FBS) and PBS buffer. Figure 1As shown in b, the quality control line probe is biotinylated BSA modified on the surface of the magnetic fluorescent nanomembrane.

[0060] A method for detecting viral glycoproteins using a kit for detecting viral glycoproteins: a method for detecting viral glycoproteins (such as Figure 4 ): Incubate the sample to be tested, the magnetic fluorescent nanofilm probe and the quality control line probe at room temperature for 2 minutes, resuspend them in the running buffer, mix them evenly and load them onto the sample pad of the immunochromatographic test strip, and contact them with the three test lines on the test strip for chromatography reaction. After the reaction is completed, use a portable fluorescence reader to read the fluorescence signal for quantitative analysis.

[0061] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0062] In the examples of the present disclosure, any specific experimental conditions not specified are based on conventional conditions well known to those skilled in the art or conditions recommended by the manufacturer.

[0063] Example 1 (1) Graphene oxide nanosheets with an average diameter of 700 nm were dispersed in water to obtain an aqueous dispersion of graphene oxide nanosheets (mass concentration of 2 mg / mL); the aqueous dispersion of graphene oxide nanosheets was mixed with a PEI aqueous solution with a mass concentration of 0.2 mg / mL in a volume ratio of 2:1, and ultrasonically treated at 20°C and 800 Hz for 30 min to obtain GO@PEI nanosheets.

[0064] (2) The aqueous dispersion of GO@PEI nanosheets (mass concentration of 2 mg / mL) and the aqueous dispersion of Fe3O4 magnetic nanoparticles (mass concentration of 10 mg / mL) were mixed in a volume ratio of 800:1 and ultrasonically treated at 20°C and 800 Hz for 30 min to obtain a GOFe magnetic nanofilm.

[0065] (3) The aqueous dispersion of GOFe nanofilm (mass concentration of 1 mg / mL) and the PEI aqueous solution (mass concentration of 0.2 mg / mL) were mixed in a volume ratio of 2:1 and ultrasonically treated at 20°C and 800 Hz for 30 min to obtain a GOFe@PEI magnetic substrate film.

[0066] (4) Add 0.04 mg of CdSe / ZnS-MPA quantum dots into 50 mL of GOFe@PEI magnetic substrate film aqueous dispersion (mass concentration is 5 mg / mL), and perform ultrasonic treatment at 20 °C and 800 Hz for 20 min to obtain GOFe@QD magnetic nanofilm; The aqueous dispersion of GOFe@QD magnetic nanofilm (mass concentration of 1 mg / mL) and the aqueous solution of the third cationic polymer (mass concentration of 0.2 mg / mL) were mixed at a volume ratio of 2:1, and ultrasonically treated at 20°C and 800 Hz for 30 min to obtain GOFe@QD@PEI magnetic nanofilm; 0.04 mg of CdSe / ZnS-MPA quantum dots were added to 50 mL of GOFe@QD@PEI magnetic nanofilm dispersion (mass concentration was 5 mg / mL), and ultrasonic treatment was performed at 20°C and 800 Hz for 20 min to obtain GOFe@DQD magnetic nanofilm.

[0067] (5) 0.05 mg of GOFe@DQD magnetic nanofilm was mixed with 10 mL of an aqueous solution of aminophenylboronic acid (mass concentration of 0.01 mg / mL) and incubated at room temperature for 3 h to obtain the magnetic fluorescent nanofilm probe S-1.

[0068] Example 2 This example is used to illustrate the preparation of a kit for detecting viral glycoproteins and a method for detecting viral glycoproteins.

[0069] The kit includes an immunochromatographic test strip, which includes a PVC bottom plate, and a sample pad, a nitrocellulose membrane and an absorption pad connected in sequence arranged above the bottom plate. The nitrocellulose membrane is provided with three detection lines and a quality control line; the quality control line is loaded with 1 mg / mL of streptavidin; the three detection lines are loaded with 1.0, 1.2, and 1.0 mg / mL of anti-SARS-CoV-2 antigen spike protein antibodies, anti-MPXVA29 envelope protein antibodies, and anti-EBOV glycoprotein antibodies, respectively. Anti-SARS-CoV-2 antigen spike protein antibodies, anti-MPXVA29 envelope protein antibodies, and anti-EBOV glycoprotein antibodies and streptavidin are sprayed on the surface of the nitrocellulose (NC) membrane, respectively, and then the modified NC membrane is placed in a constant temperature drying oven (37°C) to obtain the nitrocellulose membrane in the immunochromatographic test strip.

[0070] The kit also includes running buffer: Tween 20, fetal bovine serum (FBS), and PBS buffer.

[0071] The magnetic fluorescent nanofilm probe S-1 of Example 1 is used to detect the virus sample to be tested, and the method for detecting the virus glycoprotein (such as Figure 4 ): Incubate the sample to be tested, the magnetic fluorescent nanofilm probe and the quality control line probe at room temperature for 2 minutes. After enrichment, remove the incubation solution, resuspend in the running buffer, mix evenly and load onto the sample pad of the immunochromatographic test strip, and contact with the three detection lines on the test strip for chromatography reaction. After the reaction is completed, use a portable fluorescence reader to read the fluorescence signal for quantitative analysis.

[0072] Detect samples of anti-SARS-CoV-2 antigen spike protein antibody (SARS-CoV-2SP), anti-MPXV A29 envelope protein antibody (MPXV A29) and anti-EBOV glycoprotein antibody (EBOV GP) at different concentrations (0.001-100ng / mL). Under ultraviolet light, the visual limit of the fluorescence signal of the three detection lines is 0.01ng / mL ( Fig. 9 a). The fluorescence intensity corresponding to the entire ICA strip and the detailed fluorescence signals of the T1, T2, and T3 regions are shown in Figure 2. Fig. 9 b and 9c. The S-type calibration curves corresponding to EBOV GP (T1), MPXV A29 (T2) and SARS-CoV-2 SP (T3) were constructed, and the curves are shown as follows: Fig. 9 df. According to this calibration curve, the dynamic detection range of GOFe@DQD-APBA-ICA for the three target virus antigens is 0.01-100 ng / mL, and the correlation coefficient (R 2 ) are all greater than 0.95. In addition, the T-line fluorescence signals of EBOV GP, MPXV A29 and SARS-CoV-2 SP are linear when the target concentration is 0.5-0.001 ng / mL. The detection limits of the magnetic fluorescent nanomembrane S-1 of Example 1 for detecting EBOV, MPXV and SARS-CoV-2 are 0.93, 1.03 and 0.89 pg / mL, respectively.

[0073] This example also verifies the feasibility of using magnetic fluorescent nanomembrane probes for immunochromatography to simultaneously detect three viral glycoproteins. Figure 5 As shown, it is proved that there is no cross reaction between the three viruses detected by the immunochromatography disclosed in the present invention and between them and other pathogens. The inside of the immunochromatography test strip was observed using a scanning electron microscope, proving that the label can gather at the detection line of the immunochromatography test strip after capturing the protein. The above results verify that the magnetic fluorescent nanomembrane probe and the virus glycoprotein complex formed by it flow smoothly on the NC membrane and work stably on the immunochromatography.

[0074] This example also optimizes the concentration of the capture antibody. Figure 6 As shown, when the concentrations of anti-SARS-CoV-2 antigen spike protein antibodies, anti-MPXVA29 envelope protein antibodies, and anti-EBOV glycoprotein antibodies on the test line reached 1 mg / mL, 1.2 mg / mL, and 1 mg / mL, respectively, the signal-to-noise ratio was the highest and the test results were the best.

[0075] This example also optimizes the running buffer. Figure 7As shown in the figure, when the running buffer consisted of 10 mM PBS, 1% Tween 20 and 5% FBS, it had the highest signal-to-noise ratio and the detection results were the best.

[0076] This embodiment also optimizes the reaction time. Figure 8 As shown, the magnetic fluorescent nanomembrane probe has the highest signal-to-noise ratio and the best detection result when the immunochromatographic reaction time is 15 minutes.

[0077] Example 3 The performance of the immunochromatographic technology based on the broad-spectrum magnetic fluorescent nanomembrane probe was evaluated by detecting SARS-CoV-2 antigen spike protein, MPXVA29 envelope protein and EBOV glycoprotein samples with different concentrations (0.001-100 ng / mL).

[0078] like Fig. 9 As shown, Fig. 9 The picture of the broad-spectrum immunochromatographic strip for simultaneous detection of three target viral antigens, the corresponding fluorescence intensity of the entire ICA strip, the detailed fluorescence signals of the three detection lines, and the corresponding S-shaped calibration curve are displayed. Within the detection range of 0.01-100 ng / mL, as the concentration of SARS-CoV-2 antigen spike protein, MPXVA29 envelope protein, and EBOV glycoprotein samples increased, the fluorescence intensity gradually increased. Under ultraviolet light, the visual limit of the fluorescence signals of the three detection lines is 0.01 ng / mL. The results of the S-shaped calibration curve show that the dynamic detection range of the broad-spectrum immunochromatography for the three viral glycoproteins is 0.001-100 ng / mL, and the correlation coefficient (R 2 ) were all greater than 0.95, indicating that there was a good correlation between glycoprotein concentration (0.001-100 ng / mL) and fluorescence intensity.

[0079] Three groups of different concentrations (50, 5, and 0.5 ng / mL) of SARS-CoV-2 antigen spike protein, MPXVA29 envelope protein, and EBOV glycoprotein were used to verify the repeatability of immunochromatography based on broad-spectrum magnetic fluorescent nanomembrane probes, such as Fig.10 As shown, the fluorescence signal on the detection line changed slightly, and the RSD value of each test group was less than 12.1%, indicating that the established broad-spectrum immunochromatography technology has good repeatability.

[0080] By adding viral glycoproteins to saliva samples of healthy people to simulate clinical samples, the performance of immunochromatography based on broad-spectrum magnetic fluorescent nanomembrane probes in actual detection was evaluated. Different concentrations (100, 10, and 1 ng / mL) of SARS-CoV-2 antigen spike protein, MPXVA29 envelope protein, and EBOV glycoprotein were added to saliva samples of healthy volunteers and detected using the immunochromatographic system. Fig.11As shown, the recovery rates of three different viruses were calculated based on the fluorescence intensity of the detection line. The detection recovery rates were 84.55%-107.58%, and the RSD values ​​were 4.28%-9.27%, indicating that this method has good accuracy and reliability for the detection of real saliva samples.

[0081] In addition, the clinical application potential of the immunochromatography based on magnetic fluorescent nanomembrane probe was further studied using clinical nasopharyngeal swab specimens from patients infected with SARS-CoV-2. 34 SARS-CoV-2 positive specimens (15 strongly positive and 19 weakly positive) and 19 SARS-CoV-2 negative nasopharyngeal swabs were collected from Guangdong Provincial People's Hospital and subjected to standard enzyme-linked immunosorbent assay (ELISA) to compare the effects of the proposed immunochromatography based on broad-spectrum magnetic fluorescent nanomembrane probe. Fig.12 As shown in the figure, the test results show that the immunochromatography based on the broad-spectrum magnetic fluorescent nanomembrane probe can accurately identify SARS-CoV-2 in nasopharyngeal swabs, and the detection accuracy of clinical respiratory samples infected with SARS-CoV-2 is 100%. Deming regression analysis showed that for 19 weakly positive samples, the proposed immunochromatography based on the broad-spectrum magnetic fluorescent nanomembrane probe was highly correlated with the consistency of ELISA. The slope of the regression equation for SARS-CoV-2 weakly positive samples was 0.999 (95% confidence interval 0.996-0.999), R 2 The difference between the two methods was 0.998. In addition, the Bland-Altman plot was used to analyze the correlation between the two detection methods. The average difference between the two methods was -0.011, and the 95% confidence interval of the difference was -0.04-0.017. The intercept and slope of the Bland-Altman regression line (purple) were close to 0. The above results show that the two detection methods are equivalent for weakly positive specimens (1-0.2ng / mL).

[0082] In summary, the kit prepared by the magnetic fluorescent nanomembrane probe disclosed in the present invention can provide higher sensitivity and wider detection range when detecting clinical virus infection samples, and can shorten the detection time to 20 minutes.

[0083] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0085] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A magnetic fluorescent nanofilm probe, characterized in that: The probe comprises a magnetic substrate film layer and a quantum dot shell layer arranged in sequence along a stacking direction; The quantum dot shell layer is a multi-layer stacked structure formed by alternating quantum dot layers and third cationic polymer interlayers, and the first layer and the last layer along the stacking direction of the stacked structure are each independently the quantum dot layer; Aminophenylboronic acid is coupled to the surface of the last quantum dot layer which faces away from the graphene oxide nanosheet layer.

2. The magnetic fluorescent nanofilm probe according to claim 1, wherein: The stacked structure has 3 layers; The quantum dots in the quantum dot layer are CdSe / ZnS-MPA quantum dots.

3. The magnetic fluorescent nanofilm probe according to claim 1, wherein: The magnetic substrate film layer comprises a graphene oxide nanosheet layer, a first cationic polymer interlayer, a Fe3O4 magnetic nanoparticle layer and a second cationic polymer interlayer which are sequentially arranged along a stacking direction; Wherein, in the graphene oxide nanosheet layer, the average diameter of the graphene oxide nanosheet is 100-2000nm, preferably 500-800nm; In the Fe3O4 magnetic nanoparticle layer, the average particle size of the Fe3O4 magnetic nanoparticles is 20-200 nm, preferably 50-100 nm.

4. The magnetic fluorescent nanofilm probe according to claim 3, wherein: The first cationic polymer interlayer, the second cationic polymer interlayer and the third cationic polymer interlayer are each independently polyethyleneimine.

5. The method for preparing the magnetic fluorescent nanofilm probe according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: S1, mixing an aqueous dispersion of graphene oxide nanosheets with an aqueous solution of a first cationic polymer and performing a first ultrasonic treatment to obtain GO@PEI nanosheets; S2, mixing the aqueous dispersion of the GO@PEI nanosheets with the aqueous dispersion of Fe3O4 magnetic nanoparticles and performing a second ultrasonic treatment to obtain a GOFe magnetic nanofilm; S3, mixing the aqueous dispersion of the GOFe magnetic nanofilm with the aqueous solution of the second cationic polymer and performing a third ultrasonic treatment to obtain a GOFe@PEI magnetic substrate film; S4, adding CdSe / ZnS-MPA quantum dots to the aqueous dispersion of the GOFe@PEI magnetic substrate film and performing a fourth ultrasonic treatment to obtain a GOFe@QD magnetic nanofilm; The aqueous dispersion of the GOFe@QD magnetic nanofilm is mixed with an aqueous solution of a third cationic polymer and subjected to a fifth ultrasonic treatment to obtain a GOFe@QD@PEI magnetic nanofilm; Adding CdSe / ZnS-MPA quantum dots to the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm and performing a sixth ultrasonic treatment to obtain a GOFe@DQD magnetic nanofilm; S5. Mix the GOFe@DQD magnetic nanofilm with an aqueous solution of aminophenylboronic acid for incubation.

6. The method according to claim 5, wherein: In step S1, the average diameter of the graphene oxide nanosheets is 100-2000nm, preferably 500-800nm; the mass concentration of the aqueous dispersion of the graphene oxide nanosheets is 0.1-5mg / mL; the mass concentration of the aqueous solution of the first cationic polymer is 0.1-5mg / mL; the volume ratio of the aqueous dispersion of the graphene oxide nanosheets to the aqueous solution of the first cationic polymer is 0.5-5:1; In step S2, the average particle size of the Fe3O4 magnetic nanoparticles is 20-200 nm, preferably 50-100 nm; the mass concentration of the aqueous dispersion of the Fe3O4 magnetic nanoparticles is 5-20 mg / mL; the mass concentration of the aqueous dispersion of the GO@PEI nanosheets is 0.1-20 mg / mL; the volume ratio of the aqueous dispersion of the GO@PEI nanosheets to the aqueous dispersion of the Fe3O4 magnetic nanoparticles is 500-1000:1; In step S3, the mass concentration of the aqueous dispersion of the GOFe magnetic nanofilm is 0.1-20 mg / mL; the mass concentration of the aqueous solution of the second cationic polymer is 0.1-5 mg / mL; the volume ratio of the aqueous dispersion of the GOFe magnetic nanofilm to the aqueous solution of the second cationic polymer is 1-5:1; In step S4, the mass concentration of the aqueous dispersion of the GOFe@PEI magnetic substrate membrane is 0.1-20 mg / mL; the amount of the CdSe / ZnS-MPA quantum dots added is 0.01-0.1 mg per 100 mL of the aqueous dispersion of the GOFe@PEI magnetic substrate membrane; the mass concentration of the aqueous solution of the third cationic polymer is 0.1-5 mg / mL; the mass concentration of the aqueous dispersion of the GOFe@QD magnetic nanofilm is 0.1-20 mg / mL; the volume ratio of the aqueous dispersion of the GOFe@QD magnetic nanofilm to the aqueous solution of the third cationic polymer is 1-5:1; the mass concentration of the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm is 0.1-20 mg / mL; the amount of the CdSe / ZnS-MPA quantum dots added is 0.01-0.1 mg per 100 mL of the aqueous dispersion of the GOFe@QD@PEI magnetic nanofilm; In step S5, the mass concentration of the aqueous solution of aminophenylboronic acid is 0.01-0.5 mg / mL; the added amount of the GOFe@DQD magnetic nanofilm is 0.01-0.1 mg per 100 mL of the aqueous solution of aminophenylboronic acid.

7. Use of the magnetic fluorescent nanomembrane probe described in any one of claims 1 to 4 or the magnetic fluorescent nanomembrane probe prepared by the method described in any one of claims 5 to 6 in detecting viral glycoproteins by immunochromatography for non-disease diagnosis and / or treatment purposes, or in the preparation of products for detecting viral glycoproteins.

8. A kit for detecting viral glycoprotein, characterized in that: The kit comprises the magnetic fluorescent nanofilm probe described in any one of claims 1 to 4 or the magnetic fluorescent nanofilm probe prepared by the method described in any one of claims 5 to 6.

9. The kit according to claim 8, wherein The kit also includes an immunochromatographic test strip, which includes a sample pad, a chromatographic membrane and an absorption pad connected in sequence, and the chromatographic membrane is provided with a plurality of detection lines and quality control lines; Each of the test lines is loaded with anti-viral glycoprotein antibodies; The quality control line is loaded with streptavidin.

10. The kit according to claim 9, wherein There are three test lines, which are respectively loaded with anti-SARS-CoV-2 antigen spike protein antibodies, anti-MPXVA29 envelope protein antibodies and anti-EBOV glycoprotein antibodies.

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