Silicon shell protected magnetic quantum dot nanoprobe, preparation method, kit and application

Abstract

The invention discloses a silicon shell protected magnetic quantum dot nanoprobe, a preparation method, a kit and application. The nanoprobe comprises a nanomaterial and a specific antibody, the nanometer material is of a core-shell structure, and the outer surface of a core is sequentially coated with an interlayer and a protective shell layer outwards. The interlayer comprises a multi-layer laminated structure formed by alternately arranging a plurality of layers of positive electricity interlayers and negative electricity interlayers; the positive electric interlayer is polyethyleneimine, the negative electric interlayer is quantum dots, the quantum dots are CdSe / ZnS or carbon quantum dots, amino groups on the surface of the shell layer and a dialkynyl polyethylene ethanol solution are subjected to general coupling of specific antibodies in a click chemistry mode by taking diyne molecules as connecting arms. The magnetic nanoprobe with the core-shell structure not only has strong magnetic responsiveness and a high fluorescence signal, but also can adapt to a complex environment, after the magnetic nanoprobe is integrated with an immunochromatography test strip, to-be-detected targets including pathogenic bacteria and inflammatory markers can be rapidly captured in a whole blood sample at the same time, and the magnetic nanoprobe has huge application potential in early diagnosis of sepsis.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatographic detection, and particularly relates to a silica-shell protected magnetic quantum dot nanoprobe and a preparation method, a kit and an application thereof. Background Art

[0002] Sepsis, as a rapidly progressing and critically ill systemic infection, its early diagnosis is crucial for timely intervention and treatment. However, currently commonly used detection methods in clinics, such as blood culture, nucleic acid-based detection (such as PCR and metagenomic sequencing), and mass spectrometry technology, although having advantages in accuracy, generally have defects such as complex operation, long detection cycle (usually taking several hours to several days), and strict requirements for laboratory conditions, and it is difficult to meet the clinical needs of point-of-care testing (POCT).

[0003] Immunochromatography (ICA) has been widely used in clinical screening and on-site rapid detection in recent years due to its advantages such as simple operation, rapid detection, and low cost. However, traditional ICA test strips based on colloidal gold colorimetric signals often face problems such as insufficient sensitivity, limited quantification ability, and susceptibility to interference in complex matrices (such as whole blood) during the detection of targets such as pathogenic bacteria and inflammatory markers, resulting in unsatisfactory detection accuracy and reproducibility. For this reason, in recent years, researchers have tried to introduce nanolabels with high fluorescence intensity and excellent optical properties (such as quantum dots, upconversion fluorescent nanoparticles, etc.) to improve the performance of ICA technology. However, in the actual application process, the traditional quantum dot immunochromatographic system still has deficiencies such as signal quenching of quantum dots in complex sample environments (such as extreme pH and high salt conditions), easy detachment, and high background signals on the NC membrane.

[0004] Magnetic nanomaterials, due to their excellent magnetic responsiveness, can capture and separate targets in complex biological samples, thereby reducing the influence of interfering substances in the samples, and have been widely used to construct stable detection platforms. Existing magnetic quantum dot probes mostly adopt the method of directly adsorbing or coating quantum dots on the surface of Fe3O4 nanoparticles. Although the detection sensitivity has been improved to a certain extent, there are still many limitations in terms of structural homogeneity, fluorescence signal stability, and detachment of quantum dots during long-term storage. At the same time, traditional chemical coupling methods often affect the accuracy and reproducibility of detection results due to harsh reaction conditions, low coupling efficiency, and non-specific binding when connecting specific antibodies to nanoprobes.

[0005] In the prior art, the invention patent with the patent application number CN202211023374 discloses a homogeneous method for detecting the nucleocapsid protein of novel coronavirus based on a nanobody-monoclonal antibody sandwich. In this invention, a variety of nanobodies against the nucleocapsid protein of novel coronavirus are prepared as solid-phase antibodies, the monoclonal antibody against the nucleocapsid protein is used as the detection antibody, and the N protein of novel coronavirus is used as the detection target. A group of antibodies with excellent detection performance is screened out through an enzyme-linked immunosorbent assay, and the lowest detection limit is 0.8 ng / mL. In addition, through a one-step activation method, the nanobody and the monoclonal antibody are respectively conjugated to the surface of quantum dot microspheres and magnetic nanomicrospheres, successfully preparing two probes targeting the nucleocapsid protein of novel coronavirus, and constructing a magnetic separation immunohomogeneous method for rapid detection of the nucleocapsid protein of novel coronavirus. The lowest detection limit of the nucleocapsid protein is 0.15 ng / mL. However, this invention is easily interfered by complex sample systems and has a single detection target.

[0006] The invention patent with the patent application number CN202011409838 discloses a preparation method of a perovskite quantum dot-modified magnetic biochar composite nanomaterial. Fe3O4 magnetic nanoparticles are modified with polyethyleneimine and then adsorbed with a layer of carboxylated biochar to form a magnetic composite Fe3O4@cNDs with a biochar outer shell. Then, the magnetic composite Fe3O4@cNDs is adsorbed with amino-functionalized perovskite quantum dots to prepare a perovskite quantum dot-modified magnetic biochar composite nanomaterial Fe3O4@cNDs@CsPbX3. The application of the magnetic quantum dot label prepared from the above perovskite quantum dot-modified magnetic biochar composite nanomaterial in fluorescence immunochromatographic detection. This type of magnetic quantum dot label has good dispersibility, strong magnetic responsiveness, adjustable particle size, and is easy to prepare in large quantities. It can be used for the capture, enrichment, and highly sensitive detection of target substances in complex systems. However, this invention only adsorbs a layer of quantum dots, and the fluorescence intensity is slightly low, and the application scenario is limited.

[0007] The invention patent with the patent application number CN2016103922043 discloses a monodisperse strongly magnetic immune nanomicrosphere, and the particle size of the monodisperse strongly magnetic immune nanomicrosphere is 5 nm - 5 μm. This invention also discloses a preparation method of a monodisperse strongly magnetic immune nanomicrosphere, including the following steps: Step 1, preparation of iron oxide nanoparticles; Step 2, preparation of monodisperse silicon hydroxyl magnetic nanomicrospheres; Step 3, preparation of monodisperse amino-functionalized magnetic nanomicrospheres; Step 4, preparation of monodisperse strongly magnetic immune nanomicrospheres. The monodisperse strongly magnetic immune nanomicrosphere and its preparation method of this invention have good superparamagnetism, high magnetic content, good dispersibility, no agglomeration phenomenon, regular and standard morphology, unified and controllable particle size, and easy functionalization modification, etc. However, this invention does not have a fluorescence signal and cannot be directly applied to the immunochromatographic system.

[0008] In recent years, click chemistry, as an efficient, mild and highly specific bioconjugation technology, has been gradually applied to the fields of biosensing and immunoassay due to its advantages of fast reaction rate, stable products and no by-products. Utilizing click chemistry to achieve efficient and stable connection between antibodies and nanomaterials can significantly enhance the recognition and capture ability of probes in complex samples, providing a new technical approach for constructing sensitive, stable and fast-responsive detection platforms. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, one of the objectives of the present invention is to provide a silicon shell-protected magnetic quantum dot nanoprobe and its preparation method, kit and application. The magnetic nanoprobe disclosed by the present invention has strong magnetic responsiveness, high fluorescence signal, adjustable particle size and the ability to specifically capture target substances. At the same time, the SiO2 outer shell can improve the stability and dispersibility of the nanomaterials, and also reduce the fluorescence background signal caused by them on the test strip. The magnetic nanoprobe of this application realizes the rapid capture of substances to be detected in whole blood samples such as pathogenic bacteria and inflammatory markers, and has great application potential in the early diagnosis of sepsis.

[0010] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0011] A silicon shell-protected magnetic quantum dot nanoprobe, which includes nanomaterials and specific antibodies;

[0012] The nanomaterials have a core-shell structure, the core of the core-shell structure is Fe3O4 nanoparticles, and a sandwich layer and a protective shell layer are sequentially coated outward from the outer surface of the core;

[0013] The sandwich layer includes a multi-layer stacked structure formed by alternating multiple positive charge sandwich layers and negative charge sandwich layers. The stacked structure is 2-8 layers, the first layer in the stacking direction of the stacked structure is a positive charge sandwich layer, and the last layer is a negative charge sandwich layer;

[0014] The positive charge sandwich layer is polyethyleneimine, the negative charge sandwich layer is a quantum dot, the quantum dot is CdSe / ZnS or a carbon quantum dot, the protective shell layer is silicon dioxide, and the amino group on the surface of the shell layer is specifically conjugated with a specific antibody by click chemistry through a diyne molecule as a linker arm using a diyne-based polyalcohol solution. The specific antibody is at least one of a Pseudomonas aeruginosa antibody, a PCT antibody, and an IL6 antibody.

[0015] Preferably, 1 g of nanomaterials is conjugated with ≤1 mg of specific antibodies, and the specific antibodies are at least one of a Pseudomonas aeruginosa antibody, a PCT antibody, and an IL6 antibody.

[0016] Preferably, in any of the above solutions, the average particle size of the Fe3O4 nanoparticles is 50 - 500 nm, the thickness of each positively charged interlayer ≤ 5 nm, the average particle size of the quantum dots is 5 - 30 nm, and the thickness of the SiO2 shell layer is 5 - 30 nm.

[0017] Specifically, the average particle size of the Fe3O4 nanoparticles is any value within the range of 50 - 500 nm, such as 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm; the thickness of the positively charged interlayer is any value within the range of ≤ 5 nm, such as it can be 5 nm, 4.5 nm, 4 nm, 3.5 nm, 3 nm, 2.5 nm, 2 nm, 1.5 nm, 1 nm, 0.5 nm; the average particle size of the quantum dots is any value within the range of 5 - 30 nm, such as it can be 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm; the thickness of the SiO2 shell layer is any value within the range of 5 - 30 nm, such as it can be 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm.

[0018] The present invention also discloses a preparation method of the silicon shell - protected magnetic quantum dot nanoprobe described above. This method includes the following steps:

[0019] S1. Disperse the Fe3O4 nanoparticles in deionized water to obtain a dispersion liquid, then mix the dispersion liquid with an aqueous solution of polyethyleneimine and perform the first ultrasonic treatment to obtain Fe3O4 - PEI nanospheres;

[0020] S2. Mix the Fe3O4 - PEI nanospheres obtained in step S1 with an aqueous solution of quantum dots and perform the second ultrasonic treatment to obtain MagQDs nanospheres;

[0021] S3. Mix the MagQDs nanospheres obtained in step S2 with ammonia water and perform the third ultrasonic treatment, then add tetraethyl orthosilicate to obtain MagQDs@Si nanospheres;

[0022] S4. Mix the MagQDs@Si nanospheres obtained in step S3 with an aqueous solution of polyethyleneimine and perform the fourth ultrasonic treatment to obtain MagQDs@Si - PEI nanospheres;

[0023] S5. After activating the MagQDs@Si - PEI microspheres obtained in step S4, perform click - chemistry coupling treatment to obtain MagQDs@Si - PEI - yne nanospheres;

[0024] S6. Mix the MagQDs@Si-PEI-yne nanoparticles obtained in step S5 with a specific antibody and incubate to obtain a quantum dot nanoprobe.

[0025] Preferably, in step S1, the average particle size of the Fe3O4 nanoparticles is 50 - 500 nm, and ≤3 g of Fe3O4 nanoparticles are dispersed in every 100 mL of deionized water; the molecular weight of the polyethyleneimine is 5000 - 80000 Da, and the mass concentration of the polyethyleneimine aqueous solution is ≤2 mg / mL; the volume ratio of the dispersion liquid to the polyethyleneimine aqueous solution is 1 - 50:1.

[0026] Specifically, the mass concentration of the polyethyleneimine aqueous solution can be 2 mg / mL, 1.5 mg / mL, 1 mg / mL, 0.5 mg / mL; the volume ratio of the dispersion liquid to the polyethyleneimine aqueous solution can be 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1.

[0027] Preferably, in step S2, the average diameter of the quantum dots is 5 - 30 nm, the addition amount of Fe3O4-PEI nanoparticles in every 100 mL of the quantum dot aqueous solution is ≤4 g, and the mass concentration of the quantum dot aqueous solution is ≤10 mg / mL.

[0028] Preferably, in step S3, for every 100 mL of ammonia water, the addition amount of the MagQDs nanoparticles is ≤4 g, the addition amount of tetraethyl orthosilicate is ≤4 mL, the volume concentration of the ammonia water is ≤50 v / v, and the molar concentration of the tetraethyl orthosilicate is ≤500 mol / L.

[0029] The addition amount of the MagQDs nanoparticles can be 4 g, 3.5 g, 3 g, 2.5 g, 2 g, 1.5 g, 1 g; the addition amount of tetraethyl orthosilicate can be 4 mL, 3.5 mL, 3 mL, 2.5 mL, 2 mL, 1.5 mL.

[0030] Preferably, in step S4, the molecular weight of the polyethyleneimine is 5000 - 80000 Da, and the mass concentration of the polyethyleneimine aqueous solution is ≤2 mg / mL; after the MagQDs@Si nanoparticles are dissolved in water, they are mixed with the polyethyleneimine aqueous solution, and the volume ratio of the two is 1 - 50:1.

[0031] The volume ratio of the MagQDs@Si nanoparticle aqueous solution to the polyethyleneimine aqueous solution can be 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1.

[0032] Preferably, in any of the above solutions, in step S5, the activation treatment includes: mixing the MagQDs@Si-PEI nanospheres with a diynyl polyethylene glycol solution and performing a fifth ultrasonic treatment.

[0033] The present invention also discloses a fluorescence immunochromatography kit for jointly detecting pathogenic bacteria and inflammatory markers. The kit includes the nanoprobes described in any one of the above, the pathogenic bacteria being Pseudomonas aeruginosa, and the inflammatory markers being PCT and IL6.

[0034] Preferably, it further includes an immunochromatography test strip, which includes a sample pad, a chromatography membrane, and an absorption pad connected in sequence. There are 3 detection lines and 1 quality control line arranged on the chromatography membrane. The 3 detection lines are respectively loaded with anti-Pseudomonas aeruginosa antibody, anti-PCT antibody, and anti-IL6 antibody, and the quality control line is loaded with goat anti-mouse IgG.

[0035] The present invention also discloses the application of the above fluorescence immunochromatography kit in simultaneously detecting pathogenic bacteria and sepsis markers in whole blood, the pathogenic bacteria being Pseudomonas aeruginosa, and the inflammatory markers being PCT and IL6.

[0036] Beneficial effects

[0037] The present invention provides a silicon shell-protected magnetic quantum dot nanoprobe and its preparation method, kit and application. The nanoprobe includes a nanomaterial and a specific antibody; the nanomaterial has a multi-layer core-shell structure, the core of the core-shell structure is Fe3O4 nanoparticles, and there are multiple layers of quantum dot interlayers and a protective shell layer coated outward in sequence from the outer surface of the core; the interlayer is a multi-layer stacked structure formed by alternating positive charge interlayers and negative charge interlayers, the first layer in the stacking direction of the stacked structure is a positive charge interlayer, and the last layer is a negative charge interlayer; the positive charge interlayer is polyethyleneimine; the negative charge interlayer is a quantum dot; the protective shell layer is SiO2; the amino group of the shell layer is coupled with the specific antibody through an alkyne reaction of click chemistry.

[0038] The core-shell structured magnetic nanoprobe of the present invention not only has strong magnetic responsiveness, high fluorescence signal and the ability to specifically capture target substances, but also can adapt to complex environments. After being integrated with an immunochromatography assay (ICA) strip, it can realize the rapid enrichment and quantitative analysis of pathogenic bacteria and infectious markers, with a relatively fast detection speed and high sensitivity. At the same time, the SiO2 outer shell can improve the stability and dispersibility of the nanomaterial, and can also reduce the fluorescence background signal caused by it on the test strip, and can rapidly capture the target substances to be detected in whole blood samples, including pathogenic bacteria and inflammatory markers, in whole blood samples, and has great application potential in the early diagnosis of sepsis. Brief description of the drawings

[0039] Figure 1 Schematic diagram of the synthesis preparation method of the nanoprobe in Example 2 of the present invention;

[0040] Figure 2 Structural electron microscopy characterization and energy dispersive X-ray spectrum of the nanoprobe in Example 2 of the present invention;

[0041] Figure 3 Fluorescence enhancement effect of the nanoprobe in Example 2 of the present invention;

[0042] Figure 4 Flow chart of the detection of Pseudomonas aeruginosa, PCT and IL6 using the nanoprobe of the present invention;

[0043] Figure 5 Capture and binding effect of MagQDs@Si–ICA of the present invention on the target to be detected;

[0044] Figure 6 Optimization results of the composition of the loading buffer of MagQDs@Si–ICA of the present invention;

[0045] Figure 7 Optimization results of the membrane coating concentration of MagQDs@Si–ICA of the present invention;

[0046] Figure 8 Cross-reaction verification results of MagQDs@Si-ICA of the present invention;

[0047] Figure 9 Specificity verification results of MagQDs@Si-ICA of the present invention;

[0048] Figure 10 Multi-channel detection result diagram of the joint detection of Pseudomonas aeruginosa, PCT and IL6 using MagQDs@Si-ICA of the present invention;

[0049] Figure 11 Detection result diagram of the detection of Pseudomonas aeruginosa, PCT and IL6 by colloidal gold method;

[0050] Figure 12 Repeatability verification results of MagQDs@Si-ICA of the present invention;

[0051] Figure 13 Analysis results of MagQDs@Si-ICA of the present invention in clinical samples. Detailed implementation manners

[0052] The embodiments of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the various components described below. Various modifications can be made within the scope of the invention claimed, and embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0053] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well-known to those skilled in the art. In the examples of the present invention, where no specific experimental conditions are indicated, they are all in accordance with the conventional conditions well-known to those skilled in the art or in accordance with the conditions recommended by the manufacturer.

[0054] Example 1

[0055] The first aspect of the present disclosure provides a silicon shell-protected magnetic quantum dot nanoprobe, which nanoprobe includes a nanomaterial and a specific antibody; the nanomaterial has a core-shell structure; the core of the core-shell structure is Fe3O4 nanoparticles, and a sandwich layer and a shell layer are sequentially coated outward from the outer surface of the core.

[0056] The sandwich layer is a multi-layer stacked structure formed by alternating positive and negative sandwich layers. The first layer in the stacking direction of the stacked structure is a positive sandwich layer, and the last layer is a negative sandwich layer.

[0057] The positive sandwich layer is polyethyleneimine; the negative sandwich layer is a quantum dot; the shell layer is SiO2; the shell layer is coupled to the specific antibody through a click chemical reaction.

[0058] In this embodiment, the nanomaterial uses superparamagnetic Fe3O4 nanoparticles as the core, which provides a magnetic enrichment ability while providing a colorimetric signal; then, multiple layers of quantum dots are adsorbed on the outer surface of the Fe3O4 nanoparticles through the positive sandwich layer polyethyleneimine (PEI), and finally, a protective SiO2 shell is coated on the outermost layer of the MagQDs nanospheres through the hydrolysis and condensation of tetraethyl orthosilicate (TEOS); this nanomaterial has dual properties of superparamagnetism and strong fluorescence signal; the SiO2 shell can improve the stability and dispersibility of the nanomaterial, and also reduce the fluorescence background signal caused by it on the test strip; by treating the SiO2 shell to form a shell layer containing a large number of carboxyl sites, it can be used to couple with specific antibodies to prepare a silicon shell magnetic quantum dot probe with strong magnetic responsiveness, high fluorescence signal and specific bacterial capture ability, realizing the rapid capture of substances to be detected such as pathogenic bacteria and infectious markers in the whole blood sample to be tested.

[0059] A further optimized technical solution of this embodiment is that the Fe3O4 nanoparticles used are superparamagnetic nanoparticles, and the average particle size of the Fe3O4 nanoparticles is 50 - 500 nm, preferably 100 - 300 nm.

[0060] A further optimized technical solution of this embodiment is that the laminated structure is 2 - 8 layers, for example, it can be 2 layers, 4 layers, 6 layers or 8 layers; specifically, the laminated structure may include a first positive charge interlayer and a first negative charge interlayer; it may also include a first positive charge interlayer, a first negative charge interlayer, a second positive charge interlayer and a second negative charge interlayer.

[0061] The thickness of each positive charge interlayer can vary within a relatively large range. A further optimized technical solution of this embodiment is that the thickness of each positive charge interlayer ≤ 5 nm, preferably 0.5 - 2 nm. By controlling the thickness of each positive charge interlayer in the present invention, quantum dots can be efficiently loaded without affecting the strong magnetism of Fe3O4, thereby endowing the nanomaterial with strong magnetism and strong fluorescence signal.

[0062] A further optimized technical solution of this embodiment is that the quantum dots are carbon quantum dots, and the average particle size of the quantum dots is 5 - 30 nm, preferably 8 - 15 nm.

[0063] A further optimized technical solution of this embodiment is that the thickness of the SiO2 shell layer ≤ 30 nm, preferably 10 - 30 nm.

[0064] A further optimized technical solution of this embodiment is that relative to 1 g of the nanomaterial, ≤ 1 mg of specific antibody is conjugated, preferably 0.001 - 0.01 mg.

[0065] A further optimized technical solution of this embodiment is that the specific antibody is preferably an anti - Pseudomonas aeruginosa antibody, a PCT antibody and an IL6 antibody. This enables the nanoprobes of the present disclosure to efficiently capture, enrich and detect Pseudomonas aeruginosa, PCT and IL6 in complex samples.

[0066] The preparation method of the above - mentioned silicon - shell - protected magnetic quantum dot nanoprobe includes the following steps:

[0067] S1. Dispersing Fe3O4 nanoparticles in deionized water to obtain a dispersion liquid, and then mixing the dispersion liquid with an aqueous solution of polyethyleneimine and performing a first ultrasonic treatment to obtain Fe3O4 - PEI nanospheres;

[0068] S2. Mixing the Fe3O4 - PEI nanospheres with an aqueous solution of quantum dots and performing a second ultrasonic treatment to obtain MagQDs nanospheres;

[0069] S3. Mixing the MagQDs nanospheres with ammonia water and performing a third ultrasonic treatment, and then adding tetraethyl orthosilicate to obtain MagQDs@Si nanospheres;

[0070] S4. Dissolve the MagQDs@Si nanospheres in water, mix them with an aqueous solution of polyethyleneimine, and perform a fourth ultrasonic treatment to obtain MagQDs@Si-PEI nanospheres;

[0071] S5. Mix the MagQDs@Si-PEI nanospheres with a solution of diethynyl polyethanol and perform a fifth ultrasonic treatment for coupling to obtain MagQDs@Si-PEI-yne nanospheres;

[0072] S6. Mix the MagQDs@Si-PEI-yne nanospheres with a specific antibody and perform an incubation treatment.

[0073] In the present disclosure, based on the electrostatic self-assembly method mediated by positively charged interlayer polyethyleneimine, a multi-layer quantum dot interlayer and a layer of SiO2 protective shell are assembled layer by layer on the outer surface of superparamagnetic Fe3O4 nanoparticles. This preparation method is efficient and repeatable, and is suitable for industrial production.

[0074] In the present disclosure, the "ultrasonic treatment" can adopt the devices commonly used by those skilled in the art, which will not be elaborated here.

[0075] A further optimized technical solution of this embodiment is that in step S1, the average particle size of the Fe3O4 nanoparticles is 50 - 500 nm, preferably 100 - 300 nm; relative to every 100 mL of deionized water, the amount of Fe3O4 nanoparticles dispersed is greater than 0 and not greater than 3 g, preferably 0.5 - 1.5 g; the molecular weight of the polyethyleneimine is 5000 - 80000 Da, preferably 6000 - 70000 Da; the mass concentration of the aqueous solution of polyethyleneimine is greater than 0 and not greater than 2 mg / mL; the volume ratio of the dispersion liquid to the aqueous solution of polyethyleneimine is 1 - 50:1, preferably 40 - 50:1; the conditions of the first ultrasonic treatment include: frequency of 50 - 100 Hz, temperature of 10 - 40 °C, and time of 10 - 90 min.

[0076] A further optimized technical solution of this embodiment is that in step S2, the average particle size of the quantum dots is 5 - 30 nm, preferably 8 - 15 nm; relative to every 100 mL of the aqueous solution of quantum dots, the addition amount of the Fe3O4-PEI nanospheres is greater than 0 and not greater than 4 g, preferably 0.5 - 1.5 g; the mass concentration of the aqueous solution of quantum dots is greater than 0 and not greater than 10 mg / mL; the conditions of the second ultrasonic treatment include: frequency of 50 - 100 Hz, temperature of 10 - 40 °C, and time of 10 - 90 min.

[0077] A further optimized technical solution of this embodiment is that in step S3, relative to every 100 mL of ammonia water, the addition amount of the MagQDs nanospheres is greater than 0 and not greater than 4 g, preferably 0.5 - 1.5 g; the addition amount of tetraethyl orthosilicate (TEOS) is greater than 0 and not greater than 4 mL, preferably 0.3 - 1 mL; the volume concentration of the ammonia water is greater than 0 and not greater than 50 v / v; the molar concentration of the tetraethyl orthosilicate is greater than 0 and not greater than 500 mol / L; the conditions of the third ultrasonic treatment include: the frequency is 50 - 100 Hz, the temperature is 10 - 40 °C, and the time is 10 - 90 min.

[0078] A further optimized technical solution of this embodiment is that in step S5, the coupling treatment includes:

[0079] Mix the MagQDs@Si-PEI nanospheres with the diynyl polyethanol solution for the fifth ultrasonic treatment; in every 100 mL of the diynyl polyethanol solution, the addition amount of the MagQDs@Si-PEI nanospheres is 0.01 - 1 g, and the addition amount of the diynyl polyethanol solution (1 mM) is 1 - 10 mL; the conditions of the fifth ultrasonic treatment include: the frequency is 50 - 100 Hz, the temperature is 10 - 40 °C, and the time is 10 - 90 min.

[0080] A further optimized technical solution of this embodiment is that in step S6, for every 1 g of the MagQDs@Si-PEI-yne nanospheres, the addition amount of the specific antibody is greater than ≤1 mg, preferably 0.001 - 0.01 mg; the specific antibody is an anti-Pseudomonas aeruginosa antibody, a PCT antibody, and an IL6 antibody; the temperature of the incubation treatment is 20 - 40 °C, and the time is 20 - 180 min.

[0081] The present invention also discloses the application of the above-mentioned silicon shell-protected magnetic quantum dot nanoprobe in the preparation of detecting common pathogenic bacteria and inflammatory markers in sepsis whole blood, wherein the pathogenic bacteria is Pseudomonas aeruginosa, and the inflammatory markers are PCT and IL6.

[0082] The present invention also discloses a kit for jointly detecting pathogenic bacteria and inflammatory markers, and the kit includes the above-mentioned nanoprobe.

[0083] Applying the nanomaterial in immunochromatography (LFA) can achieve rapid enrichment and quantitative analysis of pathogenic bacteria and inflammatory markers in the sample, especially the rapid enrichment and quantitative analysis of Pseudomonas aeruginosa, PCT, and IL6, realize the magnetic enrichment effect and the multiple signal amplification of multilayer fluorescent quantum dots, eliminate matrix interference in practical applications, further improve the detection sensitivity, and have great potential in the early diagnosis of sepsis.

[0084] A further optimized technical solution of this embodiment is that the pathogenic bacterium is Pseudomonas aeruginosa; the inflammatory markers are PCT and IL6; the kit further includes an immunochromatographic test strip, which includes a sample pad, a chromatographic membrane, and an absorption pad connected in sequence. There are 3 test lines and 1 control line on the chromatographic membrane; the 3 test lines are respectively loaded with anti-Pseudomonas aeruginosa antibody, anti-PCT antibody, and anti-IL6 antibody; the control line is loaded with goat anti-mouse IgG.

[0085] A further optimized technical solution of this embodiment is that the immunochromatographic test strip further includes a bottom plate, which serves as the backing card of the immunochromatographic test strip. The material of the bottom plate is preferably PVC. The sample pad is used for loading the sample solution to be detected. The absorbent pad is used to provide capillary suction. The chromatographic membrane is a nitrocellulose membrane (NC membrane).

[0086] Specifically, anti-Pseudomonas aeruginosa antibody, PCT antibody, IL6 antibody, and goat anti-mouse IgG are respectively sprayed on the surface of the nitrocellulose membrane; 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. Among them, the concentration of the anti-Pseudomonas aeruginosa antibody is greater than 0 and not greater than 3 mg / mL, preferably 2 mg / mL; the concentration of the anti-PCT antibody is greater than 0 and not greater than 3 mg / mL, preferably 1.8 mg / mL; the concentration of the anti-IL6 antibody is greater than 0 and not greater than 3 mg / mL, preferably 2 mg / mL; the concentration of goat anti-mouse IgG is greater than 0 and not greater than 3 mg / mL, preferably 1 mg / mL. Drying is carried out in a constant temperature drying oven, the drying temperature is preferably 35°C, and the drying time is preferably 3 h.

[0087] A further optimized technical solution of this embodiment is that the kit further includes a loading buffer. Specifically, the loading buffer includes: 100% of 10 mmol / L PBS buffer, 1% of Tween 20, and 10% of fetal bovine serum (FBS).

[0088] The method for detecting a sample to be tested using the kit of the present invention is: incubating the sample to be tested with the nanoprobe, that is, shaking and mixing for 10 min, magnetically enriching and recovering bacteria through the nanoprobe, then resuspending in the loading buffer, and loading it onto the sample pad of the immunochromatographic test strip to contact the test line on the immunochromatographic test strip for chromatographic reaction. After the chromatographic reaction, the colorimetric signal on the test line of the immunochromatographic test strip is read with the naked eye, and the fluorescence signal is read through an immunofluorescence analyzer.

[0089] Example 2

[0090] As Figure 1 shown, the preparation method of the silica shell-protected magnetic quantum dot nanoprobe is similar to that of Example 1, except that it includes the following steps:

[0091] (1) Disperse 0.5 g of Fe3O4 nanoparticles (240 nm) in 50 mL of deionized water to obtain a dispersion. Then mix the dispersion with an aqueous solution of polyethyleneimine (2 mg / mL) at a volume ratio of 50:1 and perform the first ultrasonic treatment at 100 Hz and 20 °C for 30 min to obtain Fe3O4-PEI nanospheres;

[0092] (2) Mix 0.5 g of Fe3O4-PEI nanospheres with 100 mL of an aqueous solution of quantum dots (10 mg / mL) and perform the second ultrasonic treatment at 100 Hz and 20 °C for 40 min to obtain MagQDs nanospheres;

[0093] (3) Mix 0.5 g of MagQDs nanospheres with 100 mL of ammonia water (45% v / v) and perform the third ultrasonic treatment at 100 Hz and 20 °C for 10 min, then add 0.5 mL of tetraethyl orthosilicate (1 mol / L) to obtain MagQDs@Si nanospheres;

[0094] (4) Dissolve 0.5 g of MagQDs@Si nanospheres in 50 mL of water, then mix with an aqueous solution of polyethyleneimine (2 mg / mL) at a volume ratio of 50:1 and perform the fourth ultrasonic treatment at 100 Hz and 20 °C to obtain MagQDs@Si-PEI nanospheres;

[0095] (5) Mix the MagQDs@Si-PEI nanospheres with a solution of diynyl polyethanol (1 mM) and perform the fifth ultrasonic treatment for coupling to obtain MagQDs@Si-PEI-yne nanospheres;

[0096] (6) Mix the MagQDs@Si-PEI-yne nanospheres with 0.008 mg of specific antibodies (anti-Pseudomonas aeruginosa antibody, anti-PCT antibody, and anti-IL6 antibody, with the same addition amount of the three antibodies) and perform shaking incubation at 27 °C for 2 hours. Block with bovine serum albumin (10% v / v) for 1 hour.

[0097] As shown in the Figure 1 attachment, A in the figure is the synthesis process of steps 1 - 3 of MagQDs@Si nanospheres; B in the figure is the process of coupling antibodies by the alkyne reaction of MagQDs@Si nanospheres with a solution of diynyl polyethanol.

[0098] Figure 2TEM image and EDS analysis of MagQDs@Si prepared in step (3) of this example. The above images show that the synthesized MagQDs@Si has a core-shell structure with Fe3O4 nanoparticles as the core, multiple quantum dot interlayers, and a SiO2 shell as the shell layer.

[0099] Figure 3 Fluorescence enhancement effect of MagQDs@Si prepared in this example.

[0100] Example 3

[0101] The nanoprobe prepared in Example 2 was used for the detection of pathogenic bacteria and inflammatory markers. The specific process is as Figure 4 shown. The sample to be detected was incubated with the nanoprobe, and the bacteria were magnetically enriched and recovered by the nanoprobe. Then, it was resuspended in the loading buffer and loaded onto the sample pad of the immunochromatographic test strip, and contacted with the test line on the immunochromatographic test strip for chromatographic reaction. After the chromatographic reaction, the colorimetric signal on the test line of the immunochromatographic test strip was read by the naked eye, and the fluorescence signal was read by an immunofluorescence analyzer.

[0102] The detection performance of MagQDs@Si-ICA in this example depends on the capture and binding ability of the antibody conjugated to the MagQDs@Si nanoprobe to the analyte. The traditional method of conjugating antibodies usually uses EDC / NHS for activation reaction conjugation, while the present invention uses alkyne reaction based on click chemistry to conjugate antibodies. The capture efficiencies of MagQDs@Si-ICA for Pseudomonas aeruginosa, PCT, and IL6 were determined by bacterial plate counting and BCA protein assay to be 88.1%, 85.4%, and 89.7% respectively, as Figure 5 shown, and there was no significant difference compared with the traditional EDC / NHS method for conjugating antibodies.

[0103] This example optimized the operating conditions (composition of the loading buffer and membrane coating concentration) of the MagQDs@Si immunochromatographic detection platform to achieve the best detection performance of the platform. The results are as Figure 6 shown. When the composition of the loading buffer is 10 mM PBS, 1% Tween 20, and 10% FBS, the MagQDs@Si nanoprobe has the highest signal-to-noise ratio for Pseudomonas aeruginosa. Figure 7To optimize the concentration of capture antibodies on the test line, when the concentrations of anti - Pseudomonas aeruginosa antibody, anti - PCT antibody, and anti - IL6 antibody on the test line reach 2 mg / mL, 1.8 mg / mL, and 2 mg / mL respectively, the highest signal - to - noise ratio is achieved. Among them, the signal - to - noise ratio in this disclosure refers to the ratio of the fluorescence intensity of the positive to the fluorescence intensity of the negative control. The larger the signal - to - noise ratio, the greater the difference between the positive fluorescence signal and the negative fluorescence signal under this condition, and the better the detection effect.

[0104] This example verifies the multiplex detection ability of MagQDs@Si - ICA. Figure 8 Presents the photos of the T - zone of the LFIA strip and the corresponding fluorescence intensities when MagQDs@Si - ICA is used for single and combined detection of three target substances: (I) Pseudomonas aeruginosa, PCT, and IL6; (II) IL6; (III) PCT; (IV) Pseudomonas aeruginosa; (V) blank group. The obvious red fluorescence signals that appear on each T - line in the three - channel LFIA strip (I–IV) are consistent with the presence of the target antigen in the corresponding sample, and no cross - reaction is observed. In addition, no fluorescence band appears on the T - line of strip V, further confirming that the MagTQD@Si - based LFIA has a typical "on / off" detection mode and can effectively distinguish positive (presence of target antigen) from negative samples. The results show that the fluorescence signal of the T - line is derived from the complex formed by MagTQD@Si and the target antigen. Figure 9 Demonstrates the specificity of the MagTQD@Si–ICA platform. We introduced a variety of pathogenic bacteria (about 105 CFU / mL) and inflammatory markers (about 10 mg / mL) as interferents in PBS, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Influenza A virus, Influenza B virus, SARS - CoV - 2, Serum amyloid A (SAA), and C - reactive protein (CRP). These results fully demonstrate the excellent specificity and reliability of the proposed MagQDs@Si - ICA in simultaneous multiplex detection.

[0105] Example 4

[0106] This example systematically evaluates the key analytical performance of MagTQD@Si - based LFIA, including sensitivity, detection range, and repeatability. The performance of MagQDs@Si in detecting different concentrations of Pseudomonas aeruginosa (10 - 10 5 cells / mL), PCT ((0.001–10 ng / mL)), and IL6 ((0.001–10 ng / mL)) was investigated. Figure 10 Is a multi - channel detection result diagram of MagQDs@Si - ICA for combined detection of Pseudomonas aeruginosa, PCT, and IL6. Its visual detection limit by the naked eye under ultraviolet light is 102 CFU / mL, 0.05 ng / mL, and 0.01 ng / mL. We used an immunofluorescence analyzer to measure the fluorescence signals on the test line and perform analysis. Figure 10 b The 3D heatmap and the bar chart results in 11c show that the fluorescence signals on the test line are positively correlated with the sample concentration. By plotting the fluorescence signals on each T line against the antigen concentration, S-shaped calibration curves for Pseudomonas aeruginosa (T1), PCT (T2), and IL6 (T3) were constructed respectively (see Figure 10 d–f), and their correlation coefficients (R 2 ) all exceeded 0.99. Calculated according to the standard IUPAC protocol (LOD = the average fluorescence intensity of the blank control group plus 3 times the standard deviation of the blank measurement), the detection limits of MagTQD@Si-based LFIA for Pseudomonas aeruginosa, IL6, and PCT reached 7 CFU / mL, 4.44 pg / mL, and 0.94 pg / mL respectively. The results indicate that the MagQDs@Si-ICA immunochromatographic detection method based on click chemistry-coupled antibodies has high analytical performance for pathogens and inflammatory markers, with a wide detection dynamic range (5 orders of magnitude), good correlation coefficient (R 2 > 0.99), and low LOD.

[0107] Compared with Figure 11 the detection results of traditional colloidal gold in, the structure of traditional colloidal gold is a monodisperse spherical structure with a particle size of 20 - 45 nm that is prone to aggregation. The sensitivity of the MagQDs@Si immunochromatographic detection method in this application is increased by at least 100 times.

[0108] In this application, the repeatability of MagTQD@Si-ICA was evaluated by testing five independently prepared sample batches, each batch containing high concentrations (Pseudomonas aeruginosa 106 CFU / mL and PCT / IL6 10 ng / mL) and low concentrations (Pseudomonas aeruginosa 10 3 CFU / mL and PCT / IL6 0.1 ng / mL) of samples. As Figure 12 shown, all five groups of results indicate that the MagTQD@Si-based LFIA test strips always produce uniform fluorescence signals for the same target concentration, with a coefficient of variation (CV) ≤ 9.21%, indicating that the established MagTQD@Si-ICA platform has good repeatability.

[0109] In this example, the MagQDs@Si immunochromatographic detection method was further evaluated for its performance in actual clinical testing. First, the whole blood samples of healthy volunteers were diluted 10 times, and Pseudomonas aeruginosa (106, 105, and 104 CFU / mL), PCT (10, 1, and 0.1 ng / mL), and IL6 (10, 1, and 0.1 ng / mL) were added respectively. The results are asFigure 13 As shown, the fluorescence signal on the corresponding test line in the whole blood sample is stable, and the fluorescence signal intensity decreases as the concentration of the corresponding target antigen in the sample decreases. As shown in Table 1 below, this method shows high recovery rates for Pseudomonas aeruginosa (recovery rate 86.3%–88.2%), PCT (recovery rate 80.2%–86.3%), and IL6 (recovery rate 83.5%–90.3%), indicating its excellent accuracy and reliability in detecting these targets in actual whole blood samples.

[0110] Table 1 Recovery rates of MagTQDs@Si-ICA in clinical samples in Example 3

[0111]

[0112] The preferred embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present disclosure. In addition, any arbitrary combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention. The above is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any modification made by those skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, by equivalent substitution or change, should be covered within the protection scope of the present invention.

Claims

1. A silicon shell-protected magnetic quantum dot nanoprobe, characterized in that, The nano-probe includes nano-materials and specific antibodies; The nano-materials have a core-shell structure, the core of the core-shell structure is Fe3O4 nanoparticles, and a sandwich layer and a protective shell layer are sequentially coated outward from the outer surface of the core; The sandwich layer includes a multi-layer stacked structure formed by alternating positive charge sandwich layers and negative charge sandwich layers, the stacked structure is 2-8 layers, the first layer in the stacking direction of the stacked structure is a positive charge sandwich layer, and the last layer is a negative charge sandwich layer; The positive charge sandwich layer is polyethyleneimine, the negative charge sandwich layer is a quantum dot, the quantum dot is CdSe / ZnS or a carbon quantum dot, the protective shell layer is silica, and the amino group on the surface of the shell layer and the diethynyl polyalcohol solution are specifically coupled through a click chemistry method with a diyne molecule as a linker arm, and the specific antibody is at least one of a Pseudomonas aeruginosa antibody, a PCT antibody, and an IL6 antibody.

2. The silicon shell-protected magnetic quantum dot nanoprobe according to claim 1, wherein ≤1 mg of specific antibody is conjugated to 1 g of nano-materials, and the specific antibody is at least one of a Pseudomonas aeruginosa antibody, a PCT antibody, and an IL6 antibody.

3. The silicon shell-protected magnetic quantum dot nanoprobe according to claim 1, wherein The average particle size of the Fe3O4 nanoparticles is 50-500 nm, the thickness of each positive charge sandwich layer ≤5 nm, the average particle size of the quantum dots is 5-20 nm, and the thickness of the silica shell layer is 5-30 nm.

4. The preparation method of the silicon shell-protected magnetic quantum dot nanoprobe according to any one of claims 1 to 3, characterized in that, This method includes the following steps: S1. Disperse Fe3O4 nanoparticles in deionized water to obtain a dispersion liquid, then mix the dispersion liquid with an aqueous polyethyleneimine solution and perform a first ultrasonic treatment to obtain Fe3O4-PEI nano-microspheres; S2. Mix the Fe3O4-PEI nano-microspheres obtained in step S1 with an aqueous quantum dot solution and perform a second ultrasonic treatment to obtain MagQDs nano-microspheres; S3. Mix the MagQDs nano-microspheres obtained in step S2 with ammonia water and perform a third ultrasonic treatment, then add tetraethyl orthosilicate to obtain MagQDs@Si nano-microspheres; S4. Mix the MagQDs@Si nano-microspheres obtained in step S3 with an aqueous polyethyleneimine solution and perform a fourth ultrasonic treatment to obtain MagQDs@Si-PEI nano-microspheres; S5. After activating the MagQDs@Si-PEI microspheres obtained in step S4, perform click chemistry coupling treatment to obtain MagQDs@Si-PEI-yne nano-microspheres; S6. Mix the MagQDs@Si-PEI-yne nano-microspheres obtained in step S5 with a specific antibody and perform incubation treatment to obtain a quantum dot nano-probe.

5. The preparation method of the magnetic nanozyme probe according to claim 4, wherein In step S2, the average diameter of the quantum dots is 5-30 nm, the addition amount of Fe3O4-PEI nano-microspheres in every 100 mL of the aqueous quantum dot solution ≤4 g, and the mass concentration of the aqueous quantum dot solution ≤10 mg / mL.

6. The preparation method of the magnetic nanozyme probe according to claim 4, wherein, In step S3, in every 100 mL of ammonia water, the addition amount of the MagQDs nano-microspheres ≤4 g, the addition amount of tetraethyl orthosilicate ≤4 mL, the volume concentration of the ammonia water ≤50 v / v, and the molar concentration of tetraethyl orthosilicate ≤500 mol / L.

7. The preparation method of the magnetic nanozyme probe according to claim 4, wherein In step S5, the activation treatment includes: mixing the MagQDs@Si-PEI nanospheres with a diynyl polyethanol solution and performing a fifth ultrasonic treatment.

8. A fluorescence immunochromatography kit for the joint detection of pathogenic bacteria and inflammatory markers, characterized in that, The kit includes the nanoprobe according to any one of claims 1-3, the pathogenic bacterium is Pseudomonas aeruginosa, and the inflammatory markers are PCT and IL6.

9. The fluorescence immunoassay chromatography kit according to claim 8, characterized in that, It further includes an immunochromatographic test strip, which includes a sample pad, a chromatographic membrane, and an absorption pad connected in sequence. There are 3 test lines and 1 quality control line arranged on the chromatographic membrane. The 3 test lines are respectively loaded with anti-Pseudomonas aeruginosa antibodies, anti-PCT antibodies, and anti-IL6 antibodies, and the quality control line is loaded with goat anti-mouse IgG.

10. The fluorescence immunochromatographic kit according to claim 9 is used for simultaneously detecting pathogenic bacteria and sepsis markers in whole blood, the pathogenic bacterium is Pseudomonas aeruginosa, and the inflammatory markers are PCT and IL6.

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