Positively charged silicon shell modification-based magnetic fluorescent probe, preparation method and application

Abstract

The invention discloses a magnetic fluorescent probe based on positive silicon shell modification as well as a preparation method and application of the magnetic fluorescent probe. The probe adopts superparamagnetic Fe3O4 nanoparticles as an inner core and provides magnetic enrichment capacity; a cationic polymer electrostatically adsorbs multiple layers of quantum dot particles to serve as a fluorescent interlayer, and a fluorescent signal capable of being used for quantitative detection is provided; and a layer of aminated silicon dioxide shell with strong positive electricity is also arranged on the surface, so that the broad-spectrum capturing capability on bacteria with negative electricity is provided. By taking pseudomonas aeruginosa, streptococcus pneumoniae and salmonella typhimurium as examples, rapid enrichment and broad-spectrum quantitative analysis of the pseudomonas aeruginosa, streptococcus pneumoniae and salmonella typhimurium in a sample can be realized, a magnetic enrichment effect and multiple signal amplification of multiple layers of fluorescent quantum dots are realized, matrix interference in practical application is eliminated, and the detection sensitivity is high. The method has great potential in the aspects of high sensitivity and clinical monitoring of bacterial infection or pollution.

Description

Technical Field

[0001] The invention relates to the technical field of magnetic fluorescent probes, and in particular to a magnetic fluorescent probe based on positively charged silicon shell modification, a preparation method and an application thereof. Background Art

[0002] Throughout human history, pathogenic bacteria have always been the main factor threatening human life and health. Bacterial infections are closely related to many serious human diseases, such as pneumonia, infectious diarrhea, meningitis, sepsis, and urinary tract infections. Timely detection of pathogens can guide clinicians to conduct accurate treatment and rational use of drugs, and is also the most effective means of saving lives. Therefore, there is an urgent need to develop a technology suitable for rapid and on-site screening of pathogens to meet the clinical needs of early diagnosis and treatment.

[0003] Immunochromatographic assays (ICAs) have become one of the most popular point-of-care (POCT) technologies because they integrate chromatographic separation and specific antibody-antigen recognition on a simple test strip, allowing for rapid, simple and low-cost detection of various biochemical targets (e.g., proteins, nucleic acids and viruses) without relying on large instruments. Although many bacterial ICA techniques based on detection reagents have been reported, these methods suffer from low sensitivity, low detection throughput and poor versatility, mainly in two aspects: First, considering that bacteria have a large size (0.5-4 μm), the constructed ICA system usually adopts a double antibody sandwich strategy, which usually requires one antibody to label the detection nanoprobe for bacterial binding and one antibody to modify the detection line of the test strip for capturing the formed immune complex. However, the preparation and selection of high-performance antibodies for ICA is time-consuming, laborious and expensive. In addition, the antibody molecules on the surface of the nanoprobe are easily interfered by complex samples (e.g., high ion concentration and harsh pH environment). This interference reduces their biological activity and storage stability, ultimately reducing the stability and sensitivity of the detection method. Second, the performance of the current nanoprobes is not sufficient to detect bacteria. Conventional colorimetric nanoparticles (NPs), such as AuNPs and latex beads, show weak sensitivity (10 4 -10 3 Newly developed signal materials, such as Raman tags, fluorescent probes, and catalytic nanoparticles, can effectively improve the sensitivity of ICA detection of bacteria, but they still face the problems of poor stability and the need for antibody modification in complex clinical samples. In theory, the development of a high-performance antibody-free nanoprobe with signal enhancement, high stability, affinity for bacteria, and broad-spectrum binding ability can solve the challenges of sensitive and accurate detection of pathogens on the ICA platform.

[0004] In recent years, people have devoted themselves to developing antibody-free biosensors for bacterial detection, and research results have shown that ICA systems based on antibody-free nanoprobes can be used for rapid identification of pathogens. For example, Deng et al. reported that two-dimensional manganese dioxide has good bacterial adsorption ability and strong colorimetric signals, which is suitable as an antibody-free probe for ICA for the diagnosis of Salmonella enteritidis. Zhang et al. showed that Fe3O4@CuS nanostructures have a strong capture ability for bacteria, and successfully constructed colorimetric and photothermal mode ICAs for the detection of Escherichia coli O157:H7. The application of antibody-free probes reduces the dependence of ICA technology on paired antibodies, eliminates the antibody modification process, and effectively simplifies the preparation of ICA. However, the current antibody-free strategy still has the following major scientific problems to be solved: i) The mechanism of binding of antibody-free probes to bacteria is currently unclear, and the broad-spectrum detection ability of these probes has not been confirmed. ii) Existing antibody-free probe ICAs can only detect one target pathogen, and their detection volume is insufficient for rapid screening of multiple bacteria. iii) The detection limit of existing antibody-free probe ICAs for bacteria is generally higher than 10 2 / mL cell count cannot meet the actual needs of clinical diagnosis.

[0005] In the prior art, the invention patent with the patent publication number CN118393133A discloses a gold shell magnetic bead nanoparticle modified with antimicrobial peptide CP1 and its preparation method, application and kit, which relates to the field of immunoassay analysis technology. The nanoparticles of the invention include Fe3O4@Au nanoparticles and antimicrobial peptide CP1, and the antimicrobial peptide CP1 is coupled to the surface of Fe3O4@Au nanoparticles through a streptavidin-biotin system; the Fe3O4@Au nanoparticles include a Fe3O4 particle core and a polyethyleneimine layer, a gold nanoparticle layer, a DTNB first layer, a gold layer and a DTNB second layer sequentially coated on the surface of the Fe3O4 particle. The nanoparticles provided by the invention have excellent broad-spectrum capture ability, rapid magnetic enrichment function and excellent antibacterial activity, and can be used for the detection of Pseudomonas aeruginosa and Escherichia coli. However, the modification method of the patent is complicated. In addition, the probe stability is poor when bioactive molecules are used as recognition and capture molecules of antigens. Summary of the invention

[0006] In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide a magnetic fluorescent probe (MagMQD@Si + ), preparation method and application, specifically, using Fe3O4 particles (50-500nm) to load multilayer quantum dot small particles to produce magnetic quantum dots (MagQDs) with superparamagnetism and strong fluorescence signals; and through the hydrolysis reaction of tetraethyl orthosilicate and the condensation reaction of 3-aminopropyltrimethoxysilane, a layer of amino silicon shell with protective effect is coated on the surface of the magnetic quantum dots through a one-step reaction.

[0007] The second purpose of the present invention is to provide an application of a magnetic fluorescent probe modified with a positively charged silica shell, wherein the probe uses superparamagnetic Fe3O4 nanoparticles as the core to provide magnetic enrichment capability; electrostatically adsorbs multiple layers of quantum dot particles as a fluorescent interlayer through a cationic polymer polyethyleneimine (PEI) to provide a fluorescent signal that can be used for quantitative detection; and an amino-modified silica shell with a strong positive charge on the surface provides protection for the internal material and the ability to capture a broad spectrum of negatively charged bacteria.

[0008] The third purpose of the present invention is to combine the magnetic fluorescent probe modified with positively charged silica shell with ICA technology, and match it with a portable fluorescent immunoassay analyzer to achieve highly sensitive quantitative detection of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A magnetic fluorescent probe based on positively charged silica shell modification uses superparamagnetic Fe3O4 nanoparticles as the core to provide magnetic enrichment force; multiple layers of quantum dot particles are electrostatically adsorbed by cationic polymers as fluorescent interlayers, which provide fluorescent signals for quantitative detection; the surface of the fluorescent interlayer is provided with an amino-modified silica shell, which can protect the internal material and capture a wide spectrum of negatively charged bacteria.

[0011] The present invention also provides a method for preparing the positively charged silicon shell modified magnetic fluorescent probe, comprising the following steps:

[0012] (1) Using superparamagnetic Fe3O4 nanoparticles as the core, multilayer quantum dot particle-coated magnetic quantum dots (MagMQD) were prepared as the fluorescent interlayer via a cationic polymer-mediated layer-by-layer self-assembly method;

[0013] (2) The magnetic quantum dots (MagMQD) prepared in step (1) were resuspended in an ethanol solution, and then ammonia, tetraethyl orthosilicate (TEOS) and 3-aminopropyltrimethoxysilane (APTMS) were slowly added to react to form a magnetic fluorescent probe (MagMQD@Si + ).

[0014] Preferably, in step (1), the particle size of the Fe3O4 nanoparticles is 50-500nm. The particle size of the Fe3O4 nanoparticles is any value within the range of 50-500nm, such as 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm.

[0015] Preferably in any of the above schemes, in step (1), the particle size of the Fe3O4 nanoparticles is preferably 100-300nm, that is, the particle size of the Fe3O4 nanoparticles is any value in the range of 100-300nm, such as 100nm, 150nm, 200nm, 250nm, 300nm.

[0016] In any of the above schemes, preferably, in step (1), the concentration of the cationic polymer aqueous solution is ≤5 mg / mL, and after the cationic polymer is added, ultrasound is applied, and the ultrasound modification time is ≤80 min.

[0017] In any of the above schemes, it is preferred that the cationic polymer is a polyethyleneimine (PEI) aqueous solution. The molecular weight of polyethyleneimine is preferably 5000-80000Da, more preferably 6000-70000Da.

[0018] In any of the above schemes, preferably, in step (1), the particle size of a single quantum dot forming a fluorescent interlayer is 5-30 nm, the quantum dot concentration is ≤50 mg / mL, and the ultrasonic time is ≤80 min. The particle size of a single quantum dot is any value within the range of 5-30 nm, such as 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm.

[0019] Preferably, in any of the above schemes, in step (2), when preparing the amino-modified silica shell, the concentration of aqueous ammonia is ≤50% (v / v), the concentration of ethyl silicate solution is ≤600mM, the concentration range of 3-aminopropyltrimethoxysilane is ≤10% (v / v), and ultrasonication is performed after adding aqueous ammonia, ethyl silicate and 3-aminopropyltrimethoxysilane, and the ultrasonic modification time is ≤240min.

[0020] In any of the above schemes, preferably, in step (2), the shell thickness of the amino-silica is ≤50 nm, it has strong positive charge, and the Zeta potential is between 0-60 mV.

[0021] The present invention also provides the use of a probe prepared by the method for preparing the positively charged silicon shell-modified magnetic fluorescent probe in the preparation of an immunochromatography component, an immunochromatography device or an immunochromatography product.

[0022] The present invention also provides an immunochromatographic test strip using a magnetic fluorescent probe modified with a positively charged silicon shell, comprising a sample pad for loading a sample solution, a nitrocellulose membrane with an independent detection line, an absorbent pad, a positively charged magnetic fluorescent probe for detection, and a running buffer solution, wherein the positively charged magnetic fluorescent probe is obtained by using any one of the preparation methods described above.

[0023] Preferably, the detection line of the nitrocellulose membrane includes at least one of an anti-Pseudomonas aeruginosa antibody detection line, an anti-Streptococcus pneumoniae antibody detection line, and an anti-Salmonella typhimurium antibody detection line.

[0024] The mass concentration of anti-Pseudomonas aeruginosa antibodies is preferably 0-2 mg / mL, more preferably 1.5 mg / mL; the mass concentration of anti-Streptococcus pneumoniae antibodies is preferably 0-1.5 mg / mL, more preferably 0.8 mg / mL; the mass concentration of anti-Salmonella typhimurium antibodies is preferably 0-2 mg / mL, more preferably 1.2 mg / mL.

[0025] The present invention also provides an immunochromatographic test strip using any one of the positively charged silicon shell-modified magnetic fluorescent probes described above for detecting multiple bacteria in complex clinical samples.

[0026] The immunochromatographic test strip of the magnetic fluorescent probe modified with a positively charged silica shell of the present application uses a magnetic fluorescent probe modified with a positively charged silica shell to magnetically enrich and concentrate the antigens in a large volume of a sample to be tested and then perform a chromatographic reaction, thereby further improving the detection sensitivity while avoiding interference from complex matrices in the sample.

[0027] The positively charged silica shell modified magnetic fluorescent probe proposed in the present application has the functions of broad-spectrum capture and magnetic enrichment of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium and providing strong fluorescence signals. The immunochromatography technology based on the positively charged silica shell modified magnetic fluorescent probe provides two detection modes, namely, a qualitative mode and a quantitative mode. In the qualitative mode, whether a red fluorescence signal is generated on the detection line is judged by the naked eye under ultraviolet light; in the quantitative mode, highly sensitive quantitative analysis is achieved in a dry immunoassay analyzer by detecting changes in the fluorescence signal of quantum dots on the detection line.

[0028] Beneficial Effects

[0029] (1) The positively charged silica shell-modified magnetic fluorescent probe proposed in the present invention uses multiple densely packed quantum dot interlayers as the source of the fluorescent signal and successfully synthesizes a magnetic structure with a strong fluorescent signal.

[0030] (2) The multi-layer quantum dot interlayer of the positively charged silica shell modified magnetic fluorescent probe proposed in the present invention is coated with an amino SiO2 shell on the outside. The amino SiO2 shell provides protection for the positively charged silica shell modified magnetic fluorescent probe, improves its dispersibility and stability, and reduces its background signal on the nitrocellulose membrane; it can electrostatically interact with bacteria with negative charges on the surface, providing a broad-spectrum bacterial capture capability.

[0031] (3) The immunochromatographic technology based on the magnetic fluorescent probe modified with positively charged silica shell proposed in the present invention uses the magnetic fluorescent probe modified with positively charged silica shell to magnetically enrich and concentrate the antigen in the large volume of the sample to be tested, and then performs a chromatography reaction, thereby avoiding the interference of the complex matrix in the sample and further improving the detection sensitivity. First, in terms of the modification of the recognition and capture molecules of the antigen, the present application uses a one-step method to modify the amino silica shell on the surface of the magnetic quantum dots, and the modification method is simpler and the modification speed is fast; secondly, the present application does not use bioactive molecules as the recognition and capture molecules of the antigen, and the protection of the silica shell greatly improves the stability of the probe; secondly, different antimicrobial peptides have different affinities for different bacteria, and the magnetic fluorescent probe modified with positively charged silica shell in the present application has a strong and stable capture efficiency for negatively charged bacteria; finally, fluorescent quantum dots are used as the signal source for quantitative detection. Under the ultraviolet lamp with an excitation wavelength of 365nm, the probe emits a strong red fluorescence, which can greatly improve the detection sensitivity.

[0032] In summary, the present application proposes an immunochromatography technology based on a magnetic fluorescent probe modified with a positively charged silica shell. The platform uses a magnetic fluorescent probe modified with a positively charged silica shell, which can efficiently capture, enrich and detect Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium in complex samples. The positively charged silica shell-modified magnetic fluorescent probe has a superparamagnetic Fe3O4 nanoparticle as the core, providing magnetic enrichment capability; through the electrostatic adsorption of multi-layer quantum dot particles by the cationic polymer polyethyleneimine (PEI) as a fluorescent interlayer, a fluorescent signal that can be used for quantitative detection is provided; a layer of amino-modified silica shell with a strong positive charge on the surface provides protection for the internal material and the ability to capture a broad spectrum of negatively charged bacteria. The present application prepares a positively charged silica shell-modified magnetic fluorescent probe with strong magnetic responsiveness, high fluorescence signal and broad-spectrum bacterial capture capability. After the positively charged silica shell-modified magnetic fluorescent probe is integrated with the ICA test strips coated with antibodies against Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium, rapid enrichment and broad-spectrum quantitative analysis of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium in the sample can be achieved, realizing the magnetic enrichment effect and multiple signal amplification of multi-layer fluorescent quantum dots, eliminating matrix interference in practical applications, and has great potential in high-sensitivity and clinical monitoring of bacterial infection or contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of a method for preparing a magnetic fluorescent probe modified with a positively charged silicon shell according to Example 2 of the present invention;

[0034] Figure 2 This is a structural electron microscopy characterization diagram of the magnetic fluorescent probe modified with a positively charged silicon shell according to Example 2 of the present invention;

[0035] Figure 3This is a flow chart of the detection of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium using the immunochromatography technology based on the positively charged silica shell modified magnetic fluorescent probe according to Example 3 of the present invention;

[0036] Figure 4 The optimized results of the membrane concentration of the immunochromatography technique based on the magnetic fluorescent probe modified with positively charged silica shells in Example 3 of the present invention;

[0037] Figure 5 This is the incubation time optimization result of the immunochromatography technique based on the magnetic fluorescent probe modified with positively charged silica shell according to Example 3 of the present invention;

[0038] Figure 6 This is a graph showing the capture efficiency of the positively charged silica shell-modified magnetic fluorescent probe for Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium in Example 3 of the present invention;

[0039] Figure 7 This is a graph showing the detection results of detecting Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium using the immunochromatographic technique based on the magnetic fluorescent probe modified with positively charged silica shells according to Example 4 of the present invention;

[0040] Figure 8 This is a graph showing the detection results of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium using the colloidal gold method according to Example 4 of the present invention;

[0041] Fig. 9 This is a specific verification of the immunochromatographic technique based on the positively charged silica shell-modified magnetic fluorescent probe of Example 4 of the present invention;

[0042] Fig.10 This is a repeatability verification of the immunochromatography technique based on the magnetic fluorescent probe modified with positively charged silica shells according to Example 4 of the present invention;

[0043] Fig.11 The results of the analysis of the immunochromatography technique based on the magnetic fluorescent probe modified with positively charged silicon shells in Example 4 of the present invention in actual samples;

[0044] Fig.12 This is the analysis result of the immunochromatography technique based on the positively charged silica shell modified magnetic fluorescent probe in Example 4 of the present invention in clinical sputum samples. DETAILED DESCRIPTION

[0045] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the 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.

[0046] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0047] Example 1

[0048] The present invention provides a magnetic fluorescent probe based on positively charged silica shell modification, which has superparamagnetic Fe3O4 nanoparticles as the core to provide magnetic enrichment capability; multiple layers of quantum dot particles are electrostatically adsorbed by cationic polymer polyethyleneimine (PEI) as a fluorescent interlayer to provide a fluorescent signal that can be used for quantitative detection; a layer of amino-modified silica shell is provided on the surface of the fluorescent interlayer, and the silica shell has a strong positive charge, which can protect the internal material and capture a wide spectrum of negatively charged bacteria.

[0049] The positively charged silicon shell modified magnetic fluorescent probe provided by the present invention comprises superparamagnetic Fe3O4 nanoparticles. In the present invention, the Fe3O4 nanoparticles serve as a magnetic core, a fluorescent signal provider and a carrier of a multilayer quantum dot interlayer. The Fe3O4 nanoparticles are preferably superparamagnetic Fe3O4 nanoparticles, and the particle size of the Fe3O4 nanoparticles is preferably 50-500nm.

[0050] The positively charged silicon shell modified magnetic fluorescent probe provided by the present invention comprises a multi-layer cationic polymer strong positively charged interlayer, a multi-layer quantum dot fluorescent interlayer and an amino SiO2 protective shell loaded on the surface of the Fe3O4 nanoparticle; a layer of the superparamagnetic Fe3O4 nanoparticle and the multi-layer quantum dot interlayer are connected through the cationic polymer strong positively charged interlayer, and a layer of the cationic polymer self-assembly layer is in contact with the surface of the superparamagnetic Fe3O4 nanoparticle. In the present invention, the arrangement of the superparamagnetic Fe3O4 nanoparticle, the multi-layer cationic polymer strong positively charged interlayer and the multi-layer quantum dot interlayer is specifically that the surface of the superparamagnetic Fe3O4 nanoparticle is sequentially arranged with a first layer of cationic polymer strong positively charged interlayer, a layer of the quantum dot interlayer, a second layer of cationic polymer strong positively charged interlayer, a second layer of quantum dot interlayer, a third layer of cationic polymer strong positively charged interlayer and a third layer of quantum dot interlayer and an outermost layer of amino SiO2 protective shell. In the present invention, the particle size of the superparamagnetic Fe3O4 nanoparticles is preferably 50-500nm, further 100-300nm, and the particle size of a single quantum dot in the quantum dot interlayer is preferably 5-30nm, further 10nm. The cationic polymer strong positive interlayer is preferably a polyethyleneimine (PEI) layer, and the molecular weight of polyethyleneimine is preferably 5000-80000, more preferably 6000-70000.

[0051] The technical solution further optimized in this embodiment is to resuspend the obtained composite nano-luminescent material in ethanol and store it for later use.

[0052] The present invention also provides a one-step method for modifying the amino SiO2 shell on the outside of the magnetic quantum dots, that is, the protective silica shell is amino-modified so that it can be subsequently used for broad-spectrum capture of multiple negatively charged bacteria in samples.

[0053] The preparation method of the positively charged silicon shell modified magnetic fluorescent probe provided by the present invention is based on a cationic polymer-mediated electrostatic self-assembly method, in which multiple layers of the quantum dot interlayer and a layer of amino SiO2 protective shell are assembled layer by layer on the surface of superparamagnetic Fe3O4 nanoparticles. The preparation process is efficient and repeatable and suitable for industrial production.

[0054] The positively charged silica shell-modified magnetic fluorescent probe provided by the present invention can magnetically enrich and concentrate antigens in a large volume of a sample to be tested and then perform a chromatography reaction, thereby further improving the detection sensitivity while avoiding interference from complex matrices in the sample.

[0055] In the present invention, the immunochromatography technology based on the test strip of positively charged silica shell modified magnetic fluorescent probe includes a sample pad for loading sample solution, a nitrocellulose membrane with three independent detection lines (), an absorbent pad, a positively charged silica shell modified magnetic fluorescent probe for detection and a running buffer.

[0056] The sample pad is used for loading the sample solution to be detected. The absorbent pad is used to provide capillary force. The material of the base plate is preferably PVC, and the base plate serves as a backing card for the dual-signal immunochromatographic test paper. The nitrocellulose membrane is loaded with multiple test lines, preferably 3 test lines, which are respectively an anti-Pseudomonas aeruginosa antibody test line, an anti-Streptococcus pneumoniae antibody test line, and an anti-Salmonella typhimurium antibody test line, for detecting Pseudomonas aeruginosa, Streptococcus pneumoniae, and Salmonella typhimurium.

[0057] The present invention provides a method for modifying a nitrocellulose membrane in an immunochromatographic test strip of a magnetic fluorescent probe modified with a positively charged silicon shell, which is preferably the following steps:

[0058] Anti-Pseudomonas aeruginosa antibodies, anti-Streptococcus pneumoniae antibodies and anti-Salmonella typhimurium antibodies were sprayed on the surface of nitrocellulose (NC) membrane, and then the modified NC membrane was placed in a constant temperature drying oven, the drying temperature was 34°C, and the drying time was ≤5h, to obtain the nitrocellulose membrane in the immunochromatographic test paper of the positively charged silica shell modified magnetic fluorescent probe. The mass concentration of anti-Pseudomonas aeruginosa antibodies is preferably 0-2 mg / mL, more preferably 1.5 mg / mL; the mass concentration of anti-Streptococcus pneumoniae antibodies is preferably 0-1.5 mg / mL, more preferably 0.8 mg / mL; the mass concentration of anti-Salmonella typhimurium antibodies is preferably 0-2 mg / mL, more preferably 1.2 mg / mL.

[0059] The immunochromatography of the positively charged silica shell modified magnetic fluorescent probe proposed in the present invention provides two detection modes. In the visual mode, the red fluorescent signal on the detection line can be identified by the naked eye for rapid qualitative detection. In the instrumental mode, highly sensitive quantitative analysis can be achieved through the change of the fluorescent signal on the detection line.

[0060] The present invention provides a method for visual pattern detection of an immunochromatographic test strip of a positively charged silica shell modified magnetic fluorescent probe: a sample to be detected is co-incubated with a positively charged silica shell modified magnetic fluorescent probe, the bacteria-probe complex is recovered by a magnet, resuspended in a running buffer, and loaded onto a sample pad of an immunochromatographic test strip of a positively charged silica shell modified magnetic fluorescent probe, and contacted with a detection line on an immunochromatographic test strip of a positively charged silica shell modified magnetic fluorescent probe to perform a chromatographic reaction. After the chromatographic reaction, the colorimetric signal on the detection line of the immunochromatographic test strip of the positively charged silica shell modified magnetic fluorescent probe is read by naked eyes, and the fluorescent signal is read by an immunofluorescence analyzer.

[0061] Example 2

[0062] A magnetic fluorescent probe based on positively charged silica shell modification is similar to Example 1, except that superparamagnetic Fe3O4 nanoparticles are used as the core to provide magnetic enrichment capability; multiple layers of quantum dot particles are electrostatically adsorbed by cationic polymer polyethyleneimine (PEI) as a fluorescent interlayer to provide a fluorescent signal that can be used for quantitative detection; an amino-modified silica shell is arranged on the surface of the fluorescent interlayer, and the silica shell has a strong positive charge, providing protection for the internal material and the ability to capture a broad spectrum of negatively charged bacteria.

[0063] The preparation method of the positively charged silica shell modified magnetic fluorescent probe is as follows: Figure 1 As shown, the following steps are included:

[0064] (1) Using superparamagnetic Fe3O4 nanoparticles as the core, the Fe3O4 nanoparticles have a particle size of 50-500nm, adding cationic polymer polyethyleneimine aqueous solution to ultrasonically electrostatically adsorb multilayer quantum dot particles as a fluorescent interlayer, the polyethyleneimine aqueous solution concentration is 5mg / mL, the polyethyleneimine molecular weight is 6000-70000Da, the single quantum dot particle size is 5-30nm, the quantum dot concentration is 50mg / mL, the ultrasonic modification time is 80min, and the multilayer quantum dot interlayer coated magnetic quantum dots (MagMQD) are prepared by a cationic polymer PEI-mediated layer-by-layer self-assembly method;

[0065] (2) The MagMQD prepared in step (1) was resuspended in an ethanol solution, and ammonia water, ethyl silicate (TEOS) and 3-aminopropyltrimethoxysilane (APTMS) were slowly added, wherein the concentration of ammonia water was 50% (v / v), the concentration of ethyl silicate solution was 600 mM, the concentration of 3-aminopropyltrimethoxysilane was 10% (v / v), and the concentration range of 3-aminopropyltrimethoxysilane was 10% (v / v). Ammonia water, ethyl silicate and 3-aminopropyltrimethoxysilane were added in a volume ratio of 1-10:1-2:1-10, and then ultrasonicated. The ultrasonic modification time was 240 min. Through a one-step reaction, a magnetic fluorescent probe (MagMQD@Si + ).

[0066] Figure 1 Schematic diagram of the preparation method of magnetic fluorescent probe modified with positively charged silica shell. Figure 2 Electron microscopy characterization of the structure of a magnetic fluorescent probe modified with a positively charged silica shell.

[0067] Example 3

[0068] Figure 3 The flow chart of immunochromatography based on magnetic fluorescent probes modified with positively charged silica shells for detection of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium. This example optimizes the operating conditions of immunochromatography based on magnetic fluorescent probes modified with positively charged silica shells to achieve the best detection performance of the platform. Figure 4 This is the optimization result of the concentration of the capture antibody on the detection line. When the concentration of the anti-Pseudomonas aeruginosa antibody on the detection line is 1.5 mg / mL; the concentration of the anti-Streptococcus pneumoniae antibody is 0.8 mg / mL; and the concentration of the anti-Salmonella typhimurium antibody is 1.2 mg / mL, the immunochromatography based on the positively charged silica shell modified magnetic fluorescent probe has the highest signal-to-noise ratio for Pseudomonas aeruginosa, Streptococcus pneumoniae, and Salmonella typhimurium. Figure 5 This is the optimization result of the incubation time between the positively charged silica shell modified magnetic fluorescent probe and the sample. Incubating with the sample for 10 minutes can enable the immunochromatography based on the positively charged silica shell modified magnetic fluorescent probe to obtain a stronger fluorescence signal and the highest signal-to-noise ratio when detecting Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium.

[0069] The performance of the immunochromatographic technique based on the positively charged silica shell modified magnetic fluorescent probe proposed in the present invention is highly dependent on the ability of the positively charged silica shell modified magnetic fluorescent probe to capture and bind target bacteria. We used plate culture to determine the capture efficiency of the positively charged silica shell modified magnetic fluorescent probe for Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium. The results are as follows Figure 6We can conclude that the plate count results show that after 5 min of incubation, the capture efficiency of the positively charged silica shell-modified magnetic fluorescent probe for Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium is over 98%.

[0070] Example 4

[0071] This example uses different concentrations (10 5 -10 cells / mL) of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium were used to verify the detection performance of the immunochromatographic technology based on the positively charged silica shell modified magnetic fluorescent probe. Figure 7 The following is a photo of the immunochromatographic technique based on the positively charged silica shell modified magnetic fluorescent probe to detect different concentrations of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium samples. Figure 7 a It can be seen that the vLOD of the immunochromatographic platform based on the positively charged silica shell modified magnetic fluorescent probe for these three pathogens in the visual mode is 50 cells / mL, 50 cells / mL, and 100 cells / mL respectively. We use an immunofluorescence analyzer to measure the fluorescence signal on the detection line and analyze it. Figure 7 The bar graph results in b show that the fluorescence signal on the test line is positively correlated with the bacterial concentration in the sample. The calibration curve is drawn based on the relationship between the fluorescence signal on each test line and the concentration of Pseudomonas aeruginosa, Streptococcus pneumoniae, and Salmonella typhimurium, as shown in Figure 7 As shown in Figure 3, the detection limits (LOD) of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium were calculated to be 8 cells / mL, 31 cells / mL and 40 cells / mL respectively. The results showed that the immunochromatographic platform based on the magnetic fluorescent probe modified with positively charged silica shell had high analytical performance for the target bacteria, a wide dynamic range (5 orders of magnitude) and a good correlation coefficient (R 2 >0.99), low detection limit. The immunochromatographic test strip based on the magnetic fluorescent probe modified with positively charged silica shell can qualitatively screen Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium with a concentration of more than 50 cells / mL by observing the color change on the detection line under ultraviolet light, and supports the detection of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium with a concentration range of 10 5 -10 cells / mL for quantitative screening of Pseudomonas aeruginosa, Streptococcus pneumoniae, and Salmonella typhimurium. Figure 8 Compared with the detection results of traditional colloidal gold, the sensitivity of the immunochromatographic detection platform based on positively charged silica shell modified magnetic fluorescent probes is increased by 25-125 times.

[0072] At the same time, we selected common pathogens such as Shigella, Klebsiella pneumoniae, Staphylococcus epidermidis, Acinetobacter baumannii, Helicobacter pylori, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli O157:H7, Campylobacter jejuni, Streptococcus pyogenes, Vibrio cholerae, Staphylococcus aureus and Clostridium difficile (10 4 cells / mL) as bacterial interferences to test the specificity of the immunochromatographic test strip based on the magnetic fluorescent probe modified with positively charged silica shells. Fig. 9 As shown in the figure, the photo of the test strip and the fluorescent signal on the detection line clearly prove that the immunochromatographic platform based on the positively charged silica shell modified magnetic fluorescent probe has good selectivity for the target bacteria. Therefore, the specific bacterial antibodies applied on the detection line of the immunochromatographic platform based on the positively charged silica shell modified magnetic fluorescent probe are sufficient to ensure the high specificity of the detection platform. 4 cells / mL and 10 2 Tap water samples were continuously tested at a concentration of 2.174 cells / mL, and the relative standard deviation (RSD) was calculated to verify the repeatability of the immunochromatographic platform based on the positively charged silica shell modified magnetic fluorescent probe. Fig.10 These are the repeatability test results of the immunochromatography platform based on the magnetic fluorescent probe modified with positively charged silica shells. The SERS signal on the detection line changed little, and the RSD value of each test group was less than 5.54%, indicating that the established immunochromatography platform based on the magnetic fluorescent probe modified with positively charged silica shells has good repeatability.

[0073] This example evaluates the performance of the immunochromatographic platform based on positively charged silica shell-modified magnetic fluorescent probes in actual clinical testing and environmental monitoring by testing actual samples with bacterial addition. We added different concentrations (50 cells / mL to 5000 cells / mL) of Pseudomonas aeruginosa, Streptococcus pneumoniae, and Salmonella typhimurium to throat swabs of healthy people and river water, respectively. Fig.11 The results of the immunochromatographic platform based on the positively charged silica shell modified magnetic fluorescent probe for the detection of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium in actual samples show that the fluorescence signals on the corresponding detection lines in the throat swabs of healthy people and river water samples are stable, and the intensity of the fluorescence signal decreases with the decrease of the concentration of the corresponding target pathogens in the samples. We calculated the recovery rates of three different pathogens based on the fluorescence signals, and the average recovery rates of the samples were 90.82%-107.69%, with RSD values ​​less than 10%, indicating that the established platform for the quantitative detection of Pseudomonas aeruginosa, Streptococcus pneumoniae and Salmonella typhimurium has good accuracy and reliability for the quantitative detection of bacteria in complex samples.

[0074] Most bacteria (Gram-positive and Gram-negative) are electronegative over a wide pH range (4-10) because their cells have a net negative charge, which is conferred by ionized phosphoryl and carboxylic acid substituents on the macromolecules of the cell envelope. Based on this feature, in this application, we proposed a universal ICA detection platform using a carefully designed electronegative probe MagMQD@Si + The multifunctional MagMQD@Si + The probe has a 200nm Fe3O4 core with strong magnetic enrichment ability, a multilayer inner shell formed by quantum dots (QDs) with superior luminescence ability, an amino-modified SiO2 shell with broad-spectrum bacterial binding ability and high fluidity for ICA detection. + The positively charged SiO2 shell promotes strong electrostatic interactions with negatively charged pathogens, enabling the electronegative probe to rapidly capture and enrich a wide range of bacteria from complex solutions and enabling their ultrasensitive and highly specific detection on antibody-modified ICA strips. We demonstrated the universality and multiplex detection capabilities of our proposed bacterial detection method by detecting three important bacterial pathogens: Pseudomonas aeruginosa, Streptococcus pneumoniae, and Streptococcus typhi. We chose these bacteria because they represent common respiratory, nosocomial, and foodborne bacteria, respectively. Among these bacteria, Streptococcus pneumoniae is responsible for a large number of lower respiratory tract infections and deaths in newborns and young children. Salmonella typhi is strongly associated with deaths in children aged 5-14 years, causing an estimated 49,000 deaths. Pseudomonas aeruginosa commonly causes lower respiratory tract, bloodstream, and intra-abdominal infections, causing more than 500,000 deaths worldwide. The sensitivity of simultaneous detection of Pseudomonas aeruginosa (P.aeruginosa), Streptococcus pneumoniae (S.pneumoniae), and Streptococcus typhi (S.typhi) was 8 cells / mL, 31 cells / mL, and 40 cells / mL, respectively, which is about 5.2 times that of conventional double-sandwich fluorescent ICA and at least 25 times that of colloidal gold immunochromatography. Our proposed method has good accuracy and reliability in the detection of simulated clinical and environmental samples (throat swabs and lake water) and real clinical samples from 30 patients with bacterial infections, so it has great potential in the rapid identification of pathogens and early diagnosis of bacterial infections on site.

[0075] The above are only preferred specific implementation modes of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications made by any technician familiar with the technical field within the technical scope disclosed by the present invention by equivalently replacing or changing the technical solution and inventive concept of the present invention should be covered by the protection scope of the present invention.

Claims

1. A magnetic fluorescent probe based on positively charged silica shell modification, characterized in that: Superparamagnetic Fe3O4 nanoparticles are used as the core to provide magnetic enrichment force; multilayer quantum dot particles are electrostatically adsorbed by cationic polymers as a fluorescent interlayer, which provides a fluorescent signal for quantitative detection; the surface of the fluorescent interlayer is provided with an amino-containing silica shell, which can protect the internal material and capture a wide spectrum of negatively charged bacteria.

2. The method for preparing a magnetic fluorescent probe based on positively charged silica shell modification according to claim 1, characterized in that: The following steps are involved: (1) Using superparamagnetic Fe3O4 nanoparticles as the core, adding cationic polymer aqueous solution, and preparing multilayer quantum dot particles coated with magnetic quantum dots as fluorescent interlayers through cationic polymer-mediated layer-by-layer self-assembly method; (2) The magnetic quantum dots prepared in step (1) are resuspended in an ethanol solution, and then ammonia, ethyl silicate and 3-aminopropyltrimethoxysilane are slowly added to react to form a magnetic fluorescent probe protected by an amino-silica shell with a strong positive charge.

3. The method for preparing a magnetic fluorescent probe based on positively charged silica shell modification according to claim 2, characterized in that: In step (1), the particle size of the Fe3O4 nanoparticles is 50-500nm.

4. The method for preparing a magnetic fluorescent probe based on positively charged silicon shell modification according to claim 2, characterized in that: In step (1), the concentration of the cationic polymer aqueous solution is ≤5 mg / mL, and ultrasonication is performed after the cationic polymer is added, and the ultrasonic modification time is ≤80 min.

5. The method for preparing a magnetic fluorescent probe based on positively charged silica shell modification according to claim 4, characterized in that: The cationic polymer is an aqueous solution of polyethyleneimine.

6. The method for preparing a magnetic fluorescent probe based on positively charged silica shell modification according to claim 2, characterized in that: In step (1), the particle size of the quantum dot single particle forming the fluorescent interlayer is 5-30 nm, the quantum dot concentration is ≤50 mg / mL, and the ultrasonic time is ≤80 min.

7. The method for preparing a magnetic fluorescent probe based on positively charged silica shell modification according to claim 2, characterized in that: In step (2), when the amino-modified silica shell is prepared, the concentration of aqueous ammonia is ≤50% (v / v), the concentration of ethyl silicate solution is ≤600mM, the concentration of 3-aminopropyltrimethoxysilane is ≤10% (v / v), and the concentration range of 3-aminopropyltrimethoxysilane is ≤10% (v / v). After adding aqueous ammonia, ethyl silicate and 3-aminopropyltrimethoxysilane, ultrasound is performed, and the ultrasound modification time is ≤240min.

8. The method for preparing a magnetic fluorescent probe based on positively charged silica shell modification according to claim 2, characterized in that: In step (2), the shell thickness of the amino-treated silica is ≤50 nm, and the Zeta potential is between 0 and 60 mV.

9. Use of the probe prepared by the method for preparing a magnetic fluorescent probe modified with a positively charged silica shell according to any one of claims 2 to 8 in preparing an immunochromatography component, an immunochromatography device or an immunochromatography product.

10. The use according to claim 9, characterized in that: The immunochromatographic product is an immunochromatographic test strip, which includes a sample pad for loading sample solution, a nitrocellulose membrane with an independent detection line, an absorbent pad, a positive electromagnetic fluorescent probe for detection, and a running buffer. The positive electromagnetic fluorescent probe is obtained by the preparation method described in any one of claims 2 to 8.

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