Double-signal nanoprobe based on silicon dioxide interlayer protection as well as preparation method and application of double-signal nanoprobe

Abstract

The invention relates to a double-signal nanoprobe based on silicon dioxide interlayer protection as well as a preparation method and application of the double-signal nanoprobe. The nanoprobe has a core-shell structure, and the core-shell structure comprises an inner core and an outer shell layer coating the outer surface of the inner core; the inner core is SiO2 nano particles; the outer surface of the inner core comprises an outer shell layer of the core-shell structure formed by sequentially coating a first cationic polymer interlayer, a gold nanoparticle interlayer, a SiO2 inner shell layer, a second cationic polymer interlayer and a carboxylated quantum dot outer shell layer. The invention provides a double-signal nanoprobe based on silicon dioxide interlayer protection, and the nanoprobe not only has a good colorimetric signal, but also has a strong fluorescence signal. After the nanoprobe is coupled with a streptococcus pneumoniae antibody, streptococcus pneumoniae in a sample can be selectively detected, colorimetric and fluorescence signals can be output at the same time, and rapid and high-sensitivity detection of streptococcus pneumoniae is achieved.

Description

[0001] Cross-reference with related applications This application claims priority to Chinese Patent Application No. 202410960953.6, filed on July 17, 2024, the entire contents of which are incorporated herein by reference and for all purposes. Technical Field

[0002] This disclosure relates to the fields of nanomaterial synthesis and fluorescence spectroscopy analysis and detection, specifically to a dual-signal nanoprobe based on silica interlayer protection, its preparation method, and its application. Background Technology

[0003] Streptococcus pneumoniae ( S. pneumoniae Streptococcus pneumoniae (SPR) is a Gram-positive, non-motile bacterium widely distributed in nature, commonly colonizing the mucous membranes of the human upper respiratory tract. Infection with this bacterium can cause pneumonia, meningitis, otitis media, and other invasive diseases. Globally, the morbidity and mortality rates from Streptococcus pneumoniae infection are very high annually. Early detection of clinical infection and timely and accurate diagnosis in the early stages of the disease allow for appropriate treatment. However, the current gold standard for detecting Streptococcus pneumoniae is phenotypic, including culture-based, microscopic, and biochemical identification methods. The growth and identification of Streptococcus pneumoniae typically takes more than two days, and delayed diagnosis can lead to poor prognosis for individuals infected with this pathogen. Several culture-free methods for detecting Streptococcus pneumoniae, including mass spectrometry, PCR, and real-time PCR, have been developed, but all require skilled technicians and / or complex equipment, which may be unavailable in some cases.

[0004] Flow lateral immunochromatography (LFIA) is a mature point-of-care testing (POCT) technique that is simple to operate, inexpensive, and portable, meeting the needs of rapid on-site testing. Test strips based on colloidal gold nanoparticles (AuNPs) are widely used in LFIA because the results can be directly interpreted with the naked eye. However, AuNP-based LFIA has certain limitations, such as low sensitivity and inaccurate quantification due to limited colorimetric signal readings, hindering the large-scale application of conventional LFIA for rapid and accurate detection. Recently, researchers have used fluorescent materials such as upconversion particles or quantum dots (QDs) as labels, improving signal readability and reducing background interference, thereby enhancing the quantitative capability and sensitivity of LFIA. Although fluorescent probes extend LFIA to higher sensitivity, additional instruments are required to read the fluorescence signal, which greatly limits the application of fluorescent materials in POCT.

[0005] The colloidal gold and QDs two optical probes are combined to prepare a colorimetric-fluorescent double signal nano probe with easy-to-read colorimetric signal and easy-to-quantify fluorescent signal, which can be flexibly switched according to different scenes, and the application range of LFIA is expanded. However, as an energy acceptor, gold nanoparticles can quench the fluorescence of the fluorescent dye adsorbed on its surface through surface energy transfer. If only the simple combination of AuNP and QD, the fluorescence signal of quantum dots will be greatly reduced, thereby affecting the detection effect of LFIA. If you want to eliminate the influence of AuNP on the fluorescence performance, you often need to add more layers of quantum dots, but this will complicate the preparation steps of the probe.

[0006] Therefore, it is crucial to develop a rapid and accurate POCT for the timely detection of S. pneumoniae. SUMMARY

[0007] Based on the problem that the simple combination of gold nanoparticles and quantum dots reduces the fluorescence signal of quantum dots in the prior art, the present disclosure provides a double signal nano probe based on silica interlayer protection, which not only has good colorimetric signal, but also has strong fluorescent signal.

[0008] To achieve the above purpose, the first aspect of the present disclosure provides a double signal nano probe based on silica interlayer protection, the nano probe has a core-shell structure, the core-shell structure comprises an inner core and an outer shell layer coated on the outer surface of the inner core. The inner core is a SiO2 nanoparticle. The outer surface of the inner core comprises an outer shell layer of the core-shell structure formed by a first cationic polymer interlayer, a gold nanoparticle interlayer, a SiO2 inner shell layer, a second cationic polymer interlayer and a carboxylated quantum dot outer shell layer coated in turn.

[0009] Optionally, the carboxylated quantum dot outer shell layer contains CdSe@ZnS-COOH quantum dots; a specific antibody is coupled to the carboxyl end of the carboxylated quantum dot outer shell layer, and the specific antibody is an anti-S. pneumoniae antibody; relative to 1 mg of the nano probe, 0.2-1.0 mg of the specific antibody is coupled.

[0010] Optionally, the average particle size of the SiO2 nanoparticle is 100-300 nm; the average particle size of the gold nanoparticle is 3-30 nm; the average particle size of the carboxylated quantum dot is 5-30 nm; the first cationic polymer interlayer and the second cationic polymer interlayer are each independently polyethyleneimine, the thickness of the first cationic polymer interlayer is 0.5-2.0 nm, and the thickness of the second cationic polymer interlayer is 0.5-2.0 nm; the thickness of the SiO2 inner shell layer is 1-30 nm.

[0011] The second aspect of the present disclosure provides a preparation method of the nanoprobe of the first aspect, and the method comprises the following steps: S1, adding SiO2 nanoparticles into a first polyethyleneimine aqueous solution, performing first ultrasonic treatment, and obtaining SiO2-PEI nanomicrospheres; S2, adding the SiO2-PEI nanomicrospheres into a gold nanoparticle dispersion liquid, performing second ultrasonic treatment, and obtaining SiO2 / Au nanomicrospheres; S3, adding the SiO2 / Au nanomicrospheres into ammonia water, performing third ultrasonic treatment, then adding tetraethyl orthosilicate, and performing fourth ultrasonic treatment, and obtaining SiO2 / Au@Si nanomicrospheres; S4, adding the SiO2 / Au@Si nanomicrospheres into a second polyethyleneimine aqueous solution, performing fifth ultrasonic treatment, and obtaining SiO2 / Au@Si-PEI nanomicrospheres; S5, adding the SiO2 / Au@Si-PEI nanomicrospheres into a carboxylated quantum dot dispersion liquid, performing sixth ultrasonic treatment, and obtaining the nanoprobe.

[0012] Optionally, in step S1, the average particle size of the SiO2 nanoparticles is 100-300 nm; the mass concentration of the first polyethyleneimine aqueous solution is 1-10 mg / mL; and the addition amount of the SiO2 nanoparticles is 50-500 mg per 100 mL of the first polyethyleneimine aqueous solution; in step S2, the average particle size of the gold nanoparticles is 3-30 nm; the mass concentration of the gold nanoparticle dispersion liquid is 10-50 mg / mL; and the addition amount of the SiO2-PEI nanomicrospheres is 100-500 mg per 100 mL of the gold nanoparticle dispersion liquid.

[0013] Optionally, in step S3, the volume concentration of the ammonia water is 1-50 v / v; the addition amount of the SiO2 / Au nanomicrospheres is 50-500 mg and the addition amount of the tetraethyl orthosilicate is 1-10 mL per 100 mL of the ammonia water; the molar concentration of the tetraethyl orthosilicate is 50-500 mmol / L; in step S4, the mass concentration of the second polyethyleneimine aqueous solution is 1-10 mg / mL; the addition amount of the SiO2 / Au@Si nanomicrospheres is 50-500 mg per 100 mL of the second polyethyleneimine aqueous solution; in step S5, the average particle size of the carboxylated quantum dots is 5-30 nm; the mass concentration of the carboxylated quantum dot dispersion liquid is 1-10 mg / mL; and the addition amount of the SiO2 / Au@Si-PEI nanomicrospheres is 50-500 mg per 100 mL of the carboxylated quantum dot dispersion liquid.

[0014] Optionally, the method further comprises: after the activation treatment of the nanoprobe, mixing the nanoprobe with specific antibodies, and then incubating; wherein the specific antibodies are anti-streptococcus pneumoniae antibodies; and the addition amount of the specific antibodies is 0.2-1.0 mg per 1 mg of the nanoprobe.

[0015] The third aspect of the present disclosure provides an application of the nanoprobe of the first aspect or the nanoprobe prepared by the preparation method of the second aspect in the preparation of a product for detecting streptococcus pneumoniae.

[0016] The fourth aspect of the present disclosure provides a kit for detecting streptococcus pneumoniae, wherein the kit comprises the nanoprobe of the first aspect or the nanoprobe prepared by the preparation method of the second aspect.

[0017] Optionally, the kit further comprises an immunochromatography test strip, wherein the immunochromatography test strip comprises a sample pad, a chromatography membrane and an absorption pad connected in sequence, and the chromatography membrane is provided with one detection line and one quality control line; the detection line is loaded with anti-streptococcus pneumoniae antibodies; and the quality control line is loaded with goat anti-rabbit IgG.

[0018] Through the above technical solution, the present disclosure provides a double-signal nanoprobe based on a silica sandwich protection, which not only has a good colorimetric signal, but also has a strong fluorescent signal. After the nanoprobe of the present disclosure is coupled with streptococcus pneumoniae antibodies, it can selectively detect streptococcus pneumoniae in a sample, and can output colorimetric and fluorescent signals at the same time, so that it can be flexibly applied under different environmental conditions, and rapid and high-sensitivity detection of streptococcus pneumoniae is realized.

[0019] Other features and advantages of the present disclosure will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings are included to provide a further understanding of the present disclosure and constitute a part of the specification, and are used together with the following specific embodiments to explain the present disclosure, but do not constitute a limitation on the present disclosure. In the drawings: Figure 1 It is a schematic diagram of the preparation method of the SiO2 / Au@Si / QD probe of the present disclosure.

[0021] Figure 2 It is an electron microscope characterization of the structure of the SiO2 / Au@Si / QD probe of the present disclosure.

[0022] Figure 3 It is an element characterization diagram of the SiO2 / Au@Si / QD probe of the present disclosure.

[0023] Figure 4 It is an application of the SiO2 / Au@Si / QD-LFIA of the present disclosure inS. pneumoniae The detection flow chart of the SiO2 / Au@Si / QD-LFIA.

[0024] Figure 5 The antibody spraying concentration optimization results of the SiO2 / Au@Si / QD-LFIA of the present disclosure.

[0025] Figure 6 The running time optimization results of the SiO2 / Au@Si / QD-LFIA of the present disclosure.

[0026] Figure 7 The capture efficiency results of the SiO2 / Au@Si / QD of the present disclosure. S. pneumoniae

[0027] Figure 8 The detection results of the SiO2 / Au@Si / QD-LFIA of the present disclosure for detecting S. pneumoniae

[0028] The detection results of the SiO2 / Au / QD-LFIA of the present disclosure for detecting Figure 9 S. pneumoniae The detection results of the colloidal gold method of the present disclosure for detecting

[0029] Figure 10 S. pneumoniae

[0030] Figure 11 The specificity verification of the SiO2 / Au / QD-LFIA of the present disclosure.

[0031] Figure 12 The repeatability verification of the SiO2 / Au / QD-LFIA of the present disclosure.

[0032] Figure 13 The analysis results of the SiO2 / Au / QD-LFIA of the present disclosure in actual samples. DETAILED DESCRIPTION

[0033] The specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

[0034] As shown in Figure 1 , the first aspect of the present disclosure provides a double-signal nano probe based on silica interlayer protection, the nano probe has a core-shell structure, the core-shell structure includes an inner core and an outer shell layer coated on the outer surface of the inner core; The inner core is a SiO2 nanoparticle; ​​​​The outer surface of the inner core comprises an outer shell layer of the core-shell structure formed by a first cationic polymer interlayer, a gold nanoparticle interlayer, a SiO2 inner shell layer, a second cationic polymer interlayer and a carboxylated quantum dot outer shell layer in sequence.

[0035] In the present disclosure, silica (SiO2) is used as the inner core, gold nanoparticles (AuNP) providing colorimetric signals are attached to the surface of the SiO2 nanoparticles by electrostatic adsorption of polyethyleneimine (PEI), then a protective SiO2 inner shell is coated on the surface of the nanomaterial by hydrolysis of tetraethyl orthosilicate (TEOS), and finally thousands of QDs are attached to the surface of the material again by the strong positive charge of PEI. The inner shell layer formed by the SiO2 inner shell can isolate the AuNP and the quantum dots (QD), effectively avoiding the fluorescence quenching effect of the AuNP on the quantum dots QD, and effectively protecting the fluorescence performance of the QD. The nanoprobes of the present disclosure not only have good colorimetric signals, but also have strong fluorescence signals.

[0036] According to the present disclosure, the carboxylated quantum dot outer shell layer contains CdSe@ZnS-COOH quantum dots. The quantum dots have carboxyl groups for coupling with antibodies, and can output strong fluorescence signals, further improving the sensitivity of the nanoprobes of the present disclosure.

[0037] In an embodiment of the present disclosure, a specific antibody is coupled to the carboxyl end of the carboxylated quantum dot outer shell layer, and the specific antibody is an anti-Streptococcus pneumoniae antibody. In the present disclosure, the carboxyl end of the carboxylated quantum dot outer shell layer can be coupled to the amino end of the specific antibody to form a peptide bond, and then a nanoprobes modified by the specific antibody is obtained. After the nanoprobes of the present disclosure are coupled with the Streptococcus pneumoniae antibody, Streptococcus pneumoniae in a sample can be selectively detected, and rapid and high-sensitivity detection of Streptococcus pneumoniae is achieved. S. pneumoniae )antibody, Streptococcus pneumoniae in a sample can be selectively detected, and rapid and high-sensitivity detection of Streptococcus pneumoniae is achieved.

[0038] According to the present disclosure, the content of the specific antibody can be varied within a large range. In a specific embodiment of the present disclosure, 0.2-1.0 mg of the specific antibody is coupled to every 1 mg of the nanoprobes. When the content of the specific antibody is within the range of the above embodiment, false positive results caused by excessive content of the specific antibody can be avoided.

[0039] The present disclosure selects SiO2 nanoparticles with good monodispersity as the inner core of the nanoprobes. In a specific embodiment of the present disclosure, the average particle size of the SiO2 nanoparticles is 100-300 nm.

[0040] In a specific embodiment of the present disclosure, the average particle size of the gold nanoparticles is 3-30 nm; and the average particle size of the carboxylated quantum dots is 5-30 nm.

[0041] In one specific embodiment of the present disclosure, the first cationic polymer interlayer and the second cationic polymer interlayer are each independently polyethyleneimine, the thickness of the first cationic polymer interlayer is 0.5-2.0 nm, and the thickness of the second cationic polymer interlayer is 0.5-2.0 nm. When the thickness of the first cationic polymer interlayer and the thickness of the second cationic polymer interlayer are within the range of the above-mentioned embodiment, the uniformity of the nanoprobes of the present disclosure can be further ensured.

[0042] According to the present disclosure, the thickness of the SiO2 inner shell layer can vary within a large range. In one specific embodiment of the present disclosure, the thickness of the SiO2 inner shell layer is 1-30 nm. When the thickness of the SiO2 inner shell layer varies within the range of the above-mentioned embodiment, the distance between the gold nanoparticles and the quantum dots in the gold nanoparticle interlayer can be better controlled, which not only ensures that the nanoprobes of the present disclosure have good colorimetric effect, but also avoids the interference of the gold nanoparticles on the fluorescence performance of the quantum dots, thereby ensuring that the nanoprobes of the present disclosure have high fluorescence signal.

[0043] According to the present disclosure, the average particle size of the dual-signal nanoprobes protected by the silica interlayer can be controlled according to requirements. In one embodiment, the average particle size of the nanoprobes is 100-380 nm.

[0044] The second aspect of the present disclosure provides a preparation method of the nanoprobes of the first aspect, and the method comprises the following steps: S1, adding SiO2 nanoparticles into a water solution of first polyethyleneimine, performing first ultrasonic treatment to obtain SiO2-PEI nanomicrospheres; S2, adding the SiO2-PEI nanomicrospheres into a gold nanoparticle dispersion liquid, performing second ultrasonic treatment to obtain SiO2 / Au nanomicrospheres; S3, adding the SiO2 / Au nanomicrospheres into ammonia water to perform third ultrasonic treatment, then adding tetraethyl orthosilicate to perform fourth ultrasonic treatment, to obtain SiO2 / Au@Si nanomicrospheres; S4, adding the SiO2 / Au@Si nanomicrospheres into a water solution of second polyethyleneimine, performing fifth ultrasonic treatment to obtain SiO2 / Au@Si-PEI nanomicrospheres; S5, adding the SiO2 / Au@Si-PEI nanomicrospheres into a carboxylated quantum dot dispersion liquid to perform sixth ultrasonic treatment, to obtain the nanoprobes.

[0045] The preparation method of the present disclosure has high preparation efficiency and the preparation steps are repeatable, which is suitable for industrial production.

[0046] In the present disclosure, "ultrasonic treatment" can employ devices commonly used by those skilled in the art, which will not be described here.

[0047] The present disclosure selects SiO2 nanoparticles with good monodispersity as the core of the nanoprobe. In one specific embodiment of the present disclosure, the monodisperse SiO2 nanoparticles employed by the present disclosure are prepared by a modified Stöber chemical precipitation method, and the specific steps include: mixing 100 mL of anhydrous ethanol, 4 mL of ammonia water with a volume concentration of 28%, and 6 mL of deionized water, and stirring the mixture at room temperature for 20 min, then adding 4 mL of tetraethyl orthosilicate (50-500 mmol / L) at one time, sealing, and stirring magnetically at room temperature for 4 h. After the reaction is completed, the solution becomes a milky white liquid, and the purified SiO2 particles are collected by centrifugation (4800 rpm, 6 min). In one specific embodiment of the present disclosure, the average particle size of the SiO2 nanoparticles is 100-300 nm, and preferably 200-250 nm.

[0048] In the present disclosure, the mass concentration of the aqueous solution of the first polyethyleneimine has an important influence on the adsorption of gold nanoparticles. According to the present disclosure, the mass concentration of the aqueous solution of the first polyethyleneimine can be varied within a wide range. In one specific embodiment of the present disclosure, the mass concentration of the aqueous solution of the first polyethyleneimine is 1-10 mg / mL; when the mass concentration of the aqueous solution of the first polyethyleneimine is within the range of the above-mentioned embodiment, the problem of low uniformity of the prepared nanoprobe caused by the problem that too much PEI cannot be completely washed clean is avoided.

[0049] In one embodiment of the present disclosure, the addition amount of the SiO2 nanoparticles is 50-500 mg relative to 100 mL of the aqueous solution of the first polyethyleneimine; and the first ultrasonic treatment is performed under the conditions of a frequency of 50-100 Hz, a temperature of 15-25°C, and a time of 10-30 min. In the above-mentioned embodiment, by controlling the time of ultrasonic treatment, a first polyethyleneimine interlayer with a thickness of 0.5-2.0 nm can be formed.

[0050] In one specific embodiment of the present disclosure, in step S2, the average particle size of the gold nanoparticles is 3-30 nm, and preferably 20 nm; the mass concentration of the gold nanoparticle dispersion is 10-50 mg / mL; the addition amount of the SiO2-PEI nanomicrospheres is 100-500 mg relative to 100 mL of the gold nanoparticle dispersion; and the second ultrasonic treatment is performed under the conditions of a frequency of 50-100 Hz, a temperature of 15-25°C, and a time of 10-30 min.

[0051] In an embodiment of the present disclosure, in step S3, the volume concentration of the ammonia water is 1-50 v / v; the SiO2 / Au nanomicrospheres are added in an amount of 50-500 mg, and the tetraethyl orthosilicate is added in an amount of 1-10 mL per 100 mL of ammonia water; the molar concentration of the tetraethyl orthosilicate is 50-500 mmol / L. The third ultrasonic treatment is performed at a frequency of 50-100 Hz, a temperature of 15-15 ℃, and for a time of 10-30 min. The fourth ultrasonic treatment is performed at a frequency of 50-100 Hz, a temperature of 15-25 ℃, and for a time of 10-30 min. In the above embodiment, by controlling the amount of the added tetraethyl orthosilicate and the time of the ultrasonic treatment, the SiO2 inner shell layer with a thickness of 1-30 nm can be formed.

[0052] In the present disclosure, the mass concentration of the second aqueous polyethyleneimine solution has an important influence on the adsorption of the quantum dots. According to the present disclosure, the mass concentration of the second aqueous polyethyleneimine solution can be varied in a relatively large range. In an embodiment of the present disclosure, in step S4, the mass concentration of the second aqueous polyethyleneimine solution is 1-10 mg / mL; when the mass concentration of the second aqueous polyethyleneimine solution is within the range of the above embodiment, the problem of the non-uniformity of the prepared nanoprobes caused by the problem that too much PEI cannot be completely washed clean when there is too much PEI is avoided.

[0053] In an embodiment of the present disclosure, the SiO2 / Au@Si nanomicrospheres are added in an amount of 50-500 mg per 100 mL of the second aqueous polyethyleneimine solution; the fifth ultrasonic treatment is performed at a frequency of 50-100 Hz, a temperature of 15-25 ℃, and for a time of 10-30 min. In the above embodiment, by controlling the time of the ultrasonic treatment, the second polyethyleneimine interlayer with a thickness of 0.5-2.0 nm can be formed.

[0054] In the present disclosure, carboxylation of the quantum dots can enable coupling of the quantum dots with specific antibodies. The carboxylated quantum dots (CdSe@ZnS-COOH) are purchased from Suzhou Xingcang Nanometer Technology Co., Ltd.; the average particle size of the carboxylated quantum dots is 5-30 nm. In an embodiment of the present disclosure, in step S5, the mass concentration of the carboxylated quantum dot dispersion is 1-10 mg / mL; the SiO2 / Au@Si-PEI nanomicrospheres are added in an amount of 50-500 mg per 100 mL of the carboxylated quantum dot dispersion; the sixth ultrasonic treatment is performed at a frequency of 50-100 Hz, a temperature of 15-25 ℃, and for a time of 10-30 min.

[0055] In one specific embodiment of the present disclosure, the method further comprises: after the activation treatment of the nanoprobe (SiO2 / Au@Si / QD), mixing the nanoprobe with specific antibodies and then incubating; wherein the specific antibodies are anti-streptococcus pneumoniae antibodies; and the specific antibodies are added in an amount of 0.2-1.0 mg per 1 mg of the nanoprobe. Specifically, the activation treatment comprises: mixing 1 mg of SiO2 / Au@Si / QD with 100 mL of activation solution, and then ultrasonic treatment at 50-100 Hz and 15-25°C for 1-30 min; wherein the activation solution contains 10 mmol / L of 2-(N-nitro)ethanesulfonic acid (MES), 0.1 mmol / L of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) and 0.1 mmol / L of N-hydroxysuccinimide (NHS). After the activation, the SiO2 / Au@Si / QD is incubated with the specific antibodies at 20-25°C for 2-4 h, so that the carboxyl end of the SiO2 / Au@Si / QD is coupled with the amino end of the specific antibodies to form a peptide bond, and a specific antibody modified nanoprobe is obtained. In the present disclosure, the specific antibody modified nanoprobe is an anti-streptococcus pneumoniae antibody modified nanoprobe, which is a double signal probe for the capture / detection of streptococcus pneumoniae. S. pneumonia

[0056] The third aspect of the present disclosure provides an application of the nanoprobe of the first aspect or the nanoprobe prepared by the preparation method of the second aspect in the preparation of a product for detecting streptococcus pneumoniae.

[0057] In the present disclosure, the product for detecting streptococcus pneumoniae can be a kit for detecting streptococcus pneumoniae, or can be a component or device in a product for detecting streptococcus pneumoniae by immunochromatography.

[0058] The fourth aspect of the present disclosure provides a kit for detecting streptococcus pneumoniae, which comprises the nanoprobe of the first aspect or the nanoprobe prepared by the preparation method of the second aspect.

[0059] The anti-streptococcus pneumoniae antibody modified nanoprobe prepared by the present disclosure can provide both colorimetric and fluorescent signals in a lateral flow immunochromatography (LFIA) platform (SiO2 / Au@Si / QD-LFIA), meet the detection requirements under different device conditions, and realize rapid and high-sensitivity detection of streptococcus pneumoniae.

[0060] ​In one specific embodiment of this disclosure, the kit further includes an immunochromatographic test strip comprising a sample pad, a chromatography membrane, and an absorbent pad connected in sequence. The chromatography membrane has a detection line (T) and a control line (C). The detection line is loaded with anti-pneumococcal antibody; the control line is loaded with goat anti-rabbit IgG. In this disclosure, the immunochromatographic test strip is used to detect the SiO2 / Au@Si / QD probe and the running buffer.

[0061] In this disclosure, the immunochromatographic test strip also includes a backing plate, which serves as the backing card for the immunochromatographic test strip. The backing plate is preferably made of PVC. A sample pad is used for loading the sample solution to be tested. An absorbent pad is used to provide capillary force. The chromatography membrane can be a nitrocellulose membrane (NC membrane).

[0062] In one specific embodiment of this disclosure, anti-pneumococcal antibody and goat anti-rabbit IgG are sprayed onto the surface of a nitrocellulose membrane to capture the corresponding probe-bacterial complex; then the modified NC membrane is placed in a constant temperature drying oven (34°C) to obtain the nitrocellulose membrane in the immunochromatographic test strip. The concentration of anti-pneumococcal antibody is 0.5-2.0 mg / mL; the concentration of goat anti-rabbit IgG is 0.5-2.0 mg / mL.

[0063] According to this disclosure, the kit also includes a run buffer, specifically comprising: phosphate-buffered saline (PBS), 1% Tween (v / v) + 0.5% milk (w / v).

[0064] This disclosure is aimed at S. pneumonia The SiO2 / Au@Si / QD-LFIA provides two detection modes: (1) in colorimetric mode, rapid qualitative detection is performed by visually identifying the reddish-brown colorimetric signal on the detection line; (2) in fluorescence mode, detection is achieved by detecting changes in the fluorescence signal of quantum dots on the detection line. S. pneumonia Highly sensitive quantitative analysis.

[0065] The method for detecting the test sample using the kit disclosed herein includes the following steps: Mix the test sample, the anti-pneumococcal antibody-modified nanoprobe, and the running buffer; incubate at room temperature for 1-15 min; add the mixture to the sample pad of the immunochromatographic test strip; and read the colorimetric / fluorescence signals at the two detection lines of the immunochromatographic test strip after 15-20 min to achieve the detection of... S. pneumoniae Highly sensitive detection.

[0066] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0067] Unless otherwise specified, all experimental conditions in this disclosure are based on conventional conditions known to those skilled in the art or conditions recommended by the manufacturer.

[0068] Example 1 like Figure 1 As shown in the schematic diagram, the preparation method of the nanoprobe disclosed herein includes the following steps: (1) A mixture of 100 mL anhydrous ethanol, 4 mL of 28% ammonia solution and 6 mL of deionized water was sealed and stirred at room temperature for 20 min. Then, 4 mL of tetraethyl orthosilicate (100 mmol / L) was added at once, sealed, and magnetically stirred at 24 °C for 4 h. After the reaction was completed, the solution turned into a milky white liquid. The purified SiO2 particles (average particle size of 200 nm) were collected by centrifugation (4800 rpm, 6 min).

[0069] 100 mg of SiO2 nanoparticles were added to 40 mL of an aqueous solution of polyethyleneimine (8 mg / mL), and subjected to a first ultrasonic treatment at 80 Hz and 20 °C for 20 min to obtain SiO2-PEI nanospheres.

[0070] (2) 250 mg of SiO2-PEI nanospheres were added to 50 mL of gold nanoparticle dispersion (25 mg / mL), and subjected to a second ultrasonic treatment at 80 Hz and 20 °C for 30 min to obtain SiO2 / Au nanospheres.

[0071] (3) 10 mg of SiO2 / Au nanospheres were added to 4 mL of ammonia water (10 v / v) and subjected to a third ultrasonic treatment at 80 Hz and 20 °C for 20 min. Then, 0.16 mL of tetraethyl orthosilicate (100 mmol / L) was added and subjected to a fourth ultrasonic treatment at 80 Hz and 20 °C for 20 min to obtain SiO2 / Au@Si nanospheres.

[0072] (4) 250 mg of SiO2 / Au@Si nanospheres were added to 50 mL of aqueous solution of second polyethyleneimine (8 mg / mL), and subjected to fifth ultrasonic treatment at 80 Hz and 20 °C for 20 min to obtain SiO2 / Au@Si-PEI nanospheres.

[0073] (5) 100 mg of SiO2 / Au@Si-PEI nanospheres were added to 40 mL of carboxylated quantum dot dispersion (4 mg / mL) and subjected to a sixth ultrasonic treatment at 80 Hz and 20 °C for 20 min to obtain nanoprobe SiO2 / Au@Si / QD.

[0074] like Figure 2 As shown, Figure 2A and 2B are transmission electron micrograph (TEM) and enlarged transmission electron micrograph of SiO2 nanoparticles, respectively, Figure 2 C and 2D are transmission electron micrograph (TEM) and enlarged transmission electron micrograph of SiO2 / Au nanospheres, respectively, Figure 2 E and 2F are transmission electron micrograph (TEM) and enlarged transmission electron micrograph of SiO2 / Au@Si nanospheres, respectively, Figure 2 G and 2H are transmission electron micrograph (TEM) and enlarged transmission electron micrograph of SiO2 / Au@Si / QD nanoparticles, respectively. The above images show that the synthesized SiO2 / Au@Si / QD is a nanoprobe with a core-shell structure of SiO2 nanoparticles as the core, colloidal gold, silica inner shell and quantum dot outer shell.

[0075] Figure 3 is the energy dispersive X-ray spectrum (EDS) spectrum of SiO2 / Au@Si / QD prepared by the present disclosure. The above image shows that the synthesized SiO2 / Au@Si / QD contains Zn, Cd, S, Si, O and Au elements, and the Si element is obviously located in the core and outer position of the material, which proves the successful synthesis of SiO2 / Au@Si / QD.

[0076] Example 2 0.01 mg of SiO2 / Au@Si / QD was mixed with 1 mL of activation solution and treated with ultrasound at 80 Hz and 20°C for 15 min; 0.02 mg of anti-Streptococcus pneumoniae antibody was added and incubated at 20 for 3 h. The activation solution contains 10 mmol / L of MES, 0.1 mmol / L of EDC and 0.1 mmol / L of NHS, obtaining the anti-Streptococcus pneumoniae antibody modified nanoprobe.

[0077] As shown in Figure 4 , the flow chart of LFIA detection based on SiO2 / Au@Si / QD is shown in Figure 4 . S pneumoniae S pneumoniae The anti-Streptococcus pneumoniae antibody modified nanoprobe and the running buffer were mixed and incubated at room temperature for 3 min, and then added dropwise to the sample pad of the immunochromatographic test strip. After 15 min, the color signal and fluorescence signal in the T line area were used to qualitatively and quantitatively judge the anti-Streptococcus pneumoniae antibody in the sample. The dry fluorescence immunoassay instrument used in this example is FIC-S100. S. pneumoniae

[0078] Some key operating conditions of SiO2 / Au@Si / QD-LFIA were optimized in this example to achieve the best performance of the detection platform. As shown in Figure 5 , the anti-Streptococcus pneumoniae antibody modified nanoprobe was incubated at different temperatures for 3 min, and then added dropwise to the sample pad of the immunochromatographic test strip. After 15 min, the color signal and fluorescence signal in the T line area were used to qualitatively and quantitatively judge the anti-Streptococcus pneumoniae antibody in the sample. Figure 5 ​​​S. pneumoniae The results of optimization of the spray concentration of the antibody, when the anti S. pneumoniae When the antibody concentration reached 1.5 mg / mL, the SiO2 / Au@Si / QD-LFIA had the highest signal-to-noise ratio. S. pneumoniae

[0079] As Figure 6 shown, Figure 6 The results of optimization of the running time of the SiO2 / Au@Si / QD-LFIA showed that when the running time was 15 min, the SiO2 / Au@Si / QD-LFIA had the highest signal-to-noise ratio. S. pneumoniae

[0080] The performance of the SiO2 / Au@Si / QD-LFIA is highly dependent on the capture binding ability of the SiO2 / Au@Si / QD probe to the target bacteria. This embodiment used plate culture to determine the capture efficiency of the SiO2 / Au@Si / QD probe to S. pneumoniae As Figure 7 shown, Figure 7 The plate count results showed that the capture efficiency of the SiO2 / Au@Si / QD probe to S. pneumoniae reached 95%.

[0081] Example 3 This embodiment used different concentrations (0-10 5 cells / mL) of S. pneumoniae to verify the detection performance of the SiO2 / Au@Si / QD-LFIA. As Figure 8 shown, Figure 8 The detection results of the SiO2 / Au@Si / QD-LFIA for different concentrations of S. pneumoniae samples. As Figure 8 A can be seen, the visual detection limit (vLOD) of the SiO2 / Au@Si / QD-LFIA platform in the colorimetric mode was 10 3 cells / mL, and the vLOD in the fluorescent mode was 50 cells / mL, which was 50 times higher than that in the daylight colorimetric mode. We used an immunofluorescence analyzer to measure the fluorescent signal on the detection line and analyzed it. Figure 8 The bar chart results in B showed that the fluorescent signal on the detection line was positively correlated with the concentration of bacteria in the sample. By plotting the calibration curve (C) of the relationship between the fluorescent signal on each detection line and S. pneumoniae the concentration of Figure 8 S. pneumoniae ​​​The limit of detection (LOD) was determined. The results indicate that the SiO2 / Au@Si / QD-LFIA platform exhibits high analytical performance for the target bacteria, with a wide detection dynamic range (5 orders of magnitude) and good correlation coefficient (R²). 2 >0.989), low LOD ( S. pneumoniae (20 cells / mL). SiO2 / Au@Si / QD-LFIA can be observed under fluorescent light by monitoring the color change on the C-line; for concentrations exceeding 10... 3 cells / mL S. pneumoniae Qualitative screening can be performed; furthermore, the color change on the detection line can be observed under ultraviolet light to identify concentrations exceeding 50 cells / mL. S. pneumoniae Qualitative screening was performed, and fluorescence signal analysis was conducted to support concentrations in the range of 10⁻¹⁰. 5 cells / mL S. pneumoniae Quantitative screening was conducted.

[0082] Furthermore, this embodiment demonstrates that the presence of a silica shell in the middle of SiO2 / Au@Si / QD can effectively reduce the influence of AuNP on the fluorescence performance of QD. Nanomaterials without a silica shell (SiO2 / Au / QD) were tested under the same conditions at different concentrations (0-10). 5 cells / mL S. pneumoniae The result is as follows Figure 9 As shown, SiO2 / Au / QD-LFIA exhibits essentially the same colorimetric performance as SiO2 / Au@Si / QD-LFIA, but provides a significantly reduced fluorescence signal. Compared to the vLOD under sunlight conditions, the vLOD under fluorescence conditions shows no improvement. Furthermore, under sunlight colorimetric conditions, the SiO2 / Au@Si / QD-LFIA detection platform exhibits the same vLOD as colloidal gold (…). Figure 10 This indicates that the AuNP layer present in the middle of the proposed SiO2 / Au / QD provides excellent colorimetric detection performance for the SiO2 / Au@Si / QD-LFIA detection platform.

[0083] In addition, this embodiment also selects five common pathogenic bacteria (10) including Escherichia coli O157, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Legionella pneumophila. 5 The specificity of SiO2 / Au@Si / QD-LFIA was tested using cells / mL as a bacterial interfering agent. Results are as follows: Figure 11 As shown, by Figure 11 The photographs of the test strips and the fluorescence signal on the detection line clearly demonstrate the good selectivity of the SiO2 / Au@Si / QD-LFIA platform for the target bacteria. Under the same conditions, the SiO2 / Au@Si / QD-LFIA platform was used to target high concentrations (10...4.7 cell / mL), medium concentration (10 3.7 cell / mL) and low concentrations (10 2.7 cell / mL S. pneumoniae The test was conducted, and the results were as follows: Figure 12 As shown, the results indicate that the fluorescence signal changes on the detection line of the SiO2 / Au@Si / QD-LFIA platform are small, and the RSD values ​​of each test group are less than 6.06%, indicating that the established SiO2 / Au@Si / QD-LFIA platform has good repeatability.

[0084] This embodiment uses actual samples with added bacteria to evaluate the performance of the proposed SiO2 / Au@Si / QD-LFIA platform in practical clinical testing and environmental monitoring. Different concentrations (10T) of bacteria were added to throat swabs from healthy individuals and river water. 2 -10 5 cells / mL S. pneumoniae The result is as follows Figure 13 As shown, the color and fluorescence signal of the T-line are stable, and the signal intensity is directly proportional to the bacterial concentration. The bacterial recovery rate was calculated based on the fluorescence signal values ​​(80.23%–126.59%), with RSD values ​​all less than 7.33%. These results indicate that SiO2 / Au@Si / QD-LFIA has certain reliability and potential in detecting bacteria in environmental and saliva samples.

[0085] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0086] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0087] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A dual-signal nanoprobe based on silica sandwich protection, characterized in that, The nanoprobe has a core-shell structure, which includes a core and an outer shell covering the outer surface of the core. The core is SiO2 nanoparticles; The outer surface of the core includes an outer shell layer of the core-shell structure formed by sequentially covering a first cationic polymer interlayer, a gold nanoparticle interlayer, a SiO2 inner shell layer, a second cationic polymer interlayer, and a carboxylated quantum dot outer shell layer.

2. The nanoprobe according to claim 1, wherein, The carboxylated quantum dot shell contains CdSe@ZnS-COOH quantum dots; A specific antibody is coupled to the carboxyl terminus of the carboxylated quantum dot shell layer; the specific antibody is an anti-streptococcal antibody. For every 1 mg of nanoprobe, 0.2–1.0 mg of specific antibody is conjugated.

3. The nanoprobe according to claim 1, wherein, The average particle size of the SiO2 nanoparticles is 100-300 nm; the average particle size of the gold nanoparticles is 3-30 nm; and the average particle size of the carboxylated quantum dots is 5-30 nm. The first cationic polymer interlayer and the second cationic polymer interlayer are each independently made of polyethyleneimine, the thickness of the first cationic polymer interlayer is 0.5-2.0 nm, and the thickness of the second cationic polymer interlayer is 0.5-2.0 nm. The thickness of the SiO2 inner shell is 1-30 nm.

4. The method for preparing the nanoprobe according to any one of claims 1-3, characterized in that, The method includes the following steps: S1. Add SiO2 nanoparticles to an aqueous solution of a first polyethyleneimine and perform a first ultrasonic treatment to obtain SiO2-PEI nanospheres. S2. The SiO2-PEI nanospheres are added to the gold nanoparticle dispersion and subjected to a second ultrasonic treatment to obtain SiO2 / Au nanospheres. S3. The SiO2 / Au nanospheres are added to ammonia water for a third ultrasonic treatment, and then tetraethyl orthosilicate is added for a fourth ultrasonic treatment to obtain SiO2 / Au@Si nanospheres. S4. The SiO2 / Au@Si nanospheres are added to an aqueous solution of the second polyethyleneimine and subjected to a fifth ultrasonic treatment to obtain SiO2 / Au@Si-PEI nanospheres. S5. The SiO2 / Au@Si-PEI nanospheres are added to a carboxylated quantum dot dispersion and subjected to a sixth ultrasonic treatment to obtain the nanoprobe.

5. The method according to claim 4, wherein, In step S1, the average particle size of the SiO2 nanoparticles is 100-300 nm; the mass concentration of the first polyethyleneimine aqueous solution is 1-10 mg / mL; and the amount of SiO2 nanoparticles added is 50-500 mg per 100 mL of the first polyethyleneimine aqueous solution. In step S2, the average particle size of the gold nanoparticles is 3-30 nm; the mass concentration of the gold nanoparticle dispersion is 10-50 mg / mL; and the amount of SiO2-PEI nanospheres added is 100-500 mg per 100 mL of gold nanoparticle dispersion.

6. The method according to claim 4, wherein, In step S3, the volume concentration of the ammonia solution is 1-50 v / v; the amount of SiO2 / Au nanospheres added is 50-500 mg per 100 mL of ammonia solution, and the amount of tetraethyl orthosilicate added is 1-10 mL; the molar concentration of the tetraethyl orthosilicate is 50-500 mmol / L. In step S4, the mass concentration of the aqueous solution of the second polyethyleneimine is 1-10 mg / mL; the amount of SiO2 / Au@Si nanospheres added is 50-500 mg per 100 mL of the aqueous solution of the second polyethyleneimine. In step S5, the average particle size of the carboxylated quantum dots is 5-30 nm; the mass concentration of the carboxylated quantum dot dispersion is 1-10 mg / mL; and the amount of SiO2 / Au@Si-PEI nanospheres added is 50-500 mg per 100 mL of carboxylated quantum dot dispersion.

7. The method according to claim 4, wherein, The method further includes: activating the nanoprobe, mixing it with a specific antibody, and then incubating it. The specific antibody is an anti-pneumococcal antibody; The amount of specific antibody added is 0.2-1.0 mg per 1 mg of nanoprobe.

8. The application of the nanoprobe according to any one of claims 1-3 or the nanoprobe prepared by the preparation method according to claims 4-7 in the preparation of products for detecting Streptococcus pneumoniae.

9. A kit for detecting Streptococcus pneumoniae, characterized in that, The kit includes the nanoprobes according to any one of claims 1-3 or the nanoprobes prepared by the preparation methods according to claims 4-7.

10. The kit according to claim 9, wherein, The kit also includes an immunochromatographic test strip, which comprises a sample pad, a chromatography membrane, and an absorption pad connected in sequence, and the chromatography membrane is provided with a detection line and a control line; The detection line is loaded with anti-streptococcal antibodies; The quality control line was loaded with goat anti-rabbit IgG.

Images