Preparation method and application of magnetic-fluorescent hybrid nanoparticles

Abstract

The invention discloses a preparation method and application of magnetic-fluorescent hybrid nanoparticles, and the preparation method comprises the following steps: S1, synthesizing a Fe3O4-CDs-1 magnetic fluorescent carbon dot and a CDs-2, and respectively modifying Ab1 and Ab2 on the Fe3O4-CDs-1 magnetic fluorescent carbon dot and the CDs-2; in the presence of IL-8, an immune sandwich compound is formed through specific binding of Ab1 and Ab2 with IL-8; under different IL-8 concentrations, constructing a proportional fluorescence detection method to realize IL-8 detection, and establishing a standard curve; and S2, detection of IL-8 in the serum: adding Fe3O4-CDs-1 (at) SiO2-Ab1 into the serum, then carrying out magnetic separation, continuing to add CDs-2 (at) Ab2 for incubation, and finally carrying out fluorescence detection on the obtained immune complex. And substituting the obtained fluorescence ratio into the standard curve constructed in S1 to obtain the concentration of IL-8 in the serum.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent biological analysis and detection, relates to a fluorescence detection method based on magnetic fluorescent carbon dots and its application, and is a preparation method of magnetic-fluorescent hybrid nanoparticles and its application. Background Art

[0002] Interleukin-8 (IL-8) is a multifunctional proinflammatory cytokine secreted by macrophages and epithelial cells, which can attract and activate neutrophils and is produced under inflammatory stimulation. In recent years, studies have found that IL-8 is involved in almost the entire reproductive process of mammals, such as ovulation, corpus luteum formation, endometrial proliferation, decidualization, embryo implantation and its growth and development. In addition, a large number of studies have shown that abnormal increases in IL-8 concentrations are associated with a variety of cancers, such as oral cancer, melanoma, breast cancer, lung cancer, prostate cancer, pancreatic cancer, and colorectal cancer.

[0003] Therefore, the development of highly sensitive and specific IL-8 level analysis methods is crucial for the diagnosis, staging and prognosis of related diseases. Currently, enzyme-linked immunosorbent assay (ELISA) is the most mature and classic method for detecting IL-8. However, its limited sensitivity limits its application in clinical detection of IL-8.

[0004] Fluorescence immunoassay is widely used in clinical research and disease diagnosis due to its high sensitivity, simple operation, and diverse fluorescent probes. More importantly, the ratiometric fluorescence sensor with dual signals can reduce the interference of factors such as temperature, excitation intensity, and interfering molecules in the sample through self-calibration, thereby greatly improving the accuracy of detection. Obviously, the focus of ratiometric fluorescence immunoassay technology is on the preparation of fluorescent probes. Among them, carbon quantum dots (CDs) have attracted much attention due to their excellent optical properties such as high quantum yield, high stability and size adjustment, as well as low toxicity and good biocompatibility. At the same time, the simple synthesis steps and cheap and readily available synthetic raw materials of CDs make it the preferred choice among many fluorescent materials. In addition, magnetic ferroferric oxide nanoparticles Fe3O4 are classic magnetic nanomaterials that have been widely used to separate and enrich biomarkers, such as circulating tumor cells.

[0005] Therefore, if the fluorescence properties of carbon dots can be combined with the magnetism of Fe3O4, it will be widely used in biological analysis and detection. Summary of the invention

[0006] In view of the problem that enzyme-linked immunosorbent assay (ELISA) is the most mature and classic method for detecting IL-8 in the prior art, however, its limited sensitivity limits its application in clinical detection of IL-8. The present invention provides a fluorescence detection method based on magnetic fluorescent carbon dots and its application, a preparation method of magnetic-fluorescent hybrid nanoparticles and its application, and a preparation method of Fe3O4-CDs and its application. The present invention synthesizes magnetic-fluorescent hybrid nanoparticles Fe3O4-CDs. The prepared hybrid nanoparticles not only retain the excellent fluorescence properties of CDs, but also have high magnetic responsiveness, and can be widely used in biological analysis and detection. The present invention also develops a ratio-type fluorescence detection method based on magnetic fluorescent hybrid nanoparticles for sensitive and accurate detection of IL-8. The magnetic fluorescent carbon dots prepared by the present invention combine the excellent fluorescence properties of carbon dots and the excellent magnetic separation properties of Fe3O4, and can be widely used in different biological analysis and detection.

[0007] The technical principles of the present invention are as follows: A ratiometric fluorescence detection method based on magnetic fluorescent carbon dots and its application, comprising the following steps: Preparation of magnetic-fluorescent hybrid nanoparticles Fe3O4-CDs: ①Synthesize Fe3O4-CDs-1 magnetic fluorescent carbon dots and CDs-2, and modify Ab1 and Ab2 on Fe3O4-CDs-1 magnetic fluorescent carbon dots and CDs-2 respectively; ② In the presence of IL-8, Ab1 and Ab2 specifically bind to IL-8 to form an immune sandwich complex; ③ Under different IL-8 concentrations, based on the unchanged fluorescence intensity of CDs-1 and the enhanced fluorescence intensity of CDs-2, a ratiometric fluorescence detection method was constructed to detect IL-8 and establish a standard curve; Application: Detection of IL-8 in serum: ① Add Fe3O4-CDs-1@SiO2-Ab1 to the serum, then separate by magnetic field and continue to add CDs-2@Ab2 for incubation; ②Finally, the obtained immune complex is subjected to fluorescence detection; ③Substitute the obtained fluorescence ratio into the standard curve constructed in S1 to obtain the concentration of IL-8 in serum.

[0008] The purpose of the present invention is achieved through the following technical solutions: A method for preparing magnetic-fluorescent hybrid nanoparticles comprises the following steps: Preparation of S1, Fe3O4-CDs-1@SiO2 and CDs-2 nanoparticles: S1-1, dissolving ammonium ferric citrate in a solution of H2O and triethylenetetramine in a volume ratio of 9:1, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene and heating it to 200°C for 6 h, then cooling the autoclave naturally to room temperature, and obtaining magnetic Fe3O4-CDs-1 powder after the suspension is centrifuged, washed, and dried; S1-2, add cetyltrimethylammonium bromide (CTAB) to 1 mg mL -1 The synthesized Fe3O4-CDs-1 powder was ultrasonicated for 20 min; then, 25 wt% NH3·H2O was quickly added, followed by tetraethyl orthosilicate (TEOS) dropwise added under vigorous stirring, mixed evenly and heated at 40°C for 30 min. After the reaction, the mixture was centrifuged and washed with deionized water. The black powder obtained after freeze-drying was Fe3O4-CDs-1@SiO2. S1-3, adding ninhydrin and o-phenylenediamine to deionized water, and then stirring and dissolving with a magnetic stirrer for 4 hours. After the stirring is completed, the mixed solution is transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and reacted at 180°C for 12 hours. After cooling to room temperature, a dark yellow solution is obtained. The dark yellow solution is placed in a dialysis bag with a molecular weight retention of 1000 Da and dialyzed for 24 hours. The final solution is collected and protected from light at room temperature to obtain CDs-2 nanoparticles; S2. Preparation of magnetic-fluorescent hybrid nanoparticles: S2-1, disperse the Fe3O4-CDs-1@SiO2 nanoparticles obtained in S1-2 in a mixed solution of ethanol and 3-aminopropyltriethoxysilane (APTES), the ratio of Fe3O4-CDs-1@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane is (1-2) mg: (1-2) mL: (0.2-0.5) mL, and then shake the suspension at room temperature for 5 h and then perform magnetic separation to obtain amino-functionalized Fe3O4-CDs-1@SiO2; Preparation of S2-2, Fe3O4-CDs-1@SiO2-Ab1: 0.5 mL of captured IL-8 antibody (Ab1) was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at room temperature for 30 min. The concentration of IL-8 antibody (Ab1) was 20 μg mL -1, the amino-functionalized Fe3O4-CDs-1@SiO2 nanoparticles were dispersed into the solution to obtain a suspension; the suspension was incubated at 4°C with shaking for 12 h, and 100 μL of 1% bovine serum albumin (BSA) solution was used to block the excess nonspecific adsorption sites on the Fe3O4-CDs-1@SiO2 surface, and finally the Fe3O4-CDs-1@SiO2-Ab1 complex was obtained; S2-3, preparation of CDs-2@Ab2: The preparation process of CDs-2@-Ab2 complex is similar to that of S2-2. CDs-2 nanoparticles are mixed with 3-aminopropyltriethoxysilane and shaken at room temperature for 5 hours to obtain amino-functionalized CDs-2 solution. The IL-8 detection antibody Ab2 is activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at room temperature for 30 minutes, and then the amino-functionalized CDs-2 solution is added. The mixture is shaken at 4°C for 12 hours, and then blocked with 1% bovine serum albumin solution under the same conditions to obtain CDs-2@Ab2. The obtained Fe3O4-CDs-1@SiO2-Ab1 has a fluorescence emission peak of 447 nm under the optimal excitation wavelength of 360 nm. Fe3O4-CDs-1@SiO2-Ab1 has excellent magnetic response performance and can be separated from the solution within 5 seconds under the external magnet. The obtained CDs-2@Ab2 had fluorescence emission peaks of 576 nm under the optimal excitation wavelength of 420 nm; In order to facilitate detection, the optimal excitation wavelength of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 was set to 390 nm.

[0009] The present invention also relates to an application of the above-mentioned magnetic-fluorescent hybrid nanoparticles, which is a method for ratiometric fluorescence detection of IL-8 based on magnetic-fluorescent hybrid nanoparticles, comprising the following steps: Preparation of S1, Fe3O4-CDs-1@SiO2 and CDs-2 nanoparticles: S1-1, dissolving ammonium ferric citrate in a solution of H2O and triethylenetetramine in a volume ratio of 9:1, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene and heating it to 200°C for 6 h, then cooling the autoclave naturally to room temperature, and obtaining magnetic Fe3O4-CDs-1 powder after the suspension is centrifuged, washed, and dried; S1-2, add cetyltrimethylammonium bromide (CTAB) to 1 mg mL -1The synthesized Fe3O4-CDs-1 powder was ultrasonicated for 20 min; then, 25 wt% NH3·H2O was quickly added, followed by tetraethyl orthosilicate (TEOS) dropwise added under vigorous stirring, mixed evenly and heated at 40°C for 30 min. After the reaction, the mixture was centrifuged and washed with deionized water. The black powder obtained after freeze-drying was Fe3O4-CDs-1@SiO2. S1-3, adding ninhydrin and o-phenylenediamine to deionized water, and then stirring and dissolving with a magnetic stirrer for 4 hours. After the stirring is completed, the mixed solution is transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and reacted at 180°C for 12 hours. After cooling to room temperature, a dark yellow solution is obtained. The dark yellow solution is placed in a dialysis bag with a molecular weight retention of 1000 Da and dialyzed for 24 hours. The final solution is collected and protected from light at room temperature to obtain CDs-2 nanoparticles; S2. Preparation of magnetic-fluorescent hybrid nanoparticles: S2-1, disperse the Fe3O4-CDs-1@SiO2 nanoparticles obtained in S1-2 in a mixed solution of ethanol and 3-aminopropyltriethoxysilane (APTES), the ratio of Fe3O4-CDs-1@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane is (1-2) mg: (1-2) mL: (0.2-0.5) mL, and then shake the suspension at room temperature for 5 h and then perform magnetic separation to obtain amino-functionalized Fe3O4-CDs-1@SiO2; Preparation of S2-2, Fe3O4-CDs-1@SiO2-Ab1: 0.5 mL of captured IL-8 antibody (Ab1) was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at room temperature for 30 min. The concentration of IL-8 antibody (Ab1) was 20 μg mL -1 , the amino-functionalized Fe3O4-CDs-1@SiO2 nanoparticles were dispersed into the solution to obtain a suspension; the suspension was incubated at 4°C with shaking for 12 h, and 100 μL of 1% bovine serum albumin (BSA) solution was used to block the excess nonspecific adsorption sites on the Fe3O4-CDs-1@SiO2 surface, and finally the Fe3O4-CDs-1@SiO2-Ab1 complex was obtained; S2-3, preparation of CDs-2@Ab2: The preparation process of CDs-2@Ab2 complex is similar to that of S2-2. CDs-2 nanoparticles are mixed with APTES and shaken at room temperature for 5 h to obtain amino-functionalized CDs-2 solution. The IL-8 detection antibody Ab2 is activated with EDC and NHS at room temperature for 30 min, and then the amino-functionalized CDs-2 solution is added. The mixture is shaken at 4°C for 12 h, and then blocked with 1% BSA solution under the same conditions to obtain CDs-2@Ab2. The obtained Fe3O4-CDs-1@SiO2-Ab1 has a fluorescence emission peak of 447 nm under the optimal excitation wavelength of 360 nm. Fe3O4-CDs-1@SiO2-Ab1 has excellent magnetic response performance and can be separated from the solution within 5 seconds under the external magnet. The obtained CDs-2@Ab2 had fluorescence emission peaks of 576 nm under the optimal excitation wavelength of 420 nm; To facilitate detection, the optimal excitation wavelength of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 was set to 390 nm; S3, adding different concentrations of IL-8 standard solution to the Fe3O4-CDs-1@SiO2-Ab1 solution obtained in S2-2, incubating, shaking at 37℃-40℃ for 0.5h-2h, after Ab1 and IL-8 have an immune reaction, separating the Fe3O4-CDs-1@SiO2-Ab1 / IL-8 complex by magnetic separation, washing with deionized water and redispersing in deionized water; continue to add CDs-2@Ab2 to the above solution, shaking at 37℃-40℃ for 0.5h-2h and then performing magnetic separation, finally, thoroughly washing the obtained complex Fe3O4-CDs-1 / IL-8 / CDs-2@Ab2 with deionized water, and performing fluorescence detection; In the absence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the blank group (I0) is (I0) 466 / 565 ; In the presence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the experimental group (I) is (I) 466 / 565 , take the difference between the two, that is (I0-I) 466 / 565 The value of IL-8 was used to quantify the IL-8, as (I0-I) 466 / 565 The values ​​are the ordinate and the concentration of IL-8 is the abscissa to establish a standard curve; S4. Add Fe3O4-CDs-1@SiO2-Ab1 to the treated serum for incubation, oscillate at 37℃-40℃ for 0.5h-2h, then wash three times with deionized water through magnetic separation, continue to add CDs-2@Ab2, oscillate at 37℃-40℃ for 0.5h-2h, and finally transfer the obtained complex after repeated washing with deionized water to a micro-cuvette for fluorescence detection.

[0010] Furthermore, in S3 to S4, the incubation temperature was 37° C. and the incubation time was 60 min.

[0011] Furthermore, in S4, the linear range of IL-8 detection was 20 pg / mL-1000 pg / mL, and the lower limit of detection was 6.9 pg / mL.

[0012] Further, in S4, the excitation wavelength range of the fluorescence detection is 360nm~420nm; further, when the excitation wavelength is 380nm~410nm, the carbon dots have better fluorescence intensity and better detection sensitivity; most preferably, the excitation wavelength is 390nm; Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention adopts the traditional sandwich method, based on the synthesized magnetic fluorescent carbon dots, and utilizes the specific binding of Ab1 and Ab2 to IL-8 to form an immune sandwich complex. In the presence of different concentrations of IL-8, the fluorescence intensity of Fe3O4-CDs-1@SiO2-Ab1 remains unchanged, while the fluorescence intensity of CDs-2@Ab2 increases with the increase of IL-8 concentration. Based on the change of proportional fluorescence, the concentration of IL-8 can be detected with high sensitivity and accuracy. When detecting IL-8 by this fluorescence method, it has the advantages of good selectivity, high sensitivity, low detection limit, and wide detection range. It improves the existing ELISA method for detecting IL-8, such as low sensitivity and susceptibility to interference from other substances. The synthesized magnetic fluorescent carbon dots can be widely used in the separation and detection of other different target analytes.

[0013] 2. The fluorescence detection method based on magnetic fluorescent carbon dots described in the present invention is prepared by a one-step method to synthesize magnetic fluorescent carbon dots, which combines the excellent fluorescence properties of carbon dots and the excellent magnetic properties of Fe3O4, and can be widely used in biological analysis and detection. The introduction of amino groups on the surface of carbon dots by silanization is conducive to the subsequent fixation of biological molecules on the surface of carbon dots. The preparation method of magnetic nano carbon dots of the present invention is simple to operate, low in cost, fast and convenient, and can be mass-produced.

[0014] 3. The fluorescence detection method based on magnetic fluorescent carbon dots described in the present invention utilizes the fluorescence intensity of two carbon dots at different emission wavelengths to construct a ratiometric fluorescence detection method for IL-8 detection, which has the advantages of high sensitivity, low detection limit, and strong anti-interference ability. The linear range is 20 pg / mL-1000 pg / mL, and the detection limit is 6.9 pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0016] Figure 1 Schematic diagram of the principle of detecting IL-8 using a ratiometric fluorescent sensor based on magnetic fluorescent carbon dots in the present invention (where I is a schematic diagram of the preparation of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2; II is a schematic diagram of the IL-8 detection process); Figure 2 The transmission electron microscopy, high-resolution transmission electron microscopy and Fe3O4-CDs-1@SiO2 transmission electron microscopy images of Fe3O4-CDs-1 synthesized in Example 1 of the present invention are shown in FIG. Figure 2 (A) Transmission electron microscopy; Figure 2 (B) High-resolution transmission electron microscopy; Figure 2 (C) is a transmission electron microscope of Fe3O4-CDs-1@SiO2); Figure 3 The absorption, excitation and emission spectra of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the visible light region in Example 1 of the present invention (excitation wavelength: 390nm; wherein, Figure 3 (A) is Fe3O4-CDs-1@SiO2-Ab1; Figure 3 (B) is CDs-2@Ab2; Figure 3 (C) is the magnetic response diagram of Fe3O4-CDs-1@SiO2-Ab1); Figure 4 This is a fluorescence curve diagram of detecting different concentrations of IL-8 in Example 7 of the present invention (wherein, Figure 4 (A) is 0 pg·mL -1 , 20 pg·mL -1 , 50 pg·mL -1 , 100 pg mL -1 , 500 pg·mL -1 , 1000 pg mL -1 ; Figure 4 (B) is the standard curve for detecting IL-8). DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0018] Unless otherwise defined, all the professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0019] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0020] Embodiment 1: A method for preparing magnetic-fluorescent hybrid nanoparticles comprises the following steps: Preparation of S1, Fe3O4-CDs-1@SiO2 and CDs-2 nanoparticles: S1-1, dissolving ammonium ferric citrate in a solution of H2O and triethylenetetramine in a volume ratio of 9:1, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene and heating it to 200°C for 6 h, then cooling the autoclave naturally to room temperature, and obtaining magnetic Fe3O4-CDs-1 powder after the suspension is centrifuged, washed, and dried; S1-2, add cetyltrimethylammonium bromide (CTAB) to 1 mg mL -1 The synthesized Fe3O4-CDs-1 powder was ultrasonicated for 20 min; then, 25 wt% NH3·H2O was quickly added, followed by tetraethyl orthosilicate (TEOS) dropwise added under vigorous stirring, mixed evenly and heated at 40°C for 30 min. After the reaction, the mixture was centrifuged and washed with deionized water. The black powder obtained after freeze-drying was Fe3O4-CDs-1@SiO2. S1-3, adding ninhydrin and o-phenylenediamine to deionized water, and then stirring and dissolving with a magnetic stirrer for 4 hours. After the stirring is completed, the mixed solution is transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and reacted at 180°C for 12 hours. After cooling to room temperature, a dark yellow solution is obtained. The dark yellow solution is placed in a dialysis bag with a molecular weight retention of 1000 Da and dialyzed for 24 hours. The final solution is collected and protected from light at room temperature to obtain CDs-2 nanoparticles; S2. Preparation of magnetic-fluorescent hybrid nanoparticles: S2-1, disperse the Fe3O4-CDs-1@SiO2 nanoparticles obtained in S1-2 in a mixed solution of ethanol and 3-aminopropyltriethoxysilane (APTES), and then oscillate the suspension at room temperature for 5 h and then perform magnetic separation to obtain amino-functionalized Fe3O4-CDs-1@SiO2; Preparation of S2-2, Fe3O4-CDs-1@SiO2-Ab1: 0.5 mL of captured IL-8 antibody (Ab1) was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at room temperature for 30 min. The concentration of IL-8 antibody (Ab1) was 20 μg mL -1 , the amino-functionalized Fe3O4-CDs-1@SiO2 nanoparticles were dispersed into the solution to obtain a suspension; the suspension was incubated at 4°C with shaking for 12 h, and 100 μL of 1% bovine serum albumin (BSA) solution was used to block the excess nonspecific adsorption sites on the Fe3O4-CDs-1@SiO2 surface, and finally the Fe3O4-CDs-1@SiO2-Ab1 complex was obtained; S2-3, preparation of CDs-2@Ab2: The preparation process of CDs-2@-Ab2 complex was similar to that of S2-2. 1 mL of CDs-2 nanoparticles was mixed with 0.5 mL of 3-aminopropyltriethoxysilane and shaken at room temperature for 5 h to obtain amino-functionalized CDs-2 solution. 1 mL of 20 μg mL -1 1 mL of 10 mg mL -1 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were activated at room temperature for 30 minutes, and then the amino-functionalized CDs-2 solution was added. The mixture was shaken at 4°C for 12 hours and then blocked with 1% bovine serum albumin solution under the same conditions to obtain CDs-2@Ab2.

[0021] Figure 2 The transmission electron microscopy, high-resolution transmission electron microscopy and Fe3O4-CDs-1@SiO2 transmission electron microscopy images of Fe3O4-CDs-1 synthesized in Example 1 are shown in FIG. Figure 2 (A) is a transmission electron microscope, such as Figure 2 (A) shows a typical transmission electron microscope image of Fe3O4-CDs-1. Through observation, it is found that Fe3O4-CDs-1 presents uniform particle morphology and size, with a size distribution between 15-20 nm and an average size of 17.6 nm. Figure 2 (B) is a high-resolution transmission electron microscope, such as Figure 2(B) As shown, the high-resolution transmission electron microscopy image reveals the high crystallinity of Fe3O4-CDs-1; Figure 2 (C) is the transmission electron microscopy image of Fe3O4-CDs-1@SiO2. Figure 2 As shown in (C), after surface modification with SiO2, the diameter of the SiO2 layer is approximately 15 nm, which can provide sufficient silanol groups for amino modification and ultimately achieve the fixation of Ab1.

[0022] Figure 3 The absorption, excitation and emission spectra in the visible light region of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in Example 1; Figure 3 (A) is Fe3O4-CDs-1@SiO2-Ab1, such as Figure 3 As shown in (A), the maximum excitation wavelength of Fe3O4-CDs-1@SiO2-Ab1 is 360 nm. In order to obtain the fluorescence emission signals of two fluorescent groups, Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2, under the same excitation wavelength, 390 nm was selected as the excitation wavelength for immunofluorescence detection. Figure 3 (B) is CDs-2@Ab2, such as Figure 3 As shown in (B), the maximum excitation wavelength of CDs-2@Ab2 is 420 nm; in order to make the fluorescence signal of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 still reach the best after the combination of the two, we choose 390 nm as the unified excitation wavelength; Figure 3 (C) is the magnetic response diagram of Fe3O4-CDs-1@SiO2-Ab1, such as Figure 3 (C) shows a Fe3O4-CDs-1@SiO2 solution. It can be seen that the prepared Fe3O4-CDs-1@SiO2-Ab1 has good magnetic responsiveness and can be quickly separated from the solution under an external magnetic field (under an external magnet, it can be separated from the solution within 5 seconds).

[0023] Embodiment 2: A method for preparing magnetic-fluorescent hybrid nanoparticles, which is different from Example 1 in that the method for preparing Fe3O4-CDs-1 comprises the following steps: Ammonium ferric citrate (0.133 g) was dissolved in 10 mL of H2O and triethylenetetramine in a volume ratio of 9:1, and then the precursor solution was transferred to a 20 mL stainless steel autoclave lined with polytetrafluoroethylene; after sealing, the autoclave was heated to 200°C and maintained for 6 h; then the autoclave was naturally cooled to room temperature; the suspension was centrifuged (6500 rpm, 2 min), and the liquid phase was discarded by magnetic separation; the obtained solid phase was dispersed in 10 mL of acetone, ultrasonically dispersed for 3 min, and then magnetically separated; the washing process was repeated three times; finally, the black product was dried in an air circulation oven at 60°C to obtain Fe3O4-CDs-1 powder with magnetic properties.

[0024] Embodiment 3: A method for preparing magnetic-fluorescent hybrid nanoparticles, which is different from Example 1 in that the method for preparing the Fe3O4-CDs-1 surface modified with a SiO2 layer comprises the following steps, wherein the modified SiO2 layer is beneficial for the subsequent covalent fixation of antibodies on the surface of the magnetic carbon dots: Add cetyltrimethylammonium bromide (CTAB) (0.2 mM) to 10 mL (1 mg mL -1 ) was added to Fe3O4-CDs-1 synthesized by ultrasonication for 20 min; then, 0.5 mL NH3·H2O (25 wt%) was quickly added, and then 80 μL tetraethyl orthosilicate (TEOS) was added dropwise under vigorous stirring. After mixing evenly, the mixture was heated at 40°C for 30 min. After the reaction, the mixture was centrifuged and washed with deionized water until the supernatant had no UV absorption. The black powder obtained after freeze-drying was Fe3O4-CDs-1@SiO2.

[0025] Embodiment 4: A method for preparing magnetic-fluorescent hybrid nanoparticles, which is different from Example 1 in that the surface modification of Fe3O4-CDs-1@SiO2 with Ab1 comprises the following steps: First, 2 mg of Fe3O4-CDs-1@SiO2 nanoparticles were dispersed in 1 mL of ethanol and 0.2 mL of APTES. The suspension was then shaken at room temperature for 5 h and then magnetically separated. The suspension was washed with ethanol three times and deionized water twice to obtain amino-functionalized Fe3O4-CDs-1@SiO2. -1 ) EDC and NHS were used to activate Ab1 at room temperature (0.5 mL, 20 μg mL -1) After 30 min, the amino-functionalized Fe3O4-CDs-1@SiO2 nanoparticles were dispersed into the above solution; the suspension was incubated with shaking at 4°C for 12 h, and 100 μL of 1% BSA solution was used to block the excess nonspecific adsorption sites on the Fe3O4-CDs-1@SiO2 surface. The reaction condition was shaking at room temperature for 1 h; magnetic separation was performed in sequence, and the mixture was washed several times with deionized water to remove unbound Ab1 and BSA; finally, the Fe3O4-CDs-1@SiO2-Ab1 complex was obtained and dispersed in 700 μL of deionized water for later use.

[0026] Embodiment 5: A method for preparing magnetic-fluorescent hybrid nanoparticles, which differs from Example 1 in that the synthesis of CDs-2 comprises the following steps: Ninhydrin (0.16 g) and o-phenylenediamine (0.32 g) were added to 20 mL of deionized water, and then stirred and dissolved with a magnetic stirrer for 4 hours. After the stirring, the mixed solution was transferred to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene and reacted at 180°C for 12 hours. After cooling to room temperature, the dark yellow solution was placed in a dialysis bag (retention molecular weight: 1000 Da) and dialyzed for 24 hours. The final solution was collected and stored at room temperature in the dark.

[0027] Embodiment 6: A method for preparing magnetic-fluorescent hybrid nanoparticles, which is different from Example 1 in that the preparation of the CDs-2@Ab2 complex comprises the following steps: 1 mL of CDs-2 nanoparticles was mixed with 0.2 mL of APTS and shaken at room temperature for 5 h to obtain an amino-functionalized CDs-2 solution. 0.5 mL of 20 μg mL -1 Ab2 was used with 0.5 mL 10 mg mL -1 EDC and NHS were activated at room temperature for 30 min, and then 1 mL of amino-functionalized CDs-2 solution was added to 1 mL of the above solution; the mixture was shaken at 4°C for 12 h, and then blocked with 1% BSA solution under the same conditions, and the obtained CDs-2@Ab2 was directly used in subsequent experiments.

[0028] Embodiment 7: An application of magnetic-fluorescent hybrid nanoparticles comprises the following steps: Preparation of S1, Fe3O4-CDs-1@SiO2 and CDs-2 nanoparticles: S1-1, dissolving ammonium ferric citrate in a solution of H2O and triethylenetetramine in a volume ratio of 9:1, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene and heating it to 200°C for 6 h, then cooling the autoclave naturally to room temperature, and obtaining magnetic Fe3O4-CDs-1 powder after the suspension is centrifuged, washed, and dried; S1-2, add cetyltrimethylammonium bromide (CTAB) to 1 mg mL -1 The synthesized Fe3O4-CDs-1 powder was ultrasonicated for 20 min; then, 25 wt% NH3·H2O was quickly added, followed by tetraethyl orthosilicate (TEOS) dropwise added under vigorous stirring, mixed evenly and heated at 40°C for 30 min. After the reaction, the mixture was centrifuged and washed with deionized water. The black powder obtained after freeze-drying was Fe3O4-CDs-1@SiO2. S1-3, adding ninhydrin and o-phenylenediamine to deionized water, and then stirring and dissolving with a magnetic stirrer for 4 hours. After the stirring is completed, the mixed solution is transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and reacted at 180°C for 12 hours. After cooling to room temperature, a dark yellow solution is obtained. The dark yellow solution is placed in a dialysis bag with a molecular weight retention of 1000 Da and dialyzed for 24 hours. The final solution is collected and protected from light at room temperature to obtain CDs-2 nanoparticles; S2. Preparation of magnetic-fluorescent hybrid nanoparticles: S2-1, disperse the Fe3O4-CDs-1@SiO2 nanoparticles obtained in S1-2 in a mixed solution of ethanol and 3-aminopropyltriethoxysilane (APTES), and then oscillate the suspension at room temperature for 5 h and then perform magnetic separation to obtain amino-functionalized Fe3O4-CDs-1@SiO2; Preparation of S2-2, Fe3O4-CDs-1@SiO2-Ab1: 0.5 mL of captured IL-8 antibody (Ab1) was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at room temperature for 30 min. The concentration of IL-8 antibody (Ab1) was 20 μg mL -1 , disperse the amino-functionalized Fe3O4-CDs-1@SiO2 nanoparticles into the solution to obtain a suspension; the suspension was incubated at 4°C with shaking for 12 h, and 100 μL of 1% bovine serum albumin (BSA) solution was used to block the excess nonspecific adsorption sites on the Fe3O4-CDs-1@SiO2 surface, and finally the Fe3O4-CDs-1@SiO2-Ab1 complex was obtained, which was dispersed in deionized water for later use; S2-3, preparation of CDs-2@Ab2: The preparation process of CDs-2@-Ab2 complex was similar to that of S2-2. 1 mL of CDs-2 nanoparticles was mixed with 0.5 mL of 3-aminopropyltriethoxysilane and shaken at room temperature for 5 h to obtain amino-functionalized CDs-2 solution. 1 mL of 20 μg mL -1 1 mL of 10 mg mL -1 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were activated at room temperature for 30 min, and then the amino-functionalized CDs-2 solution was added. The mixture was shaken at 4 °C for 12 h, and then blocked with 1% bovine serum albumin solution under the same conditions to obtain CDs-2@Ab2. S3, adding different concentrations of IL-8 standard solution to the Fe3O4-CDs-1@SiO2-Ab1 solution obtained in S2-2, incubating, shaking at 37°C for 1 hour, after Ab1 and IL-8 have an immune reaction, separating the Fe3O4-CDs-1@SiO2-Ab1 / IL-8 complex by magnetic separation, washing with deionized water and redispersing in deionized water; continue to add CDs-2@Ab2 to the above solution, shaking at 37°C for 1 hour and then performing magnetic separation, finally, thoroughly washing the obtained complex Fe3O4-CDs-1 / IL-8 / CDs-2@Ab2 with deionized water, and performing fluorescence detection; In the absence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the blank group (I0) is (I0) 466 / 565 ; In the presence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the experimental group (I) is (I) 466 / 565 , take the difference between the two, that is (I0-I) 466 / 565 The value of IL-8 was used to quantify the IL-8, as (I0-I) 466 / 565 The values ​​are the ordinate and the concentration of IL-8 is the abscissa to establish a standard curve; S4. Fe3O4-CDs-1@SiO2-Ab1 was added to the treated serum for incubation, and the mixture was shaken at 37°C for 1 min. Then, the mixture was washed three times with deionized water by magnetic separation. CDs-2@Ab2 was added and the mixture was shaken at 37°C for 1 h. Finally, the complex was repeatedly washed with deionized water and transferred to a micro-cuvette for fluorescence detection.

[0029] Figure 1This is a schematic diagram of the principle of detecting IL-8 using a ratiometric fluorescent sensor based on magnetic fluorescent carbon dots using a magnetic-fluorescent hybrid nanoparticle according to Example 7. Figure 1 It means that a sandwich method is used, in which Ab1 and Ab2 specifically bind to IL-8 to form an immune sandwich complex. Based on the principle that the fluorescence intensity of Fe3O4-CDs-1@SiO2-Ab1 remains unchanged while the fluorescence intensity of CDs-2@Ab2 increases with the increase of IL-8 concentration, a ratio fluorescence detection method is constructed to realize the concentration detection of IL-8.

[0030] Figure 4 This is a fluorescence curve diagram of detecting different concentrations of IL-8 in Example 7, wherein Figure 4 (A) is 0 pg·mL -1 , 20pg·mL -1 , 50 pg·mL -1 , 100 pg·mL -1 , 500 pg·mL -1 , 1000 pg mL -1 ,from Figure 4 (A) It can be seen that during the detection of IL-8, the fluorescence intensity of CDs-2@Ab2 increased with the increase of IL-8 concentration, while the fluorescence intensity of Fe3O4-CDs-1@SiO2-Ab1 remained basically unchanged; (I0-I) 466 / 565 To show the before and after changes of the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2; Figure 4 (B) is the standard curve for detecting IL-8. Figure 4 (B) shows, (I0-I) 466 / 565 When the concentration of IL-8 was 20 pg·mL -1 ~1000 pg mL -1 The linear regression equation is: y =0.257x-0.06, R 2 The detection limit was 6.9 pg·mL. -1 .

[0031] Embodiment 8: An application of magnetic-fluorescent hybrid nanoparticles, which is different from Example 7 in that the preparation of the Fe3O4-CDs-1@SiO2-Ab1 / IL-8 / CDs-2@Ab2 immune sandwich complex includes the following steps: 100 μL of IL-8 solution of different concentrations was added to 100 μL of Fe3O4-CDs-1@SiO2-Ab1 solution and shaken at 37°C for 1 h. After Ab1 and IL-8 reacted immune-wise, the Fe3O4-CDs-1@SiO2-Ab1 / IL-8 complex was separated by magnetic separation, washed three times with deionized water and redispersed in 200 μL of deionized water. 150 μL of CDs-2@Ab2 was added to the above solution and shaken at 37°C for 1 h before magnetic separation. Finally, the obtained complex FCs@SiO2-Ab1 / IL-8 / CDs-2@Ab2 was thoroughly washed with deionized water and introduced into fluorescence measurement. The excitation and emission slit widths were both 10 nm, and the excitation wavelength was 390 nm. In the absence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 to CDs-2@Ab2 in the blank group (I0) was (I0) 466 / 565 ; In the presence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the experimental group (I) is (I) 466 / 565 , take the difference between the two, that is (I0-I) 466 / 565 The value was used to quantify IL-8; (I0-I) 466 / 565 The value is the vertical axis, and the concentration of IL-8 is the horizontal axis to establish a standard curve.

[0032] Embodiment 9: The application of a magnetic-fluorescent hybrid nanoparticle is different from that of Example 7 in that the detection of IL-8 in actual sample serum includes the following steps: Fe3O4-CDs-1@SiO2-Ab1 (100 μL) was added to the treated serum (50 μL) and shaken at 37°C for 60 min; then, it was washed three times with deionized water by magnetic separation, and CDs-2@Ab2 was added continuously, and shaken at 37°C for 60 min; finally, the complex obtained after repeated washing with deionized water was transferred to a micro-cuvette for fluorescence detection.

[0033] Results and Discussion: Carbon quantum dots (CDs) are widely used in bioanalysis and bioimaging due to their excellent optical properties such as high quantum yield, good stability, adjustable size, and good biocompatibility. At the same time, CDs' simple synthesis steps and cheap and readily available synthetic raw materials make it a preferred choice among many fluorescent materials. At the same time, magnetic Fe3O4 nanoparticles are widely used in the separation and enrichment of target objects. The present invention prepares and synthesizes magnetic fluorescent carbon dots through a one-step method, and the obtained magnetic fluorescent carbon dots combine the excellent fluorescence properties of carbon dots and the excellent magnetic properties of Fe3O4.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing magnetic-fluorescent hybrid nanoparticles, characterized in that: The following steps are involved: Preparation of S1, Fe3O4-CDs-1@SiO2 and CDs-2 nanoparticles: S1-1, dissolving ammonium ferric citrate in a solution of H2O and triethylenetetramine in a volume ratio of 9:1, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene and heating it to 200°C for 6 h, then cooling the autoclave naturally to room temperature, and obtaining magnetic Fe3O4-CDs-1 powder after the suspension is centrifuged, washed, and dried; S1-2, add hexadecyltrimethylammonium bromide to 1 mg mL -1 The synthesized Fe3O4-CDs-1 powder was ultrasonicated for 20 min; then, 25 wt% NH3·H2O was quickly added, and then ethyl orthosilicate was added dropwise under vigorous stirring. After mixing evenly, it was heated at 40°C for 30 min. After the reaction was completed, the mixture was centrifuged and washed with deionized water. The black powder obtained after freeze-drying was Fe3O4-CDs-1@SiO2. S1-3, adding ninhydrin and o-phenylenediamine to deionized water, and then stirring and dissolving with a magnetic stirrer for 4 hours. After the stirring is completed, the mixed solution is transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and reacted at 180°C for 12 hours. After cooling to room temperature, a dark yellow solution is obtained. The dark yellow solution is placed in a dialysis bag with a molecular weight retention of 1000 Da and dialyzed for 24 hours. The final solution is collected and protected from light at room temperature to obtain CDs-2 nanoparticles; S2. Preparation of magnetic-fluorescent hybrid nanoparticles: S2-1, disperse the Fe3O4-CDs-1@SiO2 nanoparticles obtained in S1-2 in a mixed solution of ethanol and 3-aminopropyltriethoxysilane, the ratio of Fe3O4-CDs-1@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane is (1-2) mg: (1-2) mL: (0.2-0.5) mL, and then shake the suspension at room temperature for 5 h and then perform magnetic separation to obtain amino-functionalized Fe3O4-CDs-1@SiO2; Preparation of S2-2, Fe3O4-CDs-1@SiO2-Ab1: 0.5 mL of captured IL-8 antibody Ab1 was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at room temperature for 30 min. The concentration of IL-8 antibody Ab1 was 20 μg mL -1 , the amino-functionalized Fe3O4-CDs-1@SiO2 nanoparticles were dispersed into the solution to obtain a suspension; the suspension was incubated at 4°C with shaking for 12 h, and 100 μL of 1% bovine serum albumin solution was used to block the excess nonspecific adsorption sites on the Fe3O4-CDs-1@SiO2 surface, and finally the Fe3O4-CDs-1@SiO2-Ab1 complex was obtained; S2-3, preparation of CDs-2@Ab2: The preparation process of CDs-2@-Ab2 complex is similar to that of S2-2. CDs-2 nanoparticles are mixed with 3-aminopropyltriethoxysilane and shaken at room temperature for 5 h to obtain amino-functionalized CDs-2 solution. The IL-8 antibody Ab2 is activated at room temperature for 30 min with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then the amino-functionalized CDs-2 solution is added. The mixture is shaken at 4°C for 12 h, and then blocked with 1% bovine serum albumin solution under the same conditions to obtain CDs-2@Ab2. The obtained Fe3O4-CDs-1@SiO2-Ab1 has a fluorescence emission peak of 447nm under the optimal excitation wavelength of 360nm. Fe3O4-CDs-1@SiO2-Ab1 has excellent magnetic response performance and can be separated from the solution within 5s under the external magnet. The obtained CDs-2@Ab2 had fluorescence emission peaks of 576 nm under the optimal excitation wavelength of 420 nm; The optimal excitation wavelength of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 was set to 390 nm.

2. An application of magnetic-fluorescent hybrid nanoparticles, characterized in that: The following steps are involved: Preparation of S1, Fe3O4-CDs-1@SiO2 and CDs-2 nanoparticles: S1-1, dissolving ammonium ferric citrate in a solution of H2O and triethylenetetramine in a volume ratio of 9:1, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene and heating it to 200°C for 6 h, then cooling the autoclave naturally to room temperature, and obtaining magnetic Fe3O4-CDs-1 powder after the suspension is centrifuged, washed, and dried; S1-2, add hexadecyltrimethylammonium bromide to 1 mg mL -1 The synthesized Fe3O4-CDs-1 powder was ultrasonicated for 20 min; then, 25 wt% NH3·H2O was quickly added, and then ethyl orthosilicate was added dropwise under vigorous stirring. After mixing evenly, it was heated at 40°C for 30 min. After the reaction was completed, the mixture was centrifuged and washed with deionized water. The black powder obtained after freeze-drying was Fe3O4-CDs-1@SiO2. S1-3, adding ninhydrin and o-phenylenediamine to deionized water, and then stirring and dissolving with a magnetic stirrer for 4 hours. After the stirring is completed, the mixed solution is transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene, and reacted at 180°C for 12 hours. After cooling to room temperature, a dark yellow solution is obtained. The dark yellow solution is placed in a dialysis bag with a molecular weight retention of 1000 Da and dialyzed for 24 hours. The final solution is collected and protected from light at room temperature to obtain CDs-2 nanoparticles; S2. Preparation of magnetic-fluorescent hybrid nanoparticles: S2-1, disperse the Fe3O4-CDs-1@SiO2 nanoparticles obtained in S1-2 in a mixed solution of ethanol and 3-aminopropyltriethoxysilane, the ratio of Fe3O4-CDs-1@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane is (1-2) mg: (1-2) mL: (0.2-0.5) mL, and then shake the suspension at room temperature for 5 h and then perform magnetic separation to obtain amino-functionalized Fe3O4-CDs-1@SiO2; Preparation of S2-2, Fe3O4-CDs-1@SiO2-Ab1: 0.5 mL of captured IL-8 antibody Ab1 was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at room temperature for 30 min. The concentration of IL-8 antibody Ab1 was 20 μg mL -1 , the amino-functionalized Fe3O4-CDs-1@SiO2 nanoparticles were dispersed into the solution to obtain a suspension; the suspension was incubated at 4°C with shaking for 12 h, and 100 μL of 1% bovine serum albumin solution was used to block the excess nonspecific adsorption sites on the Fe3O4-CDs-1@SiO2 surface, and finally the Fe3O4-CDs-1@SiO2-Ab1 complex was obtained; S2-3, preparation of CDs-2@Ab2: The preparation process of CDs-2@-Ab2 complex is similar to that of S2-2. CDs-2 nanoparticles are mixed with 3-aminopropyltriethoxysilane and shaken at room temperature for 5 h to obtain amino-functionalized CDs-2 solution. The IL-8 antibody Ab2 is activated at room temperature for 30 min with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then the amino-functionalized CDs-2 solution is added. The mixture is shaken at 4°C for 12 h, and then blocked with 1% bovine serum albumin solution under the same conditions to obtain CDs-2@Ab2. The obtained Fe3O4-CDs-1@SiO2-Ab1 has a fluorescence emission peak of 447nm under the optimal excitation wavelength of 360nm. Fe3O4-CDs-1@SiO2-Ab1 has excellent magnetic response performance and can be separated from the solution within 5s under the external magnet. The obtained CDs-2@Ab2 had fluorescence emission peaks of 576 nm under the optimal excitation wavelength of 420 nm; The optimal excitation wavelength of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 was set to 390 nm; S3, adding different concentrations of IL-8 standard solution to the Fe3O4-CDs-1@SiO2-Ab1 solution obtained in S2-2, incubating, shaking at 37℃-40℃ for 0.5h-2h, after Ab1 and IL-8 have an immune reaction, separating the Fe3O4-CDs-1@SiO2-Ab1 / IL-8 complex by magnetic separation, washing with deionized water and redispersing in deionized water; continue to add CDs-2@Ab2 to the above solution, shaking at 37℃-40℃ for 0.5h-2h and then performing magnetic separation, finally, thoroughly washing the obtained complex Fe3O4-CDs-1 / IL-8 / CDs-2@Ab2 with deionized water, and performing fluorescence detection; In the absence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the blank group (I0) is (I0) 466 / 565 ; In the presence of IL-8, the fluorescence intensity ratio of Fe3O4-CDs-1@SiO2-Ab1 and CDs-2@Ab2 in the experimental group (I) is (I) 466 / 565 , take the difference between the two, that is (I0-I) 466 / 565 The value of IL-8 was used to quantify the IL-8, as (I0-I) 466 / 565 The values ​​are the ordinate and the concentration of IL-8 is the abscissa to establish a standard curve; S4. Add Fe3O4-CDs-1@SiO2-Ab1 to the treated serum for incubation, oscillate at 37℃-40℃ for 0.5h-2h, then wash three times with deionized water through magnetic separation, continue to add CDs-2@Ab2, oscillate at 37℃-40℃ for 0.5h-2h, and finally transfer the obtained complex after repeated washing with deionized water to a micro-cuvette for fluorescence detection.

3. The use of a magnetic-fluorescent hybrid nanoparticle according to claim 2, characterized in that: In S3 to S4, the incubation temperature was 37° C. and the incubation time was 60 min.

4. The use of a magnetic-fluorescent hybrid nanoparticle according to claim 2, characterized in that: In S4, the linear range of IL-8 detection was 20 pg / mL-1000 pg / mL, and the detection limit was 6.9 pg / mL.

5. The use of a magnetic-fluorescent hybrid nanoparticle according to claim 2, characterized in that: In S4, the excitation wavelength range of the fluorescence detection is 360nm~420nm.

6. The use of a magnetic-fluorescent hybrid nanoparticle according to claim 5, characterized in that: In S4, the excitation wavelength of the fluorescence detection is 390 nm.

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