WO2023116586A1 - Si-FITC比率型荧光纳米探针的合成方法及其在新冠病毒抗原检测中的应用 - Google Patents

Si-FITC比率型荧光纳米探针的合成方法及其在新冠病毒抗原检测中的应用 Download PDF

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WO2023116586A1
WO2023116586A1 PCT/CN2022/139817 CN2022139817W WO2023116586A1 WO 2023116586 A1 WO2023116586 A1 WO 2023116586A1 CN 2022139817 W CN2022139817 W CN 2022139817W WO 2023116586 A1 WO2023116586 A1 WO 2023116586A1
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fitc
nps
add
nanoprobe
ratiometric fluorescent
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马英新
毛国斌
戴俊彪
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中国科学院深圳先进技术研究院
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Definitions

  • the invention relates to the technical field of fluorescent probes, in particular to a synthesis method of a Si-FITC ratiometric fluorescent nanoprobe and its application in the detection of novel coronavirus antigens.
  • Nucleic acids, antibodies, and antigens are often used as markers for virus detection.
  • Nucleic acid detection mainly includes real-time quantitative polymerase chain reaction, high-throughput gene sequencing, loop-mediated isothermal amplification and methods based on clustered regularly interspaced short palindromic repeat sequences, which have many advantages such as high sensitivity and good specificity.
  • disadvantages such as the need for skilled technicians, complex equipment, time-consuming operation, and high false positives limit its application.
  • antibody detection is simple and does not require specialized technicians, the long window period limits its development in the early diagnosis of infection. Antigen detection provides a direct and rapid method of early diagnosis without the need for expensive equipment.
  • Enzyme-linked immunosorbent assay combines the high specificity of antibody-antigen hybridization and the efficient catalytic reaction of enzymes to amplify the detection signal, and it has the advantages of high throughput, easy operation and low cost, and has been widely used in clinical diagnosis, Food safety and environmental monitoring.
  • the most commonly used formats for ELISA include colorimetric and fluorometric methods. Compared with colorimetric methods, fluorescence methods have the advantages of higher sensitivity and better selectivity.
  • Quantum dots as an excellent fluorescent nanomaterial with unique optical properties, such as broad excitation spectrum and narrow emission spectrum, high luminous efficiency and good stability, are widely used in biosensing and long-term imaging.
  • fluorescent silicon nanoparticles Si NPs
  • Si NPs fluorescent silicon nanoparticles
  • most assays are based on fluorescent probes with a single emission peak, which are easily affected by the environment and concentration.
  • the present invention proposes a synthesis method of Si-FITC ratiometric fluorescent nanoprobe and its application in the detection of novel coronavirus antigens, which solves or at least partially solves the technical defects in the prior art.
  • the present invention provides a kind of synthetic method of Si-FITC ratio type fluorescent nanoprobe, comprises the following steps:
  • the alcohol solvent includes at least one of methanol, ethanol, and isopropanol
  • the aminosilane coupling agent includes 3-aminopropyl At least one of triethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldiethoxysilane
  • the reducing agent includes trisodium citrate, ascorbic acid, sodium borohydride at least one of the
  • the synthesis method of the described Si-FITC ratiometric fluorescent nanoprobe after adding a reducing agent and an aminosilane coupling agent to the first product, stirring and mixing, react at 190 ⁇ 210°C for 1 ⁇ 3 h It also includes: mixing the reacted product with acetonitrile, centrifuging and drying to obtain the Si-FITC ratio fluorescent nanometer probe.
  • the volume ratio of the reacted product to acetonitrile is 1:(3 ⁇ 5)
  • the centrifugation rate is 7500 ⁇ 8500 rpm
  • the centrifugation time is 10 ⁇ 20 min.
  • fluorescein isothiocyanate is added to an alcohol solvent, and then an aminosilane coupling agent is added, and co-transformation is carried out in a dark and inert atmosphere.
  • the mass volume ratio of the fluorescein isothiocyanate, the alcohol solvent, and the aminosilane coupling agent is (0.3 ⁇ 0.5) g:(3 ⁇ 7) mL: (0.1 ⁇ 0.3) mL;
  • the reducing agent In the step of adding a reducing agent and an aminosilane coupling agent to the first product, stirring and mixing, and reacting at 190 ⁇ 210°C for 1 ⁇ 3 h, the reducing agent, the first product, and the aminosilane coupling
  • the mass volume ratio of the agent is (0.1 ⁇ 0.3) g:(0.05 ⁇ 0.15) mL:(0.5 ⁇ 1.5) mL.
  • the present invention also provides a Si-FITC ratiometric fluorescent nanoprobe prepared by the preparation method.
  • the present invention also provides an application of the Si-FITC ratiometric fluorescent nanoprobe in the detection of SARS-CoV-2 antigens.
  • said application comprises the following steps:
  • nucleocapsid protein into the well and incubate to form a capture antibody-nucleocapsid protein complex
  • Tris-HCl buffer solution containing H 2 O 2 into the well, add probe solution after reaction to detect and record synchronous fluorescence spectrum.
  • the application the preparation method of the conjugate of catalase and NHS-dPEG 4 -biotin, comprises the following steps:
  • the catalase and NHS-dPEG 4 -biotin are mixed, then added to PBS buffer, and the conjugate of catalase and NHS-dPEG 4 -biotin is obtained after shaking.
  • the coating buffer is a carbonate buffer with a pH of 9.6;
  • the washing buffer is a PBS solution containing Tween 20 with a pH of 7.4;
  • the pH of the Tris-HCl buffer is is 7.0.
  • the synthesis method of the Si-FITC ratiometric fluorescent nanoprobe of the present invention uses the covalent coupling reaction between the isothiocyanate group in fluorescein isothiocyanate (FITC) and the amino group in the aminosilane coupling agent
  • the first product is obtained;
  • the Si-FITC ratiometric fluorescent nanoprobe is prepared by a one-pot hydrothermal method.
  • the Si-FITC NPs prepared in this application have both the blue fluorescence of Si and the green fluorescence of FITC.
  • the probe of the peak the ratio fluorescent probe of the dual emission peak has a built-in correction function, which can eliminate the interference of environmental factors and improve the signal-to-noise ratio; Therefore, the prepared Si-FITC ratiometric fluorescent nanoprobes (Si-FITC NPs) can be directly used as ratiometric fluorescent probes to output ELISA signals for the detection of SARS-CoV-2 nucleocapsid protein;
  • the Si-FITC ratiometric fluorescent nanoprobe of the present invention can be used to detect the new coronavirus antigen, based on the principle that CAT triggers H2O2 decomposition, and Si- FITC NPs are quenched by KMnO4 to obtain a low fluorescence intensity ratio, A sensitive and selective fluorescent "off" method for the detection of nucleocapsid proteins was developed. Under optimal reaction conditions, the method was linear in the detection range of 0.02 - 50 ng/mL for nucleocapsid proteins, The lowest detection limit was 0.003 ng/mL.
  • the CAT-H 2 O 2 -KMnO 4 -Si-FITC NPs system of this application has high sensitivity, good selectivity and strong anti-interference ability for pathogen detection.
  • FIG. 1 is a schematic diagram of the synthesis method of Si-FITC ratiometric fluorescent nanoprobe and a schematic diagram of the fluorescent ELISA method of nucleocapsid protein detection of the present invention in one of the embodiments of the present invention;
  • Fig. 2 is the ultraviolet-visible light absorption spectrum and the synchronous fluorescence spectrum diagram of the Si-FITC ratio type fluorescent nanoprobe prepared in the embodiment 1 of the present invention
  • Figure 3 is a transmission microscopic image of Si-FITC NPs prepared in Example 1 of the present invention.
  • Figure 4 is an X-ray crystal diffraction pattern of Si-FITC NPs prepared in Example 1 of the present invention
  • Figure 5 is an X-ray photoelectron spectrum diagram of Si-FITC NPs prepared in Example 1 of the present invention.
  • Fig. 6 is the Fourier transform infrared spectrogram of Si-FITC NPs prepared in Example 1 of the present invention.
  • Figure 7 is the Si-FITC prepared in Example 1 of the present invention Stability of NPs
  • Figure 8 is a graph showing the influence of KMnO 4 , H 2 O 2 , and catalase (CAT) on the Si-FITC NPs prepared in Example 1;
  • Fig. 9 is the ultraviolet-visible absorption spectrum of Si-FITC NPs, KMnO 4 , Si-FITC NPs + KMnO 4 prepared in Example 1 of the present invention.
  • Fig. 10 is the fluorescence lifetime decay curve of Si-FITC NPs and Si-FITC NPs + KMnO prepared in Example 1 of the present invention
  • Fig. 11 is a graph showing the relationship between the fluorescence intensity ratio and H2O2 concentration of Si-FITC NPs prepared in Example 1 of the present invention.
  • Figure 12 is a graph of the fluorescence intensity ratio of Si-FITC NPs treated with 50 ⁇ M KMnO 4 , 100 ⁇ M H 2 O 2 and different concentrations of CAT at different pH values;
  • Figure 13 is the fluorescence intensity ratio of Si-FITC NPs treated with 50 ⁇ M KMnO 4 and 100 ⁇ M H 2 O 2 in the supernatant after adding biotin-CAT or biotin-CAT mixed with MB;
  • Figure 14 is a graph showing the relationship between the fluorescence intensity ratio of Si-FITC NPs and the reaction temperature
  • Figure 15 is a graph showing the relationship between the fluorescence intensity ratio and the reaction time of Si-FITC NPs
  • Figure 16 is a synchronous fluorescence spectrum of Si-FITC NPs under different concentrations of nucleocapsid protein
  • Figure 17 is the selectivity of nucleocapsid protein and other competing components
  • Figure 18 is the fluorescence spectrum of free fluorescein isothiocyanate (FITC) in response to different concentrations of KMnO 4 .
  • FITC free fluorescein isothiocyanate
  • the embodiment of the present application provides a method for synthesizing a Si-FITC ratiometric fluorescent nanoprobe, comprising the following steps:
  • the synthesis method of the Si-FITC ratiometric fluorescent nanoprobe of this application utilizes the covalent coupling between the isothiocyanate group in the fluorescein isothiocyanate (FITC) and the amino group in the aminosilane coupling agent.
  • the first product was obtained through the joint reaction; and the Si-FITC ratiometric fluorescent nanoprobe was prepared by a one-pot hydrothermal method. Specifically, the first product and aminosilane coupling agent were used as Si precursor and reducing agent for hydrothermal After the reaction, Si-FITC ratiometric fluorescent nanoprobes (referred to as Si-FITC NPs) can be prepared.
  • the Si-FITC NPs prepared in this application have both the blue fluorescence of Si and the green fluorescence of FITC. Compared with the probes with single emission peaks, the ratio fluorescent probes with dual emission peaks have a built-in correction function, which can eliminate the interference of environmental factors. Improve the signal-to-noise ratio.
  • the preparation method of the present application does not require further modification, and solves the problem of reduced yield of quantum dots during the coupling reaction, and the prepared Si-FITC ratiometric fluorescent nanoprobes (Si-FITC NPs) can be directly used as ratiometric fluorescence
  • the probe outputs an ELISA signal for the detection of SARS-CoV-2 nucleocapsid protein.
  • catalase catalyzes the decomposition of hydrogen peroxide, while KMnO 4 selectively quenches the fluorescence of Si in Si-FITC NPs, according to the change of the fluorescence intensity ratio of Si and FITC , can accurately measure the concentration of SARS-CoV-2 nucleocapsid protein. Therefore, this ratiometric fluorescence method combined with ELISA with good sensitivity and selectivity can be used for the detection of SARS-CoV-2 nucleocapsid protein.
  • the alcohol solvent includes at least one of methanol, ethanol, and isopropanol
  • the aminosilane coupling agent includes 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3 - at least one of aminopropylmethyldiethoxysilane
  • the reducing agent includes at least one of trisodium citrate, ascorbic acid, and sodium borohydride.
  • the alcohol is ethanol
  • the aminosilane coupling agent is 3-aminopropyltriethoxysilane (APTES)
  • the reducing agent is trisodium citrate.
  • adding a reducing agent and an aminosilane coupling agent to the first product stirring and mixing, reacting at 190-210°C for 1-3 h, and then further comprising: mixing the reacted product with acetonitrile and centrifuging and dry to obtain the Si-FITC ratiometric fluorescent nanoprobe.
  • the function of adding acetonitrile is to purify the obtained Si-FITC ratiometric fluorescent nanoprobes (Si-FITC NPs).
  • the volume ratio of the reacted product to acetonitrile is 1:(3-5), the centrifugation speed is 7500-8500 rpm, and the centrifugation time is 10-20 min.
  • trisodium citrate is first added to water to form an aqueous solution of trisodium citrate, and then trisodium citrate is added to the first product
  • the aqueous solution and aminosilane coupling agent were stirred and mixed, and then reacted at 190-210 °C for 1-3 h to obtain the Si-FITC ratiometric fluorescent nanoprobe.
  • fluorescein isothiocyanate is added to an alcohol solvent, and then an aminosilane coupling agent is added to carry out a covalent coupling reaction in the dark and inert atmosphere to obtain the first product.
  • the mass volume ratio of fluorescein cyanate, alcohol solvent, and aminosilane coupling agent is (0.3 ⁇ 0.5) g:(3 ⁇ 7) mL:(0.1 ⁇ 0.3) mL;
  • the mass-volume ratio of reducing agent, first product and aminosilane coupling agent is (0.1 ⁇ 0.3) g: (0.05 ⁇ 0.15) mL: (0.5 ⁇ 1.5) mL.
  • the inert atmosphere may be nitrogen or a rare gas (such as helium, argon, etc.).
  • the embodiment of the present application also provides the application of the above-mentioned Si-FITC ratiometric fluorescent nanoprobe in the detection of SARS-CoV-2 antigens.
  • the application of Si-FITC ratiometric fluorescent nanoprobes in the detection of novel coronavirus antigens specifically includes the following steps:
  • the preparation method of the conjugate of catalase and NHS-dPEG 4 -biotin comprises the following steps:
  • the catalase and NHS-dPEG 4 -biotin are mixed, then added to PBS buffer, and the conjugate of catalase and NHS-dPEG 4 -biotin is obtained after shaking.
  • the coating buffer is a carbonate buffer with a pH of 9.6;
  • the washing buffer is a PBS solution containing Tween 20 with a pH of 7.4; and the pH of the Tris-HCl buffer is 7.0.
  • the application of Si-FITC ratiometric fluorescent nanoprobes in the detection of nucleocapsid proteins specifically includes the following steps:
  • reporter antibody specifically, rabbit monoclonal antibody
  • Tris-HCl buffer solution (20 mM pH 7.0) containing 100 ⁇ M H 2 O 2 to the wells, react at 30 °C for 1 hour, add the probe solution to detect and record the simultaneous fluorescence spectrum.
  • the nucleocapsid protein is SARS-CoV-2 nucleocapsid-histidine recombinant protein
  • the mouse monoclonal antibody is mouse anti-SARS-CoV-2 nucleocapsid monoclonal antibody
  • the rabbit monoclonal antibody is rabbit Anti-SARS-CoV-2 nucleocapsid monoclonal antibody, among them, SARS-CoV-2 nucleocapsid-histidine recombinant protein, mouse anti-SARS-CoV-2 nucleocapsid monoclonal antibody and rabbit anti-SARS-CoV -2 nucleocapsid monoclonal antibody was purchased from Beijing Sino Biological Technology Co., Ltd. Biotinylated goat anti-rabbit secondary antibody was purchased from Beyontian Biotechnology Company, and NHS-dPEG 4 -biotin was purchased from Sigma Company.
  • a in Figure 1 shows a schematic diagram of the preparation method of Si-FITC ratiometric fluorescent nanoprobes (Si-FITC NPs) in one of the examples;
  • B in Figure 1 shows a schematic diagram of the fluorescent ELISA method for nucleocapsid protein detection of the present application .
  • Capture Ab means capture antibody
  • N Protein means nucleocapsid protein
  • Ab1 means reporter antibody
  • Ab2 means secondary antibody (i.e.
  • biotin-labeled goat anti-rabbit IgG SA means streptavidin, catalase
  • the conjugate with NHS-dPEG 4 -biotin was coupled with the secondary antibody via streptavidin as an intermediate, which can effectively decompose H 2 O 2 , and the retained KMnO 4 quenches the fluorescence of Si in Si-FITC NPs . Therefore, the Si-FITC NPs system containing KMnO4 was able to detect the concentration of nucleocapsid protein, and the fluorescence intensity ratio of Si-FITC NPs decreased with the increase of nucleocapsid protein concentration.
  • ELISA is recognized as the immunoassay technique of choice. With the advancement of technology in the field of fluorescent materials, fluorescent ELISA has been more widely used in many research fields than traditional colorimetric ELISA. Compared with organic dyes with poor photostability and single-emission quantum dots that are susceptible to environmental interference, the dual emission peak ratio fluorescent probe of the present application is expected to improve the sensitivity and accuracy of ELISA to a new level. Based on the principle that CAT triggers the decomposition of H 2 O 2 and Si-FITC NPs are quenched by KMnO 4 to obtain a low fluorescence intensity ratio, this application develops a sensitive and selective fluorescence for detecting nucleocapsid proteins"close" method.
  • the linear range of this method for the detection of nucleocapsid protein was 0.02-50 ng/mL, and the lowest detection limit was 0.003 ng/mL.
  • the CAT-H 2 O 2 -KMnO 4 -Si-FITC NPs system of this application has high sensitivity, good selectivity and strong anti-interference ability for pathogen detection.
  • the embodiment of the present application provides a method for synthesizing a Si-FITC ratiometric fluorescent nanoprobe, comprising the following steps:
  • Si-FITC NPs Si-FITC ratiometric fluorescent nanoprobes
  • a method for detecting nucleocapsid protein comprising the following steps:
  • nucleocapsid protein (0, 0.02, 0.05, 0.1, 0.5, 1, 10, 20, 40 and 50 ng/ml) to the wells and incubate at 37 °C 1 h to form the capture antibody-nucleocapsid protein complex and wash 3 times;
  • reporter antibody specifically rabbit monoclonal antibody
  • the nucleocapsid protein is SARS-CoV-2 nucleocapsid-histidine recombinant protein
  • the mouse monoclonal antibody is mouse anti-SARS-CoV-2 nucleocapsid monoclonal antibody
  • the rabbit monoclonal antibody is rabbit anti- SARS-CoV-2 nucleocapsid monoclonal antibody
  • biotinylated goat anti-rabbit secondary antibody is biotinylated goat anti-rabbit IgG.
  • Si-FITC NPs Si-FITC ratiometric fluorescent nanoprobes prepared in Example 1
  • a in Fig. 2 represents Si NPs (i.e. fluorescent silicon nanoparticles), Si-FITC NPs (i.e. Si-FITC ratio fluorescent nanoprobes prepared in Example 1), FITC (fluorescein isothiocyanate) UV -Visible absorption spectrum, where the concentration of Si NPs is 1 mg/mL, the concentration of Si-FITC NPs is 1 mg/mL, and the concentration of FITC is 5 ⁇ g/mL.
  • FITC is prepared by adding to water.
  • NPs The preparation method of NPs is specifically as follows:
  • Figure 3 is a TEM image of the Si-FITC NPs prepared in Example 1. It can be seen from Figure 3 that the Si-FITC NPs are spherical with a size of 1.6 nm and have an obvious crystal structure.
  • Fig. 4 is the XRD pattern of Si-FITC NPs and Si NPs prepared in Example 1. It can be seen from Fig. 4 that Si-FITC NPs has an obvious diffraction peak at 23o, and there is little difference from Si NPs.
  • Figure 5 is the XPS figure of the Si-FITC NPs prepared in Example 1. As can be seen from Figure 5, the XPS spectrum shows that some elements such as Si2p, S2p, C1s, N1s, O1s are distributed in the solid state of Si-FITC NPs surface.
  • Figure 6 is the FTIR spectrum of Si-FITC NPs prepared in Example 1. It can be seen from Figure 6 that in the range of 500-4000cm -1 , the strong absorption peak at 1030cm -1 is the stretching of Si-O vibration, the absorption peak at 1588 cm -1 is the bending vibration of NH, and the absorption peaks at 2931 cm -1 and 3356 cm -1 are due to the stretching vibration of OH and NH, these results indicate that the amino groups are located on the surface of Si-FITC NPs.
  • Si-FITC ratiometric fluorescent nanoprobes prepared in Example 1.
  • FIG. 7 The results of testing the photostability, pH stability and anti-interference ability of the Si-FITC NPs solution in Example 1 are shown in FIG. 7 .
  • the photostability of Si-FITC NPs was studied by continuous irradiation with 350V excitation light for 2 hours.
  • B and C in Figure 7 are the simultaneous fluorescence spectra and fluorescence intensity ratios of Si-FITC NPs solutions in Tris-HCl buffers with different pH;
  • D in Figure 7 is Si-FITC NPs solution in various ions and amino acids (including 100 ⁇ M Mn 2+ , Cu 2+ , Ca 2+ , K + , Zn 2+ , NH 4 + , Mg 2+ , Glu, Thr, Pro, Ser, Val and Tyr) immunity to interference.
  • the first step in the detection of catalase (CAT) activity is to mix different concentrations of catalase (CAT) with 100 ⁇ M H 2 O 2 was reacted for 30 minutes, then mixed with 50 ⁇ M KMnO 4 for 30 minutes, and finally Si-FITC NPs were added and simultaneous fluorescence spectra were recorded.
  • the test results are shown in Figure 8.
  • a in Figure 8 shows the simultaneous fluorescence spectra of Si-FITC NPs in response to different concentrations of KMnO 4
  • B shows the relationship between the fluorescence intensity ratio of Si-FITC NPs and the concentration of KMnO 4
  • in Figure 8 C shows the Si-FITC NPs treated with 50 ⁇ M KMnO 4 Synchronized fluorescence spectra of Si-FITC NPs in response to different concentrations of H 2 O 2
  • D represents the relationship between the fluorescence intensity ratio of Si-FITC NPs and the concentration of H 2 O 2
  • E in Figure 8 represents the response of Si-FITC NPs to different concentrations Synchronized fluorescence spectra of CAT
  • F indicates the relationship between the fluorescence intensity ratio of Si-FITC NPs and the concentration of CAT.
  • Si-FITC NPs, KMnO 4 , Si-FITC NPs + KMnO 4 represent the UV-Vis absorption spectra of different substances, in which the concentration of Si-FITC NPs is 1 mg/mL, the concentration of KMnO 4 is 75 ⁇ M, Si-FITC NPs + KMnO 4 represents the UV-Vis absorption spectrum of 1 mg/mL Si-FITC NPs and 75 ⁇ M KMnO 4 mixed in Tris-HCl buffer.
  • Si-FITC NPs and Si-FITC NPs + KMnO 4 represent the fluorescence lifetime decay curves of different substances, respectively, where the concentration of Si-FITC NPs is 1 mg/mL, and Si-FITC NPs + KMnO 4 represents the concentration of 1 mg/mL Fluorescence lifetime decay curves of Si-FITC NPs mixed with 75 ⁇ M KMnO 4 in Tris-HCl buffer.
  • Catalase CAT can effectively catalyze the decomposition of H 2 O 2 , in which the fluorescence of Si in Si-FITC NPs is quenched by KMnO 4 , after adding H 2 O 2 , KMnO 4 can be consumed to prevent the fluorescence of Si-FITC NPs from Quenching, such as adding catalase CAT to the above system, can consume H 2 O 2 , and excess KMnO 4 changes the fluorescence intensity ratio of Si-FITC NPs. In order to obtain the highest signal-to-noise ratio, the dosage of KMnO 4 and H 2 O 2 needs to be optimized.
  • the KMnO 4 concentrations of 50, 100, and 200 ⁇ M were selected, accompanied by a series of H 2 O 2 concentrations;
  • Figure 11 shows the relationship between the fluorescence intensity ratio of Si-FITC NPs and the H 2 O 2 concentration, and the KMnO 4 concentrations were 50 , 100 or 200 ⁇ M.
  • Catalase CAT and NHS-dPEG 4 -biotin were mixed in 1 mL PBS (10 mM, pH 8.0) at a molar ratio of 1:100, and shaken at 37 °C for 4 hours to obtain the concentration of catalase and NHS- dPEG 4 -biotin conjugate (referred to as biotin-CAT).
  • biotin-CAT concentration of catalase and NHS- dPEG 4 -biotin conjugate
  • magnetic beads with streptavidin were used to react with biotin-CAT in Tris-HCl buffer at 37 °C for 30 minutes, and then biotin-CAT was isolated.
  • the CAT activity of the supernatant was measured by the H 2 O 2 -KMnO 4 -Si-FITC NPs system, and the results are shown in FIG. 13 .
  • 1 is the blank control group
  • 2 is the mixed group of biotin-CAT and H 2 O 2 -KMnO 4 -Si-FITC NPs
  • 3 is the supernatant of biotin-CAT separated by streptavidin magnetic beads H 2 O 2 -KMnO 4 -Si-FITC NPs mixed group. It can be seen from Figure 13 that F 385 /F 490 in group 2 was significantly reduced, while F 385 /F 490 in group 3 was similar to that in group 1 (the blank control group without biotin-CAT), indicating that CAT and NHS-dPEG 4 - Biotin conjugation was successful.
  • biotin-CAT is linked to the secondary antibody through the intermediate action of streptavidin, thus immobilized in the 96-well plate, which is different from the free system in the 1.5 mL centrifuge tube; for nucleocapsid protein
  • the reaction temperature and time of biotin-CAT and H 2 O 2 were optimized.
  • the reaction temperatures in the control step S8 are respectively 25 °C, 30 °C, and 37 °C, and the reaction time is 1 h, wherein the concentration of H 2 O 2 is 100 ⁇ M, and the probe solution
  • the concentration of KMnO 4 in medium is 50 ⁇ M
  • the concentration of nucleocapsid protein is 50 ng/mL
  • biotin-CAT 1:100 (the dilution ratio of biotin-CAT is 1:100).
  • the relationship between the fluorescence intensity ratio of Si-FITC NPs obtained by the test and the reaction temperature is shown in Fig. 14 . It can be seen from Fig. 14 that the largest difference in the fluorescence intensity ratio occurs at 30 °C.
  • the reaction temperature in step S8 is controlled to be 30°C
  • the reaction time is 0.5 h, 1 h and 2 h, respectively
  • the concentration of H 2 O 2 is 100 ⁇ M
  • the concentration of KMnO 4 in the probe solution is 50 ⁇ M
  • nucleocapsid protein concentration 50 ng/mL is 100 ⁇ M
  • biotin-CAT is 1:100
  • the dilution ratio of biotin-CAT is 1:100.
  • the relationship between the fluorescence intensity ratio and the reaction time of the Si-FITC NPs obtained by the test is shown in Fig. 15 . It can be seen from Figure 15 that the maximum difference of the fluorescence intensity ratio appears at 1 h.
  • blank is the blank control group
  • N protein is the nucleocapsid protein
  • S protein is the new crown spike protein
  • BSA bovine serum albumin, wherein the added S protein concentration is 200 ng/mL, and the added BSA concentration is 200 ng/mL.
  • FITC free fluorescein isothiocyanate
  • the detection method of the present application shows a high ability to evaluate nucleocapsid protein in real human serum samples, and has the potential to clinically detect SARS-CoV-2.

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Abstract

本发明提供了一种Si-FITC比率型荧光纳米探针的合成方法及其在新冠病毒抗原检测中的应用，该纳米探针的制备方法利用异硫氰酸荧光素中的异硫氰酸根与氨基硅烷偶联剂中的氨基发生共价偶联反应得到第一产物；再通过一锅水热法制备得到；本申请制备得到的Si-FITC NPs同时拥有Si的蓝色荧光和FITC的绿色荧光，相对于单发射峰的探针，双发射峰的比率荧光探针具有内置校正功能，可消除环境因素干扰，提高信噪比；本申请的制备方法不需要进一步的修饰，解决了偶联反应过程中量子点产率降低的问题，制备得到的Si-FITC NPs可直接用作比率荧光探针输出ELISA信号，用于SARS-CoV-2核衣壳蛋白检测。

Description

Si-FITC比率型荧光纳米探针的合成方法及其在新冠病毒抗原检测中的应用 技术领域
本发明涉及荧光探针技术领域，特别涉及一种Si-FITC比率型荧光纳米探针的合成方法及其在新冠病毒抗原检测中的应用。
 背景技术
[根据细则26改正 21.02.2023]
COVID-19大流行突显了快速可靠的诊断技术对控制和预防病毒传播的重要性。核酸、抗体和抗原常被用作病毒检测的标志物。核酸检测，主要包括实时定量聚合酶链反应、高通量基因测序、环介导等温扩增和基于成簇规律间隔短回文重复序列的方法，具有灵敏度高、特异性好等多种优点。然而，需要熟练技术人员、设备复杂、操作费时、假阳性高等缺点限制了其应用。抗体虽然检测操作简单，不需要专门的技术人员，但长窗口期限制了其在感染早期诊断中的发展。抗原检测提供了一种直接快速的早期诊断方法，无需昂贵的设备。
酶联免疫吸附试验（ELISA）结合了抗体-抗原杂交的高特异性和酶的高效催化反应放大检测信号，并且其具有高通量、易于操作和低成本的优势，已经广泛应用于临床诊断、食品安全和环境监测。 ELISA 最常用的模式包括比色法和荧光法。与比色法相比，荧光法具有灵敏度更高、选择性更好的优点。
量子点（QDs）作为一种优良的荧光纳米材料具有独特的光学性质，如宽激发谱和窄发射谱、高发光效率和良好的稳定性，被广泛应用于生物传感和长时间成像。与II-VI族半导体纳米粒子相比，荧光硅纳米粒子（Si NPs）具有更优越的生物相容性、更低的光漂白率和更广泛的来源等优点，被证明是一种新型的有前途的荧光纳米探针。然而，大多数检测都基于具有单一发射峰的荧光探针，其易受环境和浓度的影响。
基于目前的荧光硅纳米探针存在的缺陷，有必要对此进行改进。
 技术问题
有鉴于此，本发明提出了一种Si-FITC比率型荧光纳米探针的合成方法及其在新冠病毒抗原检测中的应用，解决或至少部分解决现有技术中存在的技术缺陷。
 技术解决方案
第一方面，本发明提供了一种Si-FITC比率型荧光纳米探针的合成方法，包括以下步骤：
将异硫氰酸荧光素加入至醇溶剂中，然后加入氨基硅烷偶联剂，于避光和惰性气氛下进行共价偶联反应得到第一产物；
向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h，即得Si-FITC比率型荧光纳米探针。
优选的是，所述的Si-FITC比率型荧光纳米探针的合成方法，所述醇溶剂包括甲醇、乙醇、异丙醇中的至少一种；所述氨基硅烷偶联剂包括3-氨基丙基三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨丙基甲基二乙氧基硅烷中的至少一种；所述还原剂包括柠檬酸三钠、抗坏血酸、硼氢化钠中的至少一种。
优选的是，所述的Si-FITC比率型荧光纳米探针的合成方法，向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h后还包括：将反应后的产物与乙腈混合后离心分离，干燥即得Si-FITC比率型荧光纳米探针。
优选的是，所述的Si-FITC比率型荧光纳米探针的合成方法，反应后的产物与乙腈的体积比为1:(3~5)，离心速率为7500~8500 rpm、离心时间为10~20 min。
优选的是，所述的Si-FITC比率型荧光纳米探针的合成方法，将异硫氰酸荧光素加入至醇溶剂中，然后加入氨基硅烷偶联剂，于避光和惰性气氛下进行共价偶联反应得到第一产物的步骤中，所述异硫氰酸荧光素、所述醇溶剂、所述氨基硅烷偶联剂的质量体积比为(0.3~0.5) g:(3~7) mL:(0.1~0.3) mL；
向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h的步骤中，所述还原剂、所述第一产物、所述氨基硅烷偶联剂的质量体积比为(0.1~0.3) g:(0.05~0.15) mL:(0.5~1.5) mL。
第二方面，本发明还提供了一种Si-FITC比率型荧光纳米探针，采用所述的制备方法制备得到。
第三方面，本发明还提供了一种所述的Si-FITC比率型荧光纳米探针在新冠病毒抗原检测中的应用。
优选的是，所述的应用，包括以下步骤：
将Si-FITC比率型荧光纳米探针、KMnO 4加入至Tris-HCl缓冲液中，配制得到探针溶液；
将包被缓冲液稀释的捕获抗体包被于孔板内，孵育，之后将孔板内的液体丢弃，并用洗涤缓冲液洗涤；
向孔内加入脱脂奶粉，封闭后，使用洗涤缓冲液洗涤；
再向孔内加入核衣壳蛋白，孵育形成捕获抗体-核衣壳蛋白复合物；
向孔内加入报告抗体孵育形成夹心免疫复合物；
再向孔内加入二抗孵育；
再向孔内加入链霉亲和素，孵育，再加入过氧化氢酶与NHS-dPEG 4-生物素的偶联物；
再向孔内加入含有H 2O 2的Tris-HCl缓冲液，反应后加入探针溶液检测并记录同步荧光光谱。
优选的是，所述的应用，所述过氧化氢酶与NHS-dPEG 4-生物素的偶联物的制备方法，包括以下步骤：
将过氧化氢酶与NHS-dPEG 4-生物素混合后加入至PBS缓冲液中，振荡后即得过氧化氢酶与NHS-dPEG 4-生物素的偶联物。
优选的是，所述的应用，所述包被缓冲液为pH 为9.6的碳酸盐缓冲液；洗涤缓冲液为pH为7.4的含有Tween 20的PBS溶液；所述Tris-HCl缓冲液的pH为7.0。
 有益效果
本发明的一种Si-FITC比率型荧光纳米探针的合成方法相对于现有技术具有以下有益效果：
（1）本发明的Si-FITC比率型荧光纳米探针的合成方法，利用异硫氰酸荧光素（FITC）中的异硫氰酸根与氨基硅烷偶联剂中的氨基发生共价偶联反应得到第一产物；再通过一锅水热法制备得到Si-FITC比率型荧光纳米探针，本申请制备得到的Si-FITC NPs同时拥有Si的蓝色荧光和FITC的绿色荧光，相对于单发射峰的探针，双发射峰的比率荧光探针具有内置校正功能，可消除环境因素干扰，提高信噪比；本申请的制备方法不需要进一步的修饰，解决了偶联反应过程中量子点产率降低的问题，制备得到的Si-FITC比率型荧光纳米探针（Si-FITC NPs）可直接用作比率荧光探针输出ELISA信号，用于SARS-CoV-2核衣壳蛋白检测；
（2）本发明的Si-FITC比率型荧光纳米探针可用于检测新冠病毒抗原，基于CAT 触发H 2O 2分解，以及Si-FITC NPs被KMnO 4猝灭而得到低荧光强度比值的原理，开发了一种灵敏且选择性好的用于检测核衣壳蛋白的荧光“关闭”方法，在最佳反应条件下，该方法对核衣壳蛋白检测的线性范围为0.02 - 50 ng/mL，最低检出限为0.003 ng/mL。综上所述，本申请的CAT- H 2O 2-KMnO 4-Si-FITC NPs体系对病原体检测具有高灵敏度、良好的选择性和较强的抗干扰能力。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单的介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明其中一个实施例中Si-FITC比率型荧光纳米探针的合成方法示意图以及本发明的核衣壳蛋白检测的荧光ELISA方法示意图；
图2为本发明实施例1中制备得到的Si-FITC比率型荧光纳米探针的紫外-可见光吸收光谱和同步荧光光谱图；
图3为本发明实施例1中制备得到的Si-FITC NPs的透射显微电镜图；
图4为本发明实施例1中制备得到的Si-FITC NPs的X-射线晶体衍射图；
图5为本发明实施例1中制备得到的Si-FITC NPs的X-射线光电子能谱图；
图6为本发明实施例1中制备得到的Si-FITC NPs的傅立叶转换红外光谱图；
图7为本发明实施例1中制备得到的Si-FITC NPs的稳定性；
图8为KMnO 4、H 2O 2、过氧化氢酶（CAT）对实施例1中制备得到的Si-FITC NPs的影响图；
图9为本发明实施例1制备得到的Si-FITC NPs、KMnO 4、Si-FITC NPs + KMnO 4的紫外-可见吸收光谱图；
图10为本发明实施例1制备得到的Si-FITC NPs、Si-FITC NPs + KMnO 4的荧光寿命衰减曲线图；
图11为本发明实施例1中制备得到的Si-FITC NPs的荧光强度比值与H 2O 2浓度之间的关系图；
图12为50 μM KMnO 4、100 μM H 2O 2和不同浓度CAT处理的Si-FITC NPs在不同pH值下荧光强度比的关系图；
图13为加入biotin-CAT或biotin-CAT与MB混合后的上清液，用50 μM KMnO 4、100 μM H 2O 2处理的Si-FITC NPs的荧光强度比；
图14为Si-FITC NPs的荧光强度比与反应温度之间关系图；
图15为Si-FITC NPs的荧光强度比与反应时间之间关系图；
图16为不同浓度的核衣壳蛋白下Si-FITC NPs的同步荧光光谱图；
图17为核衣壳蛋白和其他竞争组分的选择性；
图18为游离异硫氰酸荧光素（FITC）响应不同浓度KMnO 4的荧光光谱图。
本发明的实施方式
下面详细描述本申请的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能解释为对本申请的限制。
本申请实施例提供了一种Si-FITC比率型荧光纳米探针的合成方法，包括以下步骤：
S1、将异硫氰酸荧光素加入至醇溶剂中，然后加入氨基硅烷偶联剂，于避光和惰性气氛下进行共价偶联反应得到第一产物；
S2、向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h，即得Si-FITC比率型荧光纳米探针。
需要说明的是，本申请的Si-FITC比率型荧光纳米探针的合成方法，利用异硫氰酸荧光素（FITC）中的异硫氰酸根与氨基硅烷偶联剂中的氨基发生共价偶联反应得到第一产物；再通过一锅水热法制备得到Si-FITC比率型荧光纳米探针，具体的，将第一产物、氨基硅烷偶联剂用作 Si前体与还原剂进行水热反应即可制备得到Si-FITC比率型荧光纳米探针（记为Si-FITC NPs）。本申请制备得到的Si-FITC NPs同时拥有Si的蓝色荧光和FITC的绿色荧光，相对于单发射峰的探针，双发射峰的比率荧光探针具有内置校正功能，可消除环境因素干扰，提高信噪比。本申请的制备方法不需要进一步的修饰，解决了偶联反应过程中量子点产率降低的问题，制备得到的Si-FITC比率型荧光纳米探针（Si-FITC NPs）可直接用作比率荧光探针输出ELISA信号，用于SARS-CoV-2核衣壳蛋白检测。由于核衣壳蛋白与抗体的结合，过氧化氢酶（CAT）催化过氧化氢的分解，而KMnO 4选择性地猝灭Si-FITC NPs中Si的荧光，根据Si和FITC荧光强度比的变化，可以准确测定SARS-CoV-2核衣壳蛋白的浓度。因此，该比率荧光方法结合具有良好的灵敏度和选择性的ELISA，可用于 SARS-CoV-2 核衣壳蛋白的检测。
在一些实施例中，醇溶剂包括甲醇、乙醇、异丙醇中的至少一种；氨基硅烷偶联剂包括3-氨基丙基三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨丙基甲基二乙氧基硅烷中的至少一种；还原剂包括柠檬酸三钠、抗坏血酸、硼氢化钠中的至少一种。
具体的，醇为乙醇、氨基硅烷偶联剂为3-氨基丙基三乙氧基硅烷(APTES)、还原剂为柠檬酸三钠。
在一些实施例中，向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h后还包括：将反应后的产物与乙腈混合后离心分离，干燥即得Si-FITC比率型荧光纳米探针。加入乙腈的作用是对得到的Si-FITC比率型荧光纳米探针（Si-FITC NPs）进行纯化。
在一些实施例中，反应后的产物与乙腈的体积比为1:(3~5)，离心速率为7500~8500 rpm、离心时间为10~20 min。
具体的，在一些实施例中，若采用的还原剂为柠檬酸三钠，则先将柠檬酸三钠加入至水中，配成柠檬酸三钠水溶液，再向第一产物中加入柠檬酸三钠水溶液、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h，即得Si-FITC比率型荧光纳米探针。
在一些实施例中，将异硫氰酸荧光素加入至醇溶剂中，然后加入氨基硅烷偶联剂，于避光和惰性气氛下进行共价偶联反应得到第一产物的步骤中，异硫氰酸荧光素、醇溶剂、氨基硅烷偶联剂的质量体积比为(0.3~0.5) g:(3~7) mL:(0.1~0.3) mL；
向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h的步骤中，还原剂、第一产物、氨基硅烷偶联剂的质量体积比为(0.1~0.3) g:(0.05~0.15) mL:(0.5~1.5) mL。
具体的，惰性气氛可以为氮气或稀有气体（如氦气、氩气等）等。
基于同一发明构思，本申请实施例还提供了上述的Si-FITC比率型荧光纳米探针在新冠病毒抗原检测中的应用。
在一些实施例中，Si-FITC比率型荧光纳米探针在新冠病毒抗原检测中的应用，具体包括以下步骤：
S1、将Si-FITC比率型荧光纳米探针、KMnO 4加入至Tris-HCl缓冲液中，配制得到探针溶液；
S2、将包被缓冲液稀释的捕获抗体包被于孔板内，孵育，之后将孔板内的液体丢弃，并用洗涤缓冲液洗涤；
S3、向孔内加入脱脂奶粉，封闭后，使用洗涤缓冲液洗涤；
S4、再向孔内加入核衣壳蛋白，孵育形成捕获抗体-核衣壳蛋白复合物；
S5、向孔内加入报告抗体孵育形成夹心免疫复合物；
S6、再向孔内加入二抗孵育；
S7、再向孔内加入链霉亲和素，孵育，再加入过氧化氢酶与NHS-dPEG 4-生物素的偶联物；
S8、再向孔内加入含有H 2O 2的Tris-HCl缓冲液，反应后加入探针溶液检测并记录同步荧光光谱。
在一些实施例中，过氧化氢酶与NHS-dPEG 4-生物素的偶联物的制备方法，包括以下步骤：
将过氧化氢酶与NHS-dPEG 4-生物素混合后加入至PBS缓冲液中，振荡后即得过氧化氢酶与NHS-dPEG 4-生物素的偶联物。
在一些实施例中，包被缓冲液为pH 为9.6的碳酸盐缓冲液；洗涤缓冲液为pH为7.4的含有Tween 20的PBS溶液；Tris-HCl缓冲液的pH为7.0。
具体的，在一些实施例中，Si-FITC比率型荧光纳米探针在检测核衣壳蛋白中的应用，具体包括以下步骤：
S1、将Si-FITC比率型荧光纳米探针、KMnO 4加入至Tris-HCl缓冲液中，配制得到探针溶液；
S2、将100mL包被缓冲液（50 mM 的碳酸盐缓冲液，pH 9.6）稀释的捕获抗体（具体为小鼠单克隆抗体）包被于96孔板内，在4 °C下过夜孵育，之后将孔板内的液体丢弃，并用洗涤缓冲液（10 mM 的PBS，pH 7.4，含有质量浓度为0.05% Tween 20）洗涤3次；
S3、向孔内加入300 μL质量浓度为2.5%的脱脂奶粉，在37 °C下封闭1 h，再使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S4、再向孔内加入100 μL核衣壳蛋白，在37 °C下孵育1 h形成捕获抗体-核衣壳蛋白复合物并洗涤3次；
S5、向孔内加入100 μL报告抗体（具体为兔单克隆抗体）在37 °C下孵育1h形成夹心免疫复合物并使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S6、再向孔内加入100 μL二抗（具体为生物素化山羊抗兔二抗）在37 ℃下孵育1 h并使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S7、再向孔内加入100 μL 2.5 μg/ml链霉亲和素，在37 °C下孵育0.5 h，使用洗涤缓冲液(与S2中洗涤缓冲液相同)洗涤3次；
S8、再向孔内加入100 μL过氧化氢酶与NHS-dPEG 4-生物素的偶联物，在37 ℃下孵育0.5 h，使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S9、最后向孔内加入包含100 μM H 2O 2的Tris-HCl缓冲液（20 mM pH 7.0），于30 °C下反应1 小时，加入探针溶液检测并记录同步荧光光谱。
具体的，核衣壳蛋白为SARS-CoV-2核衣壳-组氨酸重组蛋白、小鼠单克隆抗体为小鼠抗SARS-CoV-2核衣壳单克隆抗体、兔单克隆抗体为兔抗SARS-CoV-2核衣壳单克隆抗体，其中，SARS-CoV-2核衣壳-组氨酸重组蛋白、小鼠抗SARS-CoV-2核衣壳单克隆抗体和兔抗SARS-CoV-2核衣壳单克隆抗体购自北京义翘神州科技股份有限公司。生物素化山羊抗兔二抗购自碧云天生物技术公司，NHS-dPEG 4-生物素购自Sigma公司。
图1中A显示了其中一个实施例中Si-FITC比率型荧光纳米探针（Si-FITC NPs）的制备方法示意图；图1中B显示了本申请的核衣壳蛋白检测的荧光ELISA方法示意图。图1B中Capture Ab表示捕获抗体、N Protein表示核衣壳蛋白、Ab1表示报告抗体、Ab2表示二抗（即生物素标记的山羊抗兔IgG）、SA表示链霉亲和素，过氧化氢酶与NHS-dPEG 4-生物素的偶联物通过链霉亲和素作为中间体与二抗偶联，能有效地分解H 2O 2，保留的KMnO 4猝灭Si-FITC NPs中Si的荧光。因此，包含KMnO 4的Si-FITC NPs系统得以检测核衣壳蛋白的浓度，并且Si-FITC NPs的荧光强度比值随着核衣壳蛋白浓度的增加而降低。
ELISA被公认为首选的免疫测定技术。随着荧光材料领域技术的进步，荧光ELISA比传统的比色ELISA在许多研究领域得到了更广泛的应用。与光稳定性差的有机染料和易受环境干扰的单发射量子点相比，本申请的双发射峰比率荧光探针有望将ELISA的灵敏度和准确性提高到一个新的水平。本申请基于CAT 触发H 2O 2分解，以及Si-FITC NPs被KMnO 4猝灭而得到低荧光强度比值的原理，开发了一种灵敏且选择性好的用于检测核衣壳蛋白的荧光“关闭”方法。在最佳反应条件下，该方法对核衣壳蛋白检测的线性范围为0.02 -50 ng/mL，最低检出限为0.003 ng/mL。综上所述，本申请的CAT- H 2O 2-KMnO 4-Si-FITC NPs体系对病原体检测具有高灵敏度、良好的选择性和较强的抗干扰能力。
以下进一步以具体实施例说明本申请的Si-FITC比率型荧光纳米探针的合成方法和应用。
实施例1
本申请实施例提供了一种Si-FITC比率型荧光纳米探针的合成方法，包括以下步骤：
S1、将0.404g异硫氰酸荧光素（FITC）加入至5 mL乙醇中，再加入0.2 mL 3-氨基丙基三乙氧基硅烷（APTES），于避光和氮气气氛下搅拌20 h进行共价偶联反应得到第一产物（FITC-APTES）；
S2、将0.184 g柠檬酸三钠溶解在4 mL超纯水中，得到柠檬酸三钠水溶液；
S3、向0.1 mL第一产物中加入1 mL 3-氨基丙基三乙氧基硅烷（APTES）和柠檬酸三钠水溶液，搅拌10 min，然后转移至高压釜中，于200 °C下反应2 h，得到反应产物；
S4、再将反应产物与乙腈按体积比1:4的比例均匀混合，然后于8000 rpm下离心15分钟，弃去上清液后将余下的产物于60 °C下真空干燥，即得Si-FITC比率型荧光纳米探针（Si-FITC NPs）。
将50 mg步骤S4中制备得到的Si-FITC NPs溶解在1 mL去离子水中以制备终浓度为50 mg/mL的Si-FITC NPs溶液，并在4 °C下储存以备用，进行以下性能测试。
实施例2
一种检测核衣壳蛋白的方法，包括以下步骤：
S1、将实施例1中制备得到的Si-FITC比率型荧光纳米探针（Si-FITC NPs）、KMnO 4加入至Tris-HCl缓冲液中，配制得到探针溶液；
S2、将100 mL包被缓冲液（50 mM 的碳酸盐缓冲液，pH 9.6）稀释的捕获抗体（具体为小鼠单克隆抗体）包被于96孔板内，4 °C下过夜孵育，之后将孔板内的液体丢弃，并用洗涤缓冲液（10 mM 的PBS，pH 7.4，含有质量浓度为0.05% Tween 20）洗涤3次；
S3、向孔内加入300 μL质量浓度为2.5%的脱脂奶粉，在37 °C下封闭1 h，再使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S4、再向孔内加入100 μL不同浓度的核衣壳蛋白（浓度分别为0、0.02、0.05、0.1、0.5、1、10、20、40和50 ng/ml），在37 °C下孵育1 h形成捕获抗体-核衣壳蛋白复合物并洗涤3次；
S5、向孔内加入100 μL报告抗体（具体为兔单克隆抗体）在37 °C下孵育1 h形成夹心免疫复合物并使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S6、再向孔内加入100 μL二抗（具体为生物素化山羊抗兔二抗）在37 °C下孵育1 h并使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S7、再向孔内加入100 μL 2.5 μg/ml链霉亲和素，在37 °C下孵育0.5 h，使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S8、再向孔内加入100 μL过氧化氢酶与NHS-dPEG 4-生物素的偶联物，在37 °C下孵育0.5 h，使用洗涤缓冲液（与S2中洗涤缓冲液相同）洗涤3次；
S9、最后向孔内加入包含100 μM H 2O 2的Tris-HCl缓冲液（20 mM pH 7.0），于30 °C下反应1 小时，加入探针溶液检测并记录同步荧光光谱；
其中，核衣壳蛋白为SARS-CoV-2核衣壳-组氨酸重组蛋白、小鼠单克隆抗体为小鼠抗SARS-CoV-2核衣壳单克隆抗体、兔单克隆抗体为兔抗SARS-CoV-2核衣壳单克隆抗体，生物素化山羊抗兔二抗为生物素标记的山羊抗兔IgG。
性能测试
1、实施例1中制备得到的Si-FITC比率型荧光纳米探针（Si-FITC NPs）的性能表征
分别测试实施例1中制备得到的Si-FITC比率型荧光纳米探针（Si-FITC NPs）的紫外-可见光吸收光谱和同步荧光光谱，结果如图2所示。图2中A表示Si NPs（即荧光硅纳米粒子）、Si-FITC NPs（即实施例1中制备得到的Si-FITC比率型荧光纳米探针）、FITC（异硫氰酸荧光素）的紫外-可见吸收光谱图，其中Si NPs浓度1 mg/mL，Si-FITC NPs浓度为1 mg/mL，FITC浓度为5μg/mL，上述不同浓度的物质均为分别将Si NPs、Si-FITC NPs、FITC加入至水中配制得到。图2中B表示实施例1中制备得到的Si-FITC NPs的同步荧光光谱图，其中，λ ex = 350-550 nm, Δλ = 30 nm。
其中，Si NPs的制备方法具体为：
S1、将0.184 g柠檬酸三钠溶解在4 mL超纯水中，得到柠檬酸三钠水溶液；
S3、再将1 mL 3-氨基丙基三乙氧基硅烷（APTES）加入至柠檬酸三钠水溶液，搅拌10 min，然后转移至高压釜中，于200 °C下反应2 h，得到反应产物；
S4、再将反应产物与乙腈按体积比1:4的比例均匀混合，然后于8000 rpm下离心15分钟，弃去上清液后将余下的产物于60 °C下真空干燥，即得Si NPs。
从图2中可以看出，Si-FITC NPs的紫外-可见光吸收光谱在240 nm和488 nm处有两个峰，都归因于FITC；同步荧光光谱显示Si和FITC的荧光峰分别位于415 nm和520 nm，这些结果初步表明FITC与Si偶联。
图3为实施例1中制备得到的Si-FITC NPs的TEM图，从图3中可以看出Si-FITC NPs为尺寸1.6 nm的球形，具有明显的晶体结构。
图4为实施例1中制备得到的Si-FITC NPs和Si NPs的XRD图，从图4中可以看出，Si-FITC NPs位于23º具有明显的衍射峰，并且与Si NPs差距甚微。
图5为实施例1中制备得到的Si-FITC NPs的XPS图，从图5中可以看出，XPS光谱展示了一些元素如Si2p、S2p、C1s、N1s、O1s分布在Si-FITC NPs的固体表面。
图6为实施例1中制备得到的Si-FITC NPs的FTIR光谱图，从图6中可以看出，在500-4000cm -1范围内，1030cm -1处的强吸收峰为Si-O的伸缩振动，1588 cm -1处的吸收峰为NH的弯曲振动，2931cm -1和3356cm -1处的吸收峰是由于OH和NH的伸缩振动，这些结果说明氨基位于Si-FITC NPs的表面。
2、实施例1中制备得到的Si-FITC比率型荧光纳米探针（Si-FITC NPs）的稳定性。
测试实施例1中Si-FITC NPs溶液的光稳定性、pH稳定性和抗干扰能力结果如图7所示。图7中通过350V激发光连续照射2小时来研究Si-FITC NPs的光稳定性，图7中A显示了Si-FITC NPs溶液在λ em = 415 nm和λ em = 520 nm、以及游离FITC在λ em = 520 nm下的光稳定性；图7中B、C为Si-FITC NPs溶液在不同 pH的Tris-HCl 缓冲液中的同步荧光光谱和荧光强度比值；图7中D为Si-FITC NPs溶液在各种离子和氨基酸中（包括100 μM的Mn 2+、Cu 2+、Ca 2+、K +、Zn 2+、NH 4 +、Mg 2+、Glu、Thr、Pro、Ser、Val和Tyr）的抗干扰性。
从图7中A可以看出，Si-FITC NPs溶液在λ em = 415 nm下的荧光强度保留在80 %附近，而Si-FITC NPs在λ em = 520 nm下的荧光强度甚至超过100%，相比之下，游离的FITC仅保持50 %。该结果表明Si-FITC NPs中的Si和FITC均具有良好的光稳定性，且Si-FITC NPs中FITC的光稳定性优于游离的FITC，这可能是由于Si的保护。图7中C显示F 385/F 490随着pH的升高略有上升趋势，这表明Si-FITC NPs具有理想的pH稳定性。图7中D展示了探针的抗干扰能力，Si-FITC NPs在各种离子和氨基酸中荧光保持不变，这表明Si-FITC NPs具有很强的抗干扰能力。
3、KMnO 4、H 2O 2、过氧化氢酶（CAT）对实施例1中制备得到的Si-FITC NPs的影响
为了研究KMnO 4对实施例1中Si-FITC NPs的荧光响应，将4 μL 50 mg/mL的Si-FITC NPs和不同浓度的KMnO 4混合在Tris-HCl缓冲液（20 mM，pH 8.0）中，总体积为200 μL，记录同步荧光光谱，激发波长为350 ~ 600 nm，Δλ = 30 nm；
本研究中的所有荧光测量均使用同步荧光进行。与上述步骤类似，为了研究H 2O 2对Si-FITC NPs的荧光响应，将Tris-HCl缓冲液（20 mM，pH 8.0）中不同浓度的H 2O 2与50μM KMnO 4混合，然后在室温下孵育30分钟，加入4 μL 50 mg/mL Si-FITC NPs并测量同步荧光光谱。由于KMnO 4与H 2O 2的反应比过氧化氢酶（CAT）快，因此过氧化氢酶（CAT）活性检测的第一步是将不同浓度的过氧化氢酶（CAT）与100 μM H 2O 2反应30分钟，再与50 μM KMnO 4混合30分钟，最后加入Si-FITC NPs和记录同步荧光光谱。测试结果如图8所示。
图8中A表示Si-FITC NPs响应不同浓度KMnO 4的同步荧光光谱，B表示Si-FITC NPs的荧光强度比与KMnO 4浓度之间的关系；图8中C表示经50 μM KMnO 4处理的Si-FITC NPs响应不同浓度的H 2O 2的同步荧光光谱，D表示 Si-FITC NPs的荧光强度比与H 2O 2浓度之间的关系；图8中E表示Si-FITC NPs响应不同浓度CAT的同步荧光光谱；F表示Si-FITC NPs的荧光强度比与 CAT 浓度之间的关系。
从图8中A~B可以看出，随着KMnO 4浓度的增加，Si-FITC NPs中Si的荧光强度逐渐猝灭，FITC的荧光强度增加。因此，Si-FITC NPs探针的荧光强度比（F 385/F 490）与KMnO 4浓度呈负相关。
由于H 2O 2与KMnO 4能发生氧化还原反应，通过加入一系列浓度梯度的H 2O 2可以逐渐恢复Si-FITC NPs被KMnO 4猝灭的荧光强度比值。从图8中C~D中可以看出随着H 2O 2浓度的增加，Si-FITC NPs的荧光光谱逐渐恢复到原始状态，F 385/F 490与H 2O 2浓度呈正相关，H 2O 2是ELISA中过氧化氢酶CAT或辣根过氧化物酶HRP的底物。
为了探究KMnO 4猝灭Si-FITC NPs的机制，表征了紫外-可见吸收光谱和荧光寿命衰减曲线，结果如图9~10所示。
图9中Si-FITC NPs、KMnO 4、Si-FITC NPs + KMnO 4分别表示不同物质的紫外-可见吸收光谱图，其中，Si-FITC NPs浓度为1 mg/mL、KMnO 4浓度为75 μM、Si-FITC NPs + KMnO 4表示将1mg/mL的Si-FITC NPs和75 μM的KMnO 4混合在Tris-HCl缓冲液中的紫外-可见吸收光谱图。
图10中Si-FITC NPs、Si-FITC NPs + KMnO 4分别表示不同物质的荧光寿命衰减曲线，其中，Si-FITC NPs浓度为1 mg/mL、Si-FITC NPs + KMnO 4表示将1mg/mL的Si-FITC NPs和75 μM的KMnO 4混合在Tris-HCl缓冲液中的荧光寿命衰减曲线图。
从图9中可以看出，当Si-FITC NPs与KMnO 4混合时，紫外-可见吸收峰几乎没有变化，这表明KMnO 4与Si-FITC NPs不发生化学反应。从图10中可以看出，在KMnO 4存在下，Si-FITC NPs的荧光寿命显著降低，这证明在Si和KMnO 4间可能发生能量转移。综合上述结果，KMnO 4的猝灭机理为动态猝灭。
由于H 2O 2与KMnO 4能发生氧化还原反应，通过加入一系列浓度梯度的H 2O 2可以逐渐恢复Si-FITC NPs被KMnO 4猝灭的荧光强度比值。从图8中C~D中可以看出随着H 2O 2浓度的增加，Si-FITC NPs的荧光光谱逐渐恢复到原始状态，F 385/F 490与H 2O 2浓度呈正相关，H 2O 2是ELISA中过氧化氢酶CAT或辣根过氧化物酶HRP的底物。
过氧化氢酶CAT能有效地催化H 2O 2分解，其中Si-FITC NPs中Si的荧光被KMnO 4猝灭，加入H 2O 2后，可消耗KMnO 4使Si-FITC NPs的荧光免受猝灭，如在上述体系中加入过氧化氢酶CAT后，可消耗H 2O 2，过量的KMnO 4使Si-FITC NPs的荧光强度比发生变化。为了获得最高的信噪比，需要对KMnO 4和H 2O 2的用量进行优化。选择50、100、200μM的KMnO 4浓度，并伴随了一系列H 2O 2浓度；图11为Si-FITC NPs的荧光强度比值与H 2O 2浓度之间的关系，KMnO 4浓度分别为50、100 或200 μM。
从图11中可以看出，KMnO 4浓度为50 μM时，Si-FITC NPs探针的荧光强度比值能在较小的H 2O 2浓度下最快恢复到平台期。因此，最终选50 μM KMnO 4和100μM H 2O 2进行过氧化氢酶CAT活性测定。为了使过氧化氢酶CAT发挥最佳酶活，优化了CAT的反应的pH。图12为50 μM KMnO 4、100 μM H 2O 2和300 U/L浓度下CAT处理的Si-FITC NPs在不同pH值下荧光强度比的关系。从图12中可以看出，当Tris-HCl的pH值为 7时，过氧化氢酶CAT的活性最高。在此条件下，加入一系列浓度梯度的CAT，如图8中E~F所示，Si-FITC NPs荧光光谱表明中Si的荧光逐渐猝灭，F 385/F 490与CAT浓度呈负相关。
4、过氧化氢酶与NHS-dPEG 4-生物素的偶联物验证
过氧化氢酶CAT和NHS-dPEG 4-生物素以摩尔比1:100混合在1 mL PBS (10 mM, pH 8.0) 中，并37 °C下振荡4小时以获得过氧化氢酶与NHS-dPEG 4-生物素的偶联物（记为biotin-CAT）。为了验证CAT和NHS-dPEG 4-生物素的结合，使用带有链霉亲和素的磁珠与biotin-CAT在37 °C的Tris-HCl缓冲液中反应30分钟，分离然后biotin-CAT。通过H 2O 2-KMnO 4-Si-FITC NPs体系测定上清液的CAT活性，结果如图13所示。
图13中1为空白对照组，2为biotin-CAT与H 2O 2-KMnO 4-Si-FITC NPs混合组，3为biotin-CAT用链霉亲和素的磁珠分离后上清中加入H 2O 2-KMnO 4-Si-FITC NPs混合组。从图13中可以看出，2组F 385/F 490显著降低，而3组F 385/F 490与1组（未加入biotin-CAT的空白对照组）相似，表明CAT 与NHS-dPEG 4-生物素偶联成功。
5、核衣壳蛋白的检测
在核衣壳蛋白检测过程中，biotin-CAT通过链霉亲和素的中间作用连接在二抗上，从而固定在96孔板，不同于1.5 mL离心管中的游离体系；对核衣壳蛋白检测过程中，对biotin-CAT和H 2O 2的反应温度和时间进行优化。具体的参考实施例2中的方法，控制步骤S8中反应温度分别为25 °C、30 °C、37 °C，反应时间均为1 h，其中H 2O 2浓度为100 μM，探针溶液中KMnO 4浓度为50 μM，核衣壳蛋白浓度为50 ng/mL，biotin-CAT，1:100（biotin-CAT的稀释比例为1:100 ）。测试得到的Si-FITC NPs的荧光强度比与反应温度之间关系如图14所示。从图14中可以看出，荧光强度比的最大差值出现在30 °C。具体的参考实施例2中的方法，控制步骤S8中反应温度为30℃，反应时间分别为0.5 h、1 h和2 h，其中H 2O 2浓度为100 μM，探针溶液中KMnO 4浓度为50 μM，核衣壳蛋白浓度为50 ng/mL，biotin-CAT，1:100（biotin-CAT的稀释比例为1:100 ）。测试得到的Si-FITC NPs的荧光强度比与反应时间之间关系如图15所示。从图15中可以看出，荧光强度比的最大差值出现在1 h。综上，Si-FITC NPs荧光强度比的最大差值分别出现在30 °C和1 h，这是H 2O 2和biotin-CAT的最佳反应条件。在H 2O 2和biotin-CAT的最佳反应条件下，按照实施例2中的方法，测试不同浓度的核衣壳蛋白下Si-FITC NPs的同步荧光光谱，其中H 2O 2浓度为100 μM，探针溶液中KMnO 4浓度为50 μM，Δλ = 30 nm，测试结果如图16所示。从图16中A可以看出，Si-FITC NPs中Si的荧光强度随着核衣壳蛋白浓度的增加而逐渐降低。图16中B可以看出，Si-FITC NPs的荧光强度比在0.02-50 ng/mL的核衣壳蛋白之间呈线性关系，检出限为0.003 ng/mL。
为了进一步验证本申请的核衣壳蛋白检测的选择性，测试了低浓度目标核衣壳蛋白和高浓度其他非特异性蛋白。具体的，按照上述实施例2中的方法，分别检测20 ng/mL核衣壳蛋白和200 ng/mL其他竞争组分的选择性，其中H 2O 2浓度为100 μM，探针溶液中KMnO 4浓度为50 μM，测试结果如图17所示。图17中blank为空白对照组、N protein为核衣壳蛋白、S protein为新冠刺突蛋白、BSA表示牛血清蛋白，其中，加入的S protein浓度为200 ng/mL、加入的BSA浓度为200 ng/mL。
从图17中可以看出，只有核衣壳蛋白组的荧光强度比显著降低，表明本申请的检测方法对目标物具有高度特异性。
6、游离异硫氰酸荧光素（FITC）对KMnO 4的响应
图18为游离异硫氰酸荧光素（FITC）响应不同浓度KMnO 4的荧光光谱，其中FITC浓度为20 ng/mL，λ ex = 480 nm，λ em =500-650 nm。
从图18中可以看出，随着KMnO 4浓度的增加，荧光强度逐渐降低。
7、人血清样品中核衣壳蛋白的检测
按照上述实施例2中的方法，将含有不同浓度核衣壳蛋白的人血清替代纯核衣壳蛋白进行测试，并将F 385/F 490的结果代入拟合方程计算核衣壳蛋白的检测浓度。结果如下表1所示。
表1-人血清中核衣壳蛋白的检测结果
Figure dest_path_image001
从表1中可以看出，添加5 ng/mL和40 ng/mL的人血清中核衣壳蛋白测得浓度分别为5.32 ng/mL和35.18 ng/mL，回收率在理想范围内。结果表明，
采用本申请的检测方法在真实人血清样本中表现出较高的核衣壳蛋白评估能力，并具有临床检测SARS-CoV-2的潜力。
以上所述仅是本申请的部分实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。

Claims (10)

  1. 一种Si-FITC比率型荧光纳米探针的合成方法，其特征在于，包括以下步骤：
    将异硫氰酸荧光素加入至醇溶剂中，然后加入氨基硅烷偶联剂，于避光和惰性气氛下进行共价偶联反应得到第一产物；
    向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h，即得Si-FITC比率型荧光纳米探针。
  2. 如权利要求1所述的Si-FITC比率型荧光纳米探针的合成方法，其特征在于，所述醇溶剂包括甲醇、乙醇、异丙醇中的至少一种；所述氨基硅烷偶联剂包括3-氨基丙基三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨丙基甲基二乙氧基硅烷中的至少一种；所述还原剂包括柠檬酸三钠、抗坏血酸、硼氢化钠中的至少一种。
  3. 如权利要求1所述的Si-FITC比率型荧光纳米探针的合成方法，其特征在于，向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h后还包括：将反应后的产物与乙腈混合后离心分离，干燥即得Si-FITC比率型荧光纳米探针。
  4. 如权利要求3所述的Si-FITC比率型荧光纳米探针的合成方法，其特征在于，反应后的产物与乙腈的体积比为1:(3~5)，离心速率为7500~8500 rpm、离心时间为10~20 min。
  5. 如权利要求1所述的Si-FITC比率型荧光纳米探针的合成方法，其特征在于，将异硫氰酸荧光素加入至醇溶剂中，然后加入氨基硅烷偶联剂，于避光和惰性气氛下进行共价偶联反应得到第一产物的步骤中，所述异硫氰酸荧光素、所述醇溶剂、所述氨基硅烷偶联剂的质量体积比为(0.3~0.5) g:(3~7)mL:(0.1~0.3) mL；
    向第一产物中加入还原剂、氨基硅烷偶联剂搅拌混合后于190~210 °C下反应1~3 h的步骤中，所述还原剂、所述第一产物、所述氨基硅烷偶联剂的质量体积比为(0.1~0.3) g:(0.05~0.15) mL:(0.5~1.5) mL。
  6. 一种Si-FITC比率型荧光纳米探针，其特征在于，采用如权利要求1~5任一所述的制备方法制备得到。
  7. 如权利要求6所述的Si-FITC比率型荧光纳米探针在新冠病毒抗原检测中的应用。
  8. 如权利要求7所述的应用，其特征在于，包括以下步骤：
    将权利要求6中的Si-FITC比率型荧光纳米探针、KMnO 4加入至Tris-HCl缓冲液中，配制得到探针溶液；
    将包被缓冲液稀释的捕获抗体包被于孔板内，孵育，之后将孔板内的液体丢弃，并用洗涤缓冲液洗涤；
    向孔内加入脱脂奶粉，封闭后，使用洗涤缓冲液洗涤；
    再向孔内加入核衣壳蛋白，孵育形成捕获抗体-核衣壳蛋白复合物；
    向孔内加入报告抗体孵育形成夹心免疫复合物；
    再向孔内加入二抗孵育；
    再向孔内加入链霉亲和素，孵育，再加入过氧化氢酶与NHS-dPEG 4-生物素的偶联物；
    再向孔内加入含有H 2O 2的Tris-HCl缓冲液，反应后加入探针溶液检测并记录同步荧光光谱。
  9. 如权利要求8所述的应用，其特征在于，所述过氧化氢酶与NHS-dPEG 4-生物素的偶联物的制备方法，包括以下步骤：
    将过氧化氢酶与NHS-dPEG 4-生物素混合后加入至PBS缓冲液中，振荡后即得过氧化氢酶与NHS-dPEG 4-生物素的偶联物。
  10. 如权利要求8所述的应用，其特征在于，所述包被缓冲液为pH为9.6的碳酸盐缓冲液；洗涤缓冲液为pH为7.4的含有Tween 20的PBS溶液；所述Tris-HCl缓冲液的pH为7.0。
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