A preparation method for in-situ generation of carbon dots at nano-interfaces and its electrochemiluminescence analysis application

Abstract

The present application relates to the fields of chemiluminescence technology and biomedicine technology, and more specifically, to a method for preparing carbon dots generated in situ at a nano-interface and its electrochemiluminescence analysis application. The method for preparing carbon dots generated in situ at a nano-interface comprises the following steps: S1, preparing g-C3N4; S2, collecting a dispersion of g-C3N4NSs; S3, reacting to synthesize g-C3N4@CDs. The electrochemiluminescence analysis application of carbon dots generated in situ at a nano-interface uses the above-mentioned g-C3N4@CDs to prepare a g-C3N4@CDs-Ab2 signal tag and an ECL immunosensor; and constructs a sandwich-type ECL immunosensor for detecting the spike protein of the new coronavirus (SARS-CoV-2). In this application, two-dimensional graphene-phase carbon nitride nanosheets (g-C3N4NSs) are used as carriers to in situ generate carbon dots (CDs) on the surface of the g-C3N4NSs carriers to prepare carbon dot nanocomposites (g-C3N4@CDs) with uniform distribution of carbon dots on the surface of the g-C3N4NSs carriers. In the presence of the co-reactant H2O2, the obtained carbon dot nanocomposites exhibit efficient ECL performance.

Description

Technical Field

[0001] The present application relates to the fields of electrochemiluminescence technology and biomedicine technology, and more specifically, to a method for preparing carbon dots generated in situ at a nano-interface and its electrochemiluminescence analysis application. Background Art

[0002] Electrochemiluminescence (ECL) analysis has become a powerful analytical tool in clinical testing due to its high controllability, high sensitivity, and low background. Carbon dots (also known as carbon quantum dots), as an emerging class of luminescent materials, show broad application prospects in ECL bioanalysis due to their low cost, easy availability, and low toxicity. However, since the luminescence properties of carbon dots depend on size, aggregation of carbon dots can significantly reduce ECL efficiency, posing a significant challenge to the preparation of carbon dot-based nanocomposites.

[0003] In situ generation of nano-interfaces refers to a method for preparing nanocomposites by directly generating small-particle nanomaterials in situ on the surface of a nanocarrier. This method reduces the surface area of ​​the solid-liquid interface, and the surface tension that needs to be overcome during the nanoparticle generation process is smaller. Therefore, it is easier to generate uniformly distributed small-particle nanoparticles. At the same time, the nanoparticles are fixed on the carrier surface and can avoid agglomeration without the need for additional protective agents, maintaining a dispersed state at the nano-interface. Therefore, the nanoparticles can remain exposed to the greatest extent, thereby ensuring the efficiency of electron transfer.

[0004] In summary, the in situ generation of carbon dots at nanoscale interfaces effectively avoids the drawbacks of traditional synthesis-enrichment nanocomplex preparation methods that often reduce carbon dot ECL efficiency. This holds promise for preparing highly efficient ECL carbon dot nanocomplexes, potentially overcoming the key technical bottleneck limiting the application of carbon dot ECL systems in highly sensitive bioanalysis. Therefore, a method for the in situ generation of carbon dots at nanoscale interfaces remains to be developed. Summary of the Invention

[0005] In order to improve the problem of reduced ECL efficiency of carbon dots caused by the synthesis-enrichment nanocomposite preparation method, and thus facilitate the preparation of high-efficiency ECL carbon dot nanocomposites, the present application provides a preparation method for in situ generation of carbon dots at a nanointerface and its electrochemiluminescence analysis application.

[0006] In a first aspect, the present application provides a method for preparing in-situ generated carbon dots at a nano-interface, which adopts the following technical solution:

[0007] A method for preparing carbon dots generated in situ at a nanometer interface comprises the following steps:

[0008] S1. Preparation of g-C3N4: Melamine was added to a crucible and heated in a muffle furnace to obtain g-C3N4;

[0009] S2. Collecting g-C3N4NSs dispersion: dispersing g-C3N4 in deionized water, performing continuous ultrasonic treatment and then centrifuging, collecting the upper layer of g-C3N4 nanosheet dispersion, i.e., g-C3N4NSs dispersion;

[0010] S3. Reaction synthesis of g-C3N4@CDs: Take a g-C3N4NSs dispersion, add citric acid, and continue stirring to dissolve to obtain a mixed solution; then drop hydrazine hydrate into the mixed solution, stir evenly, and transfer it to a hydrothermal reactor for heating treatment. After cooling to room temperature, centrifuge, and then wash and dry to obtain a nanocomposite with carbon dots in situ generated on the surface of g-C3N4NSs, namely g-C3N4@CDs.

[0011] Preferably, the washing treatment in step S3 is washing three times with phosphate buffer, deionized water and ethanol in sequence.

[0012] Preferably, in the above-mentioned method for preparing carbon dots by in-situ generation at nano-interface, the specific process conditions of each step are as follows:

[0013] S1. Preparation of g-C3N4: 15 g of melamine was added to an alumina crucible and heated at 600 °C for 2 h at a heating rate of 5 °C / min in a muffle furnace to obtain g-C3N4.

[0014] S2. Collecting the g-C3N4NSs dispersion: Disperse g-C3N4 in deionized water at a concentration of 1 mg / mL, continuously sonicate for 16 h, and then centrifuge at 6000 rpm for 10 min to collect the upper layer of g-C3N4 nanosheet dispersion, i.e., the g-C3N4NSs dispersion.

[0015] S3. Reaction synthesis of g-C3N4@CDs: Take 10 mL of g-C3N4NSs dispersion, add 0.21 g of citric acid, continue stirring to dissolve, and obtain a mixed solution; then drop 0.1 mL of hydrazine hydrate into the mixed solution, stir evenly, and transfer it to a polytetrafluoroethylene-lined stainless steel reactor, heat at 180°C for 3 h, cool to room temperature, and centrifuge at 12000 rpm for 10 min. Wash and treat in sequence, and after drying, obtain a nanocomposite with carbon dots in situ generated on the surface of g-C3N4NSs, namely g-C3N4@CDs.

[0016] In a second aspect, the present application provides an electrochemiluminescence analysis application of nano-interface in situ generated carbon dots, using the above-prepared nano-interface in situ generated carbon dots, i.e. g-C3N4@CDs, to prepare g-C3N4@CDs-Ab2 signal tags and prepare ECL immunosensors;

[0017] A sandwich ECL immunosensor for detecting the spike protein of the new coronavirus (SARS-CoV-2) was constructed using the prepared g-C3N4@CDs-Ab2 signal tag and ECL immunosensor.

[0018] Preferably, the preparation of the g-C3N4@CDs-Ab2 signal tag comprises the following steps:

[0019] (1) Disperse g-C3N4@CDs in MES buffer containing EDC and NHS and shake to mix; after centrifugation, take the precipitate and disperse it in Na2CO3 buffer;

[0020] (2) Add SARS-COV-2 spike protein detection antibody (Ab2), shake and mix, and then centrifuge to obtain the precipitate; disperse the precipitate in PBS, add BSA solution and shake and mix, and then centrifuge to obtain the precipitate; wash the precipitate with PBS and disperse it in PBS to prepare the g-C3N4@CDs-Ab2 signal tag.

[0021] Preferably, the specific process conditions in the preparation steps of the g-C3N4@CDs-Ab2 signal tag are as follows:

[0022] (1) 2.0 mg g-C3N4@CDs was dispersed in 1.0 mL MES buffer (0.1 mol / L, pH 6.0) containing 60 mmol / L EDC and 40 mmol / L NHS, and the mixture was shaken at 4°C for 2 h. The precipitate was collected by centrifugation at 10,000 rpm for 5 min and then dispersed in 1.0 mL Na2CO3 buffer (pH 9.5).

[0023] (2) Add 10 μL of SARS-COV-2 spike protein detection antibody (Ab2), shake and mix at 4°C for 12 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate; disperse the precipitate in 1 mL of PBS (10 mmol / L, pH 7.4), add 100 μL of BSA solution (1%, w / v), shake and mix at 4°C for 1 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate; wash the precipitate three times with PBS (10 mmol / L, pH 7.4) and disperse it in 500 μL of PBS (10 mmol / L, pH 7.4) to prepare the g-C3N4@CDs-Ab2 signal tag.

[0024] Preferably, the preparation of the ECL immunosensor comprises the following steps:

[0025] (1) Polishing the glassy carbon electrode to obtain a clean electrode surface, immersing it in a glutamic acid (Glu) solution containing potassium hydrogen phthalate, and performing constant potential deposition treatment to deposit a carboxyl-rich Glu film on the electrode surface to obtain a carboxyl-functionalized glassy carbon electrode;

[0026] (2) Immerse the carboxyl-functionalized glassy carbon electrode in MES buffer containing EDC and NHS, react at room temperature, rinse with PBS, and then dropwise add SARS-COV-2 spike protein capture antibody (Ab1) and incubate at room temperature to allow Ab1 to be covalently cross-linked on the electrode surface to obtain an Ab1-modified electrode;

[0027] (3) BSA solution was added dropwise to the Ab1-modified electrode surface for incubation, and then rinsed with PBS to prepare the ECL immunosensor.

[0028] Preferably, the specific process conditions in the preparation steps of the ECL immunosensor are as follows:

[0029] (1) Polish the glassy carbon electrode to obtain a clean electrode surface, immerse it in a glutamic acid (Glu) solution (0.1 mol / L, pH 5.0) containing 10 mmol / L potassium hydrogen phthalate, and deposit it at a constant potential (-1.9 V) for 60 s to deposit a carboxyl-rich Glu film on the electrode surface to obtain a carboxyl-functionalized glassy carbon electrode;

[0030] (2) The carboxyl-functionalized glassy carbon electrode was immersed in MES buffer (0.1 mol / L, pH 6.0) containing 60 mmol / L EDC and 40 mmol / L NHS. After reacting at room temperature for 30 min, it was rinsed with PBS (pH 7.4). Then, 10 μL of SARS-COV-2 spike protein capture antibody (Ab1) was added dropwise and incubated at room temperature for 30 min to allow Ab1 to be covalently cross-linked on the electrode surface.

[0031] (3) BSA solution (0.1%) was added dropwise to the electrode surface and incubated for 1 h, followed by rinsing with PBS (pH 7.4) to prepare the ECL immunosensor.

[0032] Preferably, constructing a sandwich ECL immunosensor for detecting the spike protein of the new coronavirus (SARS-CoV-2) includes the following steps: adding different concentrations of SARS-CoV-2 spike protein to the ECL immunosensor interface and incubating, rinsing with PBS, and then adding the g-C3N4@CDs-Ab2 signal label and incubating; after rinsing to remove the unbound signal label, placing it in PBS containing H2O2, applying a voltage of 0.2~0.8 V for cyclic scanning, and measuring the ECL signal.

[0033] Preferably, the specific process conditions in the step of constructing the sandwich-type ECL immunosensor for detecting the spike protein of the new coronavirus (SARS-CoV-2) are as follows:

[0034] 10 μL of SARS-CoV-2 spike protein at different concentrations was added to the sensor interface and incubated for 1 hour. After rinsing with PBS (pH 7.4), the g-C3N4@CDs-Ab2 signal label was added and incubated for 1 hour. After rinsing to remove the unbound signal label, the sensor was placed in PBS (pH 7.4) containing 10 mmol / L H2O2, and a voltage of 0.2~0.8 V was applied for cyclic scanning to measure the ECL signal.

[0035] In summary, this application has at least the following beneficial effects:

[0036] This application utilizes two-dimensional graphene-phase carbon nitride nanosheets (g-C3N4NSs) as a carrier to in situ generate carbon dots on the surface of g-C3N4NSs. This results in a carbon dot nanocomposite (g-C3N4@CDs) with uniform carbon dot distribution on the g-C3N4NSs surface. In the presence of the co-reactant H2O2, the resulting carbon dot nanocomposite exhibits efficient ECL performance. The prepared carbon dot nanocomposite (g-C3N4@CDs) with efficient ECL performance is labeled with a detection antibody (Ab2) as an ECL signal tag to construct a sandwich ECL immunosensor for detecting the spike protein of the novel coronavirus (SARS-CoV-2). BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the ECL immunosensor for SARS-CoV-2 spike protein detection. (A) Preparation process of the g-C3N4@CDs-Ab2 signal tag.

[0038] Figure 2 : Transmission electron microscopy images of g-C3N4 nanosheets and g-C3N4@CDs nanocomposites;

[0039] Figure 2 A is the transmission electron microscope image of g-C3N4 nanosheets;

[0040] Figure 2 B is a high-angle annular dark field-scanning transmission electron microscopy image (HAADF-STEM) of g-C3N4 nanosheets;

[0041] Figure 2 C is the transmission electron microscope image of g-C3N4@CDs nanocomposite material. Figure 2 The inset in Figure C is a high-definition transmission electron microscopy image of the g-C3N4@CDs nanocomposite;

[0042] Figure 2 D is the high-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) image of g-C3N4@CDs nanocomposite.

[0043] Figure 3 : UV-visible absorption spectra of CDs, g-C3N4 nanosheets and g-C3N4@CDs.

[0044] Figure 4 : Differential charge density distribution of g-C3N4@CDs.

[0045] Figure 4 A is the top view of the differential charge density distribution of g-C3N4@CDs;

[0046] Figure 4 B is the side view of the differential charge density distribution of g-C3N4@CDs.

[0047] Figure 5 :Preparation and characterization of ECL immunosensor and feasibility analysis of detection of SARS-COV-2 spike protein;

[0048] Figure 5 A is the electrode in 5mM [Fe(CN)6] 3- / 4- Electrochemical impedance spectroscopy at different modification stages in solution;

[0049] Figure 5 B is the ECL signal response of the ECL immunosensor in the presence and absence of the target SARS-COV-2 spike protein.

[0050] Figure 6 : Performance analysis of ECL immunosensor for detecting SARS-COV-2 spike protein.

[0051] Figure 6 A is the ECL signal response of different concentrations of SARS-COV-2 spike protein;

[0052] Figure 6 B is the calibration curve of ECL signal response and logarithm of SARS-CoV-2 spike protein concentration.

[0053] Figure 7 : Specificity and stability of ECL immunosensor.

[0054] Figure 7 A is the ECL signal response of the ECL immunosensor for the specific detection of SARS-CoV-2 and other interferents;

[0055] Figure 7 B is the ECL signal response of 10 consecutive scans when the ECL immunosensor detects SARS-CoV-2 spike protein (100 fg / mL). DETAILED DESCRIPTION

[0056] The present application is further described in detail below with reference to the embodiments.

[0057] In a first aspect, the present invention discloses a method for preparing carbon dots by in-situ generation at a nano-interface, comprising the following steps:

[0058] S1. Preparation of g-C3N4: 15 g of melamine was added to an alumina crucible and heated at 600 °C for 2 h at a heating rate of 5 °C / min in a muffle furnace to obtain g-C3N4.

[0059] S2. Collecting the g-C3N4NSs dispersion: Disperse g-C3N4 in deionized water at a concentration of 1 mg / mL, continuously sonicate for 16 h, and then centrifuge at 6000 rpm for 10 min to collect the upper layer of g-C3N4 nanosheet dispersion, i.e., the g-C3N4NSs dispersion.

[0060] S3. Reaction synthesis of g-C3N4@CDs: Take 10 mL of g-C3N4NSs dispersion, add 0.21 g of citric acid, continue stirring to dissolve, and obtain a mixed solution; then drop 0.1 mL of hydrazine hydrate into the mixed solution, stir evenly, and transfer it to a polytetrafluoroethylene-lined stainless steel reactor, heat at 180°C for 3 h, cool to room temperature, centrifuge at 12000 rpm for 10 min, and wash three times with phosphate buffer (PBS, 10 mmol / L, pH 7.4), deionized water, and ethanol in sequence to obtain a nanocomposite with carbon dots in situ generated on the surface of g-C3N4 nanosheets, namely g-C3N4@CDs; after vacuum drying, store in a brown reagent bottle at 4°C for later use.

[0061] In a second aspect, the embodiments of the present application disclose an electrochemiluminescence analysis application of in situ generated carbon dots at a nano-interface. The carbon dots prepared at the nano-interface in situ, i.e., g-C3N4@CDs, are used to prepare g-C3N4@CDs-Ab2 signal tags and an ECL immunosensor. A sandwich ECL immunosensor for detecting the spike protein of the novel coronavirus (SARS-CoV-2) is constructed using the prepared g-C3N4@CDs-Ab2 signal tags and the ECL immunosensor.

[0062] The preparation of g-C3N4@CDs-Ab2 signal tag includes the following steps:

[0063] (1) 2.0 mg g-C3N4@CDs was dispersed in 1.0 mL MES buffer (0.1 mol / L, pH 6.0) containing 60 mmol / L EDC and 40 mmol / L NHS, and the mixture was shaken at 4°C for 2 h. The precipitate was collected by centrifugation at 10,000 rpm for 5 min and then dispersed in 1.0 mL Na2CO3 buffer (pH 9.5).

[0064] (2) Add 10 μL of SARS-COV-2 spike protein detection antibody (Ab2), shake and mix at 4°C for 12 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate; disperse the precipitate in 1 mL of PBS (10 mmol / L, pH 7.4), add 100 μL of BSA solution (1%, w / v), shake and mix at 4°C for 1 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate; wash the precipitate three times with PBS (10 mmol / L, pH 7.4) and disperse it in 500 μL of PBS (10 mmol / L, pH 7.4) to prepare the g-C3N4@CDs-Ab2 signal tag, which was stored in a brown reagent bottle at 4°C for later use.

[0065] The preparation of ECL immunosensor includes the following steps:

[0066] (1) The glassy carbon electrode was polished to obtain a clean electrode surface, and then immersed in a glutamic acid (Glu) solution (0.1 mol / L, pH 5.0) containing 10 mmol / L potassium hydrogen phthalate. The electrode was deposited at a constant potential (-1.9 V) for 60 s to deposit a carboxyl-rich Glu film on the electrode surface to obtain a carboxyl-functionalized glassy carbon electrode.

[0067] (2) The carboxyl-functionalized glassy carbon electrode was immersed in MES buffer (0.1 mol / L, pH 6.0) containing 60 mmol / L EDC and 40 mmol / L NHS. After reacting at room temperature for 30 min, it was rinsed with PBS (pH 7.4). Then, 10 μL of SARS-COV-2 spike protein capture antibody (Ab1) was added dropwise and incubated at room temperature for 30 min to allow Ab1 to be covalently cross-linked on the electrode surface.

[0068] (3) Add BSA solution (0.1%) to the electrode surface and incubate for 1 h, then rinse with PBS (pH 7.4) to prepare the ECL immunosensor, which was stored at 4°C until use.

[0069] The construction of a sandwich ECL immunosensor for detecting the spike protein of the novel coronavirus (SARS-CoV-2) includes the following steps:

[0070] 10 μL of SARS-CoV-2 spike protein at different concentrations was added to the sensor interface and incubated for 1 hour. After rinsing with PBS (pH 7.4), the g-C3N4@CDs-Ab2 signal label was added and incubated for 1 hour. After rinsing to remove the unbound signal label, the sensor was placed in PBS (pH 7.4) containing 10 mmol / L H2O2, and a voltage of 0.2~0.8 V was applied for cyclic scanning to measure the ECL signal.

[0071] The detection principle of the ECL immunosensor constructed in this application is as follows Figure 1 Figure A shows a method for detecting and capturing the SARS-CoV-2 spike protein. First, a carboxyl-functionalized glassy carbon electrode was cross-linked with Ab1 to recognize and capture the SARS-CoV-2 spike protein. Next, carbon dots were in situ generated on the surface of the g-C3N4 nanocarrier to create a carbon dot nanocomposite (g-C3N4@CDs). This carbon dot nanocomposite was then cross-linked with Ab2 to create an ECL signal tag (g-C3N4@CDs-Ab2). When the SARS-CoV-2 spike protein is present, Ab1 on the electrode surface can recognize and capture it. Furthermore, Ab2 on the signal tag can simultaneously recognize it, forming a sandwich structure and improving detection specificity. Finally, signal amplification is achieved based on the efficient immobilization of CDs in the g-C3N4@CDs nanocomposite and the ECL enhancement of the CDs / H2O2 system by g-C3N4. The ECL signal is proportional to the concentration of the SARS-CoV-2 spike protein.

[0072] Results and Discussion

[0073] The morphologies of g-C3N4 nanosheets and g-C3N4@CDs nanocomposites were characterized by transmission electron microscopy (TEM). Figure 2A is a high-resolution transmission electron microscopy image of g-C3N4 nanosheets, from which it can be observed that g-C3N4 presents a relatively flat sheet structure. Figure 2 B is a high-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) image of g-C3N4 nanosheets, showing uniform brightness of the g-C3N4 sheets. Figure 2 C is the high-resolution transmission electron microscope image of g-C3N4@CDs. It can be observed that a large number of particles with a diameter of about 3 nm are evenly distributed on the g-C3N4 layer structure, indicating that the method of in situ generation of CDs on the surface of g-C3N4 nanosheets can efficiently enrich CDs without particle aggregation. HAADF-STEM image ( Figure 2 In D), CDs and g-C3N4 are clearly distinguished, indicating that there is a significant difference in the atomic arrangement between CDs and g-C3N4.

[0074] The interaction between CDs and g-C3N4 nanosheets in g-C3N4@CDs nanocomposites was analyzed by UV-visible absorption spectroscopy. Figure 3 As shown in the figure, CDs exhibit characteristic absorption peaks at 210 nm and 330 nm, respectively, which are attributed to π-π* and n-π* transitions, and the absorbance is low in the long-wave region, reflecting the characteristics of the small conjugated system of CDs and the presence of structures such as C=O or C=N. Similarly, g-C3N4 nanosheets have two characteristic absorption peaks attributed to π-π* (210 nm) and n-π* (320 nm) transitions, but g-C3N4 also has a certain absorbance in the long-wave region due to its large-scale conjugated structure. When CDs are in situ generated on the g-C3N4 surface to form a g-C3N4@CDs nanocomposite, the characteristic absorption peaks of the π-π* (210 nm) and n-π* (320 nm) transitions show significant broadening, revealing the π-π interaction between CDs and g-C3N4.

[0075] Density functional theory (DFT) was used to investigate the ECL signal enhancement mechanism of g-C3N4 in the g-C3N4@CDs nanocomposite on the CDs / H2O2 system. First, a computable and traceable model with carboxyl, hydroxyl, hydrazide and pyrene cores was used to simulate the surface functional groups and graphite core of CDs. Figure 4As shown, the model CDs are non-covalently positioned nearly parallel to the model g-C3N4 sheet structure, accompanied by a significant charge rearrangement. Differential charge density analysis reveals an increase in the positive charge density around the hydrazide groups of the CDs at the g-C3N4 interface. The coreactant H2O2 generates active superoxide anions under a potential sweep of 0.2-0.8V. Because the superoxide anion is negatively charged, and the increased positive charge density of the hydrazide groups of the CDs at the g-C3N4 interface significantly enhances the interaction between the superoxide anion and the hydrazide groups on the CDs' surface, thereby increasing the reaction rate and achieving ECL signal enhancement.

[0076] Characterization of the ECL biosensor fabrication process

[0077] Electrochemical impedance spectroscopy (EIS) was used to investigate the effects of various electrode modification steps on the [Fe(CN)6] 4- / 3- The results were shown in the following table: Figure 5 Figure 1 shows the impedance curve of the bare GCE (curve a), which exhibits a small semicircle with Ret < 50 Ω. After Glu was deposited on the GCE surface, the semicircle of the impedance curve significantly widened (curve b), indicating that the Glu deposition layer increased the impedance value. Further cross-linking of Ab1 on the modified electrode surface significantly increased the impedance value (curve c). After the target SARS-CoV-2 spike protein was added to the modified electrode surface and incubated, the impedance value continued to increase (curve d). Finally, g-C3N4@CDs-Ab2 was added to the modified electrode and incubated, resulting in a further increase in the impedance value (curve e), confirming the successful fabrication of the ECL immunosensor.

[0078] Feasibility verification of ECL immunosensor for detecting SARS-COV-2 spike protein

[0079] The feasibility of biosensor detection of target SARS-COV-2 spike protein was tested using ECL characterization, and the results are as follows Figure 5 As shown in Figure B, when the prepared biosensor was used to detect a blank control, an ECL signal intensity of approximately 392 a.u. was obtained (negative, black curve). This signal originates from nonspecific adsorption of the g-C3N4@CDs-Ab2 signal tag. When the prepared biosensor was used to detect the SARS-CoV-2 spike protein target (1.0 pg / mL), the ECL intensity increased to approximately 3234 a.u. (positive, red curve). These experimental results demonstrate that the prepared immunosensor exhibits a good ECL signal response when detecting the target SARS-CoV-2 spike protein.

[0080] Analytical performance of ECL immunosensor

[0081] In order to evaluate the sensitivity of ECL immunosensor in detecting SARS-COV-2 spike protein, the prepared ECL immunosensor was used to detect standard solutions containing different concentrations of SARS-COV-2 spike protein. Figure 6 As shown in Figures A and 6B, within the SARS-CoV-2 spike protein concentration range of 1.0 fg / mL to 1.0 ng / mL, the ECL signal intensity increases with increasing SARS-CoV-2 spike protein concentration, exhibiting a linear correlation. The linear equation is I = 890lg[c / (fg / mL) + 566 (where I represents the ECL signal value and c represents the SARS-CoV-2 spike protein concentration), with a Pearson correlation coefficient of r = 0.9995. The limit of detection (LOD) of the prepared ECL immunosensor was calculated to be 0.80 fg / mL.

[0082] Specificity and stability of ECL immunosensors

[0083] SARS-CoV-2 shares over 60% similarity with MERS-CoV, SARS-CoV, HCoV-OC43, and HCoV-HKU1 in gene sequence analysis. Therefore, the four human coronaviruses were used as interfering substances to evaluate the specificity of the prepared ECL immunosensor for SARS-CoV-2 detection. Specifically, when the ECL immunosensor was incubated with the spike proteins of the four human coronaviruses (1.0 ng / mL), the ECL response of the interfering samples remained at a low level compared to the blank control group ( Figure 7 A). In contrast, when the ECL immunosensor was incubated with the target SARS-CoV-2 spike protein (10 pg / mL) and a mixture containing four interfering spike proteins (1.0 ng / mL) and the target SARS-CoV-2 spike protein (10 pg / mL), the ECL response of the mixed sample was as high as that of the sample containing only the target SARS-CoV-2 spike protein. This indicates that even when the concentration of the other interfering spike proteins is 100 times higher than that of the target SARS-CoV-2 spike protein, the interfering spike proteins have little effect on the ECL response, confirming that the prepared ECL immunosensor has high specificity for SARS-CoV-2 detection.

[0084] In addition, stability is another important parameter for evaluating the reliability of the prepared ECL immunosensor, and the stability of the sensor was verified by continuous scanning for 10 cycles. Figure 7 As shown in Figure 3B, a relatively stable ECL response was obtained during 10 continuous scanning cycles, with a relative standard deviation (RSD) of 3.16%, indicating that the prepared ECL immunosensor has good stability.

[0085] Application of ECL immunosensor in actual sample detection

[0086] The prepared ECL immunosensor was validated for detecting SARS-CoV-2 spike protein in real samples using the standard addition method. Positive samples were simulated by spiking different concentrations of SARS-CoV-2 spike protein into nasopharyngeal swabs from healthy individuals (soaked in saline). The results are shown in Table 1. Recoveries ranged from 95.70% to 110.90%, and relative standard deviations (RSDs) ranged from 0.73% to 6.56%. These results demonstrate that the constructed ECL immunosensor can be applied to the detection of SARS-CoV-2 spike protein in real biological samples with accurate and reliable results.

[0087] Table 1. Detection of SARS-CoV-2 spike protein in nasopharyngeal swab samples by standard addition method.

[0088]

[0089] This application utilizes two-dimensional graphene-phase carbon nitride nanosheets (g-C3N4NSs) as a carrier to in situ generate carbon dots on the surface of g-C3N4NSs. This results in a carbon dot nanocomposite (g-C3N4@CDs) with uniform carbon dot distribution on the g-C3N4NSs surface. In the presence of the co-reactant H2O2, the resulting carbon dot nanocomposite exhibits efficient ECL performance. The prepared carbon dot nanocomposite (g-C3N4@CDs) with efficient ECL performance is labeled with a detection antibody (Ab2) as an ECL signal tag to construct a sandwich ECL immunosensor for detecting the spike protein of the novel coronavirus (SARS-CoV-2).

[0090] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing carbon dots by in-situ generation at a nano-interface, characterized in that: The following steps are involved: S1. Preparation of g-C3N4: Melamine was added to a crucible and heated in a muffle furnace to obtain g-C3N4; S2. Collecting g-C3N4 NSs dispersion: dispersing g-C3N4 in deionized water, continuously sonicating and then centrifuging to collect the upper layer of g-C3N4 nanosheet dispersion, i.e., g-C3N4 NSs dispersion; S3. Reaction synthesis of g-C3N4@CDs: Take a g-C3N4 NSs dispersion, add citric acid, and continue stirring to dissolve to obtain a mixed solution; then drop hydrazine hydrate into the mixed solution, stir evenly, and transfer it to a hydrothermal reactor for heating treatment. After cooling to room temperature, centrifuge, wash, and dry to obtain a nanocomposite with carbon dots in situ generated on the surface of g-C3N4NSs, namely g-C3N4@CDs.

2. The method for preparing carbon dots by in-situ generation at a nano-interface according to claim 1, characterized in that: The washing treatment in step S3 is washing three times with phosphate buffer, deionized water and ethanol in sequence.

3. The method for preparing carbon dots by in-situ generation at a nano-interface according to claim 1 or 2, characterized in that: The specific process conditions of each step are as follows: S1. Preparation of g-C3N4: 15 g of melamine was added to an alumina crucible and heated at 600 °C for 2 h at a heating rate of 5 °C / min in a muffle furnace to obtain g-C3N4. S2. Collecting the g-C3N4 NSs dispersion: Disperse g-C3N4 in deionized water at a concentration of 1 mg / mL, continuously sonicate for 16 h, and then centrifuge at 6000 rpm for 10 min to collect the upper layer of g-C3N4 nanosheet dispersion, i.e., the g-C3N4 NSs dispersion; S3. Reaction synthesis of g-C3N4@CDs: Take 10 mL of g-C3N4 NSs dispersion, add 0.21 g of citric acid, continue stirring to dissolve, and obtain a mixed solution; then drop 0.1 mL of hydrazine hydrate into the mixed solution, stir evenly, and transfer it to a polytetrafluoroethylene-lined stainless steel reactor, heat at 180°C for 3 h, cool to room temperature, and centrifuge at 12000 rpm for 10 min. Wash and treat in sequence, and after drying, obtain a nanocomposite with carbon dots in situ generated on the surface of g-C3N4NSs, namely g-C3N4@CDs.

4. An electrochemiluminescence analysis application of in-situ generated carbon dots at nano-interfaces, characterized by: The nano-interface prepared according to claim 1, 2 or 3 is used to in situ generate carbon dots, i.e. g-C3N4@CDs, to prepare g-C3N4@CDs-Ab2 signal tags and to prepare ECL immunosensors; A sandwich ECL immunosensor for detecting the spike protein of the new coronavirus SARS-CoV-2 was constructed using the prepared g-C3N4@CDs-Ab2 signal tag and ECL immunosensor.

5. The electrochemiluminescence analysis application of the nano-interface in-situ generated carbon dots according to claim 4, characterized in that: The preparation of the g-C3N4@CDs-Ab2 signal tag includes the following steps: (1) Disperse g-C3N4@CDs in MES buffer containing EDC and NHS and shake to mix; after centrifugation, take the precipitate and disperse it in Na2CO3 buffer; (2) Add SARS-CoV-2 spike protein detection Ab2 antibody, shake to mix, and centrifuge to obtain the precipitate; The precipitate was dispersed in PBS, BSA solution was added and shaken to mix, and the precipitate was collected by centrifugation; the precipitate was washed with PBS and then dispersed in PBS to prepare the g-C3N4@CDs-Ab2 signal tag.

6. The electrochemiluminescence analysis application of the nano-interface in-situ generated carbon dots according to claim 5, characterized in that: The specific process conditions in the preparation steps of the g-C3N4@CDs-Ab2 signal tag are as follows: (1) 2.0 mg g-C3N4@CDs was dispersed in 1.0 mL of MES buffer (pH 6.0) containing 60 mmol / L EDC and 40 mmol / L NHS at a molar concentration of 0.1 mol / L. The mixture was shaken at 4°C for 2 h. The precipitate was collected by centrifugation at 10,000 rpm for 5 min and then dispersed in 1.0 mL of Na2CO3 buffer (pH 9.5). (2) Add 10 μL of SARS-COV-2 spike protein detection Ab2 antibody, shake and mix at 4°C for 12 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate; disperse the precipitate in 1 mL of PBS with a molar concentration of 10 mmol / L and a pH of 7.4, add 100 μL of 1% BSA solution by mass volume, shake and mix at 4°C for 1 h, and centrifuge at 10,000 rpm for 5 min to obtain the precipitate; wash the precipitate three times with PBS with a molar concentration of 10 mmol / L and a pH of 7.4, and then disperse it in 500 μL of PBS with a molar concentration of 10 mmol / L and a pH of 7.4 to prepare the g-C3N4@CDs-Ab2 signal tag.

7. The electrochemiluminescence analysis application of the nano-interface in-situ generated carbon dots according to claim 4, characterized in that: The preparation of the ECL immunosensor includes the following steps: (1) Polishing the glassy carbon electrode to obtain a clean electrode surface, immersing it in a glutamic acid solution containing potassium hydrogen phthalate, and performing constant potential deposition treatment to deposit a carboxyl-rich Glu film on the electrode surface to obtain a carboxyl-functionalized glassy carbon electrode; (2) Immerse the carboxyl-functionalized glassy carbon electrode in MES buffer containing EDC and NHS, react at room temperature, rinse with PBS, then dropwise add SARS-COV-2 spike protein to capture Ab1 antibody, incubate at room temperature, and allow Ab1 to be covalently cross-linked on the electrode surface to obtain an Ab1-modified electrode; (3) BSA solution was added dropwise to the Ab1-modified electrode surface for incubation, and then rinsed with PBS to prepare the ECL immunosensor.

8. The electrochemiluminescence analysis application of the nano-interface in-situ generated carbon dots according to claim 7, characterized in that: The specific process conditions in the preparation steps of the ECL immunosensor are as follows: (1) The glassy carbon electrode was polished to obtain a clean electrode surface, and then immersed in a glutamic acid solution with a molar concentration of 0.1 mol / L and a pH of 5.0 containing 10 mmol / L potassium hydrogen phthalate. A carboxyl-rich Glu film was deposited on the electrode surface at -1.9 V for 60 s to obtain a carboxyl-functionalized glassy carbon electrode. (2) The carboxyl-functionalized glassy carbon electrode was immersed in a MES buffer solution containing 60 mmol / L EDC and 40 mmol / L NHS at a molar concentration of 0.1 mol / L and a pH of 6.

0. After reacting at room temperature for 30 min, it was rinsed with PBS at a pH of 7.

4. Then, 10 μL of SARS-COV-2 spike protein to capture Ab1 antibody was added dropwise and incubated at room temperature for 30 min to allow Ab1 to be modified on the electrode surface through covalent cross-linking. (3) After adding a 1% BSA solution by mass volume percentage to the electrode surface and incubating for 1 hour, the electrode was rinsed with PBS at pH 7.4 to prepare an ECL immunosensor.

9. The electrochemiluminescence analysis application of the nano-interface in-situ generated carbon dots according to any one of claims 4 to 8, characterized in that: The construction of a sandwich ECL immunosensor for detecting the SARS-CoV-2 novel coronavirus spike protein includes the following steps: SARS-CoV-2 spike protein at different concentrations is added to the ECL immunosensor interface and incubated, rinsed with PBS, and then the g-C3N4@CDs-Ab2 signal label is added and incubated; after rinsing to remove the unbound signal label, the interface is placed in PBS containing H2O2, a voltage of 0.2-0.8 V is applied and cyclically scanned, and the ECL signal is measured.

10. The electrochemiluminescence analysis application of the nano-interface in-situ generated carbon dots according to claim 9, characterized in that: The specific process conditions in the steps of constructing the sandwich-type ECL immunosensor for detecting the SARS-CoV-2 novel coronavirus spike protein are as follows: 10 μL of SARS-CoV-2 spike protein at different concentrations was added to the ECL immunosensor interface and incubated for 1 h. After rinsing with PBS at pH 7.4, the g-C3N4@CDs-Ab2 signal label was added and incubated for 1 h. After rinsing to remove the unbound signal label, the sensor was placed in PBS at pH 7.4 containing 10 mmol / L H2O2, and a voltage of 0.2-0.8 V was applied for cyclic scanning to measure the ECL signal.

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