Ru(bpy)32+ luminescence promoters, their prediction, preparation and use

Abstract

The application belongs to the technical field of biosensing, and particularly relates to a Ru(bpy)3 2+ Luminescence promoter and its prediction, preparation and application. The promoter comprises Ru(bpy)3 2+ Anodic luminescence promoter CoN4@graphene and Ru(bpy)3 2+ Cathodic luminescence promoter CoC4@graphene. The application studies the 4e ‑ ORR, 2e ‑ ORR and HPRR processes, evaluates the ORR and HPRR catalytic performance, and further screens the suitable Ru(bpy)3 2+ Luminescence promoter. The application determines, through theoretical prediction, that CoC4@graphene and CoN4@graphene are potential Ru(bpy)3 2+ Luminescence promoter. A biosensor for detecting tumor marker CEA is constructed based on the promoter, and the biosensor has excellent sensitivity, linearity, selectivity and stability.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to a Ru(bpy)3 2+ Luminescence promoters and their prediction, preparation and application. Background Technology

[0002] Malignant tumors seriously threaten human physical and mental health. Current research shows that early cancer detection can effectively improve patient survival and cure rates; therefore, accurate early detection is key to curing tumors. Among numerous detection methods, electrochemiluminescence immunoassay (ECLIA) can effectively detect trace amounts of tumor markers in body fluids, making it significant in early clinical tumor detection. 2+ It is the most widely used electroluminescent emitter in clinical practice, possessing excellent spectral and potential ECL resolution. However, due to the lack of good luminescence promoters (especially cathodoluminescence promoters) and unavoidable crosstalk between different co-reactants, Ru(bpy)3... 2+ Its practical application in colorimetric detection and multi-marker detection is limited. Therefore, it is crucial to find enhancers that can improve cathode and anodic emission without mutual interference.

[0003] Searching for Ru(bpy)3 2+ Understanding the luminescence mechanism is a prerequisite for identifying luminescence promoters. Existing research indicates that reactive oxygen species (ROS), intermediates in the oxygen reduction reaction (ORR) and oxygen generation reaction (OER), can act as luminescence promoters for Ru(bpy)3. 2+ Excellent promoters for potential or spectrally resolved ECL emission. However, previous work was based on metal nanoparticle catalysts, which suffer from low catalytic efficiency, multiple side reactions, and oxidation instability, requiring further improvement. Single-atom catalysts (SACs) are widely used in battery manufacturing due to their excellent ORR and OER catalytic performance and long-term stability. Therefore, it is speculated that SACs can promote Ru(bpy)3... 2+ ECL emission from both the anodic and cathode holds immense research potential. However, due to the diversity and complexity of active metal sites, coordination, and substrates, determining suitable candidates for Ru(bpy)3 is challenging. 2+Sequencing SACs as luminescence enhancers is challenging. Researchers in this field have utilized theoretical calculations to find and design suitable SACs. For example, Xia et al. applied theoretical calculations to determine an excellent ORR catalyst for promoting the luminescence of luminol by adjusting the coordination environment; see the article "Identifying Luminol Electrochemiluminescence at the Cathode via Single-Atom Catalysts Tuned Oxygen Reduction Reaction". This work demonstrates that it is feasible to use theoretical calculations to guide the synthesis of suitable SACs as luminescence enhancers. However, due to Ru(bpy)3... 2+ The complexity of the luminescence mechanism means that theoretical calculations have not yet been used to predict Ru(bpy)3. 2+ The enhancer.

[0004] In its preliminary research, this invention discovered 1e - ORR transfer product peroxide radical (·O2H) promoted Ru(bpy)3 at -1.70V. 2+ The luminescence of the OER product hydroxyl radical (·OH) exhibits optimal Ru(bpy)3 at 1.25V. 2+ Luminescence-enhancing effect. Used to promote Ru(bpy)3 2+ The luminescent SAC not only needs to pass through 4e - The ORR process generates sufficient ROS, and 2e also needs to be considered. - The occurrence of ORR and the use of data from 2e - ORR's catalytic ability for the hydrogen peroxide reduction reaction (HPRR) with H2O2. Furthermore, 4e... - The ROS generated in the ORR process needs to be effectively retained within the system, rather than rapidly catalyzed into the final product. Existing literature has only explored the most common 4e in theoretical calculations for the development of luminescence promoters. - The ORR process, but due to Ru(bpy)3 2+ The stringent requirements on the types of free radicals, and the ORR initiation potential and Ru(bpy)3 2+ The potentials of the cathode emission potentials are quite far apart, making it inappropriate to calculate only a single path. Therefore, no theoretical calculation has yet predicted Ru(bpy)3. 2+ Successful cases of luminescence promoters.

[0005] In the prior art, invention patent CN114784297B discloses a method for preparing a single-atom cobalt ORR catalyst. This method uses gelatin as the carbon source, and a bimetallic organic framework material generated by the reaction of Co(NO3)2·6H2O, Zn(NO3)2·6H2O, and 2-methylimidazole as the cobalt source. During high-temperature pyrolysis, the presence of zinc effectively inhibits the aggregation of cobalt into cobalt nanoparticles, ultimately constructing a single-atom catalyst with a Co-NC structure. This catalyst exhibits excellent ORR catalytic performance. However, the cobalt single-atom catalyst disclosed in this patent differs from the cobalt single-atom catalyst in this application and cannot be used as a Ru(bpy)3 catalyst. 2+ Potential-resolved electrochemiluminescence promoters are used for the detection of carcinoembryonic antigens. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a Ru(bpy)3 2+ Electrochemiluminescence promoter.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Ru(bpy)3 2+ Electrochemiluminescence promoter, namely Ru(bpy)3 2+ Electrochemiluminescence promoters include Ru(bpy)3 2+ Anodic luminescence promoter and Ru(bpy)3 2+ Cathodoluminescence promoter; the Ru(bpy)3 2+ The anodic luminescence promoter is CoN4@graphene; the Ru(bpy)3 2+ The cathodoluminescence promoter is CoC4@graphene.

[0009] This invention has found that CoC4@graphene exhibits significant 3e - ORR catalytic activity ensures the accumulation and storage of ROS in the system, thereby promoting excellent cathodic luminescence; while CoN4@graphene, with its suitable OER catalytic performance and the presence of NH2 groups on its surface, exhibits superior anodic luminescence performance. Further studies confirmed that using Ru(bpy)3... 2+ When used as a luminescent material for carcinoembryonic antigen detection, CoN4@graphene was employed as Ru(bpy)3. 2+ Anodizing luminescence promoter, CoC4@graphene as Ru(bpy)3 2+ The cathodoluminescence promoter can further enhance the ECL response of the anode and cathode without interfering with each other.

[0010] Furthermore, the N content in the CoC4@graphene is 2wt%-2.5wt%, and the Co content is 0.5%-0.8%; the N content in the CoN4@graphene is 2.7wt%-3.5wt%, and the Co content is 0.9%-1.1%.

[0011] Preferably, the CoC4@graphene contains 2.3 wt% N and 0.63% Co; the CoN4@graphene contains 3.1 wt% N and 0.97% Co.

[0012] Furthermore, the coordination number of Co-C in CoC4@graphene is 3.55±0.75, and the coordination number of Co-N in CoN4@graphene is 4.4±0.37. These results indicate that both CoC4@graphene and CoN4@graphene possess a four-coordinate structure.

[0013] The second objective of this invention is to provide the aforementioned Ru(bpy)3 2+ Prediction methods for electrochemiluminescence promoters.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] Ru(bpy)3 2+ The method for predicting electrochemiluminescence promoters includes the following steps:

[0016] (1) Establish a cobalt single-atom catalyst model with different coordination structures;

[0017] (2) The 4e-ion content of the cobalt single-atom catalyst was studied using density functional theory and first-principles methods. - ORR and 2e - The ORR process was evaluated, and Ru(bpy)3 was screened to assess its ORR catalytic performance. 2+ Luminescence promoter.

[0018] This invention selects cobalt atoms, widely recognized for their excellent catalytic potential in oxygen reduction reaction (ORR) and oxygen evolution reaction (OER), as the central atom. By changing their coordinating atoms, a series of cobalt single-atom catalysts with different carbon-nitrogen ratios were designed. Their performance in ORR and HPRR catalysis was investigated based on density functional theory (DFT) and first-principles methods. The results show that in five CoC... x N 4-x In (x = 0–4), CoC4 catalyzes 3e - ORR produces ROS, while CoN4 mainly catalyzes 4e-. - ORR produces H2O, but inevitably 2e- will also occur. -ORR. Finally, we chose to synthesize CoC4@graphene and CoN4@graphene. Electrochemical experiments confirmed the accuracy of the theoretical predictions of this invention. ORR was observed in Ru(bpy)3. 2+ Satisfactory anodic and cathodic electrochemiluminescence (ECL) responses were observed in CoC4@graphene and CoN4@graphene. CoC4@graphene and CoN4@graphene are ideal Ru(bpy)3. 2+ Luminescence promoter.

[0019] Furthermore, step (1) includes: constructing a long [structure / structure] in the catalytic site region. Width high The unit cell is constructed; a defect structure is built at the geometric center of the unit cell; a Co single atom is introduced at the center of the defect structure, and the carbon at the defect is substituted according to different nitrogen coordination conditions to obtain six single-atom catalytic site models, including CoC4, CoC3N1, CoC2N2-1, CoC2N2-2, CoC3N1 and CoN4.

[0020] Furthermore, the density functional theory and first-principles method include the following steps:

[0021] (1) The cobalt single-atom catalyst model was structurally optimized based on density functional theory using a pseudopotential-plane wave algorithm to obtain the optimized model.

[0022] (2) Based on the structure optimization model obtained in step (1), construct the reaction intermediate ROS model and dock it with the central atom of the catalytic site to simulate the ROS adsorption process and obtain the intermediate adsorption state.

[0023] (3) Calculate the single-point energy of each molecular adsorption system and obtain the reaction kinetic curve.

[0024] Furthermore, in the method, all spin-polarized density functional theory calculations are performed using the Vienna First Principles Simulation Software (VASP) based on plane wave basis sets and projected enhanced wave (PAW) pseudopotentials.

[0025] Furthermore, the PBE exchange correlation functional was used at the generalized gradient approximation level.

[0026] Furthermore, the plane wave cutoff energy was set to 400 eV to expand the electronic wave function, and the Brillouin zone was structurally optimized using a 2×2×1 Monkhorst-Pack k-point grid.

[0027] Furthermore, the ground-state electron density converges to 10 -4The total energy threshold is eV, and the structure is optimized until the maximum force on any ion is less than 100 eV.

[0028] Furthermore, approximately [number] were used in the z-direction. A vacuum space is provided to avoid interaction between the periodic substrate and the repeatable image.

[0029] Furthermore, for the N-doped system, a 4×4 graphene supercell was used, in which Co atoms were embedded in double vacancies, denoted as CoC. x N 4-x x = 0 - 4.

[0030] As a preferred technical solution, the following steps are included:

[0031] (1) Model building

[0032] Catalyst models with different coordination structures were established using Materials Studio, a materials simulation software developed by Accelrys. The graphene substrate model was constructed by extracting a single layer of sheet graphite crystals from the materials library. To simulate the uniform, dispersed, and independent catalytic sites of single-atom catalysts, long-form models were constructed for the catalytic site regions. Width high The unit cell was constructed. A defect structure was built at the geometric center of the unit cell, and a single Co atom was introduced into the center of the defect. The carbon at the defect was substituted according to different nitrogen coordination conditions, resulting in six single-atom catalytic site models: CoC4, CoC3N1, CoC2N2-1, CoC2N2-2, CoC3N1, and CoN4. Among them, CoC2N2 is discussed in two configurations based on whether its structure is symmetrical or not: CoC2N2-1 and CoC2N2-2.

[0033] (2) Optimization of catalytic site structure

[0034] The VASP software structure optimization module and its supporting atomic pseudopotential file are used in the supercomputing server to perform structure optimization based on the pseudopotential-plane wave algorithm based on density functional theory to obtain a catalytic site structure close to the real state.

[0035] (3) Simulation of ROS adsorption process

[0036] The optimized model obtained in step (2) is exported, and a reaction intermediate ROS model is constructed in Materials Studio and docked with the central atom of the catalytic site. The entire adsorption model is then structurally optimized in VASP to simulate the molecular adsorption process of the catalytic reaction, resulting in an intermediate adsorption state that closely approximates reality.

[0037] (4) Energy calculation and dynamic curves

[0038] The structure-optimized model and its atomic pseudopotential file are imported into VASP. The energy calculation module in VASP is then used to calculate the total energy of each intermediate system, yielding the single-point energy of each molecular adsorption system. Combined with the reaction equations of the ORR intermediate steps, the free energy change at each step is obtained using the single-point energy, resulting in the reaction kinetic curves.

[0039] The third objective of this invention is to provide the aforementioned Ru(bpy)3 2+ Preparation method of electrochemiluminescence promoter.

[0040] To achieve the above objectives, the present invention adopts the following technical solution:

[0041] Ru(bpy)3 2+ The preparation method of the electrochemiluminescence promoter includes the following steps:

[0042] (1) Add cobalt solution to graphene to react and obtain a mixed solution;

[0043] (2) After drying the mixed solution obtained in step (1), the mixture is heated in N2 atmosphere. The product is soaked in hydrochloric acid to remove cobalt particles, and centrifuged to obtain CoC4@graphene and CoN4@graphene.

[0044] Furthermore, in step (1), the mass ratio of graphene to cobalt solution is 2-5:1, preferably 2.3:1.

[0045] Furthermore, in step (1), the solvent for graphene is methanol.

[0046] Furthermore, in step (1), the reaction conditions are: stirring in the dark for 1-3 hours; stirring until the cobalt precursor and graphene are uniformly dispersed.

[0047] Furthermore, in step (1), the concentration of the cobalt solution is preferably 2 mol / L.

[0048] Furthermore, in step (2), the drying process involves vigorously stirring the mixed solution at 60°C until it is dry.

[0049] Furthermore, in step (2), the heating conditions are: heating at 900°C for 24 hours, with or without melamine.

[0050] Furthermore, in step (2), the hydrochloric acid concentration is preferably 1M.

[0051] Furthermore, in step (2), the hydrochloric acid soaking time is preferably 24 hours.

[0052] Furthermore, in step (2), the centrifugation is performed by centrifuging multiple times with deionized water.

[0053] The fourth objective of this invention is to provide an application of a cobalt single-atom catalyst in the preparation of reagents for carcinoembryonic antigen detection.

[0054] To achieve the above objectives, the present invention adopts the following technical solution:

[0055] The application of cobalt single-atom catalysts in the preparation of reagents for carcinoembryonic antigen detection, wherein the cobalt single-atom catalysts are CoN4@graphene and CoC4@graphene; and the reagent is Ru(bpy)3. 2+ Potential-resolved electrochemiluminescence promoters.

[0056] Furthermore, the Ru(bpy)3 2+ As a luminescent material for carcinoembryonic antigen detection, CoN4@graphene acts as a Ru(bpy)3 phosphoprotein. 2+ Anodizing luminescence promoter, CoC4@graphene as Ru(bpy)3 2+ Cathodoluminescence promoter, enhancing the electrochemiluminescence response of the anode and cathode.

[0057] The fifth objective of this invention is to provide a product containing the aforementioned Ru(bpy)3 2+ Biosensors based on electrochemiluminescence promoters.

[0058] To achieve the above objectives, the present invention adopts the following technical solution:

[0059] Contains Ru(bpy)3 2+ Biosensors based on electrochemiluminescence promoters.

[0060] Furthermore, the working electrode of the biosensor is glassy carbon with surface-modified CoN4@graphene and CoC4@graphene, the reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum wire.

[0061] Furthermore, the method for preparing the biosensor is as follows:

[0062] CoN4@graphene was mixed with Ab2 (secondary antibody) and incubated for 12 hours to obtain the label; CoC4@graphene at a concentration of 2 mg / ml was dropped onto the polished electrode surface, EDC / NHS was added, and it was incubated at 37°C for 30 minutes; after washing, Ab1 (primary antibody) was added and incubated at 37°C for 1 hour; after washing, the sensor was blocked using bovine serum albumin, and the antigen and the prepared secondary antibody were added sequentially to obtain the immunosensor.

[0063] The sixth objective of this invention is to provide a method for detecting carcinoembryonic antigen.

[0064] To achieve the above objectives, the present invention adopts the following technical solution:

[0065] A method for detecting carcinoembryonic antigen (CEA) involves using the aforementioned biosensor to perform ECL detection on CEA, and qualitatively and / or quantitatively analyzing the CEA based on the obtained ECL signal intensity.

[0066] Furthermore, the detection conditions included: a scan rate of 0.1 V / s, and Ru(bpy)3 2+ The concentration was 1 mM, the pH was 6, the potential was -1.5V to 2V, and the detection environment was air or N2.

[0067] Furthermore, the sample to be tested may or may not contain interfering substances, including one or more of epidermal growth factor receptor, estrogen receptor α, and nuclear matrix protein 22.

[0068] The beneficial effects of this invention are as follows:

[0069] 1. This invention further elucidates the mechanism by which Ru(bpy)32 promotes ECL through ORR and OER.

[0070] 2. This invention successfully predicted Ru(bpy)3 based on theoretical calculations. 2+ The luminescence promoter is suitable for designing Ru(bpy)3 2+ The emission promoter provides a new approach, thereby expanding the clinical potential of ruthenium-based fluorescence systems.

[0071] 3. This invention constructs an ECL proportional immunosensor system promoted by CoSACs based on CoC4@graphene and CoN4@graphene for detecting the tumor marker CEA. This immunosensor can detect concentrations as low as 10... -15 With a detection limit as low as 33 fg / mL for CEA, this sensor exhibits high sensitivity. Furthermore, it demonstrates excellent linearity, selectivity, and stability, and is unaffected by interfering substances such as epidermal growth factor receptor, estrogen receptor α, and matrix nuclear protein 22, showing great potential for clinical applications.

[0072] 4. This invention is the first to employ computational chemistry methods to analyze Ru(bpy)3. 2+ ECL promoters were theoretically predicted. Through computational analysis, CoC4@graphene and CoN4@graphene were identified as the main candidates for synthesis. CoC4@graphene exhibited significant 3e... -ORR catalytic activity ensures the accumulation and storage of ROS in the system, thereby promoting excellent cathodic luminescence. Conversely, CoN4@graphene exhibits superior anodic luminescence performance due to its suitable OER catalytic properties and the presence of NH2 groups on its surface. Attached Figure Description

[0073] Figure 1 The diagrams show the structures of Co single-atom catalysts with different C / N coordination ratios. Figure 1 -A is the structure diagram of CoN4. Figure 1 -B is the structure diagram of CoC1N3. Figure 1 -C represents the structure of CoC2N2-1. Figure 1 -D is the structural diagram of CoC2N2-2. Figure 1 -E is the structural diagram of CoC3N1. Figure 1 -F is the structure diagram of CoC4;

[0074] Figure 2 For the calculation in CoC x N 4-x ORR free energy diagram at Co atoms with U=0;

[0075] Figure 3 A graph showing the relative charge state and corresponding ·OH adsorption energy at U=0 for the calculated ORR free energy diagram;

[0076] Figure 4 For the calculation in CoC x N 4-x HPRR free energy diagram at Co atom with U = 1.23;

[0077] Figure 5 A schematic diagram of the synthesis of Co SACs;

[0078] Figure 6 -A is the SEM image of CoC4@graphene. Figure 6 -B is the SEM image of CoN4@graphene; Figure 6 -C represents the HAADF-STEM image of CoC4@graphene. Figure 6 -D represents the HAADF-STEM image of CoN4@graphene; Figure 6 -C and Figure 6 In -D, the isolated bright points marked with red circles are Co SAs;

[0079] Figure 7 XRD spectra of CoC4@graphene, CoN4@graphene, and graphene;

[0080] Figure 8 Raman spectra of CoC4@graphene, CoN4@graphene, and graphene;

[0081] Figure 9 Co k-edge XANES spectra of CoC4@graphene, CoN4@graphene, and graphene;

[0082] Figure 10 k3-weighted Fourier transform spectra of Co foil, CoC4@graphene, and CoN4@graphene;

[0083] Figure 11 -A is a comparison of the CV curves of CoN4@graphene, CoC4@graphene and graphene on GCE in N2-saturated 0.1M KOH; Figure 11 -B is a comparison of the CV curves of CoN4@graphene, CoC4@graphene and graphene on GCE in air-saturated 0.1M KOH; Figure 11 -C is a comparison of the CV curves of CoN4@graphene, CoC4@graphene and graphene on GCE in O2-saturated 0.1M KOH;

[0084] Figure 12 -A represents bare GCE, CoC4@graphene, and CoN4@graphene in 1mM Ru(bpy)3 2+ ECL curve in solution; Figure 12 -B represents CoC4@graphene in N2, air, and O2, at 1 mM Ru(bpy)3 2+ The ECL curve in the solution, relative to the Ag line, was scanned at a rate of 0.1 V / s. Figure 12 -C represents the RDE curves of CoC4@graphene in O2-saturated 0.1M KOH at different rotational speeds (225rpm-3600rpm). Figure 12 -D represents the KL plot obtained based on RDE measurements, with all scan rates set to 50mV / s;

[0085] Figure 13 -A represents CoC4@graphene in 1mM Ru(bpy)3 2+ The graphs show the ECL intensities in solution at scan rates of 0.1 V / s, 0.05 V / s, 0.2 V / s, and 0.5 V / s. Figure 13 -B is a graph of the cathode ECL at different scan rates;

[0086] Figure 14 -A represents CoN4@graphene in 1mM Ru(bpy)3 2+ The graphs show the ECL intensities in solution at scan rates of 0.1 V / s, 0.05 V / s, 0.2 V / s, and 0.5 V / s. Figure 14 -B is a graph of the cathode ECL at different scan rates;

[0087] Figure 15 A plot of the time-dependent ECL of CoC4@graphene and CoN4@graphene;

[0088] Figure 16 For CoC4@graphene and CoN4@graphene, cyclic voltammetry ECL was used to analyze Ru(bpy)3. 2+ A diagram of cathodic emission;

[0089] Figure 17 For Ru(bpy)3 2+ A schematic diagram of the reaction mechanism of ECL generation at the cathode on CoC4@graphene / GCE and the anodic ECL generation on CoN4@graphene / GCE in PBS at pH 6.

[0090] Figure 18 This is a schematic diagram of the construction process of the immune sensor in Example 6;

[0091] Figure 19 EIS plots of naked GCE, CoC4-GCE, Ab1 / CoC4-GCE, Ag / Ab1 / CoC4-GCE, and Ab2 / Ag / Ab1 / CoC4-GCE measured in 0.1 mol / L KCl containing 5.0 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] (1:1);

[0092] Figure 20 -A represents the concentration of 1mM Ru(bpy)3 2+ In, different concentrations of CEA (10 -7 ng / ml~10 -1 ECL intensity calibration curve (ng / ml) Figure 20 -B is a graph of the ECL response of the colorimetric biosensor;

[0093] Figure 21 This is a graph showing the selective detection results of the colorimetric ECL biosensor;

[0094] Figure 22 For the calculation in CoC x N 4-x ORR free energy diagram at Co atom with U = 1.23V;

[0095] Figure 23 -A is the optimized structural diagram of OH adsorbed on CoN4; Figure 23 -B is the optimized structural diagram of OH adsorbed on CoClN3; Figure 23 -C represents the optimized structural diagram of OH adsorbed on CoC2N2-1; Figure 23 -D is the optimized structural diagram of OH adsorbed on CoC2N2-1; Figure 23 -E represents the optimized structural diagram of OH adsorbed on CoC3N1; Figure 23 -F represents the optimized structural diagram of OH adsorbed on CoC4;

[0096] Figure 24 EDS mapping for CoC4@graphene;

[0097] Figure 25 EDS mapping for CoN4@graphene;

[0098] Figure 26 These are transmission electron microscopy (TEM) images of a Co single-atom catalyst at different scales. Figure 26 -A is a TEM image of CoC4@graphene at the 200nm scale. Figure 26 -B is a TEM image of CoC4@graphene at the 100nm scale. Figure 26 -C represents the TEM image of CoC4@graphene at the 50nm scale. Figure 26 -D represents the TEM image of CoN4@graphene at the 500nm scale. Figure 26 -E represents the TEM image of CoN4@graphene at the 100nm scale. Figure 26 -F represents a TEM image of CoN4@graphene at the 50nm scale;

[0099] Figure 27 -A shows the XPS full spectra of CoC4@graphene and CoN4@graphene. Figure 27 -B represents the C2p deconvolution spectrum of CoC4@graphene. Figure 27 -C represents the C 2p deconvolution spectrum of CoN4@graphene;

[0100] Figure 28 -A is the C1s spectrum of CoC4@graphene. Figure 28 -B represents the C1s spectrum of CoN4@graphene. Figure 28 -C represents the C1s spectrum of graphene;

[0101] Figure 29 -A is a plot of the N1s region of CoC4@graphene. Figure 29 -B is a plot of the N1s region of CoN4@graphene. Figure 29 -C is a graph of the N1s region of graphene;

[0102] Figure 30 WT-EXAFS spectrum of the Co-K edge of CoC4@graphene;

[0103] Figure 31 WT-EXAFS spectrum of the Co-K edge of CoN4@graphene;

[0104] Figure 32 The WT-EXAFS spectrum of the Co K edge of the cobalt foil;

[0105] Figure 33 -A shows the results of the Co-K edge EXAFS fitting analysis of CoC4@graphene in R space; Figure 33 -B is the result of the Co K edge EXAFS fitting analysis of CoN4@graphene in R space; Figure 33 -C is the result of the EXAFS fitting analysis of the Co K edge of the Co foil in R space;

[0106] Figure 34 A plot of the k3-weighted χ(k) function of the EXAFS spectra of CoC4@graphene, CoN4@graphene, and aluminum foil;

[0107] Figure 35 The graph shows the Zeta potential measurement results for CoN4@graphene, CoC4@graphene, and graphene.

[0108] Figure 36 The graph shows the reactivity of CoN4@graphene and CoC4@graphene at 1600 rpm in O2-saturated 0.1M KOH.

[0109] Figure 37 -A is a graph showing the ring current and disk current of RRDE at 1600 rpm in O2-saturated 0.1M KOH solution; Figure 37 -B is a plot of the average number of electrons transferred (n) during the ORR of CoN4@graphene and CoC4@graphene; Figure 37 -C is a graph showing the H2O2 selectivity of CoN4@graphene and CoC4@graphene during ORR;

[0110] Figure 38 Plots of Tafel slopes for CoN4@graphene and CoC4@graphene;

[0111] Figure 39 The figure shows the electrochemical results of HPRR, where, Figure 39 -A shows the CV detection results obtained using glassy carbon (GC) electrodes modified with CoN4@graphene, CoC4@graphene, and graphene in PBS (pH=6) containing 5 mM H2O2. Figure 39 -B shows the LSVs detection results obtained using CoN4@graphene, CoC4@graphene, and graphene modified with glassy carbon (GC) electrodes in PBS (pH=6) containing 5mM H2O2; the scan rate was 50mV / s.

[0112] Figure 40 The LSV curves for CoN4@graphene, CoC4@graphene, and graphene are shown below. The detection conditions were: 0.05 MH2SO4 solution, 1.3 to 1.8 V relative to the silver line, and a scan rate of 0.1 V / s.

[0113] Figure 41 ECL plots for CoN4@graphene, CoC4@graphene, and commercial Pt / C;

[0114] Figure 42 -A represents the concentration of GCE in 1 mM Ru(bpy)3 in N2, air, and O2. 2+ Anodic ECL curves in solution (-2V to 1.5V with Ag wire, scan rate 0.1V / s); Figure 42 -B represents the concentration of CoN4@graphene in 1mM Ru(bpy)3 in N2, air, and O2. 2+ Anodic ECL curves in solution (-2V to 1.5V with Ag wire, scan rate 0.1V / s);

[0115] Figure 43 For 1mM Ru(bpy)3 2+ The graph shows the results of XPS measurements of the ratio of total carbon, oxygen, nitrogen, and cobalt atoms in CoN4@graphene on the GCE electrode before and after 2 hours of reaction.

[0116] Figure 44 -A represents the concentration of 0.1M Ru(bpy)3 in a solution containing 0.05mM benzoquinone, 0.05mM SOD, 0.05mM isopropanol, and the control group. 2+In the middle, the ECL reaction results of CoC4@Graphene are shown; Figure 44 -B represents the concentration of 0.1M Ru(bpy)3 in a solution containing 0.05mM benzoquinone, 0.05mM SOD, 0.05mM isopropanol, and the control group. 2+ In the middle, the ECL reaction results of CoN4@Graphene are shown;

[0117] Figure 45 Atomic force microscope images of each layer in the sensor architecture process;

[0118] Figure 46 To test the GCE electrodes containing 5 μL of 4 mg / mL CoN4@graphene and 5 μL of 4 mg / mL CoC4@graphene at concentrations of 0, 1 M, 2 M, 3 M, 4 M, and 5 M Ru(bpy)3, respectively. 2+ The logarithm of the ratio of anodic emission to cathodic emission in ECL testing in solution;

[0119] Figure 47 GCE electrodes containing 5 μL of 4 mg / mL CoN4@graphene and 5 μL of 4 mg / mL CoC4@graphene were respectively added to Ru(bpy)3 at pH 4, 5, 6, 7, and 8. 2+ The logarithm of the ratio of anodic emission to cathodic emission in ECL testing in solution;

[0120] Figure 48 ECL reaction diagrams for stability studies of 5 μL 4 mg / mL CoN4@graphene and 5 μL 4 mg / mL CoC4@graphene under 15 consecutive scans from -1.5 V to 2 V vs. Ag / AgCl. Detailed Implementation

[0121] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0122] In this embodiment of the invention, cobalt chloride hexahydrate (CoCl2·6H2O, 98%), phenol (0.1M), hydrochloric acid (HCl), melamine (C3H6N6), graphene powder, and isopropanol were all purchased from Sigma-Aldrich; the water used in all experiments was deionized water (DW); Ru(bpy)3 2+Materials were sourced from Suna Tech Inc.; isopropanol from Sinopharm Chemical Reagent Co., Ltd.; N-hydroxysuccinimide (NHS; Guaranteed Reagent (GR)) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; GR) from Shanghai Meipeipu Co., Ltd. (Shanghai, China); BSA (96% to 99%, GR) from Biss Inc. (Beijing, China); CEA and antibodies from Beyotime Biotechnology Co., Ltd. (Shanghai, China); human serum samples from Chongqing Medical University; PBS (0.1M, pH 7.4), KH2PO4 and Na2HPO4 were mixed with KCl (0.1M) in appropriate proportions to prepare buffer solutions for ECL detection; all other chemicals were of analytical grade and used without further purification; all aqueous solutions were freshly prepared and diluted with ultrapure water (≥18 MΩ; Milli-Q, Millipore).

[0123] In this embodiment of the invention, ECL measurements were performed on an MPI-E multifunctional electrochemiluminescence analyzer (Xi'an Ruimeisi Analytical Instrument Co., Ltd., China). The three-electrode ECL cell used modified glassy carbon as the working electrode. An Ag / AgCl (KCl saturated) electrode was used as the reference electrode, and a platinum wire was used as the counter electrode. The photomultiplier tube (PMT) bias voltage was 600 V, the scanning voltage range was 1.5 V to -2 V, and the scan rate was 100 mV / s. X-ray photoelectron spectroscopy (XPS) characterization was performed using a VG Multilab2000X instrument (Thermal Electron, USA). Fourier transform infrared spectroscopy (FT-IR) was observed on an FT-IR spectrometer (ALPHA, Bruker). Transmission electron microscopy (TEM) images were obtained using a JEM-2010 transmission electron microscope (JEOL, Japan). The obtained XAFS data were calibrated for background, leading edge, and trailing edge in Athena (version 0.9.26) [J. Synchrotron Rad. 2005, 12, 537]. Then, Fourier transform fitting was performed in Artemis (version 0.9.26) [J. Synchrotron Rad. 2005, 12, 537]. For the fitting of the Co foil, k² weighting was used, with k ranging from [value missing]. and the range of R is For sample fitting, k² weighting is used, with k ranging from 1 to 1. and the range of R is For wavelet transform analysis, χ(k) derived from Athena was imported into the Hama Fortran code. The parameters are as follows: R range is... The range of k is The weight k was 2; a Morlet function with κ = 10 and σ = 1 was used as the mother wavelet to provide the overall distribution. Raman spectra were recorded on a Rennishaw InVia spectrometer using a 100Rama scope fiber optic instrument model. Cyclic voltammetry (CVs) and electrochemical impedance spectroscopy (EIS) were performed on an electrochemical workstation (Ivium, Netherlands). CVs were recorded at a scan rate of 100 mV / s in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1 MkCl within a potential range of -0.2 V to 0.6 V. EIS measurements were performed by applying a voltage of 5 mV amplitude within a frequency range of 0.01 Hz to 106 Hz.

[0124] In this embodiment of the invention, for the oxygen reduction reaction (ORR), all tests were performed in a conventional three-electrode electrochemical cell, using a CHI 760E as the reference electrode and a counting electrode in Ag / AgCl (saturated KCl solution), and a glassy carbon (GC) electrode as the working electrode, with a diameter of 5 mm and coated with catalyst ink. To prepare the working electrode, 4 mg of catalyst was ultrasonically dispersed in 1 mL of a mixed solution comprising deionized water (700 μL), ethanol (250 μL), and... A 5% solution (50 μL) was prepared to form a homogeneous ink. Then, 5 μL of the ink was dropped onto a glassy carbon electrode and allowed to air dry at room temperature. The corresponding catalyst loading was 0.1 mg cm⁻¹. -2 Cyclic voltammetry (CV) tests were performed in a 0.1 M KOH solution saturated with oxygen, and data were statically recorded at a scan rate of 50 mV / s after the system stabilized. Rotating disk electrode (RDE) and rotating ring-disc electrode (RRDE) tests were performed in a 0.1 M KOH solution saturated with oxygen at a scan rate of 10 mV / s.

[0125] In this embodiment of the invention, the electron transfer number (n) is calculated using the following equation:

[0126]

[0127] Where Id represents disk current;

[0128] Ir represents the loop current;

[0129] N is the current collection efficiency of the Pt ring, which is 0.37.

[0130] In this embodiment of the invention, Koutecky-Levich (KL) plots (J-1 vs-1 / 2) were calculated at different potentials. The working electrode underwent cathode scanning at a rate of 10 mV / s, with the rotation speed varying from 225 to 3600 rpm. The Koutecky-Levich equation is as follows:

[0131]

[0132]

[0133] Where J is the measured current density; J k and J L These are kinetic energy and limiting current density; ω is angular velocity; n is the number of electrons transferred; F (96485 C / mol) is the Faraday constant; D0 is the concentration of O2 in 0.1 M KOH (1.9 × 10⁻⁶ mol / L). -5 The diffusion coefficient in cm / s, C0 is the volume concentration of O2 (1.2 × 10⁻⁶ cm / s), and C₀ is the diffusion coefficient in cm / s. -6 v is the kinetic viscosity of the electrolyte (0.01 cm / s), and k is the electron transfer rate constant. When the rotational speed is displayed in rpm, a constant of 0.2 is used.

[0134] In this embodiment of the invention, all spin-polarized density functional theory calculations were performed using the Vienna First Principles Simulation Package (VASP) based on plane-wave basis sets and projected enhanced wave (PAW) pseudopotentials. Perdew-Burke-Ernzerhof (PBE) exchange-correlated functionals were used at the generalized gradient approximation (GGA) level. The plane-wave cutoff energy was set to 400 eV to extend the electronic wavefunction, and the Brillouin zone was structure-optimized using a 2×2×1 Monkhorst-Pack k-point grid. The ground-state electron density converged to 10... -4 The total energy threshold is eV, and the structure is optimized until the maximum force on any ion is less than 100 eV. Approximately [amount] was used in the z-direction. A vacuum space is provided to avoid interaction between the periodic substrate and the repeatable image. For the N-doped system, a 4×4 graphene supercell is used, in which Co atoms are embedded in double vacancies (denoted as CoC). x N 4-x (x = 0 - 4).

[0135] The four reaction steps in the 4e-ORR pathway are shown below:

[0136] O2+ * +H + +e - → * OOH;

[0137] * OOH+H + +e - → * O+H2O;

[0138] * O+H + +e - → * OH;

[0139] * OH+H + +e - → * +H2O.

[0140] The four reaction steps in the HPRR pathway are shown below:

[0141] H2O2+ * → * H2O2;

[0142] * H2O2→2OH * ;

[0143] * OH+H + +e - →H2O+ * ;

[0144] * OH+H + +e - →H2O+ * .

[0145] Example 1. Theoretical calculations of ORR performance of cobalt single-atom catalysts under different coordination environments.

[0146] To investigate the ORR catalytic performance of cobalt single-atom catalysts under different coordination conditions and to further understand their effect on Ru(bpy)3 2+ The influence of anodic and cathodic luminescence was considered in this invention. Theoretical calculations based on DFT were performed to evaluate the ORR catalytic performance of a series of cobalt single-atom catalysts. Firstly, cobalt single-atom catalysts with different carbon-nitrogen ratios were designed, including CoN4, CoC1N3, CoC2N2-1, CoC2N2-2, CoC3N1, and CoC4, such as... Figure 1 As shown. It is worth noting that CoC2N2 has two different configurations. The symmetry or asymmetry of the C and N atom arrangement may affect the catalytic performance of the cobalt central atom in the intermediate process. Therefore, CoC2N2-1 and CoC2N2-2 were designed respectively. The specific methods are as follows:

[0147] (1) Model building

[0148] Catalyst models with different coordination structures were established using Materials Studio, a materials simulation software developed by Accelrys. The graphene substrate model was constructed by extracting a single layer of sheet graphite crystals from the materials library. To simulate the uniform, dispersed, and independent catalytic sites of single-atom catalysts, long-form models were constructed for the catalytic site regions. Width high The unit cell was constructed. A defect structure was built at the geometric center of the unit cell, and a single Co atom was introduced into the center of the defect. The carbon at the defect was substituted according to different nitrogen coordination conditions, resulting in six single-atom catalytic site models: CoC4, CoC3N1, CoC2N2-1, CoC2N2-2, CoC3N1, and CoN4. Among them, CoC2N2 is discussed in two configurations based on whether its structure is symmetrical or not: CoC2N2-1 and CoC2N2-2.

[0149] (2) Optimization of catalytic site structure

[0150] The Vienna Ab-initio Simulation Package (VASP) software structure optimization module and its accompanying atomic pseudopotential file were used in the supercomputing server to perform structure optimization based on density functional theory (DFT) and pseudopotential-plane wave (PP-PW) algorithm to obtain a catalytic site structure close to the real state.

[0151] (3) Simulation of ROS adsorption process

[0152] The optimized model obtained in step (2) is exported, and a reaction intermediate ROS model is constructed in Materials Studio and docked with the central atom of the catalytic site. The entire adsorption model is then structurally optimized in VASP to simulate the molecular adsorption process of the catalytic reaction, resulting in an intermediate adsorption state that closely approximates reality.

[0153] (4) Energy calculation and dynamic curves

[0154] The structure-optimized model and its atomic pseudopotential file are imported into VASP. The energy calculation module in VASP is then used to calculate the total energy of each intermediate system, yielding the single-point energy of each molecular adsorption system. Combined with the reaction equations of the ORR intermediate steps, the free energy change at each step is obtained using the single-point energy, resulting in the reaction kinetic curves.

[0155] Result: As Figure 2-4 As shown in Table 1. Figure 2 For the calculation in CoC x N4-x The ORR free energy diagram at the Co atom with U=0 is shown in Table 1. x N 4-x The Gibbs free energy values ​​of each step of the ORR at the Co atom with U=0. Through analysis of... Figure 2 Analyzing the calculation results, the most positive ΔG (free energy change) is shown in the fourth step ( * OH+H + +e - → * +H2O) indicates that this is 4e - The rate-limiting step of ORR. Simultaneously, ΔG4(4e) was observed in cobalt single-atom catalysts with different C / N ratios. - The free energy change from ·OH to H2O in ORR follows a certain pattern: the more carbon atoms involved in coordination, the larger the ΔG4 value, and the worse the ORR catalytic performance of the cobalt single-atom catalyst, and vice versa. The ΔG4 values ​​of CoN4, CoC1N3, CoC2N2-1, and CoC2N2-2 increase but are all negative (-0.455 eV, -0.443 eV, and -0.090 eV, respectively), indicating that all electron transfer steps in ORR are exothermic reactions and can successfully proceed with 4e... - The ORR process. However, for CoC4 and CoC3N1, ΔG4 is positive (0.278 eV and 0.147 eV respectively), indicating that at U = 0, 4e - ORR cannot proceed completely to produce the final product (H₂O). However, all electron transfer reactions prior to this step are energy-released, resulting in ROS such as ·OOH, ·O, and ·OH. Therefore, ORR catalyzed by CoC₄ and CoC₃N₁ leads to the accumulation of ROS in the system. Furthermore, the adsorption energy for OH (Eads(OH)) and CoC₃N₁... x N 4-x The relative charge state further confirms the above conclusion. For example... Figure 3 As shown, as the number of carbon atoms coordinated to cobalt single atoms increases, Eads(OH) increases, and the relative charge state shows a consistent trend, indicating greater stability to maintain this state and reduce the possibility of proceeding to the next step.

[0156] In order to study 2e - The existence of ORR allows us to predict CoC. x N 4-x The ability of species to generate and utilize H2O2. The step from ·OOH to H2O2 in CoN4 and CoClN3 was observed to be an exothermic process, indicating their ability to catalyze H2O2 generation. See details. Figure 2However, for the remaining configurations, this step becomes an endothermic reaction, with a positive ΔG5 (Gibbs free energy from ·OOH to H₂O₂). CoC₄ exhibits the highest ΔG5 value at 0.366 eV, exceeding its ΔG4 value of 0.278, indicating that CoC₄ rarely undergoes 2e⁻. - ORR and H2O2 are produced.

[0157] Subsequently, theoretical calculations were performed to evaluate CoC. x N 4-x The HPRR catalytic performance of CoN4 and CoC3N1 was assessed. CoN4 and CoC3N1 exhibited the lowest ΔG6 (Gibbs free energy from ·OH to H2O in HPRR) of 0.278 eV and 0.1475 eV, respectively, indicating superior HPRR catalytic performance. (See details...) Figure 4 Table 3 shows the CoC in detail. x N 4-x The Gibbs free energy values ​​for each step of the HPRR at the Co atom with U = 1.23V are given. This means that although CoN4 and CoC3N1 can produce H2O2, they can effectively catalyze the reduction of H2O2, thus accumulating very little ROS at the cathode.

[0158] Figure 22 For the calculation in CoC x N 4-x The ORR free energy diagram at the Co atom with U = 1.23 V is shown in Table 2. x N 4-x Gibbs free energy values ​​for each step of the ORR catalysis at the Co atom at U = 1.23 V. CoC at U = 1.23 V. x N 4-x The ORR catalytic ability still exhibits the same trend as when U=0, and the most positive ΔG (free energy change) is still shown in the fourth step ( * OH+H + +e - → * The more carbon atoms involved in coordination (+H2O), the larger the ΔG, and the worse the ORR catalytic performance. CoC4 has the most positive ΔG and the worst ORR catalytic performance, while CoN4 has the best ORR catalytic performance.

[0159] Figure 23 Optimized structures for OH* adsorption on CoC4, CoC3N1, CoC2N2-1, CoC2N2-2, CoC3N1, and CoN4 were presented. The key factor leading to significant differences in ORR activity is the different CoC4 and CoN4 structures for OH* adsorption. x N 4-x The adsorption form at the active site. For example... Figure 23As shown, OH* tends to connect vertically to the Co site in CoN4, indicating a strong ability to proceed to the next step. However, its connection to the Co sites in CoC2N2, CoC3N1, and CoC4 is slanted, showing a tendency to connect to the CoC bond bridge, with CoC4 being the most prominent, indicating the weakest ability to proceed to the next step.

[0160] Table 1. CoC x N 4-x Gibbs free energy of oxygen reduction reaction at U=0V

[0161] <![CDATA[ΔG1]]> -1.5325 -1.5738 -1.9934 -1.8428 -2.2354 -2.1507 <![CDATA[ΔG2]]> -0.713 -0.9761 -1.3586 -1.2568 -1.5743 -1.7511 <![CDATA[ΔG3]]> -2.2299 -1.9369 -1.4883 -1.5832 -1.2678 -1.3062 <![CDATA[ΔG4]]> -0.4546 -0.4432 -0.0897 -0.2472 0.1475 0.278 <![CDATA[ΔG5]]> -0.3057 -0.2261 0.1947 0.0446 0.4385 0.366

[0162] Table 2. CoC x N 4-x Gibbs free energy of oxygen reduction reaction at U = 1.23 V

[0163]

[0164]

[0165] Table 3. CoC x N 4-x Gibbs free energy of hydrogen peroxide reduction reaction at U = 1.23 V

[0166] <![CDATA[ΔG 1’ ]]> -0.4382 -0.3999 -0.3987 -0.3982 -0.3969 -0.3847 <![CDATA[ΔG 2’ ]]> -0.3542 -0.7868 -1.7025 -1.5756 -2.0726 -2.4797 <![CDATA[ΔG 3’ ]]> 0.7754 0.7868 1.1403 0.9828 1.3775 1.508

[0167] Example 2. Synthesis and structural characterization of CoC4@graphene and CoN4@graphene

[0168] (1) Synthesis of CoC4@graphene and CoN4@graphene

[0169] Guided by the theoretical calculations in Example 1, CoC4@graphene and CoN4@graphene were ultimately selected for synthesis. The synthesis method is a simple three-step process, as described in [reference]. Figure 5 The specific steps are as follows:

[0170] 1) Dissolve commercial graphene powder in 30 mL of methanol solution, stir vigorously, and then slowly add 3 mL of cobalt solution (CoCl2, 2 mol / L) in water. Keep stirring in the dark for at least 1 hour until the cobalt precursor and graphene are uniformly dispersed to obtain a mixed solution.

[0171] 2) At 60°C, the mixed solution was stirred vigorously until dry to obtain a homogeneous mixture of cobalt precursor and graphene.

[0172] 3) The two composites were heated at 900°C for 24 hours in a nitrogen atmosphere, with or without melamine. The products were then immersed in 1M hydrochloric acid (HCl) solution for 24 hours to remove cobalt particles. The resulting precipitate was centrifuged multiple times with deionized water to obtain CoC4@graphene and CoN4@graphene.

[0173] (2) Characterization of CoC4@graphene and CoN4@graphene

[0174] This invention employs SEM, TEM, XRD, Raman spectroscopy, and XPS to verify the successful synthesis of the two SACs. The results are as follows:

[0175] 1) Scanning electron microscope (SEM)

[0176] Scanning electron microscope (SEM) images of CoC4@graphene, CoN4@graphene, and graphene are shown below. Figure 6 -A and Figure 6 As shown in -C, hierarchical porous nanostructures with a size of approximately 100 nm are clearly visible. High-angle dark-field scanning electron microscopy (HAADF SEM) images and corresponding elemental distribution maps show that the atoms in CoC4@graphene and CoN4@graphene are uniformly distributed, represented by high-density bright spots, highlighted by red circles. See details. Figure 6 -B、 Figure 6 -D、 Figure 24 and Figure 25 The above characterization results indicate that Co species are uniformly distributed within the carbon framework and do not aggregate.

[0177] 2) Transmission electron microscopy (TEM)

[0178] TEM images such as Figure 26 As shown, the image reveals porous graphene structures observed in CoN4@graphene and CoC4@graphene, with no nanoparticle formation detected.

[0179] 3) X-ray diffraction (XRD)

[0180] like Figure 7 As shown, the XRD patterns of CoC4@graphene, CoN4@graphene, and graphene show no peak at 44.3°, indicating the absence of large crystals in the two SAC samples. The broad peak at 25.2° can be attributed to the peak of graphene carbon (002).

[0181] 4) Raman spectroscopy

[0182] This invention further confirmed the successful bonding of Co to the graphene substrate using Raman spectroscopy. Figure 8As shown, the ID / IG ratio of graphene is 1.177, while after the introduction of Co, the ID / IG ratios of CoC4@graphene and CoN4@graphene are 1.180 and 1.181, respectively. Compared with graphene, the ID / IG ratios of CoC4@graphene and CoN4@graphene increase, verifying the successful introduction and incorporation of Co.

[0183] 5) Photoelectron spectroscopy (XPS) and inductively coupled plasma mass spectrometry (ICP-MS)

[0184] This invention utilizes XPS to measure the coordination of CoC4@graphene and CoN4@graphene, and the results are as follows: Figures 27-29 As shown, a distinct peak at 283.94 eV is observed in the C1s spectrum. Considering that no other metal elements besides Co are involved in the synthesis process, this peak can be attributed to Co-C coordination. Furthermore, this peak is not present in CoN4@graphene and graphene, indicating that Co-C coordination is almost nonexistent in CoN4@graphene and graphene.

[0185] In CoN4@graphene, besides the characteristic pyridine and pyrrole nitrogen atoms in graphene, a distinct peak appears in the N 1s spectrum at 399.2 eV, which can be attributed to the formation of Co-N bonds. However, no Co-N peak was observed in the N1 spectrum of CoC4@graphene, suggesting that Co-N coordination hybrids are rarely formed. The N contents of CoC4@graphene and CoN4@graphene are 2.3 wt% and 3.1 wt%, respectively (measured by XPS). It can be seen that the N content of CoN4@graphene is greater than that of CoC4@graphene, but the difference is not significant. This is likely due to the unavoidable incorporation of N elements during the synthesis of graphene, which can be confirmed by the N content of graphene (1.5 wt%). Furthermore, inductively coupled plasma mass spectrometry analysis showed that the Co content of CoN4@graphene (0.97%) was slightly higher than that of CoC4@graphene (0.63%), which may be due to the higher affinity of N for Co, as detailed in Table 4.

[0186] Table 4. Elemental content of CoC4@graphene and CoN4@graphene determined by ICP-MS

[0187]

[0188] 6) Extended X-ray Absorption Fine Structure (EXAFS)

[0189] This invention utilizes EXAFS to further explore the atomic coordination environments of CoN4@graphene and CoC4@graphene. The results are as follows: Figure 9As shown, CoN4@graphene, represented by the red line, and CoC4@graphene, represented by the blue line, almost overlap, indicating that the valence states of Co are very similar. Furthermore, compared to Co foil, they both exhibit positive valence states because the normalized Co K-side absorption threshold of Co SACs shows a larger energy intensity. The Fourier transform (FT) k of CoC4@graphene and CoN4@graphene... 3 The weighted extended EXAFS spectrum showed a The peak value, and in The absence of a Co-Co peak indicates that no nanoparticles were formed during the synthesis process. (See details...) Figure 10 The wavelet transform (WT) of the extended EXAFS spectrum further clarifies the radial distance resolution and k-space resolution. For example... Figures 30-32 As shown, the contour plots indicate that CoC4@graphene and CoN4@graphene are approximately The maximum intensity is only found at this location, indicating the presence of a single Co atom without a Co crystal structure. Around CoC4@graphene and CoN4@graphene, the intensity is approximately... The strong intensity regions represent the Co-C and Co-N pathways, respectively. The fitting analysis results are as follows: Figure 33 , Figure 34 As shown in Table 5, the results indicate that the coordination number of Co-C in CoC4@graphene is calculated to be 3.55±0.75, and the coordination number of Co-N in CoN4@graphene is calculated to be 4.4±0.37, indicating that they both have a tetracoordinate structure.

[0190] Table 5. Co R-space EXAFS fitting parameters for Co foil, CoC4@graphene, and CoN4@graphene

[0191]

[0192] Note: The error range of the structural parameters obtained using EXAFS spectroscopy is estimated as CN ± 20%; R ± 1%; σ² ± 20%.

[0193] 7) Zeta potential

[0194] This invention utilizes Zeta potential to further explore the effect of introducing Co atoms into graphene using CoN4@graphene and CoC4@graphene, thus eliminating the possibility of cobalt nanoparticle formation. Figure 35 As shown, the Zeta potentials of CoN4@graphene and CoC4@graphene are similar to those of graphene, which means that the introduction of Co atoms has little effect on the surface properties of graphene and excludes the formation of cobalt nanoparticles.

[0195] The above results demonstrate that the present invention successfully synthesized CoN4@graphene and CoC4@graphene.

[0196] Example 3. Electrochemical testing of ORR and HPRR performance of Co SACs catalysts

[0197] This invention verifies the calculated ORR through a series of electrochemical experiments. Using Ag / AgCl as the reference electrode and 1M KOH as the electrolyte, cyclic voltammetry (CV) tests were performed under different atmospheres, with a voltage range of 0.2V to -0.8V. The results are as follows: Figure 11 As shown, the CV curves of CoC4@graphene and CoN4@graphene exhibit a significant peak at -0.2V under ambient conditions, reaching a peak at -0.35V. Notably, this peak is significantly enhanced in an O2 environment and completely suppressed in an N2 atmosphere, confirming the involvement of O2 in the catalytic reaction and indicating that ORR occurred. Conversely, graphene did not exhibit this peak in any test environment, suggesting that the Co single atom is the main active site for ORR catalysis. Furthermore, under both O2 and air conditions, the peak intensity of CoN4@graphene exceeds that of CoC4@graphene, indicating that CoN4@graphene possesses higher ORR catalytic activity relative to CoC4@graphene. These experimental results are consistent with computational chemistry findings. Rotating disk electrode (RDE) experiments performed by linear sweep voltammetry (LSV) at a rotation speed of 1600 rpm further confirmed the findings from the CV. It can be observed that the onset potentials of CoN4@graphene and CoC4@graphene are 0.01V and -0.12V respectively, compared to Ag / AgCl, and the diffusion-limiting current density (j) for each is... L The values ​​are -3.3 and -5.4 (mA cm) respectively. -2 This also demonstrates the superior catalytic performance of CoN4@graphene; see details below. Figure 36 .

[0198] Subsequently, 5 μL of CoC4@graphene and CoN4@graphene solutions were dropped onto the surface of GCEs, respectively, and subjected to 0.1 M Lu(bpy)3 at pH 6. 2+ ECL testing was performed in the solution, with a cycling voltage range of -2V to 1.5V vs Ag / AgCl. The test results are as follows: Figure 12As shown in Figure -A, satisfactory cathode ECL emission with an intensity of 11021 a.u. was observed at -1.5V. The invention then verifies the luminescent species and their promoters in the system through a simple experiment. Under saturated oxygen conditions, the luminescence intensity significantly increases to 17402 a.u., while it is significantly suppressed under saturated nitrogen conditions, as detailed in [see figure]. Figure 12 -B. This indicates that oxygen and ORR are at -1.5V for Ru(bpy)3 2+ It plays a crucial role in cathodoluminescence. Conversely, when the better ORR catalyst CoN4@graphene is used in the same system, the ECL intensity at the same potential is only 568 a.u., and it is less affected by changes in atmospheric conditions.

[0199] To further elucidate the catalytic performance of Co SACs, LSV measurements were performed on CoC4@graphene and CoN4@graphene at RDE speeds (ω) ranging from 225 to 3600 rpm. The results are shown in [reference needed]. Figure 12 -C. Based on the Koutecky-Levich (KL) equation, the number of electrons transferred (n) per oxygen molecule (O2) in CoC4@graphene is derived to be 2.5–3.3, while that in CoN4@graphene is derived to be 4.3–4.5, consistent with the calculated results. This invention also uses rotating ring-disk electrode (RRDE) measurements as a supplementary method to support the results obtained from RDE, as detailed in [link to details]. Figure 37 RRDE measurements quantify the number of transferred electrons and the H₂O₂ formation rate by analyzing the percentage contribution of four-electron processes (reflected by disk current Id) and two-electron processes (reflected by ring current Ir) to the overall reaction process. This method provides an accurate assessment for CoN₄@graphene, but for CoC₄@graphene, it cannot accurately reflect the number of transferred electrons in its ORR because it is primarily composed of 3e electrons. - ORR rarely occurs 2e - ORR (Theoretical calculations show that the conversion of OOH to H2O2 is an energy-consuming process, and ΔG5 is greater than 4e) - ORR's ΔG4).

[0200] Tafel curves are a direct criterion for distinguishing the activity of electrocatalysts. This invention further investigates the catalytic efficiency of CoN4@graphene and CoC4@graphene using Tafel curves, with results as follows: Figure 38 As shown, the slopes of the Tafel curves for CoN4@graphene and CoC4@graphene are 79 mV / dec and 97 mV / dec, respectively, indicating lower overpotential and higher ORR catalytic activity.

[0201] The HPRR activity of the two catalysts was also evaluated by CV and LSV with the same concentration of H2O2, and the results are as follows: Figure 39 As shown, the HPRR of CoN4@graphene begins at approximately 0.20 V vs. Ag / AgCl, which is more positive than the onset potential of CoC4@graphene (0.0 V vs. Ag / AgCl). These results indicate that CoN4@graphene exhibits greater catalytic activity in HPRR, while CoC4@graphene exhibits poorer HPRR catalytic performance, consistent with theoretical predictions.

[0202] Example 4. Possible mechanism by which CoN4@graphene promotes Ru(bpy)32 anodic luminescence

[0203] Regarding the mechanism of Ru(bpy)32 anodic electrochemiluminescence (ECL), we believe it is related to a precise OER reaction catalyzed by CoN4@graphene. Based on... Figure 40 The electrochemical experimental results shown indicate that CoN4@graphene and CoC4@graphene catalyze the OER reaction (H2O-e) at approximately 1.30 V and 1.34 V, respectively. - →·OH + H + Meanwhile, the oxidation potential of Ru(bpy)32 is approximately 1.13 V, indicating that Ru(bpy)32 begins to be oxidized and forms Ru(bpy)3. 3+ (Ru(bpy)3 2+ -e - →Ru(bpy)33). Therefore, since Ru(bpy)3 2+ The oxidation potential of Ru(bpy)33 precedes that of the OER reaction catalyzed by Co SACs. The accumulation of Ru(bpy)33 in the system allows for rapid utilization of the ROS (mainly ·OH, the first-step product of the OER reaction) generated by CoN4 catalysis. Subsequently, Ru(bpy)33 reacts with OH to generate excited-state Ru(bpy)32 (Ru(bpy)32... * When the excited state returns to the ground state, it emits a photon.

[0204] For CoC4@graphene, which has a weaker OER catalytic ability, its OER onset potential is 1.34V, slightly later than that of CoN4@graphene. Its weak OER catalytic performance is insufficient to generate enough ROS to utilize Ru(bpy)3. 3+ Therefore, it does not have the ability to promote Ru(bpy)3. 3+Anodic emission capability. This invention also employed the widely recognized and excellent OER catalyst, commercially available Pt / C, for electrochemical and ECL testing. The results showed that the OER catalytic ability of the Pt / C catalyst was higher than that of CoN4@graphene, but its effect on Ru(bpy)3 was less pronounced. 2+ The promoting effect of anodic ECL is only about 3000 a.u., see details. Figure 41 This can be attributed to the excessive generation of the final product O2 due to the overly strong OER catalytic performance, which extinguishes Ru(bpy)3. 2+ The anodic luminescence was observed. To demonstrate the existence of the O2 quenching effect, we used 0.1 M Ru(bpy)3... 2+ CV tests were performed on a bare GCE in the system under O2, air, and N2 conditions. The results are as follows: Figure 42 As shown, in an air environment, Ru(bpy)3 2+ The anodic emission of Ru(bpy)3 was approximately 6100 a.u., while in an O2 environment, its emission was significantly suppressed, remaining at only 1877 a.u. Conversely, in an N2 environment, ECL emission was greater than in air, approximately 7800 a.u. These results confirm the effect of O2 on Ru(bpy)3. 2+ Suppression effect of anodic luminescence.

[0205] Similarly, although the presence of CoN4@graphene enhances anodic emission, Ru(bpy)3 in an O2 environment... 2+ The anodic emission was still suppressed to approximately 2018 a.u., significantly lower than the 10100 a.u. under air conditions. On the other hand, in a nitrogen environment without O2, the emission increased to 12300 a.u. This also demonstrates that even in the presence of a promoter, O2 significantly affects the emission of Ru(bpy)3. 2+ The suppression effect of anodic luminescence still exists.

[0206] In summary, this invention has elucidated Ru(bpy)3 2+ The ECL emission mechanism at the anode. First, H₂O is converted to ·OH via a suitable OER reaction catalyzed by CoN₄@graphene. Due to its strong oxidizing power, OH excites part of Ru(bpy)₃. 2+ Formation of Ru(bpy)3 2+ * Then it returns to the ground state and emits photons. Simultaneously, XPS analysis of CoN4@graphene confirmed its similarity to Ru(bpy)3. 2+ The nitrogen content decreased from 2.66% to 0.99% after 2 hours of reaction. (See details...) Figure 43 Further speculation suggests that a small number of -NH2 groups on the CoN4@graphene surface promote the growth of Ru(bpy)3. 2+Upon entering the excited state, it resembles tripropylamine (TrA). Under anodic voltage, Ru(bpy)3 2+ A small amount of CoN4@graphene undergoes an oxidation reaction on the electrode surface, producing Ru(bpy)3. 3+ And CoN4@graphene·. Subsequently, CoN4@graphene· undergoes a deprotonation process to generate a strongly reducing intermediate (CoN4@graphene·), which reacts with Ru(bpy)3. 3+ The reaction forms Ru(bpy)3 in an excited state. 2+ This leads to the launch of the ECL.

[0207] This invention is achieved through the following equations and Figure 17 In summary, the above findings are as follows:

[0208] Pathway 1 (primary):

[0209] m Ru(bpy)3 2+ +·OH+(nm)H + →m Ru(bpy)3 2+ * +(nm)Ru(bpy)3 3+ +mH2O;

[0210] m Ru(bpy)3 2+ * →Ru(bpy)3 2+ +hv.

[0211] Pathway 2:

[0212] Ru(bpy)3 2+ -e - →Ru(bpy)3 3+ ;

[0213] CoN4@graphene(ArNRCH2R')-e - →[CoN4@graphene(ArNRCH2R')] +· ;

[0214] [CoN4@graphene(ArNRCH2R')] +· -H + →[CoN4@graphene(ArNRCHR')] · ;

[0215] [CoN4@graphene(ArNRCHR')] · +Ru(bpy)3 3+→[CoN4@graphene(ArNR=CHR')] ++ Ru(bpy)3 2+ * ;

[0216] [CoN4@graphene(ArNR=CHR')]+H2O - H + →[CoN4@graphene(ArNRH)]+R'CHO;

[0217] Ru(bpy)3 2+ * →Ru(bpy)3 2+ +hv.

[0218] Example 5. Possible mechanism by which CoC4@graphene promotes Ru(bpy)32 cathodoluminescence

[0219] This embodiment further explores the possible mechanism by which CoC4@graphene promotes the cathodoluminescence of Ru(bpy)32. Electrochemical experimental results show that CoN4@graphene and CoC4@graphene initiate the ORR catalytic process at approximately 0 V and -0.2 V, respectively. LSV and RRDE results indicate that CoN4@graphene catalyzes a 4-electron transfer ORR reaction, while CoC4@graphene promotes a 3-electron transfer ORR reaction. Specifically, CoN4@graphene can catalyze a complete ORR reaction to produce the final product H2O, while the ORR reaction catalyzed by CoC4@graphene can successfully complete the first three electron transfer steps, but fails to complete the final electron transfer (·OH). + H + +e - →H2O). This is consistent with the theoretical results calculated by DFT. Ru(bpy)32 begins to reduce at approximately -1.60V (Ru(bpy)3 2+ +e - →Ru(bpy)3). Therefore, there is a certain distance between the initiation of the ORR catalyzed by Co SACs and the formation of Ru(bpy)3 at -1.60V. Thus, the effective accumulation of ROS from low potential to -1.6V during the potential scan is crucial for exciting the Ru(bpy)32 cathode ECL. Since the 4-electron ORR reaction catalyzed by CoN4@graphene is rapid and complete, directly yielding the final product H2O, ROS cannot accumulate in the system; while CoC4@graphene regulates the incomplete 3-electron ORR reaction, thus favoring ROS accumulation in the system. This accumulated ROS helps promote the formation of Ru(bpy)3. 2+The cathode emits light.

[0220] By studying the changes in cathodic electroluminescence (ECL) intensity with varying scan rate, this invention verifies the theory of free radical accumulation. Increasing the scan rate leads to a decrease in free radical accumulation, while decreasing the scan rate leads to an increase in free radical accumulation, due to the prolonged reaction time. Figure 13 As shown, when the scan rate is 0.5 V / s, the Ru(bpy)3 promoted by CoC4... 2+ The cathode ECL only decreased to 2592 a.u. Reducing the scan rate to 0.2 V / s increased the ECL intensity to 7904 a.u, while further reduction to 0.1 V / s resulted in 16280 a.u. Further reductions in the scan rate did not significantly change the ECL intensity. This can be attributed to the balance achieved between ROS accumulation and depletion. This experiment not only demonstrates the existence of the free radical accumulation process but also confirms that 0.1 V / s is the optimal scan rate. For CoN4@graphene, the scan rate had no significant effect on the cathode ECL, indicating a lack of ROS accumulation; see [link to details]. Figure 14 .

[0221] Subsequently, this invention employed a time-dependent electroluminescence (Td ECL) method to investigate the effects of CoC4@graphene and CoN4@graphene on Ru(bpy)3 by eliminating the process of free radical accumulation. 2+ The effect of cathode ECL. First, E0 = 0V was applied for 10 seconds, then the potential was adjusted to Ered = -1.6V over the next 10 seconds, and this voltage switching cycle was repeated. With the applied Ered, ECL emission rapidly reached its maximum peak and then exhibited logarithmic decay, indicating a logarithmic decrease in ROS concentration at the electrode surface. See details... Figure 15 The results show that CoC4@graphene approximately makes Ru(bpy)3 2+ The cathode ECL was enhanced by 6326 a.u., which is lower than the 16280 a.u. of ROS accumulated by cyclic voltammetry. See details. Figure 16 Conversely, due to its stronger catalytic performance in the oxygen reduction reaction, CoN4@graphene can catalyze Ru(bpy)3. 2+ The cathode ECL enhancement is approximately 10,000 a.u. This is because in Ru(bpy)3 2+ At a reduction potential of -1.6V, both CoC4@graphene and CoN4@graphene promote the oxygen reduction reaction to produce ROS, which can be directly converted by Ru(bpy)3. 3+ Therefore, Ru(bpy)3 2+The cathode ECL depends only on the intensity of the oxygen reduction reaction and is not affected by ROS accumulation. Therefore, our proposed theory of free radical accumulation is valid.

[0222] To further investigate the effects of different types of ROS on Ru(bpy)3 2+ The contributions of anodic and cathodic luminescence were considered, a ROS scavenger was introduced into the system, and changes in ECL intensity were observed. Results Figure 44 As shown, in the presence of ROS scavengers, Ru(bpy)3 2+ The cathode ECL emission was significantly reduced. However, overall, the reduction caused by the addition of the ·OOH scavenger was greater than that caused by the ·OH scavenger. For the CoN4@graphene-promoted anodic ECL, the reduction caused by the ·OH scavenger was greater than that caused by the ·OOH scavenger. These observations suggest that ROS significantly affects Ru(bpy)3. 2+ Both cathodic and anodic electroluminescence contribute to the process, with ·OOH and ·OH playing dominant roles in promoting cathodic and anodic electroluminescence, respectively.

[0223] This embodiment provides a detailed explanation of the cathodic emission mechanism. ROS is generated and accumulated in the system. Ru(bpy)3 2+ It accepts an electron to form Ru(bpy). 3+ It then reacts with ROS to generate Ru(bpy)32 * And ECL emission occurs. Furthermore, due to the strong oxidizing power of ·OH, Ru(bpy)3 2+ It is oxidized to Ru(bpy)3 3+ Then, with Ru(bpy) 3+ Regeneration of Ru(bpy)32 * .

[0224] This invention summarizes Ru(bpy)3 2+ The cathodoluminescence mechanism is shown in the following equation and Figure 17 As shown:

[0225] O2+e - +H + →·O2H;

[0226] O2+3e - +H + →·OH;

[0227] Ru(bpy)3 2+ +e - →Ru(bpy) 3+ .

[0228] Pathway 1:

[0229] Ru(bpy) 3+ +·O2H+H + →Ru(bpy)3 2+ * +H₂O₂,Ru(bpy) 3+ +·OH+H + →Ru(bpy)3 2+ * +H2O;

[0230] Ru(bpy)3 2+ * →Ru(bpy)3 2++ hv.

[0231] Pathway 2:

[0232] Ru(bpy)3 2+ +·OH+H + →Ru(bpy)3 3+ +H2O;

[0233] Ru(bpy) 3+ +Ru(bpy)3 3+ →Ru(bpy)3 2+ * +Ru(bpy)3 2+ ;

[0234] Ru(bpy)3 2+ * →Ru(bpy)3 2+ +hv.

[0235] Example 6. Construction and characterization of a proportional immunosensor based on a Co single-atom alloy

[0236] CoC4@graphene and CoN4@graphene were dropped onto the electrode surface and loaded as markers for secondary antibody (Ab2), assembling into a sandwich-type immunosensor for detecting trace biomarkers in body fluids, with carcinoembryonic antigen (CEA) as a representative example. The fabrication process of the immunosensor is described in [link to documentation]. Figure 18 The specific steps include: mixing CoN4@graphene with Ab2 and incubating for 12 hours to obtain the label. 5 μL of CoC4@graphene (2 mg / ml) was dropped onto a polished electrode surface, followed by the addition of 5 μL of EDC / NHS, and incubation at 37°C for 30 minutes. After washing, primary antibody (Ab1) was added and incubated at 37°C for 1 hour. Following another washing step, bovine serum albumin (BSA) was used to block the sensor, and the antigen and prepared secondary antibody were added sequentially to construct an immunosensor, which can then be used to detect biomarkers.

[0237] Use 5mM [Fe(CN)6] 3- / 4- The solution was subjected to electrochemical impedance spectroscopy (EIS) testing in a three-electrode system to demonstrate the successful fabrication of the immunosensor. Figure 19 As shown, the radius of curvature of CoC4-GCE (CoC4@graphene-modified GCE) is significantly smaller than that of the bare electrode, which can be attributed to the excellent conductivity of the graphene substrate. With the addition of Ab1, Ag(CEA), and Ab2, the radius of curvature gradually increases, due to the increased resistance of the protein, validating the successful construction of the sandwich-structured immunosensor. To provide further visual evidence, atomic force microscopy (AFM) was used to observe the surface morphology and material thickness, with results as follows: Figure 45 As shown, with the sequential construction of each layer of the sensor, the surface thickness increases, and the surface roughness becomes more pronounced. These results demonstrate that the present invention successfully establishes a biosensor.

[0238] Example 7. Optimization of Detection Conditions

[0239] (1)Ru(bpy)3 2+ concentration

[0240] This invention further examines Ru(bpy)3 2+ The concentration. For example... Figure 46 As shown, it was observed in Ru(bpy)3 2+ At a concentration of 1 mM, CoC4@graphene exhibited the highest Ia / Ic ratio, indicating that it has the greatest enhancing effect on cathodic luminescence without overstimulating anodic luminescence. Meanwhile, CoN4@graphene showed the lowest Ia / Ic ratio, meaning it effectively promotes Ru(bpy)3... 2+ It exhibits anodic luminescence but also low cathodic luminescence. Ru(bpy)3 2+ Optimal cathodic or anodic luminescence enhancement selectivity cannot be achieved at concentrations above or below 1 mM. This may be because at high concentrations, Ru(bpy)3... 2+ This not only exhibits higher luminescence intensity but also leads to excessive aggregation of the excited state of the complex in solution, resulting in unavoidable side reactions and thus impairing the specific luminescence at a particular potential. On the other hand, lower concentrations of Ru(bpy)3... 2+ Restricted Ru(bpy)3 2+ The generation of excited states limits the promotion of cathode and anode luminescence. Therefore, Ru(bpy)3 was ultimately selected. 2+ The concentration is 1 mM.

[0241] (2) pH value

[0242] To determine the optimal pH value, this invention investigated the ECL signal intensity and Ig (Ia / Ic) values ​​under four pH conditions (pH = 4, 5, 6, 7), because pH < 4 may be detrimental to protein biomarkers. Figure 47 It can be observed that CoN4@graphene exhibits the highest Ig(Ia / Ic) at pH=6, while CoC4@graphene exhibits the lowest Ig(Ia / Ic) at the same pH. This means that CoN4@graphene can maximize both anodic and cathodic luminescence. Therefore, pH=6 was ultimately chosen as the pH value for the detection system.

[0243] Example 8. Linearity, Selectivity, and Stability

[0244] (1) Linear detection

[0245] Under the optimal detection conditions of Example 6, the present invention used the immunosensor constructed in Example 5 to detect different concentrations of CEA. Figure 20 As shown, with increasing CEA concentration, the cathode ECL signal of CoC4@graphene weakens, while the anodic ECL signal of CoN4@graphene strengthens accordingly. Furthermore, Ig(Ia / Ic) exhibits a satisfactory relationship with the logarithm of CEA concentration, ranging from 10... -15 ng / ml~10 -9 ng / ml. The relevant linear equation is y = 0.25246x + 2.77747, and the correlation coefficient is R. 2 =0.994; the limit of detection (LOD) is estimated to be 33 fg / mL.

[0246] (2) Selectivity

[0247] This invention selects epidermal growth factor receptor (EGFR) (10) -2 ng / mL), estrogen receptor α (ERα) (10 -2 ng / mL) and nuclear matrix protein 22 (NMP) 22 (10) -2 (ng / mL) were used as interfering substances to evaluate the selectivity of the system; these were common interfering biomolecules found in the blood of both patients and healthy individuals. For example... Figure 21 As shown, in GFR, ERα and NMP 22 In the presence of CEA, the ECL signal intensity was similar to that of the blank solution, while a high ECL response was obtained in the presence of CEA. Furthermore, the mixed solution exhibited similar ECL intensity compared to the solution containing only CEA. These results indicate that the system has good selectivity for CEA.

[0248] (3) Stability

[0249] This invention investigated the stability of the immunosensor under 15 consecutive scans from -1.5V to 2V against Ag / AgCl. The results are as follows... Figure 48 As shown, the luminescence from the cathode and anode remained relatively stable, with relative standard deviations of 5.87% and 0.38%, respectively.

[0250] (4) Real sample detection

[0251] To further evaluate the clinical detection and analytical capabilities of this immune sensor, the invention was tested on real samples. Serum samples were collected from three healthy individuals. Using a spiking method, a known concentration (1 × 10⁻⁶) of the spiking agent was added to these three samples. -12 CEA solutions were prepared and analyzed using the developed immunosensor. Ig (Ia / Ic) values ​​were recorded, and CEA concentrations were determined using a standard curve. Each sample was tested three times. Table 1 presents the mean and relative standard deviation (RSD) of the three replicate tests. The final results showed no significant difference between detected and added CEA, with RSDs fluctuating between 5.53% and 8.71%. These results demonstrate the significant potential of the sensor provided by this invention for clinical applications.

Claims

1. Ru(bpy)3 2+ Electrochemiluminescence promoter, characterized in that, The Ru(bpy)3 2+ Electrochemiluminescence promoters include Ru(bpy)3 2+ Anodic luminescence promoter and Ru(bpy)3 2+ Cathodoluminescence promoter; the Ru(bpy)3 2+ The anodic luminescence promoter is CoN4@graphene; the Ru(bpy)3 2+ The cathodoluminescence promoter is CoC4@graphene; the N content in the CoC4@graphene is 2wt%-2.5wt%, and the Co content is 0.5%-0.8%; the N content in the CoN4@graphene is 2.7wt%-3.5wt%, and the Co content is 0.9%-1.1%.

2. A method for predicting Ru(bpy)3 as described in claim 1 2+ The method for electrochemiluminescence promoters is characterized by, Includes the following steps: Establish cobalt single-atom catalyst models with different coordination structures; The 4e content of the cobalt single-atom catalyst was studied using density functional theory and first-principles methods. - ORR and 2e - The ORR process was evaluated, and Ru(bpy)3 was screened to assess its ORR catalytic performance. 2+ Luminescence promoter.

3. The method according to claim 2, characterized in that, Step (1) includes: constructing a unit cell with a length of 9.838 Å, a width of 12.780 Å, and a height of 15.000 Å in the catalytic site region; constructing a defect structure at the geometric center of the unit cell; introducing a single Co atom at the center of the defect structure, and substituting the carbon at the defect according to different nitrogen coordination conditions to obtain six single-atom catalytic site models, including CoC4, CoC3N1, CoC2N2-1, CoC2N2-2, CoC3N1, and CoN4.

4. The method according to claim 2, characterized in that, The density functional theory and first-principles approach includes the following steps: The cobalt single-atom catalyst model was structurally optimized based on density functional theory using a pseudopotential-plane wave algorithm to obtain the optimized model. Based on the structure optimization model obtained in step (1), a reaction intermediate ROS model is constructed and docked with the central atom of the catalytic site to simulate the ROS adsorption process and obtain the intermediate adsorption state. Calculate the single-point energy of each molecular adsorption system and obtain the reaction kinetic curves.

5. A method for preparing Ru(bpy)3 according to claim 1 2+ The method for electrochemiluminescence promoters is characterized by, Includes the following steps: A cobalt solution was added to graphene to produce a mixed solution. After drying the mixed solution obtained in step (1), the reaction was carried out under N2 atmosphere. The product was soaked in hydrochloric acid to remove cobalt particles, and centrifuged to obtain CoC4@graphene and CoN4@graphene.

6. The preparation method according to claim 5, characterized in that, In step (1), the mass ratio of graphene to cobalt solution is 2-5:

1.

7. The application of cobalt single-atom catalysts in the preparation of reagents for carcinoembryonic antigen detection, characterized in that, The cobalt single-atom catalyst is CoN4@graphene and CoC4@graphene as described in claim 1; the reagent is Ru(bpy)3. 2+ Potential-resolved electrochemiluminescence promoters.

8. Containing Ru(bpy)3 as described in claim 1 2+ Biosensors based on electrochemiluminescence promoters.

9. A method for detecting carcinoembryonic antigen, characterized in that, The biosensor of claim 8 is used to detect carcinoembryonic antigen (CEA) using ECL, and the ECL signal intensity is obtained; the ECL signal intensity is used to qualitatively and / or quantitatively analyze the CEA.

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