Nano-enzyme for detecting carcino-embryonic antigen

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nano-enzyme for carcino-embryonic antigen detection. The nano-enzyme is iron-based monatomic nano-enzyme dFeSA, the dFeSA is Fe and N-doped amorphous carbon nano-particles, and Fe-N3-C is taken as a core and is combined with a carbon-based carrier with nitrogen vacancy and carbon defect. According to the present invention, the sensitivity is high, the detection limit is as low as 0.017 ng / mL, the detection range is wide, the stability is excellent, the anti-interference capability is excellent, and the carcino-embryonic antigen detection method has advantages of simple operation and low cost, and is suitable for early diagnosis and prognosis monitoring of cancers.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a nanozyme for detecting carcinoembryonic antigen. Background Art

[0002] Carcinoembryonic antigen (CEA) is a key serum marker for a variety of malignant tumors, including lung cancer, colorectal cancer, and gastric cancer. Its accurate detection has important clinical value in the early diagnosis of cancer, efficacy evaluation, and prognosis monitoring. At present, the clinical detection of CEA mainly relies on technologies such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). The core principle of these methods is to use natural enzymes such as horseradish peroxidase to catalyze substrate color development. However, natural enzymes have obvious limitations: high preparation cost, poor storage stability (easy to inactivate), complex operating procedures, and the linear detection range of traditional methods is narrow (usually 0.16-10 ng / mL), which makes it difficult to meet the wide range of detection requirements for CEA concentrations in clinical samples.

[0003] In recent years, nanozymes (artificial enzyme mimics) have become a research hotspot for replacing natural enzymes due to their excellent stability, low cost and controllable catalytic activity. For example, Ir nanowires (ACS Appl. Nano Mater. 2023, 6, 13208-13215) and H-Pt3Sn nanozymes (Nano Lett. 2023, 23, 267-275) showed high sensitivity in CEA detection, with detection limits (LOD) reaching 0.92 pg / mL and 0.49 pg / mL, respectively. However, the dynamic detection range of these nanozymes is still limited (Ir: 0.0032-50 ng / mL; H-Pt3Sn: 0.001-4 ng / mL), which cannot fully cover the actual concentration range of CEA in clinical samples. In addition, the existing nanozymes have insufficient anti-interference performance in complex biological samples (such as serum), and are prone to aggregation or decreased activity during long-term storage, which seriously restricts their clinical application.

[0004] Based on the above analysis, developing a new CEA detection method with a wide linear range, high sensitivity, excellent anti-interference ability and long-term stability is still a key technical problem that needs to be broken through in the current research field. Summary of the Invention

[0005] Technical issues

[0006] To address the existing problems of CEA detection methods, such as strong dependence on natural enzymes, narrow detection range, weak anti-interference ability, and high cost, as well as the bottlenecks of low catalytic efficiency and insufficient signal amplification ability of traditional nanozymes, the present invention provides a colorimetric immunoassay method based on iron-based single-atom nanozymes (dFeSA). This method achieves highly sensitive and wide-range detection of CEA through highly active nanozyme signal amplification technology without the need for complex instruments, making it suitable for clinical serum sample analysis.

[0007] Technical Solution

[0008] The first aspect of the present invention provides a nanozyme for carcinoembryonic antigen detection, wherein the nanozyme is an iron-based single-atom nanozyme dFeSA, and the dFeSA is an amorphous carbon nanoparticle doped with Fe and N, with Fe-N3-C as the core, combined with a carbon-based carrier with nitrogen vacancies and carbon defects.

[0009] In some embodiments, the Fe-N bond length in the Fe-N3-C is The coordination number is 3.

[0010] In some embodiments, the nanozyme has an oxidase-like activity towards TMB that is more than 15 times that of the NC nanozyme in an acetic acid-sodium acetate buffer at pH 4.0.

[0011] In some embodiments, the preparation method of the nanozyme includes the following steps: mixing an iron source, an organic ligand and a template in a solvent, and synthesizing a metal-organic framework precursor by a template synthesis method; pyrolyzing the precursor at high temperature under an inert atmosphere to obtain a nanozyme with a single-atom dispersed iron active center; wherein the iron source is an aqueous solution of Fe(NO3)3, the organic ligand is a methanol solution of 2-methylimidazole and a methanol solution of Zn(NO3)2, and the template is an aqueous solution of BSA.

[0012] In some embodiments, the preparation method of the nanozyme specifically includes the following steps: a. mixing a methanol solution of 2-methylimidazole, a BSA aqueous solution and an Fe(NO3)3 aqueous solution with a volume ratio of 8:1:1 and a mass concentration ratio of 77:40:4 to obtain a mixed solution; b. then adding a methanol solution of Zn(NO3)2 of the same volume as the mixed solution in step a and a mass concentration ratio of 55.8:77 to the 2-methylimidazole in step a; c. continuously stirring at room temperature for 1 to 3 hours, centrifuging to separate the solid, washing it with methanol 1 to 3 times, and drying it at 50 to 65°C to obtain a metal-organic framework precursor Fe-BSA@ZIF-8; d. placing Fe-BSA@ZIF-8 in a tube furnace, passing argon gas and heating it to 900 to 1000°C, maintaining it for 2 to 4 hours, naturally cooling it, collecting it and grinding it to obtain a nanozyme dFeSA with a single-atom dispersed iron active center.

[0013] The second aspect of the present invention provides the use of any of the above-mentioned nanozymes in the detection of carcinoembryonic antigen.

[0014] The third aspect of the present invention provides a method for detecting carcinoembryonic antigen for non-disease diagnosis purposes, comprising the following steps: S1, coupling the nanozyme described in any one of the above items with a detection antibody to form a nanozyme-detection antibody complex; S2, constructing a double antibody sandwich immunoassay system based on the nanozyme: first, coating the capture antibody on a solid phase carrier, and then re-coating it with a blocking agent; then adding the sample to be tested and incubating it, so that the capture antibody binds to the carcinoembryonic antigen in the sample to be tested; finally, adding the nanozyme-detection antibody complex and continuing to incubate to form a capture antibody-carcinoembryonic antigen-detection antibody-nanozyme sandwich complex; S3, adding TMB (3,3',5,5'-tetramethylbenzidine) for a color reaction; S4, measuring the absorbance of the reaction solution at 652nm, and combining the measured absorbance value with the standard curve for data analysis.

[0015] In some embodiments, the capture antibody is a mouse anti-human CEA monoclonal antibody; and the detection antibody is a rabbit anti-human CEA polyclonal antibody.

[0016] In some embodiments, the amino groups of the detection antibody are covalently linked to the carboxyl groups on the surface of the nanozyme through a cross-linker to form a stable nanozyme-detection antibody complex.

[0017] In some embodiments, the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) or N-hydroxysuccinimide (NHS).

[0018] In some embodiments, the capture antibody coating condition is incubation at 4° C. for 12-16 hours; and the blocking agent re-coating condition is incubation at 37° C. for 1 hour.

[0019] In some embodiments, the solid support is a polystyrene microplate.

[0020] In some embodiments, the blocking agent is BAS.

[0021] In some embodiments, the sample to be tested is a blood product, preferably serum.

[0022] In some embodiments, the incubation condition is 37° C. for 1 hour.

[0023] In some embodiments, the temperature during the color development reaction is 23-26° C., preferably 25° C., and the color development time is 3-8 minutes, preferably 5 minutes.

[0024] In some embodiments, the color development reaction is performed in 0.1 M acetic acid-sodium acetate (HAc-NaAc) buffer at pH 4.0.

[0025] In some embodiments, the linear detection range of the method is 0.05-300 ng / mL.

[0026] In some embodiments, the limit of detection (LOD) of the method is 0.017 ng / mL.

[0027] In some embodiments, the limit of quantitation (LOQ) of the method is 0.05 ng / mL

[0028] In some embodiments, the method has no cross-reactivity to the following interferors: human serum albumin (HSA), prostate specific antigen (PSA), alpha-fetoprotein (AFP), immunoglobulin G (IgG), neuron-specific enolase (NSE), and ascorbic acid (AA).

[0029] The fourth aspect of the present invention provides a detection kit comprising: a nanozyme-detection antibody complex formed by coupling the nanozyme described in any one of the above items with a detection antibody; a solid phase carrier pre-coated with a capture antibody; a TMB colorimetric substrate solution; an acidic buffer solution; and a CEA standard.

[0030] In some embodiments, the detection kit has a detection retention rate of ≥90% after being stored at 25° C. for 21 days.

[0031] Technical Effects

[0032] 1. High Sensitivity and Wide Detection Range: This method, based on the atomic-level catalytic activity of dFeSA, has a detection limit as low as 0.017 ng / mL, significantly outperforming the traditional enzyme-linked immunosorbent assay (ELISA) (detection limit of 0.1-1 ng / mL), enabling accurate identification of low-concentration CEA in early-stage cancer patients. Furthermore, its linear detection range extends from 0.05-300 ng / mL, covering normal values (<5 ng / mL) to high concentrations in advanced cancer patients (>25 ng / mL), meeting the needs of monitoring throughout the entire disease course.

[0033] 2. Excellent stability and anti-interference ability: The detection system retains ≥90% activity after 21 days of storage at 25°C, far exceeding the activity of natural enzymes and traditional nanozyme systems. Furthermore, it has no cross-reactivity with common interfering substances in serum (such as HSA, PSA, and AA), and the recovery rate of clinical sample detection is stable at 92.7-102.6%, significantly reducing the risk of false positives.

[0034] 3. Simple operation and low cost: The detection process does not rely on natural enzymes, radioactive labels, or complex instruments, and the single detection time is shortened to 5 minutes (traditional ELISA requires more than 30 minutes). dFeSA can also be synthesized on a large scale, significantly reducing the cost of detection, making it suitable for cancer screening in primary healthcare institutions and resource-limited areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Schematic diagram of dFeSA structure (Fe-N3-C) and reaction principle;

[0036] Figure 2 : Schematic diagram of dFeSA synthesis;

[0037] Figure 3 : Morphology and structure analysis of dFeSA;

[0038] Figure 4 : Fe K-edge XANES and EXAFS spectra;

[0039] Figure 5 :Oxidase-like activity test, test condition optimization and kinetic analysis of dFeSA;

[0040] Figure 6 :Catalytic mechanism verification diagram;

[0041] Figure 7 : The detection range of CEA, its stability and anti-interference performance of this detection system. DETAILED DESCRIPTION

[0042] To facilitate the technical solution of the application, the following first provides a general explanation and definition of the terms and expressions involved in this application.

[0043] The terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0044] The detection method of the present invention is based on the synergistic effect of the atomic-level catalytic activity of dFeSA and sandwich immunoassay technology, achieving highly sensitive, wide-range detection of CEA through a signal amplification mechanism. Its core principle can be systematically explained as follows: the catalytic activity of dFeSA stems from its unique Fe-N3 coordination structure, in which single-atom iron coordinates with three nitrogen atoms to form an unsaturated active center. This structure significantly enhances the adsorption and activation of oxygen, catalyzing the conversion of O2 into superoxide radicals (·O2-). The generated ·O2- acts as a strong oxidant, efficiently oxidizing the chromogenic substrate TMB to produce a blue product (oxTMB), whose absorbance is positively correlated with CEA concentration.

[0045] The high-defect carbon-based support in the present invention refers to a nitrogen-carbon material having abundant nitrogen vacancies and carbon defects.

[0046] The double antibody sandwich immunoassay system in the present invention is an immunoassay technology based on antigen-antibody specific binding. Its core principle is to form an "antibody-antigen-antibody" sandwich structure (sandwich complex) by having two antibodies (capture antibody and detection antibody) bind to different epitopes of the target antigen respectively, thereby achieving highly specific and sensitive detection of the target antigen.

[0047] In each set of comparative experiments provided in this application, unless otherwise specified, other experimental conditions, materials, etc., except for the differences indicated in each group, are kept consistent to ensure comparability.

[0048] Unless otherwise specified, the reagents and instruments used in the embodiments of the present invention can be purchased from the market.

[0049] The following further describes the nanozyme for carcinoembryonic antigen detection provided in this application.

[0050] Example 1 Design and preparation of nanozymes

[0051] 1.1 Design of nanozymes

[0052] like Figure 1As shown, the present invention takes a single-atom iron active center (Fe-N3-C) as the core and combines it with a nanozyme design strategy for a highly defective carbon-based carrier. The designed dFeSA nanozyme is an amorphous carbon nanoparticle doped with trace amounts of Fe and N, in which Fe and N exist in the form of Fe-N3 coordination: through the atomically dispersed Fe-N3 coordination structure, the utilization of iron atoms is maximized, while the electronic regulation of Fe by N atoms is utilized to reduce the energy barrier for O2 activation and significantly enhance the oxidase-like activity. In addition, nitrogen vacancies (N-vacancy) and carbon defects are introduced into the carbon-based carrier to accelerate the diffusion of substrates and enhance mass transfer and stability. Theoretical calculations show that the adsorption energy (ΔEads) of the Fe-N3-C site for O2 is approximately 0.3 eV lower than that of the traditional Fe-N4 structure, promoting the efficient conversion of O2 to ·O2-.

[0053] 1.2 Preparation of nanozymes

[0054] The specific preparation method is as follows Figure 2 As shown, the details are as follows:

[0055] 8 mL of methanol solution containing 2-methylimidazole (0.308 g) was mixed with 1 mL of 20 mg / mL BSA aqueous solution and 1 mL of 2 mg / mL Fe(NO3)3 aqueous solution. Subsequently, 10 mL of methanol solution containing 0.279 g Zn(NO3)2 was added. After continuous stirring at room temperature for 2 h, the solid was separated by centrifugation (5000 rpm, 3 min) and washed three times with methanol. The collected solid was dried at 60 ° C to obtain Fe-BSA@ZIF-8. Fe-BSA@ZIF-8 was then placed on a quartz boat in a tube furnace, and argon gas (50 cm 3 Then, the tube furnace was heated to 900-1000°C at a heating rate of 5°C / min and maintained at the target temperature for 3 h. After natural cooling, the nanozyme dFeSA with single-atom dispersed iron active centers was obtained.

[0056] Example 2 Characterization and activity testing of nanozymes

[0057] 2.1 Morphology and structure analysis of dFeSA

[0058] The dFeSA structure was analyzed using transmission electron microscopy (TEM). Figure 3 As shown in Figure a, iron, nitrogen, and carbon elements are uniformly distributed in dFeSA. Aberration-corrected HAADF-STEM images confirm that atomically dispersed iron sites (appearing as uniformly dispersed single bright spots) are formed in dFeSA nanozymes ( Figure 3Figure b in the figure). Raman spectrum shows two characteristic peaks at about 1300 and 1580 cm-1, belonging to the D and G bands respectively ( Figure 3 (c) The ID / IG ratio of dFeSA nanozyme is 0.927, indicating that dFeSA contains abundant carbon defects. Figure 3 The N1s X-ray photoelectron spectroscopy (XPS) in the d-figure shows that the content of pyrrolic N in dFeSA is slightly higher than that in NC, which indicates that the nitrogen defect density in dFeSA is higher. Figure 3 The electron paramagnetic resonance (EPR) spectrum in the e-image further confirms this observation. The signal intensity of the representative nitrogen vacancy in dFeSA is slightly stronger than that in NC. In addition, Fe 2p XPS was used to analyze the chemical state of iron in dFeSA ( Figure 3 The peak at 711.2 eV is the Fe in Fe 2p3 / 2. 2+ and Fe 3+ ; The peak at 724.5eV is Fe in Fe 2p1 / 2 2+ and Fe 3+ .

[0059] like Figure 4 As shown in Figure a, the iron K-edge X-ray absorption near-edge structure (XANES) spectrum of the synthesized dFeSA shows an absorption edge similar to that of FePc, indicating that the valence state of iron in dFeSA is close to +2. The Fourier transform k3-weighted extended X-ray absorption fine structure (EXAFS) spectrum shows ( Figure 4 Figure b), dFeSA in There is a prominent peak at , which corresponds to the Fe-N / O bond in the first coordination sphere. Compared with Fe foil, no Fe-Fe scattering peak is observed in dFeSA, which confirms the absence of clusters and particles derived from Fe. K-space fitting results further reveal the local coordination environment of the atomically dispersed Fe sites ( Figure 4 c and d in Figure 3), the Fe-N bond length is obtained to be The coordination number is about 3.

[0060] 2.2 dFeSA oxidase-like activity test

[0061] dFeSA nanozyme rapidly oxidizes TMB to produce a blue product (oxTMB) with a characteristic absorption peak at 652 nm ( Figure 5 In contrast, the TMB oxidation activity of the NC nanozyme was almost negligible, indicating that the Fe site is the active center of dFeSA with oxidase-like performance. Quantitative analysis showed that the oxidase-like activity of dFeSA was 18.2 times that of the NC nanozyme ( Figure 5Subsequently, the catalytic activity of dFeSA was tested under different conditions, including the pH value and temperature of the measurement environment ( Figure 4 (Figures d and e in the figure). dFeSA nanozyme exhibited highly stable oxidase-like activity over a wide temperature range of 30-70°C. Even at a low temperature of 10°C, dFeSA maintained a high percentage (60%) of its maximum activity. These findings suggest that dFeSA has a wide range of potential applications at different ambient temperatures.

[0062] The steady-state kinetics of dFeSA and NC nanozymes were analyzed. By plotting the relationship between the initial reaction rate and the TMB concentration, a typical Michaelis-Menten curve was obtained ( Figure 5 f in Figure ). The Michaelis constant (KM), maximum reaction velocity (Vmax), and catalytic constant (kcat) obtained from this curve analysis were used to evaluate the binding affinity and catalytic efficiency of the nanozyme with TMB. It is worth noting that the KM value of dFeSA (0.38mM) is lower than that of NC (0.51mM), indicating that TMB has a stronger affinity for dFeSA. The Vmax value of dFeSA (0.0986μΜ / s) is 4.8 times that of NC (0.0205μM / s), and its kcat value also exceeds that of NC, indicating that dFeSA has a higher catalytic efficiency for TMB oxidation.

[0063] 2. Exploration of the oxidase-like activity mechanism of 3dFeSA

[0064] The oxidase-like mechanism of dFeSA was investigated by evaluating the catalytic performance of dFeSA nanozymes under different oxygen concentrations. Figure 6 As shown in Figure a, the oxidase-like activity of dFeSA in an inert gas environment is negligible, indicating that dFeSA itself does not act as an oxidant. Importantly, the oxidase-like activity of dFeSA in an oxygen-saturated solution is much higher than that in an air-saturated environment. Therefore, based on these control experiments, it is speculated that dFeSA catalyzes the conversion of O2 into reactive oxygen species (ROS), thereby oxidizing TMB. 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was then used as a capture agent to detect the possible involvement of ROS by EPR spectroscopy ( Figure 6 Obvious superoxide radicals (·O 2-) signal, indicating that superoxide radicals are the main oxidants. In addition, the superoxide radical signal intensity of dFeSA is much higher than that of NC, further confirming that dFeSA has stronger catalytic activity. By introducing IPA and FFA into the catalytic reaction, other free radical quenching experiments were performed at the same time. In the presence of these quenchers, the oxidase-like activity of dFeSA remained almost unchanged, indicating that almost no other free radicals were generated during the reaction except superoxide anions. In contrast, in the presence of PBQ (p-benzoquinone), the oxidase-like activity of dFeSA was significantly reduced, which is consistent with the EPR results ( Figure 6 (c in Figure 1). These findings suggest that the oxidase-like catalytic pathway of dFeSA involves the generation of superoxide anions from O2. Cyclic voltammetry (CV) further confirmed the oxidation pathway of TMB. Figure 6 As shown in Figure d, two distinct redox peaks appeared in the cyclic voltammogram of TMB in acetate buffer (pH 4.0), corresponding to two consecutive single-electron oxidation steps: (i) the formation of TMB radical intermediates; and (ii) the complete oxidation of TMB to quinone diimine.

[0065] Example 3 Construction of detection system

[0066] The detection system is based on the principle of a double-antibody sandwich immunoassay: A CEA capture antibody (Ab1) is first directionally immobilized on a 96-well plate, capturing CEA in the sample through antigen-antibody binding. A dFeSA-conjugated detection antibody (Ab2) is then introduced to form an Ab1-CEA-Ab2-dFeSA sandwich complex. Highly dispersed active sites catalyze the generation of a large amount of chromogenic product (oxTMB), amplifying the sandwich complex's signal.

[0067] 3.1 Coupling of nanozyme-antibody complex (dFeSA-Ab2)

[0068] The amino groups of Ab2 were covalently linked to the carboxyl groups on the surface of dFeSA using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) crosslinker to form a stable dFeSA-Ab2 probe.

[0069] 3.2 Optimization of reaction system

[0070] according to Figure 5 As shown in Figures a and d, when the color development reaction is carried out at a temperature of 25°C and the reaction time is 5 minutes, the background increase caused by excessive reaction can be avoided. Therefore, the conditions for the color development reaction are set as: 25°C, 5 minutes.

[0071] At the same time, according to Figure 5As shown in Figure e, the oxidase-like activity of dFeSA reached its peak when the pH value of the color development buffer (acetic acid-sodium acetate buffer) was 4.0, so the pH value was determined to be 4.0.

[0072] Example 4d Application of FeSA Nanozyme in Detecting Carcinoembryonic Antigen

[0073] 4.1 Materials and reagents

[0074] 1) Solid phase carrier

[0075] 96-well polystyrene ELISA plate

[0076] 2) Antibodies and Antigens

[0077] Mouse anti-human CEA monoclonal antibody (Ab1, capture antibody, purchased from Sangon Biotech (Shanghai) Co., Ltd.), rabbit anti-human CEA polyclonal antibody (Ab2, detection antibody, purchased from Sangon Biotech (Shanghai) Co., Ltd.), and recombinant human CEA antigen standard (concentration gradient: 0.05, 0.1, 1, 10, 100, 300 ng / mL).

[0078] 3) Buffer system

[0079] Coating buffer: pH 7.4 phosphate buffered saline (PBS, 10 mM);

[0080] Blocking agent: PBS containing 1% bovine serum albumin (BSA);

[0081] Washing solution: PBS containing 0.05% Tween-20 (PBST);

[0082] Color development buffer: pH 4.0 acetic acid-sodium acetate buffer (0.1 M);

[0083] Signal probe: dFeSA-coupled Ab2 complex (dFeSA-Ab2), prepared by covalent coupling of dFeSA and Ab2 via EDC / NHS;

[0084] Chromogenic substrate: TMB (10 mM, dissolved in DMSO).

[0085] 4.2 Testing Process

[0086] To achieve highly specific capture and sensitive detection of CEA, the present invention adopts a synergistic strategy of double-antibody sandwich immunoassay and nanozyme signal amplification: Ab1 is fixed to the surface of a 96-well plate by physical adsorption and chemical cross-linking, and bovine serum albumin (BSA) is used to block nonspecific sites to ensure stable capture of CEA antigen.

[0087] 1) Sample processing

[0088] Serum pretreatment: Collect whole blood samples, centrifuge at 3000 rpm for 10 min to separate serum, and store at -20°C until use.

[0089] Dilution strategy: High concentration samples (>300 ng / mL) need to be diluted with PBS to within the linear range for detection.

[0090] In this example, three clinical serum samples were collected for subsequent testing.

[0091] 2) Antibody immobilization

[0092] Coating with Ab1: Add 200 μL of Ab1 solution (10 μg / mL, dissolved in coating buffer) to each well of a 96-well plate and incubate at 4°C overnight (12-16 h);

[0093] Washing: Discard the liquid in the wells, add 300 μL PBST to each well, let it stand for 1 min and then discard it. Repeat 3 times.

[0094] 3) Blocking non-specific sites

[0095] Blocking: Add 300 μL of blocking solution (1% BSA) to each well and incubate at 37°C for 1 h;

[0096] Washing: Same as step 1.

[0097] 4) Antigen capture and probe binding

[0098] Add sample: Add 100 μL of the sample to be tested (serum or CEA standard) to each well and incubate at 37°C for 1 hour;

[0099] Washing: Same as step 1;

[0100] Add dFeSA-Ab2 probe: add 10 μL dFeSA-Ab2 complex (diluted in PBS containing 1% BSA) to each well and incubate at 37°C for 1 h;

[0101] Washing: Same as step 1.

[0102] 5) Color reaction and signal detection

[0103] Color development: Add 200 μL TMB color development solution (10 μM TMB dissolved in color development buffer) to each well and react at room temperature (25°C) in the dark for 5 minutes;

[0104] Reading: Immediately measure the absorbance at 652 nm using a microplate reader (reference wavelength 470 nm).

[0105] 4.3 Results Analysis

[0106] The present invention establishes a CEA standard test curve to verify the detection limit, and demonstrates the accuracy, stability, and specificity of the method through recovery experiments, stability experiments, and interference experiments.

[0107] 1) Detection of CEA concentration in the sample to be tested

[0108] Standard test: Detect the standard CEA (0.05-300ng / mL, R 2 >0.995), record the absorbance value; the result is as follows Figure 7 As shown in Figure a;

[0109] Linear fitting: With CEA concentration as the horizontal axis and absorbance (652nm) as the vertical axis, a linear equation was fitted; the results are as follows Figure 7 As shown in Figure b;

[0110] Sample Calculation: Based on the absorbance of the sample to be tested, the CEA concentration was inferred from the standard curve. The results are shown in Table 1.

[0111] 2) Detection limit verification

[0112] The detection limit of CEA detected by the method of the present invention was obtained as LOD=0.017 ng / mL through the standard detection curve.

[0113] 3) Recovery rate verification

[0114] 1.64, 2.88, and 5.35 ng / mL CEA were added to healthy human serum. The results are shown in Table 1, and the recovery rates were 92.7-102.6%.

[0115] 4) Stability verification

[0116] like Figure 7 As shown in Figure c, after the detection system was stored at 25°C for 21 days, its detection retention rate was ≥90%.

[0117] 5) Interference experiment verification

[0118] To explore the anti-interference ability of the CEA detection system, various biomarkers were added to the detection system, and then the peak intensity at 652 nm was measured after incubation for 10 minutes. Specifically, HSA, PSA, AFP, IgG, NSE, AA, or CEA at a concentration of 10 ng / mL was added to the above detection system. According to the above standard procedure, the activity of dFeSA in the presence of different biomarkers was recorded. Figure 7 As shown in Figure d, the detection method of the present invention has no cross reaction to the above-mentioned interfering substances.

[0119] Table 1 Results of CEA detection in clinical serum samples

[0120]

[0121] The present invention also uses ELISA to detect test samples for comparison. The results show that compared with commercial ELISA kits, the present invention uses dFeSA to perform CEA detection through a double antibody sandwich immunoassay system in a shorter time, lower cost, and with similar recovery rate.

[0122] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of this application in detail. It should be understood that the above are only specific implementation methods of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent replacements, improvements, etc. made on the basis of the technical solutions of this application should be included in the scope of protection of this application.

Claims

1. A nanozyme for detecting carcinoembryonic antigen, characterized in that: The nanozyme is an iron-based single-atom nanozyme dFeSA, which is an Fe- and N-doped amorphous carbon nanoparticle with Fe-N3-C as the core and combined with a carbon-based support with nitrogen vacancies and carbon defects.

2. The nanozyme according to claim 1, characterized in that The preparation method of the nanozyme comprises the following steps: An iron source, an organic ligand, and a template are mixed in a solvent to synthesize a metal-organic framework precursor by a template synthesis method; The precursor is subjected to high-temperature pyrolysis under an inert atmosphere to obtain a nanozyme having a single-atom dispersed iron active center; The iron source is an aqueous solution of Fe(NO3)3, the organic ligand is a methanol solution of 2-methylimidazole and a methanol solution of Zn(NO3)2, and the template is an aqueous solution of BSA.

3. The nanozyme according to claim 1, characterized in that The preparation method of the nanozyme specifically comprises the following steps: a. A methanol solution of 2-methylimidazole, an aqueous solution of BSA and an aqueous solution of Fe(NO3)3 were mixed in a volume ratio of 8:1:1 and a mass concentration ratio of 77:40:4 to obtain a mixed solution; b was then added to the mixed solution in step a of an equal volume, and the mass concentration of 2-methylimidazole in step a ratio of 55.8: 77 of Zn (NO3) 2 methanol solution; c. Stirring was continued at room temperature for 1 to 3 hours, and the solid was separated by centrifugation. The solid was washed with methanol 1 to 3 times and then dried at 50 to 65°C to obtain the metal-organic framework precursor Fe-BSA@ZIF-8. d. Place Fe-BSA@ZIF-8 in a tube furnace, introduce argon gas, and heat to 900-1000°C for 2-4 hours. After natural cooling, collect and grind to obtain the nanozyme dFeSA with a single-atom dispersed iron active center.

4. Use of the nanozyme according to any one of claims 1 to 3 in the detection of carcinoembryonic antigen.

5. A method for detecting carcinoembryonic antigen for non-disease diagnosis purposes, characterized in that: The following steps are involved: S1. coupling the nanozyme according to any one of claims 1 to 3 with a detection antibody to form a nanozyme-detection antibody complex; S2. Construct a nanozyme-based double antibody sandwich immunoassay system: first, coat the capture antibody on a solid phase carrier and then re-coat it with a blocking agent; then add the test sample and incubate it to allow the capture antibody to bind to the carcinoembryonic antigen in the test sample; finally, add the nanozyme-detection antibody complex and continue incubation to form a capture antibody-carcinoembryonic antigen-detection antibody-nanozyme sandwich complex; S3, adding TMB for color development; S4. Measure the absorbance of the reaction solution at 652 nm, and perform data analysis based on the measured absorbance value in combination with the standard curve.

6. The method according to claim 5, characterized in that The temperature during the color development reaction is 23-26° C., and the color development time is 3-8 minutes.

7. The method according to claim 5, characterized in that The color development reaction was carried out in 0.1 M acetic acid-sodium acetate (HAc-NaAc) buffer at pH 4.

0.

8. The method according to claim 5, characterized in that The linear detection range of the method is 0.05-300 ng / mL.

9. The method according to claim 5, characterized in that The method was non-cross-reactive with the following interferents: human serum albumin, prostate-specific antigen, alpha-fetoprotein, immunoglobulin G, neuron-specific enolase, and ascorbic acid.

10. A detection kit, characterized in that Include: A nanozyme-detection antibody complex formed by coupling the nanozyme according to any one of claims 1 to 3 with a detection antibody; a solid support pre-coated with capture antibody; TMB chromogenic substrate solution; acidic buffer; CEA standards.

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