A signal-enhanced electrochemiluminescence sensing method for rapid detection of carcinoembryonic antigen

Abstract

The present invention relates to a signal-enhanced electrochemiluminescence sensing method for rapid detection of carcinoembryonic antigen, belonging to the field of new functional material research and development and new sensor construction technology. Luminol@Au@Ni-Rh NCs as a fluorescent probe has excellent luminescence efficiency. Ti3C2T x MXene has a high specific surface area, good conductivity and active sites that are easy to modify, which effectively reduces the migration distance and migration rate of carriers. 1.93 and TiO 1.96 It has more oxygen vacancies and defects, has excellent electrocatalytic performance, and effectively promotes the decomposition of H2O2 to produce more active oxygen. x MXene@TiO 1.96 @MoS 1.93 The signal amplification effect of the substrate material was used to construct a signal-enhanced electrochemiluminescence immunosensor. Using CEA as a model analyte, an immunosensor was constructed to detect CEA with a detection range of 0.1 fg·mL. ‑1 ~100 ng·mL ‑1 The detection limit was 46.4 fg·mL ‑1 This patented invention solves the problem that traditional CEA detection methods cannot accurately detect low concentrations. This technology will provide a strong basis for future clinical medical diagnosis.

Description

Technical Field

[0001] The present invention relates to a Ti3C2T x MXene@TiO 1.96 @MoS 1.93 Preparation method of electrochemiluminescence immunosensor for rapid and sensitive detection of CEA using a composite material loaded with luminol@Au@Ni-RhNCs. x MXene@TiO 1.96 @MoS 1.93 The composite material was used as the luminescent substrate material and luminol@Au@Ni-RhNCs was used as the fluorescent probe to prepare an electrochemiluminescence immunosensor for detecting CEA, which belongs to the field of new functional materials and sensor detection technology. Background Art

[0002] Carcinoembryonic antigen (CEA) is a commonly used tumor marker, a polysaccharide-protein complex with important clinical applications. Numerous methods exist for detecting CEA, but these methods are complex, time-consuming, technically demanding, and expensive. CEA, a polysaccharide-protein complex, has been widely used as a broad-spectrum tumor marker. Serum CEA levels in normal individuals are typically below 5 ng / mL. A serum CEA concentration exceeding 20 ng / mL is considered a marker for confirming cancer. Furthermore, it is widely used in postoperative observation, efficacy assessment, and the treatment of recurrent tumors. Currently, various detection technologies have been developed, such as high-performance liquid chromatography (HPLC), mass spectrometry, and spectrophotometry. However, while these techniques are capable of detecting CEA, they have limitations due to high cost, low sensitivity, and variable response. Electrochemical sensors based on organic and inorganic electrodes have also been introduced for CEA analysis, but they suffer from low sensitivity and poor selectivity. The present invention combines the spectroscopic and electrochemical behaviors of substances to design a novel electrochemiluminescence sensor with fast analysis speed, simple operation, good stability, low detection limit, and good biocompatibility. The detection limit of the present invention for CEA reaches 46.4 fg / mL.

[0003] Electrochemiluminescence immunosensors are a type of detection device that determines the concentration of an analyte based on its electrochemiluminescence behavior. Electrochemiluminescence detection offers advantages such as simple equipment, high sensitivity, and ease of miniaturization. It has developed into an analytical method with great potential for application in food, environmental, and pharmaceutical fields.

[0004] In Ti3C2T x MXene@TiO 1.96 @MoS 1.93In the system, Ti3C2T x MXene has a layered structure, hydrophilicity, high electron transfer efficiency and ionic conductivity, technical flexibility and mechanical properties, which can adjust its electronic structure, increase the available active sites, and realize the Ti3C2T x MXene to TiO 1.96 The large amount of loading can catalyze the combination of co-reactants and luminescent materials, which is conducive to the formation of a stable ECL system. 1.96 The introduction of oxygen vacancies in TiO overcomes the problem of poor electrical conductivity of transition metal oxides and realizes the 1.96 Catalyzes the decomposition of H2O2 into more active oxygen. MoS 1.93 Phase structure plays a dominant role in its catalytic performance. 1.93 The catalytic contribution of the system cannot be ignored. The introduction of abundant defects can further improve the catalytic activity of the system and achieve the same effect as TiO 1.96 The synergistic catalytic effect of H2O2 decomposition into active oxygen is further enhanced.

[0005] Luminol is a classic electrochemiluminescent luminophore, but its electrochemiluminescence requires H2O2 as a co-reaction condition to obtain stable luminescence and the decomposition rate of H2O2 has a great influence on the luminescence of luminol. x MXene@TiO 1.96 @MoS 1.93The loaded luminol@Au@Ni-Rh NCs facilitate electron transfer during the electrochemiluminescence (ECL) process, promoting the redox behavior of the luminol@Au@Ni-Rh NCs on the electrode surface and enhancing the stability of the ECL immunosensor. The unique hollow and porous structure of Ni-Rh PBAs enables metal cations to be embedded in the interstitial sites of the framework, resulting in excellent biocompatibility, selective adsorption, and electrocatalytic activity. These properties suggest that PBAs have great potential as excellent support matrices and efficient catalysts for incorporation into noble metal nanoparticles, such as AuNPs. We synthesized luminol@Au@Ni-Rh NCs, which can be directly used as ECL emitters and signal amplifiers for readout. The addition of H2O2 as a co-reactant effectively enhances the ECL intensity of the luminol, further improving the sensor's stability, hydrophilicity, and biocompatibility, and further enhancing the material's ECL luminescence behavior. Using CEA as the target, the electrochemiluminescence intensity changes with varying concentrations, enabling ultrasensitive detection of CEA. The electrochemiluminescence immunosensor prepared by the present invention has the advantages of low cost, high sensitivity, good specificity, rapid detection, and easy preparation. It realizes rapid and ultrasensitive detection at lower CEA concentrations, effectively overcoming the shortcomings of current CEA detection methods. Summary of the Invention

[0006] One of the purposes of the present invention is to prepare Au@Ni-Rh NCs with octahedral nanocage structure and Ti3C2T nanosheet structure. x MXene@TiO 1.96 @MoS 1.93 .

[0007] The second purpose of the present invention is to improve the conductivity and electron transfer rate of Ni-Rh PBAs, hybridize with gold nanoparticles, synthesize Au@Ni-RhNCs with good conductivity, and combine it with luminol as a luminescent probe to covalently link it with the antibody to capture the antibody.

[0008] The third purpose of the present invention is to x MXene has high specific surface area, good conductivity and active sites that are easy to modify, which effectively reduces the migration distance and migration rate of carriers. 1.96 It has more oxygen vacancies and defects, which can effectively catalyze the decomposition of H2O2 to produce more active oxygen, which combines with the luminescent material to form a stable ECL response.

[0009] The fourth purpose of the present invention is to x MXene@TiO 1.96 Further loaded MoS1.93 It can capture antibody Ab1 and improve the biocompatibility of the composite material. In addition, TiO 1.96 With MoS 1.93 More defects synergistically catalyze and promote the conversion of H2O2 into more ROSs, accelerate the oxidation of luminol, and enhance the ECL response.

[0010] The fifth object of the present invention is based on Ti3C2T x MXene@TiO 1.96 @MoS 1.93 The signal amplification effect of the substrate material was used to construct a signal-enhanced electrochemiluminescence immunosensor. CEA was used as a model analyte to construct an immunosensor, and luminol@Au@Ni-RhNCs was used as a luminescent probe. An electrochemiluminescence immunosensor for detecting CEA was successfully prepared.

[0011] The sixth object of the present invention is to design and synthesize defect-rich TiO in a reducing atmosphere of citric acid and sodium citrate at high temperature. 1.96 With MoS 1.93 , and the full name of the method is inductively coupled plasma-mass spectrometry ICP-MS to finally determine the atomic stoichiometry of the material.

[0012] A seventh objective of the present invention is to demonstrate ultrasensitive detection of CEA using the constructed immunosensor, with a detection limit of 46.4 fg / mL. Compared to sensors based on TiO₂ and MoS₂, this method boasts an order of magnitude wider linear range and a lower detection limit, surpassing the capabilities of researchers in this field without extensive experimentation and innovative thinking.

[0013] The technical solutions of the present invention are as follows:

[0014] 1. A method for preparing an electrochemiluminescence sensor for rapid detection of CEA, comprising the following steps:

[0015] (1) Titanium carbide Ti3C2T x MXene in situ growth of titanium oxide TiO 1.96 and molybdenum sulfide MoS 1.93 Composite material MXene@TiO 1.96 @MoS 1.93 Preparation: 165.0 mg of sodium tetrafluoroborate NaBF4 and 10 mg of sodium citrate were weighed and dissolved in 15.0 mL of 1.0 mol / L HCl, and 150.0 mg of titanium carbide two-dimensional nanomaterial Ti3C2T was weighed. xMXene was dispersed in the above solution, and the solution was ultrasonicated for 30 min at room temperature. After the ultrasonication, the suspension was placed in a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 160 ° C for 20 h. It was washed three times by centrifugation with deionized water and ethanol, and dried in a vacuum drying oven at 60 ° C for 24 h to obtain Ti3C2T x MXene@TiO 1.96 Composite material, weigh 600.0 mg of ammonium molybdate (NH4)2MoO4 and 1600.0 mg of thiourea CH4N2S and dissolve them in 30.0 mL of deionized water to obtain a solution containing (NH4)2MoO4 and CH4N2S, weigh 130.0 mg of citric acid and 130.0 mg of Ti3C2T x MXene@TiO 1.96 The suspension was dispersed into a solution containing (NH4)2MoO4 and CH4N2S, stirred for 60 min, and placed in a polytetrafluoroethylene-lined stainless steel autoclave at 210 ° C for 18 h, washed with anhydrous ethanol and freeze-dried for 12 h to obtain Ti3C2T x MXene@TiO 1.96 @MoS 1.93 Composite materials, kept for future use;

[0016] (2) Preparation of gold hybrid nickel-rhodium alloy nanocages Au@Ni-Rh NCs 100.0-300.0 mg nickel nitrate Ni(NO3)2 and 200.0-500.0 mg sodium citrate Na3C6H5O7·2H2O were weighed and dissolved in 20.0 mL of deionized water to obtain solution A containing Ni(NO3)2 and Na3C6H5O7. 100.0-300.0 mg rhodium chloride RhCl3·3H2O were weighed and dissolved in 20.0 mL of deionized water to obtain solution B containing RhCl3. Solution A and solution B were magnetically stirred and mixed for 5 min, placed at room temperature for 24 h, washed three times with deionized water and anhydrous ethanol, respectively, and dried in a vacuum drying oven at 70 °C for 24 h to obtain Ni-Rh PBAs. 10.0-20.0 mg Ni-Rh PBAs were dissolved in 5.0 mL of anhydrous ethanol to obtain solution A. 2.5 mL of ammonia NH3·H2O was dissolved in 10.0 mL of deionized water to obtain solution B. Solution B was added to solution A and stirred at room temperature for 20 min. The solution was washed three times with deionized water and anhydrous ethanol by centrifugation, and dried in a vacuum oven at 70°C for 24 h to obtain Ni-Rh NCs. 10.0-15.0 mg of Ni-Rh NCs were weighed and stirred in 20.0 mL of anhydrous ethanol for 30 min until completely dissolved. 250 μL of 1.0% HAuCl4·6H2O was then added and stirred continuously for 30 min. While stirring, 10 μL of 1.9 mol / L NaBF4 solution was quickly added. After stirring for 1 h, the solution was washed several times with deionized water by centrifugation. The Au@Ni-RhNCs were collected, resuspended in 20.0 mL of deionized water, and sealed for later use.

[0017] (3) Preparation of luminol@Au@Ni-Rh NCs: 10.0 mL of Au@Ni-Rh NCs solution was measured and 1 mL of 10 mmol / L luminol was added to the solution. The mixture was stirred for 6 h to obtain luminol@Au@Ni-Rh NCs. The luminol@Au@Ni-Rh NCs were then packaged and stored for future use.

[0018] (4) Preparation of electrochemiluminescence sensor

[0019] ① Use Al2O3 polishing powder with particle sizes of 0.05μm and 0.03μm to polish a glassy carbon electrode (GCE) with a diameter of 4mm to obtain a mirror surface, then ultrasonicate it with ultrapure water and anhydrous ethanol, and then blow dry it with nitrogen for later use;

[0020] ②Drop coat 6μL of Ti3C2T x MXene@TiO 1.96 @MoS 1.93The suspension was added dropwise to the electrode surface and stored at room temperature until dry;

[0021] ③Continue to apply 6 μL, the concentration is 100 μg·mL -1 Ab1 solution in Ti3C2T x MXene@TiO 1.96 @MoS 1.93 Incubate on the top layer in a 4°C refrigerator overnight;

[0022] ④ Continue to drop-coat 3-6 μL of 1% bovine serum albumin solution to block nonspecific active sites. Store in a 4°C refrigerator until dry. Wash the surface of the modified electrode with PBS buffer solution and let it dry slightly.

[0023] ⑤Continue to add 6 μL of 0.1 fg mL -1 ~100 ng·mL -1 A series of CEA standard solutions with different concentrations were dropped on the electrode surface and incubated in a refrigerator at 4°C for 1 h.

[0024] ⑥ 5-10 μL of the prepared luminol@Au@Ni-Rh NCs and CEA detection antibody Ab2 complex luminol@Au@Ni-Rh NCs-Ab2 was dropped onto the electrode surface and stored in a 4°C refrigerator until dry, thereby preparing the electrochemiluminescence immunosensor for detecting CEA.

[0025] The preparation method of the electrochemiluminescence immunosensor comprises the following steps:

[0026] (1) The MPI-E electrochemiluminescence analysis system was used for the test with a three-electrode system, with a silver / silver chloride electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode, and a modified glassy carbon electrode as the working electrode;

[0027] (2) Cyclic voltammetry (CV) was used to detect CEA, with the scanning voltage set to 0-0.6 V, the scanning rate set to 0.1 V / s, the photomultiplier tube high voltage set to 600 V, and the amplification level set to 3;

[0028] (3) The measurement was performed in 10.0 mL of a buffer solution containing 1.0 to 60.0 mmol / L H2O2 at a pH of 7.4.

[0029] (4) After the electrodes are placed, obtain a luminescence spectrum every 12.0 s and record the luminescence intensity;

[0030] (5) Test the prepared CEA solutions of different concentrations and draw a working curve.

[0031] All chemical reagents required for material synthesis were purchased from local reagent stores without further processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 (A) Ti3C2Tx MXene@TiO 1.96 @MoS 1.93 (B) is the scanning electron microscopy image of Ni-Rh PBAs.

[0033] Beneficial results of the present invention

[0034] One of the beneficial results of the present invention is the preparation of Au@Ni-Rh NCs with octahedral nanocage structure and Ti3C2T nanosheet structure. x MXene@TiO 1.96 @MoS 1.93 .

[0035] The second beneficial achievement of the present invention is to improve the conductivity and electron transfer rate of Ni-Rh PBAs, hybridize with gold nanoparticles, synthesize Au@Ni-RhNCs with good conductivity, combine it with luminol as a luminescent probe and covalently link it to the antibody capture antibody.

[0036] The third beneficial result of the present invention is that Ti3C2T x MXene has high specific surface area, good conductivity and active sites that are easy to modify, which effectively reduces the migration distance and migration rate of carriers. 1.96 It has more oxygen vacancies and defects, which can effectively catalyze the decomposition of H2O2 to produce more active oxygen, which combines with the luminescent material to form a stable ECL response.

[0037] The fourth beneficial result of the present invention is that Ti3C2T x MXene@TiO 1.96 Further loaded MoS 1.93 It can capture antibody Ab1 and improve the biocompatibility of the composite material. In addition, TiO 1.96 With MoS 1.93 More defects synergistically catalyze and promote the conversion of H2O2 into more ROSs, accelerate the oxidation of luminol, and enhance the ECL response. The fifth beneficial achievement of the present invention is based on Ti3C2T x MXene@TiO 1.96 @MoS 1.93The signal amplification effect of the substrate material was used to construct a signal-enhanced electrochemiluminescence immunosensor. CEA was used as a model analyte to construct an immunosensor, and luminol@Au@Ni-Rh NCs were used as luminescent probes. An electrochemiluminescence immunosensor for detecting CEA was successfully prepared, achieving ultrasensitive detection of CEA with a detection range of 0.1 pg / mL to 100 ng / mL and a detection limit of 46.4 fg / mL. Specific implementation plan

[0038] Example 1 Titanium Carbide Ti3C2T x MXene in situ growth of titanium oxide TiO 1.96 and molybdenum sulfide MoS 1.93 Composite material MXene@TiO 1.96 @MoS 1.93 Preparation: 165.0 mg of sodium tetrafluoroborate NaBF4 and 10 mg of sodium citrate were weighed and dissolved in 15.0 mL of 1.0 mol / L HCl, and 150.0 mg of titanium carbide two-dimensional nanomaterial Ti3C2T was weighed. x MXene was dispersed in the above solution, and the solution was ultrasonicated for 30 min at room temperature. After the ultrasonication, the suspension was placed in a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 160 ° C for 20 h. It was washed three times by centrifugation with deionized water and ethanol, and dried in a vacuum drying oven at 60 ° C for 24 h to obtain Ti3C2T x MXene@TiO 1.96 Composite material, weigh 600.0 mg of ammonium molybdate (NH4)2MoO4 and 1600.0 mg of thiourea CH4N2S and dissolve them in 30.0 mL of deionized water to obtain a solution containing (NH4)2MoO4 and CH4N2S, weigh 130.0 mg of citric acid and 130.0 mg of Ti3C2T x MXene@TiO 1.96 The suspension was dispersed into a solution containing (NH4)2MoO4 and CH4N2S, stirred for 60 min, and placed in a polytetrafluoroethylene-lined stainless steel autoclave at 210 ° C for 18 h, washed with anhydrous ethanol and freeze-dried for 12 h to obtain Ti3C2T x MXene@TiO 1.96 @MoS 1.93 Composite materials, kept for future use;

[0039] Example 2 175.0 mg of nickel nitrate Ni(NO3)2 and 270 mg of sodium citrate Na3C6H5O72H2O were weighed and dissolved in 20.0 mL of deionized water to obtain solution A containing Ni(NO3)2 and Na3C6H5O7. 135.0 mg of rhodium chloride RhCl3·3H2O were weighed and dissolved in 20.0 mL of deionized water to obtain solution B containing RhCl3. Solution A and solution B were magnetically stirred and mixed for 5 min, allowed to stand at room temperature for 24 h, and washed three times with deionized water and anhydrous ethanol, respectively, and dried in a vacuum drying oven at 70°C for 24 h to obtain Ni-Rh PBAs. 12.0 mg of Ni-Rh PBAs were weighed and dissolved in 5.0 mL of anhydrous ethanol to obtain solution A. 3.0 mL of ammonia NH3 was measured. H2O was dissolved in 10.0 mL of deionized water to obtain solution B, which was added to solution A and stirred at room temperature for 20 min. The solution was washed three times with deionized water and anhydrous ethanol by centrifugation, and dried in a vacuum oven at 70°C for 24 h to obtain Ni-RhNCs. 10.0 mg of Ni-RhNCs was weighed and stirred in 20.0 mL of anhydrous ethanol for 30 min until completely dissolved. 250 μL of HAuCl46H2O (1.0 wt%) was then added and stirred continuously for 30 min. While stirring, 10 μL of 1.9 mol / L NaBF4 solution was quickly added. After stirring for 1 h, the solution was washed several times with deionized water by centrifugation, and the Au@Ni-RhNCs were collected, resuspended in 20.0 mL of deionized water, and sealed for later use.

[0040] Example 3 Preparation of Luminol-Loaded Gold Hybrid Nickel-Rhodium Alloy Nanocage Luminescent Material luminol@Au@Ni-RhNCs

[0041] Measure 10.0 mL of Au@Ni-Rh NCs solution, add 1 mL of 10 mmol / L luminol to the above solution, stir for 6 h, and obtain luminol@Au@Ni-RhNCs. Then, package and store them for future use.

[0042] Example 4 Preparation of buffer solution

[0043] 4.54 g of KH2PO4 was weighed and dissolved in 500.0 mL of deionized water to obtain a 1 / 15 mol / L KH2PO4 mother solution A. 5.94 g of Na2HPO42H2O was weighed and dispersed in 500.0 mL of deionized water to obtain a 1 / 15 mol / L Na2HPO4 mother solution B. Mother solutions A and B were mixed in different proportions to obtain a buffer solution PBS with a pH range of 6.8 to 8.4.

[0044] Example 5 Preparation of CEA solution

[0045] Measure 100-200 μL of 1 mg / mL CEA and dissolve it in 1.0-10.0 mL of PBS buffer solution. Ultrasonicate for 1 min to prepare a 0.0001-100 ng / mL solution. Store in a refrigerator at 4°C until ready to use.

[0046] Example 6 Preparation of electrochemiluminescence sensor

[0047] ① Use Al2O3 polishing powder with particle sizes of 0.05μm and 0.03μm to polish a glassy carbon electrode (GCE) with a diameter of 4mm to obtain a mirror surface, then ultrasonicate it with ultrapure water and anhydrous ethanol, and then blow dry it with nitrogen for later use;

[0048] ②Drop coat 6μL of Ti3C2T x MXene@TiO 1.96 @MoS 1.93 The suspension was added dropwise to the electrode surface and stored at room temperature until dry;

[0049] ③Continue to apply 6 μL, the concentration is 100 μg·mL -1 Ab1 solution in Ti3C2T x MXene@TiO 1.96 @MoS 1.93 Incubate on the top layer in a 4°C refrigerator overnight;

[0050] ④ Continue to drop-coat 3-6 μL of 1% bovine serum albumin solution to block nonspecific active sites. Store in a 4°C refrigerator until dry. Wash the surface of the modified electrode with PBS buffer solution and let it dry slightly.

[0051] ⑤Continue to add 6 μL of 0.1 fg mL -1 ~100 ng·mL -1 A series of CEA standard solutions with different concentrations were dropped on the electrode surface and incubated in a refrigerator at 4°C for 1 h.

[0052] ⑥ 5-10 μL of the prepared luminol@Au@Ni-Rh NCs and CEA detection antibody Ab2 complex luminol@Au@Ni-Rh NCs-Ab2 was dropped onto the electrode surface and stored in a 4°C refrigerator until dry, thereby preparing the electrochemiluminescence immunosensor for detecting CEA.

Claims

1. A method for preparing an electrochemiluminescence immunosensor for rapid detection of carcinoembryonic antigen (CEA), characterized in that: The following steps are involved: (1) Titanium carbide Ti3C2T x MXene in situ growth of titanium oxide TiO 1.96 and molybdenum sulfide MoS 1.93 Composite material MXene@TiO 1.96 @MoS 1.93 Preparation Weigh 165.0 mg of sodium tetrafluoroborate NaBF4 and 10 mg of sodium citrate and dissolve them in 15.0 mL of 1.0 mol / L HCl. Weigh 150.0 mg of titanium carbide two-dimensional nanomaterial Ti3C2T x MXene was dispersed in the above solution, and the solution was ultrasonicated for 30 min at room temperature. After the ultrasonication, the suspension was placed in a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 160 ° C for 20 h. It was washed three times by centrifugation with deionized water and ethanol, and dried in a vacuum drying oven at 60 ° C for 24 h to obtain Ti3C2T x MXene@TiO 1.96 Composite material, weigh 600.0 mg of ammonium molybdate (NH4)2MoO4 and 1600.0 mg of thiourea CH4N2S and dissolve them in 30.0 mL of deionized water to obtain a solution containing (NH4)2MoO4 and CH4N2S, weigh 130.0 mg of citric acid and 130.0 mg of Ti3C2T x MXene@TiO 1.96 The suspension was dispersed into a solution containing (NH4)2MoO4 and CH4N2S, stirred for 60 min, and placed in a polytetrafluoroethylene-lined stainless steel autoclave at 210 ° C for 18 h, washed with anhydrous ethanol and freeze-dried for 12 h to obtain Ti3C2T x MXene@TiO 1.96 @MoS 1.93 Composite materials, kept for future use; (2) Preparation of gold hybrid nickel-rhodium alloy nanocages Au@Ni-Rh NCs 100.0-300.0 mg of nickel nitrate Ni(NO3)2 and 200.0-500.0 mg of sodium citrate Na3C6H5O7·2H2O were weighed and dissolved in 20.0 mL of deionized water to obtain solution A containing Ni(NO3)2 and Na3C6H5O7. 100.0-300.0 mg of rhodium chloride RhCl3·3H2O were weighed and dissolved in 20.0 mL of deionized water to obtain solution B containing RhCl3. Solution A and solution B were magnetically stirred and mixed for 5 minutes, placed at room temperature for 24 hours, centrifuged and washed three times with deionized water and anhydrous ethanol, respectively, and dried in a vacuum drying oven at 70°C for 24 hours to obtain Ni-Rh nanoalloy cubic blocks Ni-Rh PBAs. 10.0-20.0 mg of Ni-RhPBAs were dissolved in 5.0 mL of anhydrous ethanol to obtain solution A. 2.5 mL of ammonia NH3·H2O was dissolved in 10.0 mL of deionized water to obtain solution B. Solution B was added to solution A and stirred at room temperature for 20 min. The solution was washed three times with deionized water and anhydrous ethanol by centrifugation, and dried in a vacuum oven at 70°C for 24 h to obtain Ni-Rh NCs. 10.0-15.0 mg of Ni-Rh NCs were weighed and stirred in 20.0 mL of anhydrous ethanol for 30 min until completely dissolved. 250 μL of 1.0% HAuCl4·6H2O was then added and stirred continuously for 30 min. 10 μL of 1.9 mol / L NaBF4 solution was quickly added while stirring. After stirring for 1 h, the solution was washed several times with deionized water by centrifugation. The Au@Ni-RhNCs were collected, resuspended in 20.0 mL of deionized water, and sealed for later use. (3) Preparation of luminol@Au@Ni-Rh NCs, a gold hybrid nickel-rhodium alloy nanocage luminescent material loaded with luminol Measure 10.0 mL of Au@Ni-Rh NCs solution, add 1 mL of 10 mmol / L luminol to the above solution, stir for 6 h, and obtain luminol@Au@Ni-RhNCs. Then, package and store them for future use. (4) Preparation of electrochemiluminescence sensor ① Use Al2O3 polishing powder with particle sizes of 0.05μm and 0.03μm to polish a glassy carbon electrode (GCE) with a diameter of 4mm to obtain a mirror surface, then ultrasonicate it with ultrapure water and anhydrous ethanol, and then blow dry it with nitrogen for later use; ②Drop coat 6μL of Ti3C2T x MXene@TiO 1.96 @MoS 1.93 The suspension was added dropwise to the electrode surface and stored at room temperature until dry; ③Continue to apply 6 μL, the concentration is 100 μg·mL -1 Ab1 solution in Ti3C2T x MXene@TiO 1.96 @MoS 1.93 Incubate on the top layer in a 4°C refrigerator overnight; ④ Continue to drop-coat 3-6 μL of 1% bovine serum albumin solution to block nonspecific active sites. Store in a 4°C refrigerator until dry. Wash the surface of the modified electrode with PBS buffer solution and let it dry slightly. ⑤Continue to add 6 μL of 0.1 fg mL -1 ~100 ng·mL -1 A series of CEA standard solutions with different concentrations were dropped on the electrode surface and incubated in a refrigerator at 4°C for 1 h. ⑥ 5-10 μL of the prepared luminol@Au@Ni-Rh NCs and CEA detection antibody Ab2 complex luminol@Au@Ni-Rh NCs-Ab2 was dropped onto the electrode surface and stored in a 4°C refrigerator until dry, thereby preparing the electrochemiluminescence immunosensor for detecting CEA.

2. The preparation method of the electrochemiluminescence immunosensor obtained by the preparation method according to claim 1, wherein the detection method comprises the following steps: (1) The MPI-E electrochemiluminescence analysis system was used for the test with a three-electrode system, with a silver / silver chloride electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode, and a modified glassy carbon electrode as the working electrode; (2) Cyclic voltammetry (CV) was used to detect CEA, with the scanning voltage set to 0-0.6 V, the scanning rate set to 0.1 V / s, the photomultiplier tube high voltage set to 600 V, and the amplification level set to 3; (3) The measurement was performed in 10.0 mL of a buffer solution containing 1.0 to 60.0 mmol / L H2O2 at a pH of 7.

4. (4) After the electrodes are placed, obtain a luminescence spectrum every 12.0 s and record the luminescence intensity; (5) Test the prepared CEA solutions of different concentrations and draw a working curve.

Images