Preparation method and application of photoelectrochemical immunosensor for detecting carcinoembryonic antigen

Abstract

The application discloses a preparation method of a photoelectrochemical immunosensor for detecting carcinoembryonic antigen + The signal amplification strategy of liposomes and Bi2S3@BiOI heterojunction as a base material are used to successfully construct a PEC immunosensor of Bi2S3@BiOI / Ag2S ternary heterojunction, and the PEC immunosensor is used for detecting the content of carcinoembryonic antigen. Specifically, a sandwich immunocomplex is formed through specific recognition of the aptamer fixed on the magnetic beads, the aptamer labeled on the liposomes and CEA, methanol as a demulsifier is added, the liposomes are broken to release a large number of signal molecules Ag + . The released Ag + is transferred to the surface of the electrode modified by Bi2S3@BiOI to form Ag2S with a narrow band gap in situ, the ternary heterojunction of Bi2S3@BiOI / Ag2S is formed due to the existence of the energy level matching relationship, the light absorption range is expanded, the separation of photoinduced electron-hole pairs is accelerated, the photocurrent signal is enhanced, and the purpose of signal amplification is achieved.

Description

Technical Field

[0001] This invention belongs to the field of photoelectrochemical biosensor technology, and specifically discloses a novel split-type photoelectrochemical (PEC) immunosensor for detecting the tumor marker carcinoembryonic antigen (CEA) and its preparation method. Background Technology

[0002] Cancer is a malignant disease caused by gene misplacements and mutations, posing a serious threat to human health and having a high mortality rate. Related research indicates that the quantitative analysis of certain biomarkers is crucial for clinical diagnosis and cancer treatment. Carcinoembryonic antigen (CEA) is considered an important tumor biomarker, widely present in various cancers such as pancreatic cancer, breast cancer, liver cancer, and lung cancer. Normally, the level of CEA in normal human serum is far below 5 ng / mL. In cancer patients, inflammation can significantly increase serum CEA levels, making it a predictive indicator for cancer diagnosis and related treatments. Therefore, there is an urgent need to develop a highly sensitive and specific bioanalytical method for the accurate detection of low-abundance CEA.

[0003] Among the currently published patents related to the detection of carcinoembryonic antigen (CEA), CN114942263A describes a method for preparing a split-type photoelectrochemical (PEC) sensor for detecting CEA. This method changes the polarity of the photocurrent, avoiding false positive or false negative results caused by interfering substances in the actual sample, thus achieving highly sensitive detection of CEA. However, it still suffers from the problem of poor photocatalytic activity of the photoelectrode.

[0004] CN107064509A describes the preparation and application of a PEC immunosensor for detecting carcinoembryonic antigen (CEA). It utilizes a zinc oxide-based multi-component sensitization structure to initially amplify the signal. Further signal amplification is achieved by combining the weakened sensitization effect resulting from the specific recognition of the aptamer by CEA, causing the DNA probe to dishybridize and detach from the electrode surface, with the steric hindrance effect of the conjugate formed between CEA and the aptamer. This leads to sensitive detection of CEA. However, the material synthesis is complex, and the material stability is poor.

[0005] CN110082530A describes a hydrogel formulation based on quantum dots and gold nanorods, its preparation method, and its application. The method involves first binding quantum dots to a carcinoembryonic antigen (CEA) aptamer; then binding gold nanorods to the CEA aptamer; finally, mixing the CEA-modified quantum dots and CEA-modified gold nanorods with a hydrogel solution to obtain the final product. The prepared hydrogel formulation can detect CEA, exhibiting a photoquenching efficiency of 1224% when co-incubated with 16 μL of CEA at a concentration of 1 mg / mL. Although quantum dots possess high fluorescence efficiency and photostability, they are prone to aggregation, leading to a decrease in fluorescence intensity. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for preparing a split photoelectrochemical (PEC) immunosensor based on a Bi2S3@BiOI / Ag2S ternary heterojunction, and its application in detecting the tumor marker carcinoembryonic antigen (CEA).

[0007] The present invention adopts the following technical solution:

[0008] A method for preparing a photoelectrochemical immunosensor for detecting carcinoembryonic antigen includes the following steps:

[0009] (1) Preparation of Bi2S3@BiOI heterojunction;

[0010] (2) Preparation of the magnetically controlled immunoprobe MB-Apt;

[0011] (3) Preparation of the signal probe ALL-Apt;

[0012] (4) Construct the GCE / Bi2S3@BiOI sensing interface;

[0013] (5) Construct a photoelectrochemical immunosensor to detect carcinoembryonic antigen. By forming a Bi2S3@BiOI / Ag2S ternary heterojunction in situ on the electrode surface through the specific recognition of the target substance with the magnetically controlled immunoprobe and the signal probe, sensitive detection of carcinoembryonic antigen can be achieved.

[0014] Further, the preparation of the Bi2S3@BiOI heterojunction in step (1) specifically includes the following steps:

[0015] (1.1) Synthesis of Bi2S3 nanorods: The synthesis was carried out using a one-step hydrothermal method.

[0016] First, disperse 0.0001~10 g of Bi(NO3)3·5H2O in 5~50 mL of ethylene glycol and stir magnetically for 10~40 min to obtain solution A; at the same time, add 0.0001~10 g of Na2S·9H2O to 5~50 mL of water and stir magnetically for 10~40 min to obtain solution B;

[0017] Next, 0.0001~10 g of urea was ultrasonically dispersed in 5~50 mL of water to obtain solution C;

[0018] Solution B was rapidly added dropwise to solution A to obtain a large amount of black suspension with an irritating odor. Then, solution C was mixed with the above suspension and ultrasonically dispersed for 10-40 minutes to obtain a black mixed solution.

[0019] The resulting black mixed solution was transferred into the lining of a 10-100 mL high-temperature reactor and heated at 100-200 °C. After the reaction was completed, the solution was allowed to cool naturally to room temperature. After washing several times with ultrapure water and ethanol by centrifugation, the solution was dried under vacuum at 60 °C to obtain the black product Bi2S3 nanorods.

[0020] (1.2) Synthesis of BiOI nanoflowers:

[0021] First, dissolve 0.0001~10 g Bi(NO3)3·5H2O in 5~50 mL of water using ultrasound to obtain solution D. At the same time, dissolve 0.0001~10 g potassium iodide in 5~50 mL of water using ultrasound to obtain solution E.

[0022] Subsequently, under magnetic stirring, solution E was added dropwise to solution D. This process produced a large amount of orange-red precipitate. After vigorous stirring at room temperature, the precipitate was washed several times with ultrapure water and ethanol, and then vacuum dried at 80°C to obtain BiOI nanoflowers.

[0023] (1.3) The prepared Bi2S3 nanorods and BiOI nanoflowers were mixed and dispersed in an ethanol solution at a mass ratio of 2:1. The mixture was ultrasonically treated at room temperature. The brown product obtained by ultrasonication was centrifuged and washed at 5000~12000 rpm and dried in a vacuum oven at 60℃ to obtain Bi2S3@BiOI heterojunction.

[0024] Further, step (2) of preparing the magnetically controlled immunoprobe MB-Apt specifically includes the following steps:

[0025] Sonicate the 2-20 mg / mL SMB stock solution for 2 min to disperse it evenly;

[0026] Take 50-500 μL of SMB stock solution that is ultrasonically dispersed evenly, wash it three times with PBS buffer solution with a concentration of 10 mmol / L and pH=7.4, and then redisperse it in 200-2000 μL of PBS solution.

[0027] Then, add 50-200 μL of biotin-modified CEA aptamer Bio-Apt at a concentration of 50 μmol / L, and incubate with shaking at 37°C for 60 min. Remove excess unbound Bio-Apt by magnetic separation and washing, and redisperse it in 200-2000 μL of PBS solution and store it at 4°C for use.

[0028] Further, step (3) of preparing the signal probe ALL-Apt specifically includes the following steps:

[0029] (3.1) Encapsulation of Ag with biotinylated liposomes +Synthesize ALL:

[0030] Weigh 0.0001~10 mg of dipalmitoylphosphatidylcholine (DPPC), 0.0001~10 mg of distearate-phosphatidylethanolamine-polyethylene glycol-biotin (DSPE-PEG-Biotin), and 0.0001~10 mg of cholesterol and add them to a round-bottom flask containing 3-30 mL of chloroform. Dissolve the dissolved material by sonication and evaporate the chloroform under reduced pressure at 45°C. A uniform, transparent film is then formed and adheres to the inner wall of the flask.

[0031] Then add 5-50 mL of silver nitrate solution with a concentration of 2-20 mmol / L, stir magnetically for 60 min in a constant temperature water bath at 45℃, sonicate for 5 min, and then put it into a 1 KD dialysis bag for dialysis to obtain ALL, and store it at 4℃ in the dark for later use.

[0032] (3.2) Preparation of ALL-Apt:

[0033] Take 20-2000 μL of ALL and 20-200 μL of streptavidin (SA) at a concentration of 100 ng / mL and incubate on a shaker at 37℃ and 1300 rpm for 10 min. Then add 50-200 μL of Bio-Apt at a concentration of 50 μmol / L and continue shaking and incubating for 30 min to obtain ALL-Apt, which can be stored at 4℃ for use.

[0034] Furthermore, step (4) of constructing the GCE / Bi2S3@BiOI sensing interface specifically includes the following steps:

[0035] Glassy carbon electrode (GCE) was pretreated on chamois with 5 μm alumina powder, polished to a mirror finish, rinsed with ultrapure water, and then ultrasonically washed with ethanol and ultrapure water in sequence. It was then dried at room temperature for later use.

[0036] Take 0.0001~10 mg of Bi2S3@BiOI heterojunction prepared in step (1) and ultrasonically disperse it in 0.1~10 mL of ethanol solution. Take 1~15 μL of Bi2S3@BiOI dispersion and uniformly drop it onto the surface of GCE electrode. Let it dry completely at room temperature and store it in the dark for later use.

[0037] Furthermore, step (5) of constructing a photoelectrochemical immunosensor for detecting carcinoembryonic antigen specifically includes the following steps:

[0038] First, add 10-100 μL of CEA solution of different concentrations to 10-100 μL of MB-Apt, incubate at room temperature for 30 min, and remove excess unbound CEA by magnetic separation and washing twice. PBS (10 mmol / L, pH=7.4) solution was used for magnetic separation and washing during the experiment.

[0039] Next, add 10~100 μL of ALL-Apt and incubate at room temperature for 30 min to obtain MB-Apt-CEA-ALL-Apt sandwich immune complex. Magnetic separation and washing are used to remove excess unbound ALL-Apt.

[0040] Then, 10–100 μL of methanol was added and the mixture was incubated at room temperature for 15 min to induce liposome lysis and release Ag. + Magnetic separation and retention of the supernatant;

[0041] Subsequently, 20–200 μL of the lysis buffer was transferred using a pipette and incubated on the GCE / Bi₂S₃@BiOI electrode in the dark for 5 min. After rinsing with ultrapure water, the GCE / Bi₂S₃@BiOI / Ag electrode was obtained. + electrode;

[0042] Take 2~200 μL of Na2S with a concentration of 0.1 mol / L and incubate it on the electrode for 2 min to generate Ag2S in situ. Then rinse with ultrapure water to obtain GCE / Bi2S3@BiOI / Ag2S electrode. Dry it at room temperature and modify it with 2~200 μL of 0.5% perfluorosulfonic acid polymer (nafion) solution to prevent the electrode surface material from falling off.

[0043] Finally, the modified electrode was immersed in PBS (10 mmol / L, pH=7.4) containing ascorbic acid (AA) at a concentration of 0.0001~10 mol / L for PEC testing. A xenon lamp of 200~800 W was used as the excitation source, with the lamp on-off for 20 s and the applied potential of 0.2 V.

[0044] During detection, the intensity of the photocurrent signal gradually increases with the increase of CEA concentration. The relationship between CEA concentration and photocurrent signal can be used to detect carcinoembryonic antigen.

[0045] Furthermore, the photoelectrochemical immunosensor prepared using the method of this invention was used to detect the tumor marker carcinoembryonic antigen (CEA). During detection, the photocurrent signal intensity gradually increased with increasing CEA concentration, due to the introduction of Ag through sandwich immunization. +The amount of [agent] will increase, thus increasing the amount of Ag2S generated in situ on the Bi2S3@BiOI surface. Furthermore, there is a certain energy level matching relationship between Bi2S3@BiOI and Ag2S. The construction of this ternary heterojunction can effectively suppress the recombination of photogenerated electron-hole pairs, thereby enhancing the photocurrent signal.

[0046] The preparation method of this invention is based on Ag. + Based on a liposome signal amplification strategy and using Bi2S3@BiOI heterojunctions as the substrate material, a PEC immunosensor based on a Bi2S3@BiOI / Ag2S ternary heterojunction was successfully constructed for the first time and used to detect the level of the tumor marker carcinoembryonic antigen (CEA). It forms a sandwich immune complex through the specific recognition of aptamers immobilized on magnetic beads, aptamers labeled on liposomes, and CEA. Upon addition of methanol as a demulsifier, the liposomes rupture, releasing a large amount of the signaling molecule Ag. + Released Ag + The transfer of Ag2S to the electrode surface modified with Bi2S3@BiOI forms a narrow bandgap Ag2S in situ. Due to the energy level matching relationship, a Bi2S3@BiOI / Ag2S ternary heterojunction is formed, which expands the light absorption range, accelerates the separation of photogenerated electron-hole pairs, and enhances the photocurrent signal, thereby achieving the purpose of signal amplification.

[0047] The split-type PEC immunosensor constructed in this invention has a wide linear range (0.005~50 ng / mL) and a low detection limit (1.21 pg / mL), and also exhibits good selectivity, reproducibility and stability, providing a novel sensing strategy for the detection of clinical tumor markers. Attached Figure Description

[0048] Figure 1 The photocurrent response curves of the photoelectrochemical immunosensor prepared for this example for detecting different concentrations of CEA are shown in the figure.

[0049] Figure 2 The graph shows the logarithmic linear relationship between the sensor photocurrent signal intensity and the CEA concentration under different CEA concentrations in the example. Detailed Implementation

[0050] The present invention will be further described below with reference to the embodiments and accompanying drawings, but this is not intended to limit the scope of the invention. Example

[0051] A method for preparing a photoelectrochemical immunosensor for detecting carcinoembryonic antigen includes the following steps:

[0052] (1) Preparation of Bi2S3@BiOI heterojunction;

[0053] (2) Preparation of the magnetically controlled immunoprobe MB-Apt;

[0054] (3) Preparation of the signal probe ALL-Apt;

[0055] (4) Construct the GCE / Bi2S3@BiOI sensing interface;

[0056] (5) Construct a photoelectrochemical immunosensor to detect carcinoembryonic antigen. By forming a Bi2S3@BiOI / Ag2S ternary heterojunction in situ on the electrode surface through the specific recognition of the target substance with the magnetically controlled immunoprobe and the signal probe, sensitive detection of carcinoembryonic antigen can be achieved.

[0057] Step (1) involves preparing the Bi2S3@BiOI heterojunction, specifically including the following steps:

[0058] (1.1) Synthesis of Bi2S3 nanorods: The synthesis was carried out using a one-step hydrothermal method.

[0059] First, 2 g of Bi(NO3)3·5H2O was dispersed in 20 mL of ethylene glycol and magnetically stirred for 20 min to obtain solution A; at the same time, 5 g of Na2S·9H2O was added to 30 mL of water and magnetically stirred for 25 min to obtain solution B.

[0060] Next, 5 g of urea was ultrasonically dispersed in 20 mL of water to obtain solution C;

[0061] Solution B was rapidly added dropwise to solution A to obtain a large amount of black suspension with an irritating odor. Then, solution C was mixed with the black suspension and ultrasonically dispersed for 25 minutes to obtain a black mixed solution.

[0062] The black mixed solution was transferred into the lining of a 15 mL high-temperature reactor and heated at 120 °C for 24 h. After the reaction was completed, it was naturally cooled to room temperature, washed several times with ultrapure water and ethanol by centrifugation, and then vacuum dried at 60 °C for 6 h to obtain the black product Bi2S3 nanorods.

[0063] (1.2) Synthesis of BiOI nanoflowers:

[0064] First, 5 g of Bi(NO3)3·5H2O was ultrasonically dissolved in 15 mL of water to obtain solution D, and 5 g of KI was ultrasonically dissolved in 20 mL of water to obtain solution E.

[0065] Subsequently, under magnetic stirring, solution E was added dropwise to solution D. This process produced a large amount of orange-red precipitate. After vigorous stirring at room temperature for 1 h, the solution was washed several times with ultrapure water and ethanol, and then vacuum dried at 80 °C to obtain BiOI nanoflowers.

[0066] (1.3) The prepared Bi2S3 nanorods and BiOI nanoflowers were mixed and dispersed in an ethanol solution at a mass ratio of 2:1. The mixture was ultrasonically treated for 30 min at room temperature. The brown product obtained by ultrasonication was centrifuged and washed at 8000 rpm and dried in a vacuum oven at 60℃ to obtain Bi2S3@BiOI heterojunction.

[0067] Step (2) involves preparing the magnetically controlled immunoprobe MB-Apt, which includes the following steps:

[0068] Sonicate the 15 mg / mL SMB stock solution for 2 min to disperse it evenly.

[0069] Take 80 μL of SMB stock solution that is ultrasonically dispersed evenly, wash it three times with PBS (10 mmol / L, pH=7.4) buffer solution, and redisperse it in 500 μL of PBS solution.

[0070] Then add 150 μL of Bio-Apt at a concentration of 50 μmol / L, incubate with shaking at 37 °C for 60 min, remove excess unbound Bio-Apt by magnetic separation and washing, redisperse it in 500 μL of PBS solution and store it at 4 °C for use.

[0071] Step (3) Prepare the signal probe ALL-Apt, which specifically includes the following steps:

[0072] (3.1) Encapsulation of Ag with biotinylated liposomes + The synthesis of ALL:

[0073] Weigh 5 mg of DPPC, 5 mg of DSPE-PEG-Biotin and 0.2 mg of cholesterol and add them to a round-bottom flask containing 5 mL of chloroform. Dissolve by sonication and evaporate under reduced pressure at 45°C. Chloroform evaporates, and a uniform, transparent film is obtained that adheres to the inner wall of the flask.

[0074] Then add 20 mL of silver nitrate solution with a concentration of 15 mmol / L, stir magnetically for 60 min in a constant temperature water bath at 45℃, sonicate for 5 min, and then put it into a 1 KD dialysis bag for dialyzing to obtain ALL, and store it at 4℃ in the dark for later use.

[0075] (3.2) Preparation of ALL-Apt:

[0076] Take 100 μL of ALL and 100 μL of streptavidin (SA) and incubate them on a shaker at 37℃ and 1300 rpm for 10 min. Then add 100 μL of Bio-Apt at a concentration of 50 μmol / L and continue shaking and incubating for 30 min to obtain ALL-Apt. Store at 4℃ for later use.

[0077] Step (4) Constructing the GCE / Bi2S3@BiOI sensing interface, specifically including the following steps:

[0078] The GCE electrode is pretreated on suede with 5 μm alumina powder, polished to a mirror finish, rinsed with ultrapure water, and then ultrasonically washed with ethanol and ultrapure water in sequence. It is then dried at room temperature for later use.

[0079] Take 0.1 mg of Bi2S3@BiOI heterojunction and ultrasonically disperse it in 1 mL of ethanol solution. Take 9 μL of Bi2S3@BiOI dispersion and uniformly drop it onto the surface of GCE electrode. Let it dry completely at room temperature and store it in the dark for later use.

[0080] Step (5) Constructing a photoelectrochemical immunosensor for detecting carcinoembryonic antigen (CEA), specifically including the following steps:

[0081] First, 20 μL of CEA solution of different concentrations was added to 20 μL of MB-Apt and incubated at room temperature for 30 min. Excess unbound CEA was removed by magnetic separation and washing twice. PBS (10 mmol / L, pH=7.4) solution was used for magnetic separation and washing during the experiment.

[0082] Next, 20 μL of ALL-Apt was added and incubated at room temperature for 30 min to obtain MB-Apt-CEA-ALL-Apt sandwich immune complex. Magnetic separation and washing were used to remove excess unbound ALL-Apt.

[0083] Then, 10 μL of methanol was added and the mixture was incubated at room temperature for 15 min to induce liposome lysis and release Ag. + Magnetic separation and retention of the supernatant;

[0084] Subsequently, 50 μL of the lysis buffer was transferred onto the GCE / Bi2S3@BiOI electrode and incubated in the dark for 5 min, followed by rinsing with ultrapure water to obtain GCE / Bi2S3@BiOI / Ag. + electrode;

[0085] Another 60 μL of 0.1 mol / L Na2S was incubated on the electrode for 2 min to generate Ag2S in situ. The electrode was then rinsed with ultrapure water to obtain the GCE / Bi2S3@BiOI / Ag2S electrode. The electrode was dried at room temperature and modified with 100 μL of 0.5% Nafion solution to prevent the electrode surface material from falling off.

[0086] Finally, the modified electrode was immersed in a PBS (10 mmol / L, pH=7.4) solution containing 0.001 mol / L ascorbic acid (AA) for PEC testing, using a 200 W xenon lamp as the excitation source, with the lamp on-off for 20 s and an applied potential of 0.2 V.

[0087] During detection, the intensity of the photocurrent signal gradually increases with the increase of CEA concentration. The relationship between CEA concentration and photocurrent signal can be used to detect carcinoembryonic antigen.

[0088] The method of this invention analyzes and measures different concentrations of CEA using the PEC immunosensing platform. (Refer to...) Figure 1 The graph shows the relationship between the CEA content detected by the photoelectrochemical immunosensor prepared in the examples, where the CEA concentrations are 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 10, and 50 ng / mL, respectively.

[0089] like Figure 1 As shown, the photocurrent signal intensity gradually increases with increasing CEA concentration. This is due to the introduction of Ag through a sandwich immunoreaction. + The amount of photogenerated electrons will increase, which will also increase the amount of Ag2S generated in situ on the Bi2S3@BiOI surface. Furthermore, there is a certain energy level matching relationship between Bi2S3@BiOI and Ag2S. The formation of Bi2S3@BiOI / Ag2S ternary heterojunction can effectively suppress the recombination of photogenerated electron-hole pairs, thereby enhancing the photocurrent signal.

[0090] like Figure 2 As shown, the photoelectrochemical immunosensor detects carcinoembryonic antigen (CEA) in a linear range of 0.005–50 ng / mL, and within this concentration range, the photocurrent signal intensity exhibits a linear relationship with the logarithm of the CEA concentration. The linear equation is as follows: I =212.97×logC [CEA] +820.39 (R) 2 =0.9977), and the limit of detection (LOD) is 1.21 pg / mL (where LOD = 3SD / K, SD is the standard deviation of the PEC current signal measured in 11 blank samples, and K is the slope of the linear equation).

Claims

1. A method for preparing a photoelectrochemical immunosensor for detecting carcinoembryonic antigen, characterized in that, Includes the following steps: (1) Preparation of Bi2S3@BiOI heterojunction; (2) Preparation of the magnetically controlled immunoprobe MB-Apt; (3) Prepare the signal probe ALL-Apt, which includes the following steps; (3.1) Encapsulation of Ag with biotinylated liposomes + Synthesize ALL: Weigh 0.0001~10 mg of DPPC, 0.0001~10 mg of DSPE-PEG-Biotin and 0.0001~10 mg of cholesterol and add them to a round-bottom flask containing 3-30 mL of chloroform. Dissolve the chloroform by ultrasonication and evaporate it under reduced pressure at 45℃. A uniform and transparent film is obtained and adheres to the inner wall of the flask. Then add 5-50 mL of silver nitrate solution with a concentration of 2-20 mmol / L, stir magnetically for 60 min in a constant temperature water bath at 45℃, sonicate for 5 min, and then put it into a 1 KD dialysis bag for dialysis to obtain ALL, and store it at 4℃ in the dark for later use. (3.2) Preparation of ALL-Apt: Take 20-2000 μL of ALL and 20-200 μL of streptavidin at a concentration of 100 ng / mL, and incubate on a shaker at 37℃ and 1300 rpm for 10 min. Then add 50-200 μL of Bio-Apt at a concentration of 50 μmol / L and continue shaking and incubating for 30 min to obtain ALL-Apt, which can be stored at 4℃ for use. (4) Construct the GCE / Bi2S3@BiOI sensing interface; (5) Constructing a photoelectrochemical immunosensor, specifically including the following steps: First, add 10-100 μL of CEA solution of different concentrations to 10-100 μL of MB-Apt, incubate at room temperature for 30 min, and remove excess unbound CEA by magnetic separation and washing twice. The magnetic separation and washing solution used in the experiment is 10 mmol / L PBS solution with pH=7.

4. Next, add 10~100μL of ALL-Apt and incubate at room temperature for 30 min to obtain MB-Apt-CEA-ALL-Apt sandwich immune complex. Magnetic separation and washing are used to remove excess unbound ALL-Apt. Then, 10–100 μL of methanol was added and the mixture was incubated at room temperature for 15 min to induce liposome lysis and release Ag. + Magnetic separation and retention of the supernatant; Subsequently, 20–200 μL of the lysis buffer was transferred using a pipette and incubated on the GCE / Bi₂S₃@BiOI electrode in the dark for 5 min, followed by rinsing with ultrapure water to obtain GCE / Bi₂S₃@BiOI / Ag + electrode; Take 2~200 μL of Na2S with a concentration of 0.1 mol / L and incubate it on the electrode for 2 min to generate Ag2S in situ. Then rinse with ultrapure water to obtain GCE / Bi2S3@BiOI / Ag2S electrode. Dry it at room temperature and modify it with 2~200 μL of 0.5% Nafion solution to prevent the electrode surface material from falling off. Finally, the modified electrode was immersed in a 10 mmol / L PBS solution containing 0.0001–10 mol / L ascorbic acid at pH 7.4 for PEC testing. A 200–800 W xenon lamp was used as the excitation source, with the lamp on and off for 20 s and an applied potential of 0.2 V.

2. The preparation method according to claim 1, characterized in that, The preparation of Bi2S3@BiOI heterojunction in step (1) includes the following steps: (1.1) Synthesis of Bi2S3 nanorods: Bi2S3 nanorods were synthesized using a one-step hydrothermal method. First, disperse 0.0001~10 g of Bi(NO3)3·5H2O in 5~50 mL of ethylene glycol and stir magnetically for 10~40 min to obtain solution A; at the same time, add 0.0001~10 g of Na2S·9H2O to 5~50 mL of water and stir magnetically for 10~40 min to obtain solution B. Next, 0.0001~10 g of urea was ultrasonically dispersed in 5~50 mL of water to obtain solution C; Solution B was added dropwise to solution A to obtain a large amount of black suspension with an irritating odor. Then, solution C was mixed with the black suspension and ultrasonically dispersed for 10-40 minutes to obtain a black mixed solution. The black mixed solution was transferred into the lining of a 10-100 mL high-temperature reactor and heated to 100-200 °C. After the reaction was completed, it was naturally cooled to room temperature. After washing several times with ultrapure water and ethanol by centrifugation, it was vacuum dried at 60 °C to obtain the black product Bi2S3 nanorods. (1.2) Synthesis of BiOI nanoflowers: First, dissolve 0.0001~10 g Bi(NO3)3·5H2O in 5~50 mL of water using ultrasound to obtain solution D. At the same time, dissolve 0.0001~10 g potassium iodide in 5~50 mL of water using ultrasound to obtain solution E. Subsequently, under magnetic stirring, solution E was added dropwise to solution D. This process produced a large amount of orange-red precipitate. After vigorous stirring at room temperature, the precipitate was washed several times with ultrapure water and ethanol, and then vacuum dried at 80°C to obtain BiOI nanoflowers. (1.3) The prepared Bi2S3 nanorods and BiOI nanoflowers were mixed and dispersed in an ethanol solution at a mass ratio of 2:

1. The mixture was ultrasonically treated at room temperature. The brown product obtained by ultrasonication was centrifuged and washed at 5000~12000 rpm and dried in a vacuum oven at 60℃ to obtain Bi2S3@BiOI heterojunction.

3. The preparation method according to claim 1, characterized in that, The preparation of the magnetically controlled immunoprobe MB-Apt in step (2) includes the following steps: Sonicate the 2-20 mg / mL SMB stock solution for 2 min to disperse it evenly; Take 50-500 μL of SMB stock solution that is ultrasonically dispersed evenly, wash it three times with PBS buffer solution with a concentration of 10 mmol / L and pH=7.4, and then redisperse it in 200-2000 μL of PBS solution. Then, add 50-200 μL of biotin-modified CEA aptamer Bio-Apt at a concentration of 50 μmol / L, and incubate with shaking at 37 °C for 60 min. Remove excess unbound Bio-Apt by magnetic separation and washing, and redisperse it in 200-2000 μL of PBS solution and store it at 4 °C for use.

4. The preparation method according to claim 1, characterized in that, Step (4) involves constructing the GCE / Bi2S3@BiOI sensing interface, which includes the following steps: The GCE electrode was pretreated on suede with 5 μm alumina powder, polished to a mirror finish, rinsed with ultrapure water, and then ultrasonically washed with ethanol and ultrapure water in sequence. It was then dried at room temperature for later use. Take 0.0001~10 mg of Bi2S3@BiOI heterojunction prepared in step (1) and ultrasonically disperse it in 0.1~10 mL of ethanol solution. Take 1~15 μL of Bi2S3@BiOI dispersion and uniformly drop it onto the surface of GCE electrode. Let it dry completely at room temperature and store it in the dark for later use.

5. The preparation method according to claim 1, characterized in that, The photoelectrochemical immunosensor described above has a linear range of 0.005~50 ng / mL for detecting carcinoembryonic antigen and a detection limit of 1.21 pg / mL.

6. A photoelectrochemical immunosensor prepared by the method according to any one of claims 1-5, characterized in that, The photoelectrochemical immunosensor is used to detect carcinoembryonic antigen (CEA).

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