Construction method and application of ratio type electrochemical luminescence biosensor based on antifouling erythrocyte membrane

Abstract

The invention discloses a construction method and application of a ratio-dependent electrochemical luminescence (ECL) biosensor based on an antifouling red cell membrane, and belongs to the technical fields of photoelectric analysis, biosensing and interface antifouling. In the invention, the erythrocyte membrane with super-hydrophilicity and low membrane protein content is used as an efficient antifouling interface, and the good biocompatibility of the erythrocyte membrane avoids the influence of a chemical antifouling coating on the biological activity of the ECL sensor. Meanwhile, the electropolymerized polyaniline nanowire is embedded into the antifouling erythrocyte membrane, so that the chemical stability and the conductivity of the antifouling erythrocyte membrane are enhanced, and the application of the antifouling erythrocyte membrane in ECL analysis is expanded. C3N4 is introduced as an interior label for eliminating background signals and external interference, so that the detection precision of the biosensor is improved. On the basis, the constructed ECL antifouling biosensor realizes ultra-sensitive detection of human breast cancer cells MCF-7 in human serum, and the detection limit is as low as 3 cells / mL.

Description

Technical Field

[0002] The invention discloses a method for constructing a ratiometric electrochemiluminescent biosensor based on antifouling erythrocyte membrane and its application in detecting human breast cancer cells MCF-7, belonging to the technical fields of photoelectric analysis, biosensing and interface antifouling. Background Art

[0003] Circulating tumor cells (CTCs) are cells that shed from primary tumors into the blood and spread throughout the body. They are considered to be key biomarkers for determining the occurrence and metastasis of malignant tumors. Since the abundance of CTCs in the blood is low, about 10 to 200 cells / mL, it is still a challenging task to develop efficient analytical methods to achieve sensitive determination of CTCs. Among them, electrochemiluminescence (ECL) technology has the advantages of high sensitivity, fast response speed, low cost, and simple operation. It is an ideal choice for trace detection of various serological biomarkers.

[0004] There are a large number of interfering biomolecules in serum, and their nonspecific adsorption on the sensing interface will interfere with the luminescent signal, thereby affecting the detection sensitivity and service life of the biosensor. Therefore, designing a sensing platform with efficient antifouling ability to resist the interfacial adsorption of the above-mentioned interfering biomolecules is crucial for trace detection of biomarkers in serum. At present, widely used antifouling materials include polyethylene glycol, hydrogels, peptides and zwitterions. In addition, natural cell membranes also have good application prospects in antifouling systems due to their inherent superhydrophilicity and high biocompatibility given by the phospholipid bilayer. Among them, the erythrocyte membrane not only retains a rich lipid layer and biological functions, but also has fewer membrane proteins compared to other cell membranes, making it more advantageous as an antifouling interface. However, the poor stability and conductivity of the erythrocyte membrane limit its application in biological analysis. In order to solve the above problems, the present invention electrodeposits a layer of polyaniline nanowires on the surface of the electrode so that it is evenly embedded in the erythrocyte membrane. Due to the high conductivity of the polyaniline nanowires and the effective fixation of the erythrocyte membrane, the conductivity and chemical stability of the erythrocyte membrane are greatly improved.

[0005] The present invention has developed an ultra-sensitive ratiometric ECL biosensor with polyaniline nanowire-fixed erythrocyte membrane as an antifouling interface for the precise detection of human breast cancer cells MCF-7 in serum. Specifically, the capture antibody is modified to the surface of the erythrocyte membrane by utilizing the interaction between the lipid bilayer membrane and 1,2-dioleoyl-SN-glycerol-3-phosphoethanol-polyethylene glycol, human breast cancer cells MCF-7 are selected as the test target, and luminol-modified labeled antibodies are used as signal probes. At the same time, C3N4 is introduced as an internal standard, and the self-calibration of the internal standard signal and the detection signal is used to reduce the background and instrument interference to improve the detection accuracy of the ECL biosensor. Based on the introduction of highly bioactive antifouling erythrocyte membranes and self-calibration ratio sensing strategies, the ECL biosensor constructed by the present invention realizes trace detection of human breast cancer cells MCF-7, with a detection limit as low as 3 cells / mL, which promotes the sensitive and accurate determination of CTCs in complex serum media and the early screening and timely warning of related cancers. Summary of the invention

[0006] One of the technical tasks of the present invention is to make up for the deficiencies of the prior art, and to prepare a polyaniline nanowire-fixed erythrocyte membrane as an antifouling interface, which greatly improves the antifouling ability and working life of the ECL sensor while ensuring its biological activity;

[0007] The second technical task of the present invention is to utilize the anti-fouling properties of erythrocyte membrane and ratio sensing strategy to construct a ratiometric ECL biosensor based on anti-fouling erythrocyte membrane, which improves the sensitivity and accuracy of biological target detection, uses low cost raw materials, has a simple preparation process, and a safe operation process;

[0008] The third technical task of the present invention is to provide the use of the ratiometric ECL biosensor based on the anti-fouling red blood cell membrane constructed by the construction method, namely, for the sensitive and accurate determination of human breast cancer cells MCF-7 in serum medium; the constructed ratiometric ECL biosensor has a low detection limit and a wide linear range for the detection of human breast cancer cells MCF-7, which promotes the trace detection of CTCs and the early screening and early warning of related cancers in a complex serum environment, and has certain industrial application prospects.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] 1. Construction of a ratiometric ECL biosensor based on antifouling erythrocyte membrane

[0011] The glassy carbon electrode was polished with Al2O3 slurry and ultrasonically cleaned; C3N4 was added to the treated electrode surface, and then the modified electrode was immersed in aniline containing HClO4 and electrodeposited for 90 minutes to form polyaniline nanowires; 10μL of erythrocyte membrane vesicle solution was added to the electrode surface; 10μL of 1mg / mL 1,2-dioleoyl-SN-glycerol-3-phosphoethanol-polyethylene glycol-capture antibody solution was added and incubated for 30 minutes; then different concentrations of human breast cancer cell MCF-7 solution were added to the electrode surface and incubated for 30 minutes; 8-10μL of luminol-detection antibody solution was further modified on the electrode surface to construct a ratiometric ECL biosensor based on anti-fouling erythrocyte membrane;

[0012] The erythrocyte membrane vesicle solution is prepared by suspending rabbit erythrocytes in a 0.25× phosphate buffered saline solution and placing the solution in an ice bath for 20 minutes; then centrifuging the solution at 8000 g to remove hemoglobin released during hypotonic treatment; resuspending the purified erythrocyte membrane in a 1× phosphate buffered saline solution containing 0.2 mM ethylenediaminetetraacetic acid; and ultrasonically treating the erythrocyte membrane vesicle solution to obtain the erythrocyte membrane vesicle solution, which is then stored at -80°C.

[0013] The human breast cancer cell MCF-7 solutions of different concentrations are prepared by mixing human breast cancer cell MCF-7 at 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, 5×10 6 The concentration of cells / mL was uniformly dispersed in 1× phosphate buffered saline to obtain human breast cancer cell MCF-7 solutions of different concentrations;

[0014] The luminol-detection antibody solution is prepared by adding a mixed solution containing 0.4M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1M N-hydroxysuccinimide to 10 μg / mL of the detection antibody, and incubating the mixture at 4°C for 1 hour to activate the carboxyl group; then adding the activated detection antibody solution to a luminol solution with a concentration of 7 mg / mL and reacting the mixture for 4 hours to obtain a luminol-detection antibody solution;

[0015] In each step of the electrode interface modification process, ultrapure water is used for rinsing to remove incompletely bound reagents.

[0016] 2. Application of the ratiometric ECL biosensor based on antifouling erythrocyte membrane constructed by the construction method for detecting human breast cancer cells MCF-7 in serum

[0017] The invention uses the constructed ECL biosensor as the working electrode, the calomel electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode to design a three-electrode detection system; uses a phosphate buffered saline solution containing 100-300 mM K2S2O8 and 5-7 mM H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8-1.8 V, the scanning speed to 0.1 V / s, the amplification level to 3, and the photomultiplier tube voltage to 800 V; draws a linear curve based on the ECL response, and obtains that the constructed biosensor has an applicable detection range of 10-5×10 6 cells / mL, and the detection limit was 3 cells / mL. The results showed that the constructed ECL biosensor had excellent sensitivity and accuracy, stability, specificity and reproducibility for the detection of human breast cancer cells MCF-7, and was suitable for trace detection of human breast cancer cells MCF-7 in serum media.

[0018] Beneficial technical effects of the present invention:

[0019] 1. The present invention prepares a polyaniline nanowire-fixed erythrocyte membrane as an efficient antifouling interface for ECL biosensing analysis; the inherent phospholipid bilayer of the erythrocyte membrane makes it super-hydrophilic and highly biocompatible, and compared with other cell membranes, it has less membrane protein, which is more advantageous as an antifouling interface; a layer of polyaniline nanowires is electropolymerized on the electrode surface to be uniformly embedded in the antifouling erythrocyte membrane. Due to the high conductivity of the polyaniline nanowires and the effective fixation of the erythrocyte membrane, the conductivity and chemical stability of the antifouling erythrocyte membrane are greatly improved;

[0020] 2. The present invention constructs a ratiometric ECL biosensor based on antifouling erythrocyte membrane; the combination of high bioactivity antifouling erythrocyte membrane and self-calibration ratio sensing strategy improves the detection sensitivity and accuracy of the ECL biosensor, effectively reducing false positive and false negative results;

[0021] 3. The ratiometric ECL biosensor based on anti-fouling red blood cell membrane constructed in the present invention has a low detection limit and a wide detection range for the detection of human breast cancer cells MCF-7, high biological activity and a long working life, as well as excellent stability, specificity and reproducibility. It is suitable for trace detection of CTCs in complex serum environments and early screening and timely warning of related cancers; the constructed ECL biosensor has a simple preparation process and low cost, and has certain industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1Flow chart of the construction method of the ratiometric ECL biosensor based on antifouling erythrocyte membrane (glassy carbon electrode: GCE; polyaniline nanowires: PANI NWs; erythrocyte membrane: RBCM; capture antibody: Ab1; detection antibody: Ab2). Figure 2 Scanning electron microscopy images of (A) PANI NWs and (B) RBCM / PANI NWs; and (C) elemental mapping of RBCM / PANI NWs. Figure 3 Static water contact angle diagrams of (A) GCE and (B) RBCM / PANI NWs / GCE. Figure 4 (A) UV-visible absorption spectrum of BCA protein assay standard sample and (B) corresponding calibration curve. Figure 5 The differential pulse voltammetry curves of (A) PANI NWs / C3N4 / GCE and (B) RBCM / PANI NWs / C3N4 / GCE incubated in different concentrations of bovine serum albumin and (C) the corresponding signal suppression rate bar graph; and the differential pulse voltammetry curves of (D) PANI NWs / C3N4 / GCE and (E) RBCM / PANI NWs / C3N4 / GCE incubated in different concentrations of human serum and (F) the corresponding signal suppression rate bar graph. Figure 6 Fluorescence imaging of the bare electrode and RBCM / PANI NWs modified electrode after incubation in fluorescein-conjugated bovine serum albumin solution for 90 min (indium tin oxide: ITO). Figure 7 This is a graph showing the concentration optimization results for RBCM. Figure 8 Characterization diagram of the construction process of the ratiometric ECL biosensor based on antifouling red blood cell membrane according to (A) differential pulse voltammetry and (B) electrochemical impedance spectroscopy tests; where: a: GCE; b: C3N4 / GCE; c: PANI NWs / C3N4 / GCE; d: RBCM / PANI NWs / C3N4 / GCE; e: Ab1 / RBCM / PANI NWs / C3N4 / GCE; f: MCF-7 / Ab1 / RBCM / PANI NWs / C3N4 / GCE; g: Ab2-luminol / MCF-7 / Ab1 / RBCM / PANI NWs / C3N4 / GCE. Fig. 9These are the ECL intensity-time curves of GCE, PANI NWs / C3N4 / GCE, Ab2-luminol / MCF-7 / Ab1 / RBCM / PANI NWs / C3N4 / GCE in phosphate buffered saline containing K2S2O8 and H2O2. Fig.10 (A) The ECL intensity-time curve of the ratiometric ECL biosensor based on antifouling erythrocyte membrane after incubation with different concentrations of human breast cancer cells MCF-7 and (B) the corresponding calibration curve; the concentration gradients are 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, 5×10 6 cells / mL. Fig.11 (A) The ECL intensity-time curve of the ECL biosensor without internal standard after incubation with different concentrations of human breast cancer cells MCF-7 and (B) the corresponding calibration curve; the concentration gradients are 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL. Fig.12 (A) Specificity, (B) signal stability, (C) storage stability, and (D) reproducibility of the ratiometric ECL biosensor based on antifouling erythrocyte membrane. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto, and all changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0023] Example 1 A method for constructing a ratiometric ECL biosensor based on antifouling erythrocyte membranes: A glassy carbon electrode was polished with Al2O3 slurry and ultrasonically cleaned; C3N4 was dripped onto the treated electrode surface, and then the modified electrode was immersed in aniline containing HClO4 and electrodeposited for 90 minutes to form polyaniline nanowires; 10 μL of erythrocyte membrane vesicle solution was dripped onto the electrode surface; 10 μL of 1,2-dioleoyl-SN-glycerol-3-phosphoethanol-polyethylene glycol-capture antibody solution with a concentration of 1 mg / mL was continuously dripped and incubated for 30 minutes; then different concentrations of human breast cancer cell MCF-7 solutions were dripped onto the electrode surface and incubated for 30 minutes; 8 μL of luminol-detection antibody solution was further modified on the electrode surface to construct a ratiometric ECL biosensor based on antifouling erythrocyte membranes;

[0024] The erythrocyte membrane vesicle solution is prepared by suspending rabbit erythrocytes in a 0.25× phosphate buffered saline solution and placing the solution in an ice bath for 20 minutes; then centrifuging the solution at 8000 g to remove hemoglobin released during hypotonic treatment; resuspending the purified erythrocyte membrane in a 1× phosphate buffered saline solution containing 0.2 mM ethylenediaminetetraacetic acid; and ultrasonically treating the erythrocyte membrane vesicle solution to obtain the erythrocyte membrane vesicle solution, which is then stored at -80°C.

[0025] The human breast cancer cell MCF-7 solutions of different concentrations are prepared by mixing human breast cancer cell MCF-7 at 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, 5×10 6 The concentration of cells / mL was uniformly dispersed in 1× phosphate buffered saline to obtain human breast cancer cell MCF-7 solutions of different concentrations;

[0026] The luminol-detection antibody solution is prepared by adding a mixed solution containing 0.4M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1M N-hydroxysuccinimide to 10 μg / mL of the detection antibody, and incubating the mixture at 4°C for 1 hour to activate the carboxyl group; then adding the activated detection antibody solution to a luminol solution with a concentration of 7 mg / mL and reacting the mixture for 4 hours to obtain a luminol-detection antibody solution;

[0027] In each step of the electrode interface modification process, ultrapure water is used for rinsing to remove incompletely bound reagents.

[0028] Example 2 A method for constructing a ratiometric ECL biosensor based on antifouling erythrocyte membranes: A glassy carbon electrode was polished with Al2O3 slurry and ultrasonically cleaned; C3N4 was dripped onto the treated electrode surface, and then the modified electrode was immersed in aniline containing HClO4 and electrodeposited for 90 minutes to form polyaniline nanowires; 10 μL of erythrocyte membrane vesicle solution was dripped onto the electrode surface; 10 μL of 1 mg / mL 1,2-dioleoyl-SN-glycerol-3-phosphoethanol-polyethylene glycol-capture antibody solution was continued to be dripped and incubated for 30 minutes; then different concentrations of human breast cancer cell MCF-7 solutions were dripped onto the electrode surface and incubated for 30 minutes; 9 μL of luminol-detection antibody solution was further modified on the electrode surface to construct a ratiometric ECL biosensor based on antifouling erythrocyte membranes;

[0029] The erythrocyte membrane vesicle solution is prepared by suspending rabbit erythrocytes in a 0.25× phosphate buffered saline solution and placing the solution in an ice bath for 20 minutes; then centrifuging the solution at 8000 g to remove hemoglobin released during hypotonic treatment; resuspending the purified erythrocyte membrane in a 1× phosphate buffered saline solution containing 0.2 mM ethylenediaminetetraacetic acid; and ultrasonically treating the erythrocyte membrane vesicle solution to obtain the erythrocyte membrane vesicle solution, which is then stored at -80°C.

[0030] The human breast cancer cell MCF-7 solutions of different concentrations are prepared by mixing human breast cancer cell MCF-7 at 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, 5×10 6 The concentration of cells / mL was uniformly dispersed in 1× phosphate buffered saline to obtain human breast cancer cell MCF-7 solutions of different concentrations;

[0031] The luminol-detection antibody solution is prepared by adding a mixed solution containing 0.4M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1M N-hydroxysuccinimide to 10 μg / mL of the detection antibody, and incubating the mixture at 4°C for 1 hour to activate the carboxyl group; then adding the activated detection antibody solution to a luminol solution with a concentration of 7 mg / mL and reacting the mixture for 4 hours to obtain a luminol-detection antibody solution;

[0032] In each step of the electrode interface modification process, ultrapure water is used for rinsing to remove incompletely bound reagents.

[0033] Example 3 A method for constructing a ratiometric ECL biosensor based on antifouling erythrocyte membranes: A glassy carbon electrode was polished with Al2O3 slurry and ultrasonically cleaned; C3N4 was dripped onto the treated electrode surface, and then the modified electrode was immersed in aniline containing HClO4 and electrodeposited for 90 minutes to form polyaniline nanowires; 10 μL of erythrocyte membrane vesicle solution was dripped onto the electrode surface; 10 μL of 1 mg / mL 1,2-dioleoyl-SN-glycerol-3-phosphoethanol-polyethylene glycol-capture antibody solution was continued to be dripped and incubated for 30 minutes; then different concentrations of human breast cancer cell MCF-7 solutions were dripped onto the electrode surface and incubated for 30 minutes; 10 μL of luminol-detection antibody solution was further modified on the electrode surface to construct a ratiometric ECL biosensor based on antifouling erythrocyte membranes;

[0034] The erythrocyte membrane vesicle solution is prepared by suspending rabbit erythrocytes in a 0.25× phosphate buffered saline solution and placing the solution in an ice bath for 20 minutes; then centrifuging the solution at 8000 g to remove hemoglobin released during hypotonic treatment; resuspending the purified erythrocyte membrane in a 1× phosphate buffered saline solution containing 0.2 mM ethylenediaminetetraacetic acid; and ultrasonically treating the erythrocyte membrane vesicle solution to obtain the erythrocyte membrane vesicle solution, which is then stored at -80°C.

[0035] The human breast cancer cell MCF-7 solutions of different concentrations are prepared by mixing human breast cancer cell MCF-7 at 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, 5×10 6 The concentration of cells / mL was uniformly dispersed in 1× phosphate buffered saline to obtain human breast cancer cell MCF-7 solutions of different concentrations;

[0036] The luminol-detection antibody solution is prepared by adding a mixed solution containing 0.4M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1M N-hydroxysuccinimide to 10 μg / mL of the detection antibody, and incubating the mixture at 4°C for 1 hour to activate the carboxyl group; then adding the activated detection antibody solution to a luminol solution with a concentration of 7 mg / mL and reacting the mixture for 4 hours to obtain a luminol-detection antibody solution;

[0037] In each step of the electrode interface modification process, ultrapure water is used for rinsing to remove incompletely bound reagents.

[0038] Example 4 Application of the ratiometric ECL biosensor based on antifouling erythrocyte membrane constructed by the construction methods described in Examples 1, 2 and 3 for the detection of human breast cancer cells MCF-7 in serum

[0039] The invention adopts the constructed ECL biosensor as the working electrode, the calomel electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode to design a three-electrode detection system; uses a phosphate buffered saline solution containing 100mM K2S2O8 and 5mM H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8 to 1.8V, the scanning speed to 0.1V / s, the amplification level to 3, and the photomultiplier tube voltage to 800V; draws a linear curve based on the ECL response, and obtains that the constructed biosensor has an applicable detection range of 10 to 5×10 6 cells / mL, and the detection limit was 3 cells / mL. The results showed that the constructed ECL biosensor had excellent sensitivity and accuracy, stability, specificity and reproducibility for the detection of human breast cancer cells MCF-7, and was suitable for trace detection of human breast cancer cells MCF-7 in serum media.

[0040] Example 5 Application of the ratiometric ECL biosensor based on antifouling erythrocyte membrane constructed by the construction methods described in Examples 1, 2 and 3 for the detection of human breast cancer cells MCF-7 in serum

[0041] The invention adopts the constructed ECL biosensor as the working electrode, the calomel electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode to design a three-electrode detection system; uses a phosphate buffered saline solution containing 200mM K2S2O8 and 6mM H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8 to 1.8V, the scanning speed to 0.1V / s, the amplification level to 3, and the photomultiplier tube voltage to 800V; draws a linear curve based on the ECL response, and obtains that the constructed biosensor has an applicable detection range of 10 to 5×10 6 cells / mL, and the detection limit was 3 cells / mL. The results showed that the constructed ECL biosensor had excellent sensitivity and accuracy, stability, specificity and reproducibility for the detection of human breast cancer cells MCF-7, and was suitable for trace detection of human breast cancer cells MCF-7 in serum media.

[0042] Example 6 Application of the ratiometric ECL biosensor based on antifouling erythrocyte membrane constructed by the construction methods described in Examples 1, 2 and 3 for the detection of human breast cancer cells MCF-7 in serum

[0043] The invention adopts the constructed ECL biosensor as the working electrode, the calomel electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode to design a three-electrode detection system; uses a phosphate buffered saline solution containing 300mM K2S2O8 and 7mM H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8 to 1.8V, the scanning speed to 0.1V / s, the amplification level to 3, and the photomultiplier tube voltage to 800V; draws a linear curve based on the ECL response, and obtains that the constructed biosensor has an applicable detection range of 10 to 5×10 6 cells / mL, and the detection limit was 3 cells / mL. The results showed that the constructed ECL biosensor had excellent sensitivity and accuracy, stability, specificity and reproducibility for the detection of human breast cancer cells MCF-7, and was suitable for trace detection of human breast cancer cells MCF-7 in serum media.

Claims

1. A method for constructing a ratiometric electrochemiluminescence biosensor based on antifouling erythrocyte membrane, characterized in that: The glassy carbon electrode was polished with Al2O3 slurry and ultrasonically cleaned; C3N4 was added to the treated electrode surface, and then the modified electrode was immersed in aniline containing HClO4 and electrodeposited for 90 minutes to form polyaniline nanowires; 10μL of erythrocyte membrane vesicle solution was added to the electrode surface; 10μL of 1mg / mL 1,2-dioleoyl-SN-glycero-3-phosphoethanol-polyethylene glycol-capture antibody solution was continued to be added and incubated for 30 minutes; then different concentrations of human breast cancer cell MCF-7 solution were added to the electrode surface and incubated for 30 minutes; 8-10μL of luminol-detection antibody solution was further modified on the electrode surface to construct a ratiometric electrochemiluminescence biosensor based on anti-fouling erythrocyte membrane.

2. The method for constructing a ratiometric electrochemiluminescence biosensor based on antifouling erythrocyte membrane according to claim 1, characterized in that: The erythrocyte membrane vesicle solution is prepared by suspending rabbit erythrocytes in 0.25× phosphate buffered saline solution and placing the solution in an ice bath for 20 minutes; then centrifuging the solution at 8000 g to remove hemoglobin released during hypotonic treatment; and resuspending the purified erythrocyte membrane in 1× phosphate buffered saline solution containing 0.2 mM ethylenediaminetetraacetic acid. After sonication, the erythrocyte membrane vesicle solution was obtained and stored at -80°C.

3. The method for constructing a ratiometric electrochemiluminescence biosensor based on antifouling erythrocyte membrane according to claim 1, characterized in that: The human breast cancer cell MCF-7 solutions of different concentrations are prepared by mixing human breast cancer cell MCF-7 at 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, 5×10 6 The concentration of cells / mL was evenly dispersed in 1× phosphate buffered saline to obtain human breast cancer cell MCF-7 solutions of different concentrations.

4. The method for constructing a ratiometric electrochemiluminescence biosensor based on antifouling erythrocyte membrane according to claim 1, characterized in that: The luminol-detection antibody solution is prepared by adding a mixed solution containing 0.4M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1M N-hydroxysuccinimide to 10μg / mL of the detection antibody, and incubating the mixture at 4°C for 1h to activate the carboxyl group; then, the activated detection antibody solution is added to a luminol solution with a concentration of 7mg / mL and the mixture is shaken for reaction for 4h to obtain a luminol-detection antibody solution.

5. The method for constructing a ratiometric electrochemiluminescence biosensor based on antifouling erythrocyte membrane according to claim 1, characterized in that: In each step of the electrode interface modification process, ultrapure water is used for rinsing to remove incompletely bound reagents.

6. Use of the ratiometric electrochemiluminescence biosensor based on antifouling erythrocyte membrane constructed by the construction method according to claim 1 for detecting human breast cancer cells MCF-7.

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