Construction method and application of a ratiometric electrochemiluminescence biosensor based on anti-fouling erythrocyte membranes
By electrodepositing polyaniline nanowires on the electrode surface and combining the red blood cell membrane and lipid bilayer membrane, modifying the capture antibody and luminol-labeled antibodies, a ratio-type ECL biosensor with anti-fouling red blood cell membrane was constructed, which solved the problem of insufficient detection sensitivity and stability of electrochemiluminescence biosensors in serum, and achieved high sensitivity and accuracy detection of human breast cancer cells.
Patent Information
- Application Number
- CN202411409781.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2025-08-01
- Estimated Expiration
- 2044-10-10
AI Technical Summary
When existing electrochemiluminescence biosensors detect circulating tumor cells in serum, there are problems of decreased detection sensitivity and lifespan caused by interfering with the nonspecific adsorption of biomolecules, and the stability and conductivity of the red blood cell membrane are insufficient.
Polyaniline nanowires were electrodeposited on the electrode surface, combining the red blood cell membrane and the lipid bilayer membrane, modifying the capture antibody and luminol-labeled antibody, introducing C3N4 as an internal standard, and using a ratio sensing strategy to construct a ratio-type ECL biosensor for anti-fouling red blood cell membrane.
It has achieved high sensitivity and accuracy detection of human breast cancer cell MCF-7, with a detection limit of as low as 3 cells/mL. It is suitable for trace detection of CTCs in complex serum media, promoting early screening and early warning of cancer.
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Abstract
Description
Technical Field
[0001] The present invention discloses a construction method of a ratio-type electrochemiluminescence biosensor based on an anti-fouling erythrocyte membrane and its application in the detection of human breast cancer cell MCF-7, belonging to the technical fields of optoelectronic analysis, biosensing, and interfacial anti-fouling technology. Background Art
[0002] Circulating tumor cells (CTCs) are cells that shed from primary tumors into the blood and spread throughout the body, and are considered key biomarkers for determining the occurrence and metastasis of malignant tumors. Due to the low abundance of CTCs in the blood, approximately 10 - 200 cells / mL, developing efficient analytical methods for sensitive determination of CTCs remains a challenging task. Among them, electrochemiluminescence (ECL) technology has advantages such as high sensitivity, fast response speed, low cost, and simple operation, and is an ideal choice for realizing trace detection of various serum biomarkers.
[0003] There are a large number of interfering biomolecules in serum, and their non-specific adsorption on the sensing interface will interfere with the luminescence signal, thus affecting the detection sensitivity and service life of the biosensor. Therefore, designing a sensing platform with high anti-fouling ability to resist the interfacial adsorption of the above interfering biomolecules is crucial for the trace detection of biomarkers in serum. Currently, widely used anti-fouling materials include polyethylene glycol, hydrogel, polypeptide, and zwitterion. In addition, natural cell membranes, due to their inherent super-hydrophilicity and high biocompatibility conferred by the phospholipid bilayer, also have good application prospects in the anti-fouling system. Among them, erythrocyte membranes not only retain a rich lipid layer and biological functions, but also have fewer membrane proteins compared with other cell membranes, making them more advantageous as anti-fouling interfaces. However, the poor stability and conductivity of erythrocyte membranes limit their application in bioanalysis. To solve the above problems, a layer of polyaniline nanowires was electrodeposited on the electrode surface to uniformly embed them into the erythrocyte membrane. Due to the high conductivity of polyaniline nanowires and the effective fixation of erythrocyte membranes, the conductivity and chemical stability of erythrocyte membranes have been greatly improved.
[0004] The present invention develops a highly sensitive ratiometric ECL biosensor with a red blood cell membrane immobilized by polyaniline nanowires as an anti-fouling interface for the precise detection of human breast cancer cells MCF-7 in serum. Specifically, by utilizing the interaction between the lipid bilayer membrane and 1,2-dioleoyl-sn-glycero-3-phosphoethanol-polyethylene glycol, the capture antibody is modified onto the surface of the red blood cell membrane. Human breast cancer cells MCF-7 are selected as the test target, and the labeled antibody modified with luminol is used as the signal probe. Meanwhile, C3N4 is introduced as an internal standard, and the self-calibration of the internal standard signal and the detection signal is used to reduce background and instrument interference, thereby improving the detection accuracy of the ECL biosensor. Based on the introduction of a highly bioactive anti-fouling red blood cell membrane and a self-calibration ratiometric sensing strategy, the ECL biosensor constructed by the present invention realizes the trace detection of human breast cancer cells MCF-7, with a detection limit as low as 3 cells / mL, promoting the sensitive and precise determination of CTCs in complex serum media and the early screening and timely warning of related cancers. Summary of the Invention
[0005] One of the technical tasks of the present invention is to make up for the deficiencies of the prior art by preparing a red blood cell membrane immobilized by polyaniline nanowires as an anti-fouling interface, which greatly improves its anti-fouling ability and working life while ensuring the biological activity of the ECL sensor.
[0006] The second technical task of the present invention is to construct a ratiometric ECL biosensor based on an anti-fouling red blood cell membrane by utilizing the anti-fouling characteristics of the red blood cell membrane and the ratiometric sensing strategy, which improves the sensitivity and accuracy of the detection of biological targets, with low raw material costs, simple preparation processes, and safe operation procedures.
[0007] The third technical task of the present invention is to provide the use of the ratiometric ECL biosensor based on an anti-fouling red blood cell membrane constructed by the described construction method, that is, for the sensitive and precise determination of human breast cancer cells MCF-7 in serum media; 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, promoting the trace detection of CTCs in a complex serum environment and the early screening and warning of related cancers, and having a certain industrial application prospect.
[0008] To achieve the above object, the technical scheme adopted by the present invention is as follows:
[0009] 1. A construction method of a ratiometric ECL biosensor based on an anti-fouling red blood cell membrane
[0010] A 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. 10 μL of erythrocyte membrane vesicle solution was added to the electrode surface. 10 μL of 1 mg / mL 1,2-dioleoyl-SN-glycero-3-phosphoethanol-polyethylene glycol-capture antibody solution was then added and incubated for 30 minutes. Different concentrations of human breast cancer MCF-7 cell solutions were then added to the electrode surface and incubated for 30 minutes. The electrode surface was further modified with 8 to 10 μL of luminol-detection antibody solution to construct a ratiometric ECL biosensor based on anti-fouling erythrocyte membranes.
[0011] The erythrocyte membrane vesicle solution is prepared by suspending rabbit erythrocytes in 0.25× phosphate buffered saline 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 membranes in 1× phosphate buffered saline containing 0.2 mM ethylenediaminetetraacetic acid; and ultrasonically treating the solution to obtain the erythrocyte membrane vesicle solution, which is then stored at -80°C.
[0012] The human breast cancer cell MCF-7 solutions of different concentrations were prepared by adding 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;
[0013] The luminol-detection antibody solution is prepared by adding a mixed solution containing 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1 M N-hydroxysuccinimide to 10 μg / mL of the detection antibody, incubating with shaking at 4°C for 1 hour to activate the carboxyl groups; then adding the activated detection antibody solution to a 7 mg / mL luminol solution and shaking for 4 hours to obtain a luminol-detection antibody solution.
[0014] Each step of the electrode interface modification process is rinsed with ultrapure water to remove incompletely bound reagents.
[0015] 2. Application of the ratiometric ECL biosensor based on antifouling erythrocyte membrane constructed by the construction method described above for the detection of human breast cancer cells MCF-7 in serum
[0016] The present 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 buffer solution containing 100-300 mM of K2S2O8 and 5-7 mM of H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8 to 1.8 V, the scanning speed to 0.1 V / s, the amplification factor to 3, and the photomultiplier tube voltage to 800 V; draws a linear curve based on the ECL response, and the applicable detection range of the constructed biosensor for human breast cancer cells MCF-7 can be obtained as 10-5×10 6 cells / mL, the detection limit is 3 cells / mL. The results show that the constructed ECL biosensor has excellent sensitivity, accuracy, stability, specificity and reproducibility for the detection of human breast cancer cells MCF-7, and is applicable to the trace detection of human breast cancer cells MCF-7 in serum medium.
[0017] The beneficial technical effects of the present invention:
[0018] 1. The present invention prepares a polyaniline nanowire-fixed red blood cell membrane as an efficient anti-fouling interface for ECL biosensing analysis; the inherent phospholipid bilayer of the red blood cell membrane makes it have super hydrophilicity and high biocompatibility, and compared with other cell membranes, it has fewer membrane proteins and has more advantages as an anti-fouling interface; a layer of polyaniline nanowires is electro-polymerized on the electrode surface to be uniformly embedded in the anti-fouling red blood cell membrane. Due to the high conductivity of the polyaniline nanowires and the effective fixation of the red blood cell membrane, the conductivity and chemical stability of the anti-fouling red blood cell membrane are greatly improved;
[0019] 2. The present invention constructs a ratio-type ECL biosensor based on the anti-fouling red blood cell membrane; the combination of the highly bioactive anti-fouling red blood cell membrane and the self-calibrating ratio sensing strategy improves the detection sensitivity and accuracy of the ECL biosensor, and effectively reduces false positive and false negative results;
[0020] 3. The ratio-type ECL biosensor based on the anti-fouling red blood cell membrane constructed by 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 long working life, as well as excellent stability, specificity and reproducibility, and is applicable to the trace detection of CTCs in complex serum environment and the early screening and timely warning of related cancers; the preparation process of the constructed ECL biosensor is simple and the cost is low, and it has a certain industrial application prospect. Description of the Drawings
[0021] Figure 1Flowchart of the construction method of a ratiometric ECL biosensor based on an anti-fouling red blood cell membrane (glassy carbon electrode: GCE; polyaniline nanowires: PANI NWs; red blood cell membrane: RBCM; capture antibody: Ab1; detection antibody: Ab2).
[0022] Figure 2 Scanning electron microscope images of (A) PANI NWs and (B) RBCM / PANI NWs; and (C) elemental mapping of RBCM / PANI NWs.
[0023] Figure 3 Static water contact angle images of (A) GCE and (B) RBCM / PANI NWs / GCE.
[0024] Figure 4 Ultraviolet-visible absorption spectra and (B) corresponding calibration curves of BCA protein assay standard samples.
[0025] Figure 5 Differential pulse voltammograms and (C) corresponding signal inhibition rate histograms of (A) PANI NWs / C3N4 / GCE and (B) RBCM / PANI NWs / C3N4 / GCE incubated in different concentrations of bovine serum albumin; and differential pulse voltammograms and (F) corresponding signal inhibition rate histograms of (D) PANI NWs / C3N4 / GCE and (E) RBCM / PANI NWs / C3N4 / GCE incubated in different concentrations of human serum.
[0026] Figure 6 Fluorescence imaging of a bare electrode and an RBCM / PANI NWs modified electrode after incubation in a fluorescein-conjugated bovine serum albumin solution for 90 min (indium tin oxide: ITO).
[0027] Figure 7 Graph of the RBCM concentration optimization results.
[0028] Figure 8 Characterization diagrams of the construction process of a ratiometric ECL biosensor based on an anti-fouling red blood cell membrane according to (A) differential pulse voltammetry and (B) electrochemical impedance spectroscopy; 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.
[0029] Figure 9 ECL intensity-time curves of GCE, PANI NWs / C3N4 / GCE, Ab2-luminol / MCF-7 / Ab1 / RBCM / PANI NWs / C3N4 / GCE in phosphate buffer solution containing K2S2O8 and H2O2.
[0030] Figure 10 Are (A) ECL intensity-time curves and (B) corresponding calibration curves of the ratiometric ECL biosensor based on antifouling red blood cell membrane after incubation with different concentrations of human breast cancer cells MCF-7; where 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.
[0031] Figure 11 Are (A) ECL intensity-time curves and (B) corresponding calibration curves of the control ECL biosensor without internal standard after incubation with different concentrations of human breast cancer cells MCF-7; where 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.
[0032] Figure 12 Are (A) specificity, (B) signal stability, (C) storage stability, (D) reproducibility of the ratiometric ECL biosensor based on antifouling red blood cell membrane. Detailed implementation manners
[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. Any changes made by professionals in this field to the technical solutions of the present invention shall fall within the protection scope of the present invention.
[0034] Example 1. A construction method of a ratiometric ECL biosensor based on an anti-fouling erythrocyte membrane. The glassy carbon electrode was polished with Al2O3 slurry and ultrasonically cleaned; C3N4 was dropped onto the surface of the treated electrode, and then the modified electrode was immersed in aniline containing HClO4 for 90 min of electrodeposition to form polyaniline nanowires; 10 μL of the erythrocyte membrane vesicle solution was dropped onto the electrode surface; 10 μL of a 1,2-dioleoyl-sn-glycero-3-phosphoethanol-polyethylene glycol-capture antibody solution with a concentration of 1 mg / mL was continuously dropped and incubated for 30 min; then solutions of human breast cancer cells MCF-7 with different concentrations were dropped onto the electrode surface and incubated for 30 min; 8 μL of a luminol-detection antibody solution was further modified on the electrode surface to construct a ratiometric ECL biosensor based on an anti-fouling erythrocyte membrane;
[0035] The erythrocyte membrane vesicle solution was prepared by suspending rabbit erythrocytes in 0.25× phosphate buffered saline and placing them in an ice bath for 20 min; then centrifuged at 8000 g to remove the hemoglobin released during the hypotonic treatment; the purified erythrocyte membrane was resuspended in 1× phosphate buffered saline containing 0.2 mM ethylenediaminetetraacetic acid; after sonication, the erythrocyte membrane vesicle solution was obtained and stored at -80 °C;
[0036] The solutions of human breast cancer cells MCF-7 with different concentrations were prepared by uniformly dispersing human breast cancer cells MCF-7 at concentrations of 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 in 1× phosphate buffered saline to obtain solutions of human breast cancer cells MCF-7 with different concentrations;
[0037] The luminol-detection antibody solution was prepared by adding a mixed solution containing 0.4 M of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1 M of N-hydroxysuccinimide to a detection antibody at 10 μg / mL, and incubating with shaking at 4 °C for 1 h to activate the carboxyl group; then the activated detection antibody solution was added to a luminol solution at a concentration of 7 mg / mL and reacted with shaking for 4 h to obtain the luminol-detection antibody solution;
[0038] In each step of the electrode interface modification process, ultrapure water was used for rinsing to remove unbound reagents completely.
[0039] Example 2. A construction method of a ratiometric ECL biosensor based on an anti-fouling red blood cell membrane. The glassy carbon electrode is polished with Al2O3 slurry and ultrasonically cleaned; C3N4 is dropped onto the surface of the treated electrode, and then the modified electrode is immersed in aniline containing HClO4, and electrodeposited for 90 min to form polyaniline nanowires; 10 μL of red blood cell membrane vesicle solution is dropped onto the electrode surface; 10 μL of 1,2-dioleoyl-sn-glycero-3-phosphoethanol-polyethylene glycol-capture antibody solution with a concentration of 1 mg / mL is continuously dropped and incubated for 30 min; then solutions of human breast cancer cells MCF-7 with different concentrations are dropped onto the electrode surface and incubated for 30 min; 9 μL of luminol-detection antibody solution is further modified on the electrode surface to construct a ratiometric ECL biosensor based on an anti-fouling red blood cell membrane;
[0040] The red blood cell membrane vesicle solution is prepared by suspending rabbit red blood cells in 0.25× phosphate buffered saline and placing them in an ice bath for 20 min; then centrifuging at 8000 g to remove the hemoglobin released during the hypotonic treatment; the purified red blood cell membrane is resuspended in 1× phosphate buffered saline containing 0.2 mM ethylenediaminetetraacetic acid; after ultrasonic treatment, the red blood cell membrane vesicle solution is obtained and stored at -80 °C;
[0041] The solutions of human breast cancer cells MCF-7 with different concentrations are prepared by uniformly dispersing human breast cancer cells MCF-7 at concentrations of 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 in 1× phosphate buffered saline to obtain solutions of human breast cancer cells MCF-7 with different concentrations;
[0042] The luminol-detection antibody solution is prepared by adding a mixed solution containing 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1 M N-hydroxysuccinimide to the detection antibody at 10 μg / mL, and incubating with shaking at 4 °C for 1 h to activate the carboxyl group; then the activated detection antibody solution is added to the luminol solution at a concentration of 7 mg / mL and reacted with shaking for 4 h to obtain the luminol-detection antibody solution;
[0043] In each step of the electrode interface modification process, ultrapure water is used for rinsing to remove the unreacted reagents that are not completely bound.
[0044] Example 3. A method for constructing a ratiometric ECL biosensor based on an anti-fouling erythrocyte membrane. The glassy carbon electrode is polished with Al2O3 slurry and ultrasonically cleaned. C3N4 is dropped onto the surface of the treated electrode, and then the modified electrode is immersed in aniline containing HClO4 and electro-deposited for 90 min to form polyaniline nanowires. 10 μL of the erythrocyte membrane vesicle solution is dropped onto the electrode surface. Then, 10 μL of a 1,2-dioleoyl-sn-glycero-3-phosphoethanol-polyethylene glycol-capture antibody solution with a concentration of 1 mg / mL is continuously dropped and incubated for 30 min. Then, human breast cancer cell MCF-7 solutions with different concentrations are dropped onto the electrode surface and incubated for 30 min. Further, 10 μL of a luminol-detection antibody solution is modified on the electrode surface to construct a ratiometric ECL biosensor based on an anti-fouling erythrocyte membrane;
[0045] For the erythrocyte membrane vesicle solution, rabbit erythrocytes are suspended in 0.25× phosphate buffered saline and placed in an ice bath for 20 min. Subsequently, hemoglobin released during the hypotonic treatment is removed by centrifugation at 8000 g. The purified erythrocyte membrane is resuspended in 1× phosphate buffered saline containing 0.2 mM ethylenediaminetetraacetic acid. After ultrasonic treatment, the erythrocyte membrane vesicle solution is obtained and stored at -80 °C;
[0046] For the human breast cancer cell MCF-7 solutions with different concentrations, human breast cancer cells MCF-7 are uniformly dispersed in 1× phosphate buffered saline at concentrations of 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, and 5×10 6 cells / mL respectively to obtain human breast cancer cell MCF-7 solutions with different concentrations;
[0047] For the luminol-detection antibody solution, a mixed solution containing 0.4 M of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1 M of N-hydroxysuccinimide is added to the detection antibody at 10 μg / mL, and the mixture is shaken and incubated at 4 °C for 1 h to activate the carboxyl group. Then, the activated detection antibody solution is added to a luminol solution with a concentration of 7 mg / mL and shaken and reacted for 4 h to obtain the luminol-detection antibody solution;
[0048] In each step of the electrode interface modification process, ultrapure water is used for rinsing to remove unreacted reagents that have not completely bound.
[0049] Example 4. Use of the ratiometric ECL biosensor based on an anti-fouling erythrocyte membrane constructed by the construction methods described in Example 1, Example 2, and Example 3 for the detection of human breast cancer cell MCF-7 in serum
[0050] The present 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 buffer solution containing 100 mM of K2S2O8 and 5 mM of H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8 to 1.8 V, the scanning speed to 0.1 V / s, the amplification factor to 3, and the photomultiplier tube voltage to 800 V; draws a linear curve based on the ECL response, and the applicable detection range of the constructed biosensor for human breast cancer cells MCF-7 is 10 to 5×10 6 cells / mL, and the detection limit is 3 cells / mL. The results show that the constructed ECL biosensor has excellent sensitivity, accuracy, stability, specificity, and reproducibility for the detection of human breast cancer cells MCF-7, and is suitable for the trace detection of human breast cancer cells MCF-7 in serum medium.
[0051] Example 5 Application of the ratio-type ECL biosensor based on an anti-fouling erythrocyte membrane constructed by the construction method described in Example 1, Example 2, and Example 3 for the detection of human breast cancer cells MCF-7 in serum
[0052] The present 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 buffer solution containing 200 mM of K2S2O8 and 6 mM of H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8 to 1.8 V, the scanning speed to 0.1 V / s, the amplification factor to 3, and the photomultiplier tube voltage to 800 V; draws a linear curve based on the ECL response, and the applicable detection range of the constructed biosensor for human breast cancer cells MCF-7 is 10 to 5×10 6 cells / mL, and the detection limit is 3 cells / mL. The results show that the constructed ECL biosensor has excellent sensitivity, accuracy, stability, specificity, and reproducibility for the detection of human breast cancer cells MCF-7, and is suitable for the trace detection of human breast cancer cells MCF-7 in serum medium.
[0053] Example 6 Application of the ratio-type ECL biosensor based on an anti-fouling erythrocyte membrane constructed by the construction method described in Example 1, Example 2, and Example 3 for the detection of human breast cancer cells MCF-7 in serum
[0054] The present 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 buffer solution containing 300 mM of K2S2O8 and 7 mM of H2O2 as the detection solution; uses an ECL analyzer to perform signal testing, sets the scanning voltage range to -1.8 to 1.8 V, the scanning speed to 0.1 V / s, the amplification factor to 3, and the photomultiplier tube voltage to 800 V; draws a linear curve based on the ECL response, and the applicable detection range of the constructed biosensor for human breast cancer cells MCF-7 can be obtained as 10 to 5×10 6 cells / mL, and the detection limit is 3 cells / mL. The results show that the constructed ECL biosensor has excellent sensitivity, accuracy, stability, specificity and reproducibility for the detection of human breast cancer cells MCF-7, and is suitable for the trace detection of human breast cancer cells MCF-7 in serum medium.
Claims
1. A method for constructing a ratiometric electrochemiluminescence biosensor based on an anti-fouling erythrocyte membrane, characterized in that, The glassy carbon electrode was polished with Al2O3 slurry and ultrasonically cleaned; C3N4 was dropped onto the surface of the treated electrode, and then the modified electrode was immersed in aniline containing HClO4, and electrodeposited for 90 min to form polyaniline nanowires; 10 μL of the erythrocyte membrane vesicle solution was dropped onto the electrode surface; 10 μL of a 1,2-dioleoyl-sn-glycero-3-phosphoethanol-polyethylene glycol-capture antibody solution with a concentration of 1 mg / mL was continuously dropped and incubated for 30 min; then human breast cancer cell MCF-7 solutions with different concentrations were dropped onto the electrode surface and incubated for 30 min; 8 - 10 μL of the luminol-detection antibody solution was further modified on the electrode surface to construct a ratiometric electrochemiluminescence biosensor based on the antifouling erythrocyte membrane.
2. The construction method of a ratiometric electrochemiluminescence biosensor based on an anti-fouling erythrocyte membrane according to claim 1, characterized in that, The erythrocyte membrane vesicle solution was prepared by suspending rabbit erythrocytes in 0.25× phosphate buffered saline and placing them in an ice bath for 20 min; Subsequently, hemoglobin released during the hypotonic treatment was removed by centrifugation; the purified erythrocyte membrane was resuspended in 1× phosphate buffered saline containing 0.2 mM ethylenediaminetetraacetic acid; After ultrasonic treatment, the erythrocyte membrane vesicle solution was obtained and stored at -80 °C.
3. The construction method of a ratiometric electrochemiluminescence biosensor based on an anti-fouling erythrocyte membrane as claimed in claim 1, characterized in that, The human breast cancer cell MCF-7 solutions with different concentrations are prepared by uniformly dispersing human breast cancer cells MCF-7 at concentrations of 10 cells / mL, 50 cells / mL, 500 cells / mL, 5000 cells / mL, 5×10 4 cells / mL, 5×10 5 cells / mL, and 5×10 6 cells / mL in 1× phosphate buffered saline solution to obtain human breast cancer cell MCF-7 solutions with different concentrations.
4. The construction method of a ratiometric electrochemiluminescence biosensor based on an anti-fouling erythrocyte membrane as claimed in claim 1, wherein The luminol-detection antibody solution was prepared by adding a mixed solution containing 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.1 M N-hydroxysuccinimide to the detection antibody at 10 μg / mL, and incubating with shaking at 4 °C for 1 h to activate the carboxyl group; then the activated detection antibody solution was added to the luminol solution at a concentration of 7 mg / mL and reacted with shaking for 4 h to obtain the luminol-detection antibody solution.
5. The construction method of a ratiometric electrochemiluminescence biosensor based on an anti-fouling red blood cell membrane as claimed in claim 1, wherein, In each step of the electrode interface modification process, ultrapure water was used for rinsing to remove unbound reagents.