Electrochemically actuated biosensor for actively capturing tumor markers and detection method

Abstract

The invention discloses an electrochemically actuated biosensor for actively capturing tumor markers and a detection method. The electrochemical actuation biosensor comprises an electrochemical driver with a three-layer structure, wherein the electrochemical driver is composed of two identical MXene-Au electrode layers and a middle inert film; according to the MUC1 actuated biosensing detection method of the electrochemical driver based on the MXene-Au electrode layer, under the action of electrochemical CV circulation, the electrochemical driver with AuNPs coupled with the MUC1 aptamer on the surface of the electrode layer can actively capture a tumor marker MUC1 far away from an electrode area. According to the application, the actuation performance and the biosensing performance can be simultaneously improved through the AuNPs subjected to in-situ reduction on MXene, an actuation and biosensing dual-promotion effect is achieved, the binding efficiency of the bendable electrode and biomolecules is improved, further, more target molecules MUC1 are recognized, and ultra-sensitive biological detection is realized.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical actuation and biosensor detection technology, and in particular to an electrochemical actuation biosensor and a detection method for actively capturing tumor markers. Background Art

[0002] Electrochemical biosensor detection technology is widely used in biological detection due to its advantages such as low cost, easy preparation, rapid sensitivity, and high specificity. Conventional electrochemical biodetection is passive detection through the binding of biological molecules to electrodes through fixed electrodes such as glassy carbon electrodes and indium tin oxide electrodes. The binding ability of target molecules is weak, which prolongs the detection time and reduces the detection efficiency.

[0003] The electrochemical actuator, which also works in electrolyte solution, is a flexible and bendable actuator that converts electrical energy into chemical energy and then into mechanical energy by reversibly inserting and removing ions in the electrolyte solution into the electrode. It has the advantages of stable operation, light weight and easy production. During the detection process, it can actively capture the target molecule tumor marker MUC1 far away from the electrode area, improve the binding efficiency of the flexible electrode and biological molecules, and thus achieve rapid and ultra-sensitive biological detection.

[0004] MXene's own good electrical conductivity, photothermal conversion performance, reversible ion intercalation, chemical stability and other properties make it an ideal electrode material for making flexible actuators and sensors. Considering the low capacitance caused by the self-stacking effect of MXene, gold nanoparticles (AuNPs) are in situ reduced on MXene to make MXene-Au electrodes. On the one hand, the electrode conductivity and charge storage capacity are enhanced to improve the electrochemical performance and driving performance. On the other hand, AuNPs have excellent conductivity, chemical inertness and biocompatibility, and can be chemically modified in a variety of ways and widely used in electrochemical biological detection. Therefore, in situ reduced AuNPs can simultaneously improve the actuation and biosensing performance, thereby obtaining better detection effects.

[0005] The Chinese invention patent "Application No. CN202311808630.7" based on conventional electrochemical biodetection discloses a magnetically induced self-assembly electrochemical biosensor for detecting MUC1. Through magnetically induced self-assembly, MUC1 aptamers and ssDNA are attached to Fe3O4 / α-Fe2O3@Au nanoparticles. The aptamer specifically captures the MUC1 protein, and ssDNA is released to generate electrical signals during the capture process, thereby achieving high-sensitivity detection. The Chinese invention patent "Application No. CN201810189289.4" discloses a method for preparing a proportional signal amplification aptamer sensor based on three-dimensional graphene. The method uses a gold nano-three-dimensional graphene complex as a modified electrode substrate material, and then ferrocene-labeled mucin aptamers are grafted onto gold nanoparticles on the surface of three-dimensional graphene. When the ferrocene-labeled aptamer captures mucin on the electrode surface, the mucin aptamer-gold nanocomplex labeled with methyl blue is co-incubated with it and then used for electrochemical detection. It has the advantages of high sensitivity, good stability, simple method, and high cost-effectiveness. The Chinese invention patent "Application No. CN201811237939.4" discloses a competitive electrochemical aptamer sensor based on MXene for the detection of mucin MUC1. MXene can directly bind to DNA molecules through electrostatic attraction. The large surface area of ​​MXene provides a large number of attachment sites for signal molecules, plays a role in signal amplification, and has specificity for the detection of MUC1, with high stability and sensitivity. Summary of the invention

[0006] In order to overcome the technical defects in traditional electrochemical biological detection, the present invention proposes an electrochemically actuated biosensor and detection method for actively capturing tumor markers. Considering the low capacitance caused by the self-stacking effect of MXene, gold nanoparticles (AuNPs) are in situ polymerized and reduced on MXene to make a MXene-Au electrode layer. On the one hand, the electrode conductivity and charge storage capacity are enhanced to improve the electrochemical performance and driving performance. On the other hand, AuNPs have excellent conductivity, chemical inertness and biocompatibility, which can simultaneously improve the actuation and biosensing performance. Furthermore, under the action of electrochemical CV cycles, the actuator of AuNPs coupled to the MUC1 aptamer on the surface of the electrode layer can actively capture the tumor marker MUC1 far away from the electrode area, realizing ultra-sensitive biological detection.

[0007] To achieve the above object, the technical solution adopted by the present invention is: An electrochemically actuated biosensor for actively capturing tumor markers, characterized in that it includes an actuating device and a MUC1 aptamer, wherein the actuating device includes an electrochemical driver, and the electrochemical driver is a three-layer structure, which is respectively composed of two identical electrode layers and an intermediate inert layer, wherein the two electrode layers are respectively coated on the upper and lower surfaces of the intermediate inert layer, wherein the electrode layer is a MXene-Au electrode layer composed of MXene-Au; and the intermediate inert layer is an intermediate inert film composed of a polyacetamide film, and an electrode area is formed between the two electrode layers. The MUC1 aptamer is dripped onto the surface of the electrode layer, and after drying, the surface of the electrode layer is washed with PBS buffer to remove the unabsorbed MUC1 aptamer. Under the cyclic action of electrochemical cyclic voltammetry, AuNPs on the surface of the electrode layer of the electrochemical actuator are coupled to the MUC1 aptamer, so that the electrochemical actuator can actively capture the tumor marker MUC1 far away from the electrode area.

[0008] As a preferred technical solution of the present invention: the sequence of the MUC1 aptamer is: 5'-SH-C6-GCAGTTGATCCTTTGGATACCCTGG-3'.

[0009] 3. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 1, characterized in that the preparation process of the electrochemical actuator is as follows: S1. Preparation of MXene solution: The LiF powder and HCl solution are completely dissolved to prepare an etching solution, and then Ti3AlC2 powder is added and stirred with a magnetic stirrer for 12-48 h. The solution is repeatedly separated and washed by a centrifuge until it is neutral, and then ultrasonicated and centrifuged to extract the supernatant to obtain a uniformly layered MXene solution.

[0010] S2. Preparation of MXene-Au solution: 1-3 ml of MXene solution and 0.2-0.3 ml of HAuCl4⋅3H2O solution were mixed and stirred with a magnetic stirrer for 20-60 min to prepare MXene-Au solution.

[0011] S3. Preparation of electrochemical actuator: After uniform stirring, 2-3 ml of MXene-Au solution was poured onto a 90-120μm thick PI film in a mold with outer dimensions of 40-80 mm, 40-80 mm, 4-8 mm, and inner dimensions of 30-50 mm, 30-50 mm, 4-8 mm, respectively. The film was dried at a constant temperature of 40-80 ℃ in a drying oven and cut into electrochemical actuators with a length of 20-30 mm, a width of 3-5 mm, and a thickness of approximately 100-150μm.

[0012] As a preferred technical solution of the present invention: in S1, the mass of LiF powder in the preparation of the MXene solution is 3-6 g.

[0013] As a preferred technical solution of the present invention: in S1, the HCl solution in the preparation of the MXene solution is 50-80 ml 9M HCl solution.

[0014] As a preferred technical solution of the present invention: in S1, the Ti3C2Tx powder in the preparation of the MXene solution is 2-4 g.

[0015] As a preferred technical solution of the present invention: in S1, the concentration of the obtained MXene solution is 10-20 mg / ml.

[0016] As a preferred technical solution of the present invention: in S2, the concentration of the HAuCl4⋅3H2O solution is 10-30 mg / ml.

[0017] The detection method of the electrochemically actuated biosensor for actively capturing tumor markers is characterized by comprising the following steps: 10-20 μL of 1 μM MUC1 aptamer is dripped onto the surface of the electrode layer, dried at room temperature, and the surface of the electrode layer is dripped with a PBS buffer solution with a pH of 7.2-7.4 to remove the unabsorbed MUC1 aptamer, and dried at room temperature; An electrochemical workstation three-electrode system was used, with a platinum wire electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and an electrochemical driver as the working electrode. The electrochemical cyclic voltammetry method was performed at a scanning speed of 50-200 mV / s from -2 V to 2 V for 5-20 cycles. The electrochemical driver reciprocated in an electrolyte solution containing different concentrations of the tumor marker MUC1 and a pH=7.2-7.4 PBS solution to fully capture the surrounding target molecule MUC1. After the cycle, the electrode surface was rinsed with PBS buffer at pH 7.2-7.4 to remove unadsorbed MUC1, and then 2.5-5 mM [Fe(CN) 6 ] 3- / 4-MUC1 was detected by differential pulse voltammetry in PBS solution with pH=7.2-7.4 and 0.1-1M KCl. In the three-electrode system of the electrochemical workstation, the electrochemical actuator produces a cyclical bending motion due to the reversible insertion and removal of cations in the electrode in the electrolyte; while AuNPs are coupled with the MUC1 aptamer on the surface of the electrode layer, and the bent electrode in the actuated state can actively capture the tumor marker MUC1 away from the electrode area, thereby realizing biological detection.

[0018] As a preferred technical solution of the present invention: the setting parameters of the differential pulse voltammetry are as follows: initial potential: -1V; end potential: 1 V; potential increment: 0.004 V; amplitude: 0.05 V; pulse width: 0.06 s; pulse period: 0.5 s.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. On the one hand, AuNPs reduced in situ on MXene can enhance the electrode conductivity and charge storage capacity to improve the electrochemical performance and driving performance. On the other hand, the rich chemical modification of AuNPs can adapt to various biorecognition elements; 2. AuNPs have excellent conductivity, chemical inertness and biocompatibility, and can improve both actuation and biosensing performance, having a dual-promoting effect of actuation and biosensing; 3. The symmetrical structure of the electrochemical actuator composed of MXene-Au electrodes has good actuation stability; 4. During the detection process, the flexible electrochemical actuator can actively capture the target molecule MUC1 far away from the electrode area, improve the binding efficiency between the flexible electrode and MUC1, and thus achieve ultra-sensitive biological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the structure of the electrochemical driver in the present invention.

[0021] Figure 2 This is a schematic diagram of the process of active capture of MUC1 by the electrochemical driver in the present invention.

[0022] List of reference numerals: 1. MXene-Au; 2. PI film; 3. Three-electrode system of electrochemical workstation; 4. Working electrode; 5. Reference electrode; 6. Counter electrode; 7. AuNPs; 8. MXene; 9. MUC1 aptamer; 10. MUC1; 11. Cation. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0025] like Figure 1 As shown, the present invention proposes an electrochemically actuated biosensor for actively capturing tumor markers, including an actuating device and a MUC1 aptamer 9, wherein the actuating device includes an electrochemical driver, and the electrochemical driver is a three-layer structure, which is respectively composed of two identical electrode layers and an intermediate inert layer, and the two electrode layers are respectively coated on the upper and lower surfaces of the intermediate inert layer, and the electrode layer is a MXene-Au electrode layer composed of MXene-Au1; the intermediate inert layer is an intermediate inert film composed of a polyacetamide film, and an electrode area is formed between the two electrode layers. The MUC1 aptamer 9 is dripped onto the surface of the electrode layer, and after drying, the surface of the electrode layer is washed with PBS buffer to remove the unabsorbed MUC1 aptamer 9. Under the cyclic action of electrochemical cyclic voltammetry, AuNPs7 on the surface of the electrode layer of the electrochemical actuator couples with the MUC1 aptamer 9, so that the electrochemical actuator can actively capture the tumor marker MUC110 far away from the electrode area.

[0026] The preparation process of the electrochemical actuator is as follows: S1. Preparation of MXene solution: The LiF powder and HCl solution are completely dissolved to prepare an etching solution, and then Ti3AlC2 powder is added and stirred with a magnetic stirrer for 12-48 h. The solution is repeatedly separated and washed by a centrifuge until it is neutral, and then ultrasonicated and centrifuged to extract the supernatant to obtain a uniformly layered MXene solution.

[0027] S2. Preparation of MXene-Au solution: 1-3 ml of MXene solution and 0.2-0.3 ml of HAuCl4⋅3H2O solution were mixed and stirred with a magnetic stirrer for 20-60 min to prepare MXene-Au solution.

[0028] S3. Preparation of electrochemical actuator: After uniform stirring, 2-3 ml of MXene-Au solution was poured onto a 90-120μm thick PI film 2 in a mold with outer dimensions of 40-80 mm, 40-80 mm, 4-8 mm, and inner dimensions of 30-50 mm, 30-50 mm, 4-8 mm, respectively. The film was dried at a constant temperature of 40-80 °C in a drying oven and cut into electrochemical actuators with a length of 20-30 mm, a width of 3-5 mm, and a thickness of approximately 100-150μm. Example

[0029] 4.8 g LiF powder and 60 ml 9M HCl solution were completely dissolved to prepare etching solution, then 3 g Ti3C2Tx powder was added, stirred with a magnetic stirrer for 24 h, separated and washed repeatedly by a centrifuge until neutral, then subjected to ultrasonication and centrifugation, and the supernatant was extracted to obtain a uniformly layered 15 mg / ml MXene 8 (Ti3C2Tx) solution. Then 2 ml MXene8 (15 mg / ml) and 0.25 ml HAuCl4⋅3H2O (20 mg / ml) solution were mixed and stirred with a magnetic stirrer for 30 min to prepare a MXene-Au1 solution.

[0030] like Figure 1 As shown, 2.25 ml of MXene-Au 1 solution was poured onto a 100 μm thick PI 2 film in a mold after uniform stirring. The mold was a square hollow structure with an outer layer having dimensions of 60 mm, 60 mm, and 5 mm in length, width, and height, respectively, and an inner layer having dimensions of 40 mm, 40 mm, and 5 mm in length, width, and height, respectively. The mold was dried at 60 °C in a drying oven and then cut into an electrochemical actuator having a length of 30 mm, a width of 4 mm, and a thickness of approximately 120 μm.

[0031] like Figure 2 Schematic diagram of the process of active capture of MUC1 by electrochemical actuator. First, 20 μL of 1 μM MUC1 aptamer 10 was dripped on the surface of the electrode layer to couple with AuNPs 7 on the surface of MXene 8, and then dried at room temperature. Then, the electrode surface was washed with PBS buffer at pH = 7.4 to remove the unadsorbed MUC1 aptamer 9, and then dried at room temperature. Among them, the sequence of MUC1 aptamer 9 is: 5'-SH-C6-GCAGTTGATCCTTTGGATACCCTGG-3', and MUC1 10 is MUC1 peptide (APDTRPAPG).

[0032] Secondly, an electrochemical workstation three-electrode system 3 was used, the reference electrode 5 was an Ag / AgCl electrode, the counter electrode 6 was a platinum wire electrode, the working electrode 4 was an electrochemical driver based on a MXene-Au electrode layer, and the electrolyte solution was a PBS solution with a pH of 7.4, and the solution contained different concentrations of tumor markers MUC1 10; 10 cycles were performed from -2 V to 2 V at a scanning speed of 100 mV / s in cyclic voltammetry (CV), and the MXene-Au 1 electrochemical driver reciprocated in the electrolyte solution to fully capture the surrounding target molecules MUC1 10.

[0033] After the last cycle, the electrode surface was rinsed with PBS buffer at pH 7.4 to remove unadsorbed MUC1 10, and then the electrode surface was incubated in 5 mM [Fe(CN) 6 ] 3- / 4- Differential pulse voltammetry (DPV) was used to detect MUC110 in 0.1 M KCl, 0.1 M KCl, and PBS solution with pH = 7.4. The setting parameters of differential pulse voltammetry were: initial potential: -1 V; end potential: 1 V; potential increment: 0.004 V; amplitude: 0.05 V; pulse width: 0.06 s; pulse period: 0.5 s.

[0034] The electrochemical actuation-biosensing detection principle is that in the three-electrode system 3 of the electrochemical workstation, the electrochemical actuator produces a cyclical bending motion due to the reversible insertion and removal of cations 11 in the electrode in the electrolyte; and AuNPs 7 are coupled to the MUC1 aptamer 9 on the surface of the electrode layer. The curved electrode in the actuated state can actively capture the tumor marker MUC1 10 away from the electrode area, thereby realizing ultra-sensitive biological detection. Example

[0035] 3 g LiF powder and 60 ml 9M HCl solution were completely dissolved to prepare etching solution, then 2 g Ti3C2Tx powder was added, stirred with a magnetic stirrer for 36 h, separated and washed repeatedly by a centrifuge until neutral, then subjected to ultrasound and centrifugation, and the supernatant was extracted to obtain a uniformly layered 10 mg / ml MXene 8 (Ti3C2Tx) solution. Then 1 ml MXene8 (10 mg / ml) and 0.2 ml HAuCl4⋅3H2O (10 mg / ml) solution were mixed and stirred with a magnetic stirrer for 20 min to prepare a MXene-Au1 solution.

[0036] like Figure 1As shown, 2 ml of MXene-Au 1 solution was poured onto a 90 μm thick PI 2 film in a mold after uniform stirring. The mold was a square hollow structure with outer dimensions of 40 mm, 40 mm, and 4 mm in length, width, and height, respectively, and inner dimensions of 30 mm, 30 mm, and 4 mm in length, width, and height, respectively. The mold was dried at 45 °C in a drying oven and then cut into an electrochemical actuator with a length of 20 mm, a width of 3 mm, and a thickness of about 120 μm. .

[0037] like Figure 2 The schematic diagram of the electrochemical actuation-biosensing process of active capture of MUC1 is shown. First, 10 μL of 1 μM MUC1 aptamer 10 is dripped on the surface of the electrode layer to couple with AuNPs 7 on the surface of MXene 8, and then dried at room temperature. Then, the electrode surface is dripped with PBS buffer at pH = 7.2 to remove the unadsorbed MUC1 aptamer 9, and then dried at room temperature. Among them, the sequence of MUC1 aptamer 9 is: 5'-SH-C6-GCAGTTGATCCTTTGGATACCCTGG-3', and MUC1 10 is MUC1peptide (APDTRPAPG).

[0038] Secondly, an electrochemical workstation three-electrode system 3 was used, the reference electrode 5 was an Ag / AgCl electrode, the counter electrode 6 was a platinum wire electrode, the working electrode 4 was a MXene-Au electrochemical driver, and the electrolyte solution was a PBS solution with a pH of 7.2, and the solution contained different concentrations of tumor markers MUC1 10; 5 cycles were performed from -2 V to 2 V at a scanning speed of 50 mV / s in cyclic voltammetry (CV), and the electrochemical driver based on the MXene-Au electrode layer reciprocated in the electrolyte solution to fully capture the surrounding target molecules MUC1 10.

[0039] After the last cycle, the electrode surface was rinsed with PBS buffer at pH 7.2 to remove the unadsorbed MUC1 10, and then 2.5 mM [Fe(CN) 6 ] 3- / 4- Differential pulse voltammetry (DPV) was used to detect MUC110 in 0.2 M KCl, 0.2 M KCl, and PBS solution with pH = 7.2. The setting parameters of differential pulse voltammetry were: initial potential: -1 V; end potential: 1 V; potential increment: 0.004 V; amplitude: 0.05 V; pulse width: 0.06 s; and pulse period: 0.5 s.

[0040] The electrochemical actuation-biosensing detection principle is that in the three-electrode system 3 of the electrochemical workstation, the electrochemical actuator produces a cyclical bending motion due to the reversible insertion and removal of cations 11 in the electrode in the electrolyte; and AuNPs 7 are coupled to the MUC1 aptamer 9 on the surface of the electrode layer. The curved electrode in the actuated state can actively capture the tumor marker MUC1 10 away from the electrode area, thereby realizing ultra-sensitive biological detection. Example

[0041] 6 g LiF powder and 60 ml 9M HCl solution were completely dissolved to prepare etching solution, then 4 g Ti3C2Tx powder was added, stirred with a magnetic stirrer for 48 h, separated and washed repeatedly by a centrifuge until neutral, then subjected to ultrasound and centrifugation, and the supernatant was extracted to obtain a uniformly layered 20 mg / ml MXene 8 (Ti3C2Tx) solution. Then 3 ml MXene8 (20 mg / ml) and 0.3 ml HAuCl4⋅3H2O (30 mg / ml) solution were mixed and stirred with a magnetic stirrer for 60 min to prepare a MXene-Au1 solution.

[0042] like Figure 1 As shown, 3 ml of MXene-Au 1 solution was poured onto a 120 μm thick PI 2 film in a mold after uniform stirring. The mold was a square hollow structure with an outer layer with dimensions of 80 mm, 80 mm, and 5 mm in length, width, and height, respectively, and an inner layer with dimensions of 50 mm, 50 mm, and 5 mm in length, width, and height, respectively. The film was dried at 60 °C in a drying oven and then cut to produce an electrochemical actuator based on a MXene-Au 1 electrode layer with a length of 25 mm, a width of 3.5 mm, and a thickness of approximately 140 μm.

[0043] like Figure 2 Shown is a schematic diagram of the process of active capture of MUC1 by electrochemical actuator.

[0044] A detection method for an electrochemically actuated biosensor that actively captures tumor markers comprises the following steps: 10-20 μL of 1 μM MUC1 aptamer 9 is dripped onto the surface of the electrode layer, dried at room temperature, and the surface of the electrode layer is dripped with a PBS buffer solution with a pH of 7.2-7.4 to remove the unabsorbed MUC1 aptamer 9, and dried at room temperature; An electrochemical workstation three-electrode system was used, with a platinum wire electrode as the counter electrode 6, an Ag / AgCl electrode as the reference electrode 5, and an electrochemical driver as the working electrode. During electrochemical cyclic voltammetry (CV), 5-20 cycles were performed from -2 V to 2 V at a scanning speed of 50-200 mV / s. The electrochemical driver reciprocated in an electrolyte solution containing different concentrations of the tumor marker MUC110 and a pH=7.2-7.4 PBS solution to fully capture the surrounding target molecule MUC110. After the cycle, the electrode surface was rinsed with PBS buffer at pH 7.2-7.4 to remove the unadsorbed MUC110, and then 2.5-5 mM [Fe(CN) 6 ] 3- / 4- , 0.1-1M KCl pH = 7.2-7.4 PBS solution using differential pulse voltammetry for MUC110 detection, In the three-electrode system of the electrochemical workstation, the electrochemical actuator produces a cyclical bending motion due to the reversible insertion and removal of cation 11 in the electrode in the electrolyte; while AuNPs7 is coupled with MUC1 aptamer 9 on the surface of the electrode layer, and the bent electrode in the actuated state can actively capture the tumor marker MUC110 away from the electrode area, thereby realizing biological detection.

[0045] The setting parameters of the differential pulse voltammetry are as follows: initial potential: -1 V; end potential: 1 V; potential increment: 0.004 V; amplitude: 0.05 V; pulse width: 0.06 s; pulse period: 0.5 s.

[0046] First, 15 μL of 1 μM MUC1 aptamer 10 was dripped onto the electrode surface to couple with AuNPs 7 on the MXene 8 surface, and then dried at room temperature. Then, the electrode surface was washed with PBS buffer at pH 7.3 to remove the unadsorbed MUC1 aptamer 9, and then dried at room temperature. The sequence of MUC1 aptamer 9 is: 5'-SH-C6-GCAGTTGATCCTTTGGATACCCTGG-3', and MUC1 10 is MUC1 peptide (APDTRPAPG).

[0047] Secondly, an electrochemical workstation three-electrode system 3 was used, the reference electrode 5 was an Ag / AgCl electrode, the counter electrode 6 was a platinum wire electrode, the working electrode 4 was an electrochemical driver, and the electrolyte solution was a PBS solution with a pH of 7.3, and the solution contained different concentrations of tumor markers MUC1 10; 20 cycles were performed from -2 V to 2 V at a scanning speed of 200 mV / s in cyclic voltammetry (CV), and the electrochemical driver reciprocated in the electrolyte solution to fully capture the surrounding target molecules MUC1 10.

[0048] After the last cycle, the electrode surface was rinsed with PBS buffer at pH 7.3 to remove unadsorbed MUC1 10, and then 3 mM [Fe(CN) 6 ] 3- / 4- Differential pulse voltammetry (DPV) was used to detect MUC110 in 0.5 M KCl, 0.5 M KCl, and PBS solution with pH = 7.3. The setting parameters of differential pulse voltammetry were: initial potential: -1 V; end potential: 1 V; potential increment: 0.004 V; amplitude: 0.05 V; pulse width: 0.06 s; and pulse period: 0.5 s.

[0049] The principle of electrochemically actuated biosensor detection is that in the three-electrode system 3 of the electrochemical workstation, the electrochemical driver produces a cyclical bending motion due to the reversible insertion and separation of cations 11 in the electrode in the electrolyte; and AuNPs 7 are coupled to the MUC1 aptamer 9 on the surface of the electrode layer. The curved electrode in the actuated state can actively capture the tumor marker MUC1 10 away from the electrode area, thereby realizing ultra-sensitive biological detection.

[0050] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. Therefore, the present invention defined by the attached claims is not limited to the specific details described in the above description, and many obvious changes that do not depart from the purpose or scope of the present invention may also achieve the purpose of the present invention.

Claims

1. An electrochemically actuated biosensor for actively capturing tumor markers, characterized in that: The invention comprises an actuator and a MUC1 adapter (9), wherein the actuator comprises an electrochemical driver, wherein the electrochemical driver has a three-layer structure, and is respectively composed of two identical electrode layers and an intermediate inert layer, wherein the two electrode layers are coated on the upper and lower surfaces of the intermediate inert layer, respectively, wherein the electrode layers are MXene-Au electrode layers composed of MXene-Au (1); and the intermediate inert layer is an intermediate inert film composed of a polyacetamide film, and an electrode region is formed between the two electrode layers. The MUC1 aptamer (9) is dripped onto the surface of the electrode layer, and after drying, the surface of the electrode layer is rinsed with PBS buffer to remove the unabsorbed MUC1 aptamer (9). Under the cyclic action of electrochemical cyclic voltammetry, the AuNPs (7) on the surface of the electrode layer of the electrochemical actuator are coupled to the MUC1 aptamer (9), so that the electrochemical actuator can actively capture the tumor marker MUC1 (10) far away from the electrode area.

2. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 1, characterized in that: The sequence of the MUC1 aptamer (9) is: 5'-SH-C6-GCAGTTGATCCTTTGGATACCCTGG-3'.

3. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 1, characterized in that: The preparation process of the electrochemical actuator is as follows: S1. Preparation of MXene (8) solution: The LiF powder and HCl solution are completely dissolved to prepare an etching solution, and then Ti3AlC2 powder is added and stirred with a magnetic stirrer for 12-48 h. The solution is repeatedly separated and washed by a centrifuge until it is neutral, and then subjected to ultrasonication and centrifugation. The supernatant is extracted to obtain a uniformly layered MXene (8) solution. S2. Preparation of MXene-Au (1) solution: 1-3 ml of MXene (8) solution and 0.2-0.3 ml of HAuCl4⋅3H2O solution were mixed and stirred with a magnetic stirrer for 20-60 min to prepare a MXene-Au (1) solution; S3. Preparation of electrochemical actuator: After uniform stirring, 2-3 ml of MXene-Au (1) solution was poured onto a 90-120 μm thick PI film (2) in a mold with an outer layer having dimensions of 40-80 mm, 40-80 mm, and 4-8 mm, and an inner layer having dimensions of 30-50 mm, 30-50 mm, and 4-8 mm, respectively. The film was dried at a constant temperature of 40-80 °C in a drying oven and cut into electrochemical actuators with a length of 20-30 mm, a width of 3-5 mm, and a thickness of approximately 100-150 μm.

4. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 3, characterized in that: In S1, the mass of LiF powder in the preparation of the MXene (8) solution is 3-6 g.

5. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 3, characterized in that: In S1, the HCl solution in the preparation of the MXene (8) solution is 50-80 ml of 9M HCl solution.

6. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 3, characterized in that: In S1, the Ti3C2Tx powder in the preparation of the MXene (8) solution is 2-4 g.

7. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 3, characterized in that: In S1, the obtained MXene (8) solution concentration was 10-20 mg / ml.

8. The electrochemically actuated biosensor for actively capturing tumor markers according to claim 3, characterized in that: In S2, the concentration of the HAuCl4⋅3H2O solution is 10-30 mg / ml.

9. The detection method of the electrochemically actuated biosensor for actively capturing tumor markers according to any one of claims 1 to 8, characterized in that: The steps include: 10-20 μL of 1 μM MUC1 aptamer (9) is dripped onto the surface of the electrode layer, dried at room temperature, and the surface of the electrode layer is washed with a PBS buffer solution having a pH of 7.2-7.4 to remove the unadsorbed MUC1 aptamer (9), and dried at room temperature; An electrochemical workstation three-electrode system was used, with a platinum wire electrode as the counter electrode (6), an Ag / AgCl electrode as the reference electrode (5), and an electrochemical driver (4). During electrochemical cyclic voltammetry, 5 to 20 cycles were performed from -2 V to 2 V at a scanning speed of 50 to 200 mV / s. The electrochemical driver reciprocated in an electrolyte solution containing different concentrations of the tumor marker MUC1 (10) and a pH=7.2-7.4 PBS solution, fully capturing the surrounding target molecule MUC1 (10); After the cycle, the electrode surface was rinsed with PBS buffer at pH 7.2-7.4 to remove the unadsorbed MUC1 (10), and then 2.5-5 mM [Fe(CN) 6 ] 3- / 4- MUC1 (10) was detected by differential pulse voltammetry in PBS solution with 0.1-1 M KCl and pH = 7.2-7.

4. In the three-electrode system (3) of the electrochemical workstation, the electrochemical actuator produces a cyclic bending motion due to the reversible insertion and removal of cations (11) in the electrode in the electrolyte; and AuNPs (7) are coupled to the MUC1 aptamer (9) on the surface of the electrode layer. The bent electrode in the actuated state can actively capture the tumor marker MUC1 (10) away from the electrode area, thereby realizing biological detection.

10. The detection method of the electrochemically actuated biosensor for actively capturing tumor markers according to claim 9, characterized in that: The setting parameters of the differential pulse voltammetry are as follows: initial potential: -1 V; end potential: 1 V; potential increment: 0.004 V; Amplitude: 0.05 V; Pulse width: 0.06 s; Pulse period: 0.5 s.

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