Electrochemical Sensor for Digoxin Antibody Based on Atom Transfer Radical Polymerization

Abstract

The invention discloses a construction method, a construction structure and an application of a digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction, including designing a cascaded strand displacement cyclic amplification strategy, regulating atom transfer radical polymerization reaction, constructing an electrochemical sensing interface for detecting digoxin antibody, and detecting the antibody in serum samples. In the electrochemical sensor for detecting antibody of the invention, a DNA probe modified with a small molecule antigen determinant is used for specific recognition, a cascaded strand displacement cyclic amplification reaction is designed, and the growth of electrochemical signal is regulated by means of atom transfer radical polymerization reaction, so as to realize the sensitive detection of digoxin antibody in serum samples, and the detection limit is as low as 1.5 pM. This research has important application value in the prevention, diagnosis and treatment of human immune diseases.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical biosensors, and particularly to a construction method, a construction structure and an application of a digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction. Background Art

[0002] Antibodies are glycoproteins produced by the body's immune system, which can fight against pathogen and foreign body invasions and are closely related to the pathogenesis of autoimmune diseases. The change in antibody content can be used as an indicator for diagnosing and treating diseases such as systemic lupus erythematosus and rheumatoid arthritis. Currently, the techniques for detecting antibodies usually involve the binding of antibody and antigen, such as Western blotting, radioimmunoassay and enzyme-linked immunosorbent assay. Although these methods can achieve the detection of antibodies, they cannot overcome the problems such as the need to introduce a variety of exogenous reagents for natural antigens and easy denaturation, and the experimental operations are relatively complex and time-consuming, which limits the practical applicability of these methods. Therefore, constructing simple and sensitive sensors for detecting antibodies is an urgent need for clinical diagnosis and medical research.

[0003] Studies have shown that using DNA-modified small antigenic determinants to recognize and detect antibodies can improve the limitations of traditional methods. These methods usually use signal probes modified with electroactive substances on the electrode. In the absence of the target antibody, the signal probe collides with the electrode surface, generating a strong current signal. When the antigenic determinant on the signal probe binds to the target antibody, the steric hindrance will reduce the collision efficiency of the probe and inhibit the current response signal. Although these methods have made certain progress, they still have the problem of high background signals, which limits the improvement of sensitivity. In addition, using chemical modification operations to pre-label electrochemical signals increases the experimental cost and the complexity of the operation. To address these problems, the present invention uses DNA probes modified with small antigenic determinants for specific recognition of target antibodies, designs cascade strand displacement cycles for amplification, and uses atom transfer radical polymerization reaction to regulate the growth of electrochemical signals to achieve efficient and sensitive detection of digoxin antibodies in serum samples. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a construction method, a construction structure and an application of a digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction, which uses DNA probes modified with small antigenic determinants for specific recognition, introduces cascade strand displacement cycles for amplification, and uses atom transfer radical polymerization reaction to regulate the growth of electrochemical signals to achieve sensitive detection of digoxin antibodies in serum samples.

[0005] To solve the above technical problem, the technical solution of the present invention is: a digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction, and its innovation lies in: the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction includes the following steps:

[0006] Step S1: Modify the fixed probe IP onto the surface of the gold electrode AuE, and block it with mercaptohexanol MCH to obtain MCH / IP / AuE;

[0007] Among them, the sequence of the IP is 5’-SH-(CH2)6-TTTTTTTTTTTT-N3-3’;

[0008] Step S2: Hybridize the substrate probe SP with the auxiliary probe AMP and the blocking probe BP to obtain the double-stranded SP / AMP-BP, and further modify this double-stranded onto the surface of the magnetic bead MB to obtain SP / AMP-BP / MB;

[0009] Among them, the sequence of the SP is 5’-AGAATGTAGAGTTACGAGCAAATAAGGGGTAATCTAGTGAGCATTTTTTT-biotin-3’; the sequence of the AMP is 5’-GACTGACTAGCATGCTCACTAGATTAC-3’; the sequence of the BP is 5’-CCCTTATTTGCTCGTAA CT-3’;

[0010] Step S3: Prepare copper oxide (CuO) nanoparticles by solvothermal method, and modify the displacement probe DP onto the surface of CuO to obtain the DP-CuO complex;

[0011] Among them, the sequence of the DP is 5’-TCTCTCTCTCTCTCTCTGCTCACTAGATTACCCCTTATTTGCTCGTAACT-3’;

[0012] Step S4: Mix different concentrations of digoxin antibodies with the recognition probes RP1, RP2, SP / AMP-BP / MB, and DP-CuO for reaction, then add hydrobromic acid HBr to dissolve CuO, transfer it to the surface of MCH / IP / AuE, and carry out click chemical reaction with 2-bromoisobutyric acid 3-butynyl ester BBriB and ascorbic acid AA to connect BBriB to the electrode surface;

[0013] Among them, the sequence of the RP1 is 5’-Dig-TTTTTTATCTAGTGAGCATTTCTACATT CT-3’; the sequence of the RP2 is 5’-TATTTGCTCGTAACTTTTTGCTAGTCAGTCT TTTTT-Dig-3’;

[0014] Step S5: Place the electrode after the reaction in Step S4 in the substrate solution for polymerization reaction, and carry out atom transfer radical polymerization reaction at a constant voltage;

[0015] Preferably, the operation of preparing the sensing interface in step S1 is as follows: First, react the immobilized probe IP with tris(2-carboxyethyl)phosphine for 1 hour, drop it onto the electrode surface, and react in the dark for 10 hours. After rinsing, drop mercaptohexanol MCH onto the electrode and seal for 2 hours to obtain MCH / IP / AuE.

[0016] Preferably, the experimental operation in step S2 is as follows: Mix the substrate probe SP with the auxiliary probe AMP and the blocking probe BP, heat at 95°C for 10 minutes, then cool to 25°C at a rate of 1°C per minute to obtain the double-stranded SP / AMP-BP. Subsequently, mix it with streptavidin-modified magnetic beads MB and stir at room temperature for 120 minutes. After centrifugation, seal it in bovine serum albumin to obtain SP / AMP-BP / MB.

[0017] Preferably, the experimental operation in step S3 is as follows: Dissolve the divalent copper salt (such as copper acetate) with an organic solvent (such as ethanol), transfer it to a polytetrafluoroethylene reaction kettle, react at a high temperature of 180°C for 1 hour, then perform centrifugation and drying. Subsequently, mix it with the DP chain at room temperature and react for 2 hours to obtain DP-CuO.

[0018] Preferably, the experimental operation in step S4 is as follows: Mix the equimolar ratio of the recognition probes RP1, RP2, SP / AMP-BP / MB, DP-CuO with digoxin antibodies at different concentrations, react at room temperature for 100 minutes, then add hydrogen bromide HBr to dissolve CuO. Subsequently, transfer it to the surface of MCH / IP / AuE, and add 2-bromo-2-methylpropionic acid 3-butynyl ester BBriB and ascorbic acid to react for 50 minutes to connect BBriB to the electrode surface through click chemistry.

[0019] Preferably, the operation in step S5 is as follows: Place the electrode after the reaction in step S4 in a dimethylformamide solution containing copper / tris(2-dimethylaminoethyl)amine Cu II / Me6TREN, ferrocenylmethyl methacrylate FcMMA, potassium bromide KBr, potassium hexafluorophosphate KPF6, and apply a voltage of -0.56V through an electrochemical workstation to react for 60 minutes to perform atom transfer radical polymerization and generate polymerized FcMMA on the electrode surface.

[0020] The construction structure of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction, applying the construction method of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction, the construction structure of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction is an electrochemical sensor obtained by using the construction method of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction.

[0021] Application of digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction, and the application of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction is the application of the electrochemical sensor in detecting the content of digoxin antibody in serum samples.

[0022] The advantages of the present invention are as follows: Compared with the prior art, the present invention uses a DNA probe modified with a small molecule antigen determinant for specific recognition of antibodies, designs a cascade strand displacement cycle amplification strategy, and regulates the growth of electrochemical signals by means of atom transfer radical polymerization reaction, providing a construction method, construction structure and application of an electrochemical sensor for efficient detection of antibodies, which has important application value in the prevention, diagnosis and treatment of human immune diseases. Brief Description of the Drawings

[0023] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0024] Figure 1 is a schematic diagram of the principle of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction of the present invention;

[0025] Figure 2 is a feasibility characterization diagram of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction of the present invention;

[0026] Figure 3 is a characterization diagram of polymerized FcMMA in the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction of the present invention: A. Cyclic voltammetry characterization; B. Linear relationship diagram of current and scan rate; C. Scanning electron microscopy image; D-F. Elemental characterization diagrams (scale bar is 500 nm);

[0027] Figure 4 is a quantitative detection diagram of digoxin antibody in the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction of the present invention: A. Current response caused by digoxin antibodies at different concentrations; B. Linear relationship diagram of current and logarithm of concentration;

[0028] Figure 5 is a detection diagram of serum samples in the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction of the present invention. Specific Embodiments

[0029] The digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction of the present invention includes the following steps:

[0030] Step S1: Modify the fixed probe IP onto the surface of the gold electrode AuE, and block it with mercaptohexanol MCH to obtain MCH / IP / AuE; wherein, the sequence of the IP is 5’-SH-(CH2)6-TTTTTTTTTTTT-N3-3’;

[0031] Step S2: Hybridize the substrate probe SP with the auxiliary probe AMP and the blocking probe BP to obtain the double-stranded SP / AMP-BP, and further modify the double-stranded onto the surface of the magnetic bead MB to obtain SP / AMP-BP / MB; wherein, the sequence of the SP is 5’-AGAATGTAGAGTTACGAGCAAATAAGGGGTAATCTAGTGAGCATTTTTTT-biotin-3’; the sequence of the AMP is 5’-GACTGACTAGCATGCTCACTAGATTAC-3’; the sequence of the BP is 5’-CCCTTATTTGCTCGTAACT-3’;

[0032] Step S3: Prepare copper oxide (CuO) nanoparticles by solvothermal method, and modify the displacement probe DP onto the surface of CuO to obtain the DP-CuO complex; wherein, the sequence of the DP is 5’-TCTCTCTCTCTCTCTGCTCACTAGATTACCCCTTATTTGCTCGTAACT-3’;

[0033] Step S4: Mix different concentrations of digoxin antibody with the recognition probes RP1, RP2, SP / AMP-BP / MB, and DP-CuO for reaction, then add hydrogen bromide HBr to dissolve CuO, transfer it to the surface of MCH / IP / AuE, and perform click chemical reaction with 2-bromo-2-methylpropionic acid 3-butynyl ester BBriB and ascorbic acid AA to connect BBriB to the electrode surface; wherein, the sequence of the RP1 is 5’-Dig-TTTTTTATCTAGTGAGCATTTCTACATTCT-3’; the sequence of the RP2 is 5’-TATTTGCTCGTAACTTTTTGCTAGTCAGTCTTTTT-Dig-3’;

[0034] Step S5: Place the electrode after the reaction in Step S4 in the substrate solution for polymerization reaction, and perform atom transfer radical polymerization reaction at a constant voltage.

[0035] The present invention uses a DNA probe modified with a small molecule antigen determinant for specific recognition of antibodies, designs a cascaded strand displacement cyclic amplification strategy, and regulates the growth of electrochemical signals by means of atom transfer radical polymerization reaction, providing a construction method, a construction structure and an application of an electrochemical sensor for efficient detection of antibodies, which has important application value in the prevention, diagnosis and treatment of human immune diseases.

[0036] The operation of preparing the sensing interface in the above step S1 is as follows: First, react the immobilized probe IP with tris(2-carboxyethyl)phosphine for 1 hour, drop it onto the electrode surface and react in the dark for 10 hours. After rinsing, drop mercaptohexanol MCH onto the electrode and block it for 2 hours to obtain MCH / IP / AuE.

[0037] The experimental operation of the above step S2 is as follows: Mix the base probe SP, the auxiliary probe AMP and the blocking probe BP, heat them at 95°C for 10 minutes, then cool them to 25°C at a rate of 1°C / minute to obtain the double strand SP / AMP-BP. Subsequently, mix it with streptavidin-modified magnetic beads MB and stir at room temperature for 120 minutes. After centrifugation, block it in bovine serum albumin to obtain SP / AMP-BP / MB.

[0038] The experimental operation of the above step S3 is as follows: Dissolve a divalent copper salt (such as copper acetate) with an organic solvent (such as ethanol), transfer it to a polytetrafluoroethylene reaction kettle and react at a high temperature of 180°C for 1 hour. Subsequently, perform centrifugation and drying, and then mix and react with the DP chain at room temperature for 2 hours to obtain DP-CuO.

[0039] The experimental operation of the above step S4 is as follows: Mix the recognition probes RP1, RP2, SP / AMP-BP / MB, DP-CuO in equimolar ratio with digoxin antibodies at different concentrations, react at room temperature for 100 minutes, then add hydrogen bromide HBr to dissolve CuO, and then transfer it to the surface of MCH / IP / AuE. Then add 2-bromo-2-methylpropionic acid 3-butynyl ester BBriB and ascorbic acid and react for 50 minutes to connect BBriB to the electrode surface through click chemical reaction.

[0040] The operation of the above step S5 is as follows: Place the electrode after the reaction in step S4 in a dimethylformamide solution containing copper / tris(2-dimethylaminoethyl)amine Cu II / Me6TREN, ferrocene methanol methacrylate FcMMA, potassium bromide KBr, potassium hexafluorophosphate KPF6, and apply a voltage of -0.56V through an electrochemical workstation to react for 60 minutes to perform atom transfer radical polymerization reaction and generate polymerized FcMMA on the electrode surface.

[0041] The constructed structure of the electrochemical sensor for detecting digoxin antibody is an electrochemical sensor obtained by using the construction method of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction. The application of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction is the application of the electrochemical sensor in detecting the content of digoxin antibody in serum samples.

[0042] The specific principle is as follows:

[0043] In this invention, the substrate chain IP and mercaptohexanol MCH are fixed on the surface of the gold electrode as the sensing substrate MCH / IP / AuE( Figure 1 ). The 5' and 3' ends of the recognition probes RP1 and RP2 are respectively modified with the antigenic determinant Dig. The digoxin antibody anti-Dig can bind to the recognition probes RP1 and RP2 simultaneously to form a complex AD-2RP, which makes the recognition probes approach each other and then triggers a strand displacement reaction (Cycle I). On the surface of the magnetic bead MB, AD-2RP first binds to the toehold at the end of SP to displace the blocking probe BP, exposing a new toehold in the middle of SP. DP-CuO undergoes a strand displacement reaction by means of the middle toehold to displace AD-2RP and the auxiliary probe AMP, so that AD-2RP can be recycled. In addition, AMP contains the complementary sequences of the recognition probes RP1 and RP2 and can hybridize with each other to form an AMP-2RP complex. AMP-2RP further triggers a strand displacement reaction similar to Cycle I (Cycle II). After multiple cycles, a large amount of SP / DP-CuO / MB complexes can be obtained. Subsequently, magnetic separation is carried out, CuO is dissolved with hydrobromic acid HBr, and it is transferred to the surface of the MCH / IP / AuE electrode. Ascorbic acid AA is added to reduce divalent copper to cuprous ions. The 3' end of the IP chain is modified with an azide group, and the BBriB initiator can be connected to the electrode surface under the catalysis of cuprous ions. Finally, Cu II / Me6TREN is added and a constant voltage is applied to reduce it to Cu I / Me6TREN, and Cu I / Me6TREN reacts with BBriB on the electrode surface to generate free radicals to further polymerize FcMMA on the electrode surface, obtaining a significantly amplified current signal and realizing the sensitive detection of digoxin antibody.

[0044] To prove the feasibility of the sensor for detecting antibodies in this invention, we carried out square wave pulse voltammetry SWV tests. From Figure 2It can be seen that in the absence of the target antibody, the current response of the sensor is very small (curve a). In the presence of digoxin antibody, after the sensing electrode MCH / IP / AuE reacts with SP / AMP-BP / MB, RP1, RP2 and DP-CuO, a significantly increased peak current is obtained at 0.28 V (curve b). In contrast, in the three control experiments including without SP / AMP-BP / MB (curve c), without RP1 and RP2 (curve d), and without DP-CuO (curve e), the current signal does not change significantly. The above experimental results prove that the sensor can be used for the detection of digoxin antibody.

[0045] The cyclic voltammetry technique and scanning electron microscopy were used to characterize the polymerization growth of the electroactive substance FcMMA at the electrode interface. First, the CV responses of the sensing electrode at different scan rates were tested. The results are as Figure 3 shown in A-B. The peak current increases with the increase of the scan rate, and there is a good linear relationship between the peak current value and the scan rate, indicating that FcMMA is connected to the electrode surface. Figure 3 The scanning electron micrograph of C shows a large number of dense nanoparticles. From Figure 3 the elemental distribution characterizations of D-F, it can be seen that P, Br and Fe are uniformly distributed on the electrode, indicating the presence of DNA, BBriB and FcMMA on the electrode respectively, further proving that the electroactive substance FcMMA has successfully polymerized and grown on the electrode surface.

[0046] To investigate the sensitivity of the sensor, we recorded and analyzed the current responses caused by different concentrations of digoxin antibody. As can be seen from Figure 4 A, the SWV current signal increases with the increase of the digoxin antibody concentration. Figure 4 The linear analysis of B found that in the range of 5 pM to 200 nM, there is a good linear relationship between the SWV peak current value and the logarithm of the digoxin antibody concentration, and the linear equation is i = 29.77 + 2.5811lgc (R 2 = 0.9935). Calculated by the 3-fold standard deviation rule (LOD = 3S b / m), the detection limit is 1.5 pM, where S b is the standard deviation of the blank response and m is the slope of the calibration curve. In addition, the relative standard deviation obtained by repeating the detection of 50 nM digoxin antibody four times by the sensor is 3.1%, indicating that the sensor has excellent reproducibility.

[0047] By analyzing the feasibility of the sensor for detecting human serum samples, the application of this method for detecting actual biological samples was investigated. The serum sample was diluted 10 times with a buffer solution in advance, and different concentrations of digoxin antibody (50 pM, 1 nM, 10 nM and 200 nM) were added. The experimental results are as Figure 5As shown, the current response caused by the blank serum sample is very small (column a). The addition of digoxin antibody enables the sensor to obtain an increased current signal (columns b - e), and the current signal increases with the increase in the concentration of digoxin antibody. At the same time, we conducted the analysis of the recovery rate of spiked samples and obtained that the recovery rate was between 93.6% and 107.3%, and the relative standard deviation was between 2.1% and 4.5%, indicating that this method has good applicability in the detection of actual samples.

[0048] It should be noted that in the description of this specification, the terms are only used for descriptive purposes and to distinguish similar objects. There is no sequence between the two, nor can it be understood as indicating or implying relative importance. In addition, in the description of this specification, unless otherwise stated, the meaning of "a plurality of" is two or more.

[0049] Any numerical value cited in this article includes all values from the lower value to the upper value increasing by one unit between the lower limit value and the upper limit value. There should be at least a two - unit interval between any lower value and any higher value. These are just examples to clearly express, and it can be considered that all possible combinations of the numerical values listed between the lowest value and the highest value are clearly described in this specification in a similar manner.

[0050] It should be understood that the above description is for illustrative purposes rather than for limitation. By reading the above description, many embodiments and many applications other than the provided examples will be obvious to those skilled in the art. Therefore, the scope of this teaching should not be determined by reference to the above description, but should be determined by reference to the appended claims and the full scope of the equivalents of these claims. For the sake of comprehensiveness, all articles and references including patent applications and published announcements are incorporated herein by reference. Omitting any aspect of the subject matter disclosed herein in the foregoing claims is not to abandon such subject matter, nor should it be considered that the inventor did not consider such subject matter as part of the disclosed inventive subject matter.

Claims

1. A digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction, characterized in that: The preparation of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization reaction includes the following steps: Step S1: modifying the fixed probe IP onto the surface of the gold electrode AuE, and blocking it with mercaptohexanol MCH to obtain MCH / IP / AuE; Wherein, the sequence of the IP is 5'-SH-(CH2)6-TTTTTTTTTTTT-N3-3'; Step S2: hybridizing the substrate probe SP with the auxiliary probe AMP and the blocking probe BP to obtain a double-stranded SP / AMP-BP, and further modifying the double-stranded SP / AMP-BP / MB on the surface of the magnetic bead MB; Wherein, the sequence of the SP is 5'-AGAATGTAGAGTTACGAGCAAATAAGGGGTAATCTAGTGAGCATTTTTTT-biotin-3'; the sequence of the AMP is 5'-GACTGACTAGCATGCTCACTAGATTAC-3'; the sequence of the BP is 5'-CCCTTATTTGCTCGTAACT-3'; Step S3: preparing CuO nanoparticles by solvothermal method, modifying the displacement probe DP onto the surface of CuO to obtain DP-CuO complex; Wherein, the sequence of the DP is 5'-TCTCTCTCTCTCTCTCTGCTCACTAGATTACCCCTTATTTGCTCGTAACT-3'; Step S4: digoxin antibodies of different concentrations were mixed with recognition probes RP1, RP2, SP / AMP-BP / MB, and DP-CuO for reaction, and then hydrogen bromide HBr was added to dissolve CuO, and then transferred to the MCH / IP / AuE surface, and BBriB was connected to the electrode surface by click chemistry reaction with 2-bromoisobutyric acid-3-butynyl ester BBriB and ascorbic acid AA; The 5' and 3' ends of the recognition probes RP1 and RP2 are modified with antigenic determinant clusters Dig respectively; the digoxin antibody can bind to the recognition probes RP1 and RP2 at the same time; The sequence of RP1 is 5'-Dig-TTTTTTATCTAGTGAGCATTTCTACATTCT-3'; the sequence of RP2 is 5'-TATTTGCTCGTAACTTTTTGCTAGTCAGTCTTTTTT-Dig-3'; Step S5: placing the electrode after the reaction in step S4 in a substrate solution for polymerization reaction, and performing an atom transfer radical polymerization reaction under a constant voltage; The experimental operation of step S5 is to place the electrode after the reaction in step S4 in a solution containing copper / tris(2-dimethylaminoethyl)amine Cu II / Me6TREN, ferrocenylmethanol methacrylate FcMMA, potassium bromide KBr, potassium hexafluorophosphate KPF6 in a dimethylformamide solution, and an electrochemical workstation was used to apply a voltage of -0.56 V for 60 minutes to carry out atom transfer radical polymerization reaction to generate polymerized FcMMA on the electrode surface.

2. The digoxin antibody electrochemical sensor based on atom transfer radical polymerization as claimed in claim 1, characterized in that: The experimental operation of step S1 is to first react the fixed probe IP with tri(2-carboxyethyl)phosphine for 1 hour, then drip it onto the electrode surface to react for 10 hours in the dark, and then drip mercaptohexanol MCH onto the electrode to seal it for 2 hours after rinsing, so as to obtain MCH / IP / AuE.

3. The digoxin antibody electrochemical sensor based on atom transfer radical polymerization as claimed in claim 1, characterized in that: The experimental operation of step S2 is to mix the base probe SP with the auxiliary probe AMP and the blocking probe BP, heat them at 95°C for 10 minutes, then cool them to 25°C at a rate of 1°C / min to obtain double-stranded SP / AMP-BP, then mix them with streptavidin-modified magnetic beads MB and stir them at room temperature for 120 minutes. After centrifugation, place them in bovine serum albumin for blocking to obtain SP / AMP-BP / MB.

4. The digoxin antibody electrochemical sensor based on atom transfer radical polymerization as claimed in claim 1, characterized in that: The experimental operation of step S3 is to dissolve the divalent copper salt with an organic solvent, transfer it to a polytetrafluoroethylene reactor and place it at a high temperature of 180° C. for reaction for 1 hour, then centrifuge and dry it, and then mix it with the DP chain at room temperature for reaction for 2 hours, so as to obtain DP-CuO.

5. The digoxin antibody electrochemical sensor based on atom transfer radical polymerization as claimed in claim 1, characterized in that: The experimental operation of step S4 is to mix the recognition probes RP1, RP2, SP / AMP-BP / MB, DP-CuO in equal molar ratios with digoxigenin antibodies of different concentrations, react at room temperature for 100 minutes, then add hydrogen bromide HBr to dissolve CuO, and then transfer it to the MCH / IP / AuE surface, and add 2-bromoisobutyric acid-3-butynyl ester BBriB and ascorbic acid to react for 50 minutes, and connect BBriB to the electrode surface through click chemistry reaction.

6. Use of the digoxin antibody electrochemical sensor based on atom transfer radical polymerization as described in any one of claims 1 to 5 in detecting the content of digoxin antibodies.

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