CEA Electrochemical Detection Kit and Detection Method Based on eATRP Signal Amplification Strategy

Abstract

The present invention discloses a CEA electrochemical detection kit and a detection method based on the eATRP signal amplification strategy. The kit mainly includes the following raw materials: Apt1, Apt2, MCH, PEI, BMP, Me6TREN, CuBr2, KPF6, FMMA, LiClO4, EDC, NHS. In the present invention, FMMA is used as the electrochemical signal unit, and eATRP is adopted as the signal amplification strategy to achieve cascaded signal amplification with the macromolecular polymer PEI. Apt1 self-assembles onto the gold electrode surface through gold-sulfur bonds, and gradually forms a sandwich structure of Apt1-CEA-Apt2 through the specific recognition of aptamer-antigen. PEI is pre-connected to Apt2 through amide bonds, and the amino groups on PEI provide a large number of active sites for the connection of the eATRP initiator BMP. In the eATRP reaction solution, the bromine groups on the initiator BMP induce the occurrence of eATRP reaction on the electrode surface, and a large number of electrochemically active substances FMMA are grafted onto the electrode. Finally, the SWV is used to detect the current response value, thereby realizing the highly sensitive detection of CEA.

Description

Technical Field

[0001] The present invention relates to a CEA electrochemical detection kit and a detection method based on an eATRP signal amplification strategy, belonging to the technical field of bioanalysis. Background Art

[0002] With the rapid development of social economy, people's living standards have been significantly improved. However, at the same time, the number of cancer patients globally is gradually increasing, seriously threatening human health and life safety. Therefore, it is of great significance to establish a method for quickly, accurately, and sensitively detecting tumors.

[0003] Tumor markers (TM) are substances produced by the body in response to cell carcinogenesis, showing abnormal levels. Detecting tumor markers can help with early diagnosis and extend the survival period of patients. Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker present on the surface of cancer cells differentiated from endodermal cells. After being transmitted through the cell membrane, it is secreted extracellularly, and CEA components can be detected in body fluids such as peripheral serum and gastric juice. This component shows relatively high levels in malignant tumors such as lung cancer, breast cancer, cervical cancer, and gastric cancer, and has wide clinical applications. It plays an important role in the early diagnosis, treatment evaluation, development, monitoring, and prognosis of various types of cancer. However, the content of early disease markers in body fluids is relatively low, and the human body environment is complex, making it difficult to directly detect, and having high requirements for the detection limit of the detection method.

[0004] Currently, many methods have been reported for detecting CEA, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), chemiluminescent enzyme immunoassay (CLEIA), etc. As a typical product of the cross-disciplinary and development of multiple disciplines, biosensors, as an emerging detection method, have received extensive attention due to their advantages such as high sensitivity, simple operation, and strong specificity. It mainly consists of three parts: a molecular recognition element, a signal conversion element, and a signal amplification device. The molecular recognition element usually uses molecules such as antibodies, enzymes, and DNA that have the ability to specifically bind to the target substance to increase the specificity of the sensor. The signal conversion element can convert the generated biological signal into a measurable electrical signal or fluorescence signal. On the target molecule of a traditional sensor, only one signal molecule can be connected, resulting in the sensitivity of the sensor being difficult to meet the detection requirements. In order to increase the sensitivity of the sensor, the signal amplification device further amplifies the generated signal, thereby achieving highly sensitive detection of trace markers.

[0005] Common signal amplification strategies mainly include means such as nanomaterials, natural enzymes, and polymer chains. However, metal nanomaterials are expensive and have a complex preparation process, and the properties of enzymes are unstable and are easily affected by external environments such as pH and temperature. The polymer chain method can effectively graft single signal molecules onto the polymer backbone, thereby significantly increasing the loading amount of signal molecules on the electrode, and it is a new type of signal amplification strategy that is efficient, simple, and economical. Currently, there are various polymerization methods: atom transfer radical polymerization (ATRP), ring-opening polymerization (ROP), reversible addition-fragmentation chain transfer polymerization (RAFT), click polymerization, etc. The present invention aims to construct an electrochemical detection kit based on the eATRP signal amplification strategy for highly sensitive detection of CEA. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a CEA electrochemical detection kit and detection method based on the eATRP signal amplification strategy, which overcomes the shortcoming that the catalyst in the traditional ATRP reaction is sensitive to air, and has high sensitivity, good specificity, and simple operation, and has good applicability in human serum detection.

[0007] In order to achieve the above purpose, one of the technical solutions of the present invention is:

[0008] A CEA electrochemical detection kit based on the eATRP signal amplification strategy, comprising the following raw materials: Apt1, Apt2, MCH, PEI, BMP, Me6TREN, CuBr2, KPF6, FMMA, LiClO4, EDC, NHS, DMSO, H2SO4, absolute ethanol, PBS buffer solution, ultrapure water. Among them,

[0009] Apt1 sequence: 5’-SH-(CH2)6-ATACCAGCTTATTCAATT-3’

[0010] Apt2 sequence: 5’-AGGGGGTGAAGGGATACCC-3’.

[0011] Furthermore, when in use, some of the raw materials are formulated into solutions. The concentration of the Apt1 solution is 1 μM, the concentration of the Apt2 solution is 100 μM, the concentration of the EDC solution is 100 μM, the concentration of the NHS solution is 100 μM, the concentration of the MCH solution is 2 mM, the concentration of the PEI solution is 2 mg / mL, the concentration of the BMP solution is 2 mM, the concentration of the KPF6 solution is 0.1 M, the concentration of the FMMA solution is 10 mM, and the concentrations of CuBr2 and Me6TREN in the CuBr2 / Me6TREN solution are both 10 mM, and the concentration of the LiClO4 solution is 1.0 M.

[0012] One of the technical solutions of the present invention is: a method for detecting CEA, comprising the following steps:

[0013] (1) Electrode modification

[0014] ① Drop the Apt1 solution onto the gold electrode, react, wash, and dry;

[0015] ② Immerse the electrode obtained in step ① in the MCH solution, react, wash, and dry;

[0016] ③ Drop the sample to be detected onto the surface of the electrode obtained in step ②, react, wash, and dry;

[0017] ④ Drop the Apt2-PEI solution onto the electrode obtained in step ③, react, wash, and dry;

[0018] ⑤ Drop the BMP solution onto the surface of the electrode obtained in step ④, react, wash, and dry;

[0019] ⑥ Immerse the electrode obtained in step ⑤ in the eATRP reaction solution, and perform electrochemical polymerization with an i-t curve at a constant potential. Subsequently, treat the electrode with the LSV method to remove surface impurities;

[0020] (2) Electrochemical determination

[0021] Immerse the modified electrode in the LiClO4 solution, and perform electrochemical detection with the SWV method. Analyze the CEA content according to the magnitude of the electrical signal.

[0022] Furthermore, the gold electrode is pretreated first. The pretreatment method is: polish the surface of the gold electrode with 0.3μm and 0.05μm alumina powder respectively. Then, ultrasonically clean the electrode successively with ultrapure water, absolute ethanol, and ultrapure water. Immerse the cleaned electrode in the freshly prepared piranha solution for 15 minutes, and repeat the above cleaning steps. After the cleaning is completed, take out the electrode, and treat the electrode with cyclic voltammetry in a 0.5M H2SO4 solution. The potential range is set to -0.3 to 1.5V, the scanning rate is 0.1V / s, and repeat the scanning until a coincident CV diagram is obtained. Finally, clean the electrode with ultrapure water and dry it with nitrogen.

[0023] Furthermore, the reaction temperature in step ① is 37°C and the time is 2h; the reaction temperature in step ② is 37°C and the time is 0.5h; the reaction temperature in step ③ is 37°C and the time is 1h; the reaction temperature in step ④ is 37°C and the time is 1h; the reaction temperature in step ⑤ is 37°C and the time is 25 minutes; the polymerization temperature in step ⑥ is room temperature and the time is 40 minutes; the scanning range of SWV in step (2): 0 to 0.8V, the scanning rate: 1.0V / s, and the potential increment: 4mV.

[0024] Further, the preparation method of the Apt2-PEI solution is as follows:

[0025] Mix the Apt2 solution, EDC solution, and NHS solution in equal volumes, then add them to PBS buffer and activate by shaking to obtain the activated Apt2 solution; then add an equal volume of PEI solution and mix, and react by shaking to obtain the Apt2-PEI solution.

[0026] Further, the preparation method of the eATRP reaction solution is as follows:

[0027] ① Dissolve CuBr2 and Me6TREN in DMSO to prepare a CuBr2 / Me6TREN solution with both the concentrations of CuBr2 and Me6TREN being 10 mM.

[0028] ② Mix 1.8 mL of DMSO, 0.1 mL of the CuBr2 / Me6TREN solution, 0.1 mL of the FMMA solution, and 8.0 mL of the KPF6 solution to obtain the eATRP reaction solution.

[0029] One of the technical solutions of the present invention is: an application of the above kit in detecting CEA.

[0030] The preparation method and detection method principle of the modified electrode of the present invention are as Figure 1 shown.

[0031] First, the CEA aptamer 1 (Apt1) modified with a thiol group at one end is self-assembled onto the gold electrode surface through an "Au-S" covalent bond, and the unbound sites of the gold electrode are blocked with 6-mercaptohexanol (MCH). Then, through the specific recognition of the aptamer and the antigen, CEA and Apt2-PEI are successively connected to the gold electrode. A large number of amino groups on PEI are connected to the initiator 2-bromo-2-methylpropionic acid (BMP) through amide bonds, thereby introducing a large number of eATRP initiation sites. Then, the electrode is immersed in the eATRP reaction solution, and electrocatalytic polymerization is carried out under the electrochemical method i-t, and the signal unit ferrocenemethanol methacrylate (FMMA) is polymerized onto the electrode; the polymerized electrode is immediately treated with linear sweep voltammetry (LSV) to remove the impurities adsorbed on the electrode surface. Finally, the prepared electrode is placed in a lithium perchlorate electrolyte solution, and the performance of the detection is tested by square wave voltammetry (SWV).

[0032] The electrochemically mediated ATRP process uses BMP as the initiator, CuBr2 as the catalyst, and Me6TREN as the ligand, and the reaction involves the reversible conversion between the low-valent Cu I activator and the high-valent Cu II deactivator. Cu II Br / Me6TREN + is reduced to Cu at a negative potential voltageI Br / Me6TREN, Cu I Br / Me6TREN further dissociates into Cu I / Me6TREN + and Br - 。Using Cu I / Me6TREN + as an activator, it reacts with the active site C-Br on the initiator to generate the initial radical (R·) and Cu II Br / Me6TREN + passivator. Then R· reacts with FMMA to generate the chain radical R-FMMA. R-FMMA reacts with Cu II Br / Me6TREN + to produce the target product R-FMMA-Br. The passivator is converted into Cu I / Me6TREN + by reacting with the chain radical or through electrochemical reduction, and can initiate a new round of reaction again. The entire catalytic system establishes a reversible dynamic equilibrium between the active species and the dormant species through redox reactions. As the signal monomer FMMA is continuously added to the chain radical, a large number of target molecules are grafted onto the electrode surface, enabling highly sensitive detection of CEA.

[0033] Advantages of the present invention:

[0034] 1. The present invention uses the macromolecular polymer PEI to amplify the signal, avoiding the complex synthesis process required for using nanomaterials and the influence of external environment, temperature and other factors when using biological enzymes, so that while amplifying the signal, the detection efficiency and stability are not affected.

[0035] 2. The present invention adopts the electro-mediated atom transfer radical polymerization (eATRP) strategy. Based on the advantages of a wide range of available monomers and ordered polymer structures in traditional ATRP, the process of the polymerization reaction can be regulated by adjusting the potential, and the use of metal catalysts is reduced, which is more effective and environmentally friendly.

[0036] 3. In this invention, ferrocenylmethyl methacrylate (FMMA) is used as the electrochemistry signal unit, and eATRP is adopted as the signal amplification strategy to achieve cascade signal amplification with the macromolecular polymer PEI. Apt1 self-assembles onto the gold electrode surface through gold-sulfur bonds, and gradually forms a sandwich structure of Apt1-CEA-Apt2 through the specific recognition of aptamer-antigen. PEI is pre-connected to Apt2 through amide bonds, and the amino groups on PEI provide a large number of active sites for the connection of the eATRP initiator BMP. In the eATRP reaction solution, the bromine group on the initiator BMP induces the occurrence of eATRP reaction on the electrode surface, and a large number of electrochemically active substances FMMA are grafted onto the electrode. Finally, square wave voltammetry (SWV) is used to detect the current response value, so as to achieve highly sensitive detection of CEA. The experimental results show that in the range of 10 -3 ~10 2 ng·mL -1 , there is a good linear relationship between the current signal intensity and the CEA concentration. The obtained linear regression equation is: I = 1.1165lg C CEA +4.8054 (R 2 = 0.998), where I represents the current intensity (μA), and C CEA is the CEA concentration (ng·mL -1 ), and the detection limit is: 70.17 fg·mL -1 (S / N = 3). The experimental results show that this invention has good stability and reproducibility, and this invention shows excellent detection performance in the detection of actual samples, and is expected to become a new method for clinical detection of CEA. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A is the preparation method diagram of the modified electrode of this invention, Figure 1 B is the schematic diagram of the eATRP principle.

[0038] Figure 2 A is the SWV signal diagram of the electrode under different modification conditions; Figure 2 B is the CV curve of different scanning rates; Figure 2 C is the EIS impedance curve of the electrode after each step of modification; Figure 2 D is the CV curve of the electrode after each step of modification.

[0039] Figure 3 are the atomic force microscope photos of the electrode surface before and after eATRP modification.

[0040] Figure 4 are the contact angle photos of the electrode surface in different modification states.

[0041] Figure 5A is for the optimization of the BMP reaction time; B is for the optimization of the eATRP reaction time.

[0042] Figure 6 A is the SWV response signal of different concentrations of CEA; B is the linear relationship diagram between the CEA concentration and the current intensity.

[0043] Figure 7 A is for the kit to 10 ng·mL -1 CEA, CY, BSA, CTnI and 100 mU·mL -1 The selectivity of ALP; B is the signal intensity of different concentrations of CEA in human serum and PBS buffer, respectively. Specific embodiments

[0044] The following further elaborates on the specific embodiments of the present invention in conjunction with the examples.

[0045] Carcinoembryonic antigen (CEA) and all synthetic oligonucleotides, including CEA1 (Apt1: 5'-SH-(CH2)6-ATACCAGCTTATTCAATT-3', SEQ ID NO.1) and CEA2 (Apt2: 5'-AGGGGGTGAAGGGATACCC-3', SEQ ID NO.2), were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0046] Example 1: Kit

[0047] The CEA electrochemical detection kit based on the eATRP signal amplification strategy includes the following raw materials: Apt1, Apt2, 6-mercapto-1-hexanol (MCH), polyethyleneimine (PEI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-bromo-2-methylpropionic acid (BMP), tris(2-dimethylaminoethyl)amine (Me6TREN), CuBr2, potassium hexafluorophosphate (KPF6), ferrocenylmethanol methacrylate (FMMA), LiClO4, dimethyl sulfoxide (DMSO), H2SO4, absolute ethanol, PBS buffer, ultrapure water.

[0048] Some of the raw materials were formulated into solutions. The concentration of the Apt1 solution was 1 μM, the concentration of the Apt2 solution was 100 μM, the concentration of the EDC solution was 100 μM, the concentration of the NHS solution was 100 μM, the concentration of the MCH solution was 2 mM, the concentration of the PEI solution was 2 mg / mL, the concentration of the BMP solution was 2 mM, the concentration of the KPF6 solution was 0.1 M, the concentration of the FMMA solution was 10 mM, and the concentrations of CuBr2 and Me6TREN in the CuBr2 / Me6TREN solution were both 10 mM. The concentration of the LiClO4 solution was 1.0 M.

[0049] Example 2: Construction of the kit

[0050] (1) Electrode pretreatment

[0051] The surface of the gold electrode was polished with 0.3 μm and 0.05 μm alumina powder respectively. Then, the electrode was ultrasonically cleaned successively with ultrapure water, absolute ethanol and ultrapure water. The cleaned electrode was immersed in freshly prepared piranha solution for 15 min, and the above cleaning steps were repeated. After the cleaning was completed, the electrode was immediately taken out and treated in 0.5 M H2SO4 solution by cyclic voltammetry. The potential range was set to -0.3~1.5 V, the scanning rate was 0.1 V / s, and the scanning was repeated until a coincident CV diagram was obtained. Finally, the electrode was cleaned with ultrapure water and dried with nitrogen. The preparation method of the piranha solution is: 98% H2SO4 and H2O2 are mixed at a volume ratio of 3:1;

[0052] (2) Modified electrode

[0053] ① 10 μL of Apt1 solution (1 μM) was dropped onto the electrode and reacted at 37 °C for 2 h, washed and dried.

[0054] ② The electrode from step ① was immersed in 300 μL of MCH solution (2 mM) and reacted at 37 °C for 0.5 h, washed and dried.

[0055] ③ 10 μL of the solution to be detected (containing CEA) was dropped onto the electrode from step ② and reacted at 37 °C for 1 h, washed and dried.

[0056] ④ 10 μL of the prepared Apt2-PEI solution (1 μM) was dropped onto the electrode from step ③ and reacted at 37 °C for 1 h, washed and dried.

[0057] ⑤ 10 μL of BMP solution (2 mM) was dropped onto the electrode from step ④ and reacted at 37 °C for 25 min, washed and dried.

[0058] ⑥ The electrode from step ⑤ was immersed in freshly prepared eATRP reaction solution (10 mL). Under a constant potential, it was electrochemically polymerized for 40 min (at room temperature) using an i-t curve (constant potential: -0.5 V; standing time: 3 s; sampling interval: 0.1 s). Subsequently, the electrode was immediately treated by linear sweep voltammetry (LSV) (starting potential: 0 V; ending potential: 0.2 V; scanning rate: 1 V / s) to remove surface impurities.

[0059] (3) Electrochemical determination

[0060] The modified electrode was immersed in 1.0 M LiClO4 solution (10 mL), and electrochemical detection was carried out by SWV (scan range: 0 - 0.8 V, scan rate: 1.0 V / s, potential increment: 4 mV). The CEA content was analyzed according to the magnitude of the electrical signal.

[0061] The preparation method of the Apt2-PEI solution was as follows:

[0062] Take 10 μL of Apt2 solution (100 μM) and mix it with an equal volume of EDC solution (100 μM) and NHS solution (100 μM), then add it to 470 μL of PBS buffer solution, and place it in a constant temperature shaker at 37 °C for 2 h of activation to obtain 0.5 mL of activated Apt2 solution (2 μM); then add an equal volume of PEI solution (2 mg / mL) and mix, and place it in a constant temperature shaker at 37 °C for 1 h of reaction to obtain Apt2-PEI solution (1 μM).

[0063] The preparation method of the eATRP reaction solution was as follows:

[0064] ① Dissolve CuBr2 and Me6TREN in DMSO to prepare a CuBr2 / Me6TREN solution with both the concentrations of CuBr2 and Me6TREN being 10 mM.

[0065] ② Mix 1.8 mL of DMSO, 0.1 mL of CuBr2 / Me6TREN solution, 0.1 mL of FMMA solution (10 mM), and 8.0 mL of KPF6 solution (0.1 M) to prepare the eATRP reaction solution.

[0066] Example 3: Feasibility verification

[0067] To evaluate the feasibility of detecting CEA by the present invention, SWV (1.0 M LiClO4, potential range 0 - 0.8 V) was used to measure the redox current signals of a series of modified electrodes and compare them. The results are as Figure 2 shown in A. Curves b - e in the figure reflect that when Apt1, CEA, Apt2, and BMP are not added during the electrode construction process, there is no obvious oxidation current within the potential range of ferrocene. This is because the initiator of eATRP is not grafted onto the electrode, resulting in the non-occurrence of electrocatalytic polymerization. Curve f reflects that when CuBr2 / Me6TREN is not added, there is no obvious oxidation current, indicating that the eATRP reaction does not occur without a catalyst and a ligand. Similarly, curve g shows that the lack of an electroactive probe (FMMA) also does not result in an obvious signal peak. When all the above components are continuously modified onto the electrode surface, an obvious electrochemical signal can be detected (curve a). These experiments confirm that this strategy is feasible for CEA detection.

[0068] Example 4: Characterization

[0069] The freshly prepared electrode was characterized by CV in 1.0 M KNO3 solution at different scan rates from 0.01 to 1.0 V / s and potentials from 0 to 0.6 V. Figure 2 As shown in Figure B, the redox current has a good linear relationship with the scan rate, indicating that the electroactive polymer is fixed to the electrode through covalent bonds rather than relying on diffusion.

[0070] EIS can characterize the modification of the electrode surface during the electrode construction process. In the presence of 5 mM [Fe(CN)6] 3- / 4- In the electrolyte solution, the EIS spectra of the same electrode in the continuous preparation process are collected step by step. In the Nyquist plot, the diameter of the semicircle represents the charge transfer resistance (Rct), which is the obstacle encountered when electrons transfer on the electrode surface. Figure 2 As can be seen from C, the Rct of the bare gold electrode (curve a) in the impedance spectrum is only 281Ω, indicating that the electrode surface is clean and electrons can be quickly transferred between the electrode and the solution interface. After Apt1 is fixed to the electrode through "Au-S", the phosphorylation site of Apt1 and [Fe(CN)6] 3- / 4- Due to the electrostatic repulsion between them, Rct gradually increases (~954Ω, curve b). Subsequently, the excess binding sites on the electrode surface are occupied by MCH, resulting in a further increase in Rct (~1551Ω, curve c). Next, CEA recognizes Apt1 and forms a protein layer on the electrode surface, which hinders the efficiency of electron transfer, resulting in an increase in Rct (~2596Ω, curve d). When Apt2-PEI is connected to the electrode, Rct (~231Ω, curve e) is significantly reduced, which is due to the fact that a large number of amino groups on PEI promote electron transfer efficiency. The initiator BMP is fixed to the electrode, and Rct (~406Ω, curve f) increases again. Finally, Rct (~9692Ω, curve g) after eATRP increases significantly, which is because a large amount of FMMA is grafted to the electrode surface, causing a sharp increase in steric hindrance. The results show that the construction process of the present invention is effective.

[0071] The characteristics of the electrode surface at different modification steps were evaluated by CV. Figure 2As shown in D, as Apt1, MCH, and CEA were gradually modified onto the electrode, the peak current on the electrode surface gradually decreased (55.4 - 34.7 μA, curves a - d). When Apt2 - PEI adhered to the electrode surface, the peak current increased significantly (57.7 μA, curve e). Then, when the initiator was modified onto the electrode, the peak current further decreased (56.2 μA, curve f). Finally, through the polymerization reaction, a large amount of FMMA was grafted onto the electrode, and the peak current decreased significantly (32.3 μA, curve g). The CV results were consistent with the EIS trend, indicating that the electrode was successfully constructed.

[0072] The electrode surface was characterized by atomic force microscopy (AFM) and water contact angle (WCA). As Figure 3 shown in A, the height of the Apt1 / MCH / CEA / Apt2 - PEI / BMP - modified gold electrode was 17.9 nm. After eATRP occurred, due to the formation of the polymer on the gold electrode, the height of the gold electrode increased to 34.0 nm ( Figure 3 B). This result indicates that the polymer can be formed on the electrode and FMMA was successfully grafted onto the electrode surface.

[0073] Since the hydrophilicity of the electrode surface changes after modifying the material, WCA can be used to study the hydrophilicity of the modified gold electrode. Figure 4 The changes in WCA during different modification steps are shown in Figure 4 A. As can be seen from Figure 4 A, since the gold electrode is hydrophobic, the WCA of the bare electrode is 95.3°. When Apt1 was immobilized on the gold electrode, the WCA decreased to 92.3° ( Figure 4 B), which was caused by the hydrophilic groups on Apt1. After blocking the unbound sites on the surface with MCH, the change in WCA was not significant, only decreasing to 91.2° ( Figure 4 C), because MCH has both hydrophobic and hydrophilic groups. Since there are hydrophilic amino and carboxyl groups in proteins, when CEA specifically recognized Apt1, the WCA decreased to 86.8° ( Figure 4 D). Similarly, due to the presence of a large number of amino groups on PEI, after Apt2 - PEI was connected to the electrode, the WCA decreased significantly to 76.5° ( Figure 4 E). After BMP was connected to the electrode through an amide bond, due to the action of the amide bond and halogen, the WCA decreased to 72.6° ( Figure 4 F). Finally, when the polymer chain with FMMA was grafted onto the electrode, due to the hydrophilic methacrylate in the polymer, the contact angle of the electrode decreased to 71.8° (

[0074] Example 5: Optimization of Detection Conditions

[0075] To optimize the performance of the kit, important conditions during the electrode construction process were optimized, including the BMP reaction time, eATRP time, etc., to improve the analytical performance of the kit.

[0076] (1) BMP reaction time

[0077] As an initiator for eATRP, the amount of BMP attached to the electrode directly affects the occurrence of the polymerization reaction. As the reaction time extends, more and more initiators attach to the electrode, providing more reaction sites for polymerization, and the current intensity gradually increases. Figure 5 Figure A shows the relationship between the BMP time and the current intensity. Within the first 25 min, the current increases with the reaction time. After 25 min, the current intensity almost remains unchanged because the reaction reaches equilibrium. Therefore, 25 min was selected as the optimal reaction duration.

[0078] (2) eATRP reaction time

[0079] Similarly, in the polymerization reaction, the polymerization amount of FMMA is also affected by the eATRP reaction time. As shown in Figure 5 Figure B, as the polymerization time extends, the current intensity gradually increases and reaches the maximum value at about 40 min. This is because a large amount of FMMA is grafted onto the electrode, and the steric hindrance on the electrode surface gradually increases, thus restricting the progress of the polymerization reaction. Therefore, the optimal reaction time for eATRP is 40 min.

[0080] Example 6: Analytical performance

[0081] Under the optimal conditions, the constructed electrode was used to detect different concentrations of CEA in a 1 M LiClO4 solution by SWV method to study the detection range and detection limit of the electrode. As shown in Figure 6 it can be seen that within the range of 10 -3 ~10 2 ng·mL -1 , there is a good linear relationship between the CEA concentration and the current intensity. The linear regression equation is: I (μA) = 1.1165 lg C CEA + 4.8054 (R2 = 0.998), and the detection limit is: 70.17 fg·mL -1 (S / N = 3).

[0082] Compared with several other methods, the method proposed in the present invention for detecting CEA has a larger detection range and a lower detection limit, indicating that the kit of the present invention has potential application value in the early detection of cancer (the following table).

[0083]

[0084] Example 7: Specificity, anti-interference ability, stability and reproducibility of the kit

[0085] To verify the specificity of the present invention for different proteins and ensure its detection performance, under the optimal conditions, the selectivity of the kit for 10 ng·mL -1 CEA, cytokeratin 19 fragment (CYFRA 21-1, CY), bovine serum albumin (BSA), cardiac troponin (CTnI), and 100 mU·mL -1 alkaline phosphatase (ALP) was compared. As can be seen from Figure 7 A, there are obvious differences in the signals generated by CEA and other enzymes and proteins. The current intensities of CY, CTnI, and ALP only account for 8.96%, 7.26%, and 7.15% of that of CEA respectively. This is because there is good specificity between Apt and CEA, but there may be partial bases recognized by BSA, resulting in a relatively high signal of BSA, reaching 28.65% of that of CEA, but there are also obvious differences in its current intensity from that of CEA.

[0086] To study the anti-interference ability of the present invention in human serum, the signal intensities of different concentrations of CEA in 10% human serum were detected respectively, and the signal of CEA in the human serum sample was compared with that in PBS buffer. As can be seen from Figure 7 B: The signals of 10 ng·mL -1 , 100 pg·mL -1 , and 5 pg·mL -1 CEA measured in human serum were 3.63%, 1.12%, and 8.37% smaller than those measured in PBS buffer respectively. It shows that the present invention has a certain anti-interference ability and has potential for clinical application.

[0087] To evaluate the stability of the present invention, the signal intensities of the newly constructed electrode and the electrode after storage for a period of time were compared. Two groups of modified electrodes were prepared under the same conditions. One group detected the signal intensity immediately after construction, and the other group detected the prepared electrode after storing at 4°C for 14 days. The results showed that the signal intensity measured after two weeks of storage was 93.8% of that of the newly prepared electrode, indicating that the electrode has good stability.

[0088] In addition, the reproducibility of the present invention was also explored under the same experimental conditions. The experimental results showed that the relative standard deviations within the group and between groups were 1.78% and 3.09% respectively. It shows that the present invention has good reproducibility.

[0089] Example 8: Practical application value

[0090] Finally, to verify the practical application value of the kit, 5 different clinical serum samples were detected. The experimental results are shown in the following table. The relative errors between the measured results and the clinically measured data are all less than 5%, indicating that the kit can detect actual clinical samples and has certain clinical application value.

[0091] Sequence Listing <110> Henan University of Chinese Medicine <120> CEA Electrochemical Detection Kit and Detection Method Based on eATRP Signal Amplification Strategy <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence () <400> 1 shchatacca gcttattcaa tt 22 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence () <400> 2 agggggtgaa gggataccc 19

Claims

1. CEA electrochemical detection kit based on eATRP signal amplification strategy, characterized in that, It includes the following raw materials: Apt1, Apt2, MCH, PEI, BMP, Me6TREN, CuBr2, KPF6, FMMA, LiClO4, EDC, NHS; Apt1 sequence: 5’-SH-(CH2)6-ATACCAGCTTATTCAATT-3’ Apt2 sequence: 5’-AGGGGGTGAAGGGATACCC-3’.

2. The kit according to claim 1, wherein It also includes: DMSO, H2SO4, absolute ethanol, PBS buffer solution, ultrapure water.

3. The kit according to claim 1, characterized in that, When in use, part of the raw materials are formulated into solutions. The concentration of the Apt1 solution is 1 µM, the concentration of the Apt2 solution is 100 µM, the concentration of the EDC solution is 100 µM, the concentration of the NHS solution is 100 µM, the concentration of the MCH solution is 2 mM, the concentration of the PEI solution is 2 mg / mL, the concentration of the BMP solution is 2 mM, the concentration of the KPF6 solution is 0.1 M, the concentration of the FMMA solution is 10 mM, the concentrations of CuBr2 and Me6TREN in the CuBr2 / Me6TREN solution are both 10 mM, and the concentration of the LiClO4 solution is 1.0 M.

4. Use of the kit according to any one of claims 1-3 in the preparation of a CEA detection product.

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