CN114778839A - CEA electrochemical detection kit based on eATRP signal amplification strategy and detection method - Google Patents

Abstract

The invention discloses a CEA electrochemical detection kit based on an eATRP signal amplification strategy and a detection method, wherein the kit mainly comprises the following raw materials: apt1, Apt2, MCH, PEI, BMP, Me₆TREN、CuBr₂、KPF₆、FMMA、LiClO₄EDC, NHS. The invention takes FMMA as an electrochemical signal unit, adopts eATRP as a signal amplification strategy, and realizes cascade signal amplification with a macromolecular polymer PEI. Apt1 self-assembles to the surface of gold electrode by gold-sulfur bond, and gradually forms a sandwich structure of Apt1-CEA-Apt2 through the specificity recognition of aptamer-antigen. PEI was previously linked to Apt2 via an amide linkage, with the amino groups on the PEI providing a number of active sites for the attachment of the etatr initiator BMP. In the eATRP reaction solution, bromine radicals on initiator BMP induce eATRP reaction on the surface of an electrodeA large amount of the electrochemically active substance FMMA is grafted onto the electrode. And finally, detecting the current response value by using the SWV, thereby realizing the high-sensitivity detection of the CEA.

Description

CEA electrochemical detection kit and detection method based on eATRP signal amplification strategy

technical field

The 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 biological analysis.

Background technique

With the rapid development of social economy, people's living standards have been significantly improved, but at the same time, the number of cancer patients in the world is gradually increasing, which is a serious threat to human health and life safety. Therefore, it is of great significance to establish a rapid, accurate and sensitive method for tumor detection.

Tumor markers (TM) are substances that stimulate the body to produce during the process of cell carcinogenesis, and their content is abnormal. Detection of tumor markers can help early diagnosis and prolong patient survival. Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker that exists on the surface of cancer cells differentiated from endoderm cells. It is secreted to the outside of the cell after passing through the cell membrane. CEA can be detected in peripheral serum, gastric juice and other body fluids. Element. This component shows a high level in lung cancer, breast cancer, cervical cancer, gastric cancer and other malignant tumors, and is widely used in clinical practice. 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 markers of disease in body fluids is relatively low, and the human environment is complex, which makes direct detection difficult and requires high detection limits for detection methods.

Various methods have been reported for the detection of CEA, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), chemiluminescence enzyme immunoassay (CLEIA) and so on. Biosensors are a typical product of multidisciplinary crossover and development. As an emerging detection method, it has received extensive attention due to its high sensitivity, simple operation, and strong specificity. It is mainly composed of three parts: molecular recognition element, signal conversion element and signal amplification device. Molecular recognition element usually uses antibodies, enzymes, DNA and other molecules with specific binding ability to increase the specificity of the sensor. The signal conversion element can convert the generated biological signal into a measurable electrical or fluorescent signal. The target molecule of traditional sensors can only be connected to one signal molecule, which makes it difficult for the sensitivity of the sensor to meet the detection requirements. In order to increase the sensitivity of the sensor, the signal amplification device further amplifies the generated signal, so as to achieve highly sensitive detection of trace markers.

Commonly used signal amplification strategies mainly include nanomaterials, natural enzymes, and polymer chains. However, metal nanomaterials are expensive, complicated in preparation process, and unstable in properties of enzymes, which are easily affected by external environments such as pH and temperature. The polymer chain method can effectively graft a single signal molecule onto the polymer backbone, thereby significantly increasing the loading of the signal molecule on the electrode, which is an efficient, simple and economical new signal amplification strategy. At present, there are many 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

In view of 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 is sensitive to air in the traditional ATRP reaction, and has high sensitivity and specificity. It is good, simple to operate, and has good applicability in the detection of human serum.

In order to achieve the above object, one of the technical solutions of the present invention is:

CEA electrochemical detection kit based on eATRP signal amplification strategy, including the following raw materials: Apt1, Apt2, MCH, PEI, BMP, Me ₆ TREN, CuBr ₂ , KPF ₆ , FMMA, LiClO ₄ , EDC, NHS, DMSO, H ₂ SO ₄ , absolute ethanol, PBS buffer, ultrapure water. in,

Apt1 sequence: 5'-SH-(CH ₂ ) ₆ -ATACCAGCTTATTCAATT-3'

Apt2 sequence: 5'-AGGGGGTGAAGGGATACCC-3'.

Further, when using, some raw materials are prepared into solutions, the concentration of Apt1 solution is 1 μM, the concentration of Apt2 solution is 100 μM, the concentration of EDC solution is 100 μM, the concentration of NHS solution is 100 μM, the concentration of MCH solution is 2mM, and the concentration of PEI solution is 2 mg/mL, The concentration of BMP solution is 2 mM, the concentration of KPF ₆ solution is 0.1 M, the concentration of FMMA solution is 10 mM, the concentration of CuBr ₂ and Me ₆ TREN in CuBr ₂ /Me ₆ TREN solution is both 10 mM, and the concentration of LiClO ₄ solution is 1.0 M.

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

(1) Electrode modification

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

② Soak the electrode of step ① in MCH solution, react, wash and blow dry;

③ drop the sample to be detected on the electrode surface of step ②, react, wash, and blow dry;

④ Add the Apt2-PEI solution dropwise to the electrode in step ③, react, wash, and blow dry;

⑤ Add the BMP solution dropwise to the electrode surface of step ④, react, wash, and blow dry;

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

(2) Electrochemical determination

The modified electrode was immersed in _LiClO4 solution, electrochemical detection was performed by SWV method, and the CEA content was analyzed according to the magnitude of the electrical signal.

Further, the gold electrode is pretreated first. The pretreatment method is as follows: polishing the surface of the gold electrode with 0.3 μm and 0.05 μm alumina powder respectively, and then ultrasonically cleaning the electrode with ultrapure water, anhydrous ethanol and ultrapure water in sequence. Soak the cleaned electrode in the freshly prepared piranha acid solution for 15min, and repeat the above cleaning steps, take it out after cleaning, and treat the electrode with cyclic _voltammetry in _0.5MH2SO4 solution, and the potential range is set to -0.3~1.5V, the scan rate is 0.1V/s, and the scanning is repeated until the overlapping CV diagrams are obtained. Finally, the electrodes are washed with ultrapure water and dried with nitrogen.

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

Further, the preparation method of Apt2-PEI solution is:

Mix equal volumes of Apt2 solution, EDC solution and NHS solution, then add to PBS buffer, shake and activate to obtain activated Apt2 solution; then add equal volume of PEI solution to mix, shake and react to obtain Apt2-PEI solution.

Further, the preparation method of eATRP reaction solution is:

① Dissolve CuBr ₂ and Me ₆ TREN in DMSO to prepare a CuBr ₂ /Me ₆ TREN solution with both CuBr ₂ and Me ₆ TREN concentrations of 10 mM;

② Mix 1.8 mL of DMSO, 0.1 mL of CuBr ₂ /Me ₆ TREN solution, 0.1 mL of FMMA solution and 8.0 mL of KPF ₆ solution to prepare an eATRP reaction solution.

One of the technical solutions of the present invention is: an application of the above-mentioned kit in detecting CEA.

The principle of the preparation method and detection method of the modified electrode of the present invention is shown in FIG. 1 .

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

The electrochemically mediated ATRP process uses BMP as the initiator, CuBr ₂ as the catalyst, and Me ₆ TREN as the ligand, and the reaction involves a reversible transformation between a low-valence Cu ^I activator and a high-valence Cu ^II inactivator. Cu ^II Br/Me ₆ TREN ⁺ was reduced to Cu ^I Br/Me ₆ TREN under negative potential voltage, and Cu ^I Br/Me ₆ TREN was further dissociated into Cu ^I /Me ₆ TREN ⁺ and Br ⁻ . Using Cu ^I /Me ₆ TREN ⁺ as an activator, it reacted with the active site C-Br on the initiator to generate initial radicals (R·) and Cu ^II Br/Me ₆ TREN ⁺ passivation agent. Then R· reacts with FMMA to generate chain radical R-FMMA. R-FMMA reacts with Cu ^II Br/Me ₆ TREN ⁺ to generate the target product R-FMMA-Br. The passivating agent can be converted into Cu ^I /Me ₆ TREN ⁺ by reacting with chain radicals or by electrochemical reduction, which can start a new round of reactions again. The entire catalytic system establishes a reversible dynamic equilibrium between active and dormant species through redox reactions. With the continuous addition of the signal monomer FMMA into the chain free radicals, the target molecules are grafted on the electrode surface in large quantities, which can realize the highly sensitive detection of CEA.

Beneficial effects of the present invention:

1. The present invention uses the macromolecular polymer PEI to amplify the signal, avoiding the complex synthesis process required for the use of nanomaterials and the use of biological enzymes that are easily affected by factors such as external environment, temperature, etc. affect the efficiency and stability of detection.

2. The present invention adopts an electrically mediated atom transfer radical polymerization (eATRP) strategy. Based on the advantages of traditional ATRP, such as a wide range of available monomers and an ordered polymer structure, 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.

3. The present invention uses ferrocene methanol methacrylate (FMMA) as the electrochemical signal unit, adopts eATRP as the signal amplification strategy, and realizes cascade signal amplification with the macromolecular polymer PEI. Apt1 self-assembles onto the surface of the gold electrode through gold-sulfur bonds, and gradually forms a sandwich structure of Apt1-CEA-Apt2 through the specific recognition of aptamer-antigen. PEI was previously linked to Apt2 through an amide bond, and the amino group on PEI provided a large number of active sites for the attachment of the eATRP initiator BMP. In the eATRP reaction solution, the bromine group on the initiator BMP induced the eATRP reaction on the electrode surface, and a large amount of electrochemically active material FMMA was grafted onto the electrode. Finally, square wave voltammetry (SWV) is used to detect the current response value, thereby realizing highly sensitive detection of CEA. The experimental results show that in the range of 10 ^-3 to 10 ² ng·mL ^-1 , there is a good linear relationship between the current signal intensity and the CEA concentration. The linear regression equation obtained is: I=1.1165lg C _CEA +4.8054(R ² =0.998), where I represents the current intensity (μA), 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 the present invention has good stability and reproducibility, and the present invention exhibits excellent detection performance in actual sample detection, and is expected to become a new method for clinical detection of CEA.

Description of drawings

FIG. 1A is a schematic diagram of the preparation method of the modified electrode of the present invention, and FIG. 1B is a schematic diagram of the principle of eATRP.

Figure 2A is the SWV signal diagram of the electrode under different modification conditions; Figure 2B is the CV curve of the different scan rate; Figure 2C is the EIS impedance curve of the electrode modified in each step; Figure 2D is the CV of the modified electrode in each step curve.

Figure 3 is the atomic force microscope pictures of the electrode surface before and after eATRP modification.

Figure 4 shows the contact angle photographs of the electrode surfaces in different modified states.

Figure 5A shows the optimization of the BMP reaction time; B shows the optimization of the eATRP reaction time.

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

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

Detailed ways

The specific embodiments of the present invention will be further described in detail below with reference to the examples.

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 Bioengineering (Shanghai) Co., Ltd.

Example 1: Kit

CEA electrochemical detection kit based on eATRP signal amplification strategy, including the following raw materials: Apt1, Apt2, 6-mercapto-1-hexanol (MCH), polyethyleneimine (PEI), 1-(3-dimethylaminopropyl) yl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-bromo-2-methylpropionic acid (BMP), tris(2-dimethylamino) Ethyl)amine (Me ₆ TREN), CuBr ₂ , potassium hexafluorophosphate (KPF ₆ ), ferrocene methanol methacrylate (FMMA), LiClO ₄ , dimethyl sulfoxide (DMSO), H ₂ SO ₄ , Absolute ethanol, PBS buffer, ultrapure water.

Part of the raw materials were prepared into solutions, the concentration of Apt1 solution was 1 μM, the concentration of Apt2 solution was 100 μM, the concentration of EDC solution was 100 μM, the concentration of NHS solution was 100 μM, the concentration of MCH solution was 2 mM, the concentration of PEI solution was 2 mg/mL, and the concentration of BMP solution was 2 mM. , the concentration of KPF ₆ solution is 0.1M, the concentration of FMMA solution is 10mM, the concentration of CuBr ₂ and Me ₆ TREN in CuBr ₂ /Me ₆ TREN solution is both 10mM, and the concentration of LiClO ₄ solution is 1.0M.

Example 2: Construction of the kit

(1) Electrode pretreatment

The surface of the gold electrode was polished with 0.3 μm and 0.05 μm alumina powder, respectively. Then, the electrodes were ultrasonically cleaned with ultrapure water, absolute ethanol and ultrapure water in sequence. Soak the cleaned electrode in the freshly prepared piranha acid solution for 15 min, and repeat the above cleaning steps. Immediately after cleaning, the electrodes were taken out and treated with cyclic voltammetry in a 0.5MH ₂ SO ₄ solution. The potential range was set to -0.3 to 1.5 V, and the scan rate was 0.1 V/s. The scan was repeated until overlapping CV maps were obtained. Finally, the electrode was rinsed with ultrapure water and dried with nitrogen. The preparation method of piranha acid solution is as follows: 98% H ₂ SO ₄ and H ₂ O ₂ are mixed in a volume ratio of 3:1;

(2) Modified electrode

① Add 10 μL of Apt1 solution (1 μM) dropwise to the electrode, react at 37°C for 2 h, wash and dry.

② Soak the electrode of step ① in 300 μL MCH solution (2 mM), react at 37° C. for 0.5 h, wash and dry.

③ Add 10 μL of the solution to be detected (containing CEA) dropwise to the electrode in step ②, react at 37° C. for 1 hour, wash and dry.

④ Add 10 μL of the prepared Apt2-PEI solution (1 μM) dropwise to the electrode in step ③, react at 37° C. for 1 h, wash and dry.

⑤ Add 10 μL of BMP solution (2 mM) dropwise to the electrode in step ④, react at 37° C. for 25 min, wash and dry.

⑥ Immerse the electrode of step ⑤ in the newly prepared eATRP reaction solution (10 mL), and conduct electrochemical polymerization with i-t curve (constant potential: -0.5V; standing time: 3s; sampling interval: 0.1s) under constant potential 40min (normal temperature). Immediately thereafter, the electrodes were treated with linear sweep voltammetry (LSV) (start potential: 0 V; stop potential: 0.2 V; scan rate: 1 V/s) to remove surface impurities.

(3) Electrochemical determination

The modified electrode was immersed in 1.0 M LiClO ₄ solution (10 mL), and electrochemical detection was carried out by SWV (scanning range: 0-0.8 V, scanning rate: 1.0 V/s, potential increment: 4 mV), according to the electrical signal Size analysis of CEA content.

The preparation method of Apt2-PEI solution is:

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

The preparation method of eATRP reaction solution is:

① Dissolve CuBr ₂ and Me ₆ TREN in DMSO to prepare a CuBr ₂ /Me ₆ TREN solution with both CuBr ₂ and Me ₆ TREN concentrations of 10 mM;

② Mix 1.8 mL of DMSO, 0.1 mL of CuBr ₂ /Me ₆ TREN solution, 0.1 mL of FMMA solution (10 mM) and 8.0 mL of KPF ₆ solution (0.1 M) to prepare an eATRP reaction solution.

Example 3: Feasibility Verification

In order to evaluate the feasibility of the present invention to detect CEA, SWV (1.0M LiClO ₄ , potential range of 0-0.8V) was used to measure the redox current signals of a series of modified electrodes and compared. The results are shown in Figure 2A. The curves b–e in the figure reflect that there is no obvious oxidation current in the potential range of ferrocene when Apt1, CEA, Apt2, and BMP are not added during the electrode construction. This is because the initiator of eATRP is not grafted onto the electrode, resulting in the electrocatalytic polymerization not taking place. Curve f reflects that there is no obvious oxidation current when CuBr ₂ /Me ₆ TREN is not added, indicating that the eATRP reaction does not occur without catalyst and ligand. Likewise, curve g shows that in the absence of the electroactive probe (FMMA) there is no distinct signal peak. When all of the above components were continuously modified to the electrode surface, a distinct electrochemical signal could be detected (curve a). These experiments confirmed the feasibility of this strategy for CEA detection.

Example 4: Characterization

The freshly prepared electrodes were characterized by CV in 1.0 M _KNO3 solution with different scan rates ranging from 0.01 to 1.0 V/s and potentials from 0 to 0.6 V. As shown in Figure 2B, the redox current has a good linear relationship with the scan rate, indicating that the electroactive polymer is immobilized to the electrode through covalent bonds rather than diffusion-dependent connections.

EIS can characterize the modification of the electrode surface during the electrode construction. In 5mM [Fe(CN) ₆ ] ^3-/4- electrolyte solution containing 0.1M KNO ₃ EIS spectrum. In the Nyquist diagram, the diameter of the semi-circle represents the charge transfer resistance (Rct), which is the resistance to electron transfer across the electrode surface. It can be seen from Figure 2C that 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 rapidly transferred between the electrode and the solution interface. After Apt1 was immobilized on the electrode via "Au-S", Rct gradually increased due to the electrostatic repulsion between Apt1 at the phosphorylation site and [Fe(CN) ₆ ] ^3-/4- (~954Ω, curve b ). Subsequently, excess binding sites on the electrode surface were occupied by MCH, resulting in a further increase in Rct (∼1551 Ω, curve c). Next, CEA recognizes Apt1 and forms a protein layer on the electrode surface that hinders the efficiency of electron transfer, resulting in an increase in Rct (∼2596 Ω, curve d). When Apt2-PEI was attached to the electrode, Rct (~231 Ω, curve e) was significantly reduced, which was due to the large number of amino groups on PEI promoting electron transfer efficiency. The initiator BMP is immobilized on the electrode and Rct (~406Ω, curve f) increases again. Finally, the Rct (~9692 Ω, curve g) increases significantly after eATRP, which is due to the dramatic increase in steric hindrance due to the large amount of FMMA being grafted to the electrode surface. The results show that the construction process of the present invention is effective.

The properties of the electrode surface at different modification steps were evaluated by CV. As shown in Figure 2D, 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 was attached to the electrode surface, the peak current was significantly increased (57.7 μA, curve e). Then, the initiator was modified onto the electrode, and the peak current decreased further (56.2 μA, curve f). Finally, the polymerization reaction grafted a large amount of FMMA onto the electrode, the peak current was significantly reduced (32.3 μA, curve g), and the CV results were consistent with the EIS trend, indicating that the electrode was successfully constructed.

The electrode surfaces were characterized by atomic force microscopy (AFM) and water contact angle (WCA). It can be seen from Figure 3A that the height of the Apt1/MCH/CEA/Apt2-PEI/BMP modified gold electrode is 17.9 nm. After eATRP occurred, the gold electrode height grew to 34.0 nm due to polymer formation on the gold electrode (Fig. 3B). This result indicated that the polymer could be formed on the electrode, and FMMA was successfully grafted to the electrode surface.

Since the hydrophilicity of the electrode surface will change after the material is modified, WCA can be used to study the hydrophilicity of the modified gold electrode. Figure 4 shows the change of WCA in different modification steps. As can be seen in Figure 4A, the WCA of the bare electrode is 95.3° since the gold electrode is hydrophobic. When Apt1 was immobilized on the gold electrode, the WCA decreased to 92.3° (Fig. 4B), which was caused by the hydrophilic groups on Apt1. After blocking the unbound sites on the surface with MCH, the WCA did not change much, only dropping to 91.2° (Fig. 4C), which is due to the presence of both hydrophobic and hydrophilic groups on MCH. Due to the presence of hydrophilic amino and carboxyl groups in the protein, WCA decreased to 86.8° when CEA specifically recognized Apt1 (Fig. 4D). Likewise, WCA was significantly reduced to 76.5° after Apt2-PEI was attached to the electrode due to the presence of a large number of amino groups on PEI (Fig. 4E). After the BMP was attached to the electrode via an amide bond, the WCA was reduced to 72.6° due to the action of the amide bond and halogen (Fig. 4F). Finally, the polymer chains with FMMA were grafted onto the electrode, and the contact angle of the electrode dropped to 71.8° due to the hydrophilic methacrylate in the polymer (Fig. 4G). The above results prove that the present invention is feasible for detecting CEA.

Example 5: Optimization of detection conditions

In order to maximize the performance of the kit, important conditions in the electrode construction process were optimized, including BMP reaction time, eATRP time, etc., to improve the analytical performance of the kit.

(1) BMP reaction time

As the initiator of eATRP, the amount of BMP attached to the electrode directly affects the occurrence of the polymerization reaction. With the prolongation of the reaction time, more and more initiators were attached to the electrodes, providing more reaction sites for polymerization, and the current intensity gradually increased. Figure 5A shows the relationship between BMP time and current intensity. In the first 25 min, the current increased with the reaction time, and the current intensity remained almost unchanged after 25 min, because the reaction reached equilibrium. Therefore, 25 min was chosen as the optimal reaction time.

(2) eATRP reaction time

Similarly, in the polymerization reaction, the polymerization amount of FMMA is also affected by the reaction time of eATRP. It can be seen from Fig. 5B that with the extension of the polymerization time, the current intensity gradually increases and reaches the maximum value at about 40 min. This is because a large amount of FMMA was grafted onto the electrode, and the steric hindrance on the electrode surface gradually increased, thus limiting the progress of the polymerization reaction. Therefore, the optimal reaction time of eATRP was 40 min.

Example 6: Analyzing Performance

Under optimal conditions, the constructed electrode was used to detect different concentrations of CEA in 1M _LiClO4 solution by SWV method to study the detection range and detection limit of this electrode. It can be seen from Figure 6 that in the range of 10 ^-3 to 10 ² ng·mL ^-1 , the CEA concentration and the current intensity have a good linear relationship. The linear regression equation is: I(μA)=1.1165lg C _CEA +4.8054(R ₂ =0.998), the detection limit was: 70.17 fg·mL ^-1 (S/N=3).

Compared with several other methods, the method proposed in the present invention detects CEA with a larger detection range and lower detection limit, which indicates that the kit of the present invention has potential application value in the early detection of cancer (table below).

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

In order to verify the specificity of the present invention for different proteins to ensure its detection performance, under optimal conditions, the kits were compared for 10 ng·mL ^-1 CEA, cytokeratin 19 fragment (CYFRA 21-1, CY), Selectivity of bovine serum albumin (BSA), cardiac troponin (CTnI) and 100mU·mL ^-1 alkaline phosphatase (ALP). It can be seen from Figure 7A that there are significant differences in the signals produced 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 CEA, respectively. This is due to the good specificity between Apt and CEA, but there may be some bases that recognize BSA, resulting in a relatively high signal of BSA, reaching 28.65% of CEA, but there is also a significant difference between its current intensity and CEA.

In order 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 respectively detected, and the signal of CEA in human serum samples was compared with the signal in PBS buffer. It can be seen from Figure 7B that the signals of 10ng·mL ^-1 , 100pg·mL ^-1 , and 5pg·mL ^-1 of CEA measured in human serum are 3.63% smaller than those measured in PBS buffer, respectively. 1.12%, 8.37%. It shows that the invention has certain anti-interference ability when detecting CEA, and has clinical application potential.

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

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

Example 8: Practical application value

Finally, in order to verify the practical application value of the kit, 5 different clinical serum samples obtained were tested. The experimental results are shown in the following table. The relative error between the measured results and the clinically measured data is less than 5%, indicating that the kit can detect actual clinical samples and has certain clinical application value.

sequence listing

<110> Henan University of Traditional Chinese Medicine

<120> CEA electrochemical detection kit and detection method based on eATRP signal amplification strategy

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 22

<212> DNA

<213> artificial sequence()

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shchatacca gcttattcaa tt 22

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<211> 19

<212> DNA

<213> artificial sequence()

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agggggtgaa gggataccc 19

Claims (10)

1. CEA electrochemical detection kit based on eATRP signal amplification strategy, is characterized in that, comprises the following raw materials: Apt1, Apt2, MCH, PEI, BMP, Me ₆ TREN, CuBr ₂ , KPF ₆ , FMMA, LiClO ₄ , EDC, NHS. 2 . The kit according to claim 1 , further comprising: DMSO, H ₂ SO ₄ , absolute ethanol, PBS buffer, and ultrapure water. 3 . 3. test kit according to claim 1, is characterized in that, Apt1 sequence: 5'-SH-(CH ₂ ) ₆ -ATACCAGCTTATTCAATT-3' Apt2 sequence: 5'-AGGGGGTGAAGGGATACCC-3'. 4. The test kit according to claim 1, characterized in that, during use, some raw materials are prepared into solutions, the Apt1 solution concentration is 1 μM, the Apt2 solution concentration is 100 μM, the EDC solution concentration is 100 μM, and the NHS solution concentration is 100 μM, The concentration of MCH solution is 2 mM, the concentration of PEI solution is 2 mg/mL, the concentration of BMP solution is 2 mM, the concentration of KPF ₆ solution is 0.1 M, the concentration of FMMA solution is 10 mM, the concentration of CuBr ₂ /Me ₆ TREN solution is CuBr ₂ and Me ₆ TREN Both are 10 mM, and the LiClO ₄ solution concentration is 1.0 M. 5. a method utilizing the test kit described in any one of claims 1-4 to detect CEA, is characterized in that, comprises the following steps: (1) Electrode modification ①Drop the Apt1 solution onto the gold electrode, react, wash, and dry; ② Soak the electrode of step ① in MCH solution, react, wash and blow dry; ③ drop the sample to be detected on the electrode surface of step ②, react, wash, and blow dry; ④ Add the Apt2-PEI solution dropwise to the electrode in step ③, react, wash, and blow dry; ⑤ Add the BMP solution dropwise to the electrode surface of step ④, react, wash, and blow dry; ⑥ Immerse the electrode in step ⑤ in the eATRP reaction solution, perform electrochemical polymerization with the i-t curve at a constant potential, and then treat the electrode with the LSV method to remove surface impurities; (2) Electrochemical determination The modified electrode was immersed in _LiClO4 solution, electrochemical detection was performed by SWV method, and the CEA content was analyzed according to the magnitude of the electrical signal. 6 . The method according to claim 5 , wherein the gold electrode is pretreated first, and the pretreatment method is as follows: polishing the surface of the gold electrode with 0.3 μm and 0.05 μm alumina powder respectively, and then sequentially using ultrapure The electrodes were ultrasonically cleaned with water, absolute ethanol and ultrapure water. The cleaned electrodes were immersed in the newly prepared piranha _acid solution for ₁₅ min, and the above cleaning steps were repeated. The electrodes were treated with cyclic voltammetry, the potential range was set to -0.3 to 1.5 V, the scan rate was 0.1 V/s, and the scanning was repeated until overlapping CV patterns were obtained. Finally, the electrodes were washed with ultrapure water and dried with nitrogen. 7. method according to claim 5 is characterized in that, the reaction temperature of step 1. is 37 ℃, and the time is 2h; the reaction temperature of step 2. is 37 ℃, and the time is 0.5h; the reaction temperature of step 3. is 37 ℃ , the time is 1h; the reaction temperature of step ④ is 37 ℃, the time is 1h; the reaction temperature of step ⑤ is 37 ℃, the time is 25min; the polymerization temperature of step ⑥ is room temperature, the time is 40min; the SWV of step (2) Scanning range: 0~0.8V, scanning rate: 1.0V/s, potential increment: 4mV. 8. method according to claim 5, is characterized in that, the preparation method of Apt2-PEI solution is: Mix equal volumes of Apt2 solution, EDC solution and NHS solution, then add to PBS buffer, shake and activate to obtain activated Apt2 solution; then add equal volume of PEI solution to mix, shake and react to obtain Apt2-PEI solution. 9. method according to claim 5, is characterized in that, the preparation method of eATRP reaction solution is: ① Dissolve CuBr ₂ and Me ₆ TREN in DMSO to prepare a CuBr ₂ /Me ₆ TREN solution with both CuBr ₂ and Me ₆ TREN concentrations of 10 mM; ② Mix 1.8 mL of DMSO, 0.1 mL of CuBr ₂ /Me ₆ TREN solution, 0.1 mL of FMMA solution and 8.0 mL of KPF ₆ solution to prepare an eATRP reaction solution. 10. A use of the kit according to any one of claims 1-4 in detecting CEA.

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