A High-Sensitivity cTnI Detection Kit Based on ARGET ATRP and ROP Dual Polymerization Cascade Amplification and Its Application

Abstract

The application discloses a high-sensitivity cTnI detection kit based on ARGET ATRP and ROP double polymerization cascade amplification, a use method and application thereof, and the kit comprises a gold electrode, Apt1, Apt2, MCH, BIBB, an ARGET ATRP reaction solution and NCA-Fc.The principle is that the Apt1 probe is self-assembled on the surface of the gold electrode through an Au-S bond, cTnI is captured through specific recognition, a sandwich structure is formed by introducing Apt2, the initiator BIBB is connected on the Apt2 through a bromination reaction, the generated ATRP polymer is used as a macromolecular initiator of a ROP reaction, and a large number of electroactive polymer chains are grafted to the electrode surface through the ROP reaction initiated by the amino group, so that the sensitivity of detection is improved.The linear range of detection is 100fg / mL-100ng / mL, and the detection limit is as low as 32.24fg / mL.Meanwhile, the application also has good accuracy and applicability when analyzing real serum samples, and lays a foundation for the detection of cTnI in the medical field and the early diagnosis of AMI in the later stage.

Description

Technical Field

[0001] This invention relates to a highly sensitive cTnI detection kit based on ARGET ATRP and ROP dual polymerization cascade amplification, its usage method, and its application, belonging to the field of bioanalytical technology. Background Technology

[0002] Myocardial infarction has become one of the most life-threatening cardiovascular diseases, with more than half of patients diagnosed with acute myocardial infarction (AMI). Some AMI patients experience few or no symptoms in the early stages, such as chest or back pain, shortness of breath, and heartburn. Therefore, developing sensitive and efficient methods for detecting AMI is crucial. AMI can be diagnosed using various cardiac biomarkers, including cardiac troponin I (cTnI), cardiac troponin T, creatine kinase MB, and myoglobin. Among these, cTnI is widely recognized as the preferred biomarker for diagnosing AMI. The high sensitivity and selectivity of cTnI detection are of great significance for the early diagnosis, disease assessment, and prognostic analysis of AMI.

[0003] Currently, various methods have been reported for the detection of cTnI, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, chemiluminescent immunoassay, fluorescence immunoassay, surface plasmon resonance (SPR), colorimetry, and electrochemical biosensors. Electrochemical biosensors are a typical product of multidisciplinary integration and development. As an emerging detection method, they have attracted widespread attention due to their advantages such as high sensitivity, simple operation, and strong specificity. In particular, aptamer-based electrochemical sensors are increasingly favored by researchers due to their advantages such as high specificity to antibodies, low immunogenicity, small size, good chemical stability, and ease of modification of electrochemical units.

[0004] Traditional electrochemical methods couple an electroactive label to a target biomolecule, offering simplicity and low cost, but their sensitivity is limited and cannot achieve highly sensitive detection. Studies have shown that compared to signal amplification via nanotechnology, polymerization reactions, by controlling the dynamic growth of polymer chains, can effectively improve detection sensitivity. Furthermore, they offer advantages such as mild reaction conditions and ease of operation. Among these methods, atom transfer radical polymerization (ATRP) has a wide range of applicable monomers and a narrow molecular weight distribution, making it suitable for preparing graft copolymers and other desirable properties. ARGET ATRP reactions are primarily based on using organic reducing agents such as ascorbic acid and glucose to balance CuI and CuII, reducing the Cu catalyst concentration from 10,000 ppm to 10 ppm, allowing for further controlled polymerization and showing great promise in industrial and biomedical applications. Ring-opening polymerization (ROP) is a highly efficient living polymerization reaction with advantages of atom economy and chemical specificity. The polymers prepared exhibit good biocompatibility and biodegradability, attracting widespread attention in agriculture, medicine, and pharmaceuticals.

[0005] In summary, the purpose of this invention is to develop an electrochemical reagent kit that uses NCA-Fc as a monomer and the generated ATRP polymer as a macromolecular initiator for the ROP reaction, based on the ARGET ATRP and ROP dual polymerization cascade amplification, which has the characteristics of high sensitivity and good selectivity, and can be used for highly sensitive detection of cTnI. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a highly sensitive cTnI detection kit based on ARGET ATRP and ROP dual polymerization cascade amplification, its usage method, and its application. This kit features good selectivity and high sensitivity, and also exhibits good accuracy and applicability when analyzing real serum samples.

[0007] To achieve the above objectives, one of the technical solutions of the present invention is as follows:

[0008] A highly sensitive cTnI detection kit based on ARGET ATRP and ROP dual polymerization cascade amplification is characterized by comprising: gold electrode, Apt1, Apt2, MCH, BIBB, ARGET ATRP reaction solution, NCA-Fc, LiClO4, DMSO, DMF, ethanol, and PBS buffer.

[0009] Furthermore, the sequences of Apt1 and Apt2 are as follows:

[0010] Apt1:5'-SH-(CH2)6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'

[0011] Apt2: 5'-NH2-(CH2)6-CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3'.

[0012] Furthermore, the preparation method of the ARGET ATRP reaction solution is as follows:

[0013] ① Dissolve CuBr2 and Me6TREN in DMSO to prepare a CuBr2 / Me6TREN solution with a concentration of 10 mM for both CuBr2 and Me6TREN;

[0014] ② Dissolve the monomeric NAMA in PBS buffer to prepare a 40 mM monomeric NAMA solution;

[0015] ③ Dissolve AA in PBS buffer to prepare an AA solution with a concentration of 2 mM;

[0016] ④ Take 0.4 mL of each of the solutions from ①②③ above, add 2.8 mL of PBS buffer and mix well to obtain the ARGET ATRP reaction solution.

[0017] Furthermore, the synthesis method of NCA-Fc is as follows:

[0018] (1) Weigh ferrocene carboxylic acid and dissolve it in DCM, add triethylamine dropwise, then add HBTU and HOBT in sequence. After the reaction, evaporate to dryness by rotation to obtain the product Fc-OBT.

[0019] (2) Add TFA dropwise to the DCM solution of Boc-L-Lys NCA. After the reaction, move the mixture to room temperature and stir overnight to obtain the NCA product.

[0020] (3) Triethylamine was added dropwise to the prepared Fc-OBT, and then the prepared NCA was added. The reaction was carried out at room temperature, and the reaction process was monitored by thin-layer chromatography. After the reaction was completed, post-processing was performed to obtain the monomer NCA-Fc.

[0021] Furthermore, in steps (1) and (2), the molar ratio of ferroceneic acid:triethylamine:HBTU:HOBT:Boc-L-Lys NCA is 1:2:1:1:1;

[0022] In step (2), the ratio of TFA:Boc-L-Lys NCA:DCM is 0.41 mL:1 mmol:3 mL;

[0023] The amount of triethylamine used in steps (1) and (3) is the same;

[0024] The dropping conditions in steps (1)-(3) are all 0℃; the reaction time in step (1) is 1h, and the reaction time in step (2) is 1h.

[0025] In step (3), the post-treatment involves washing with saturated NaHCO3 solution, 0.5M hydrochloric acid solution, saturated NaHCO3 solution and ultrapure water in sequence to separate the DCM organic phase, drying with anhydrous Na2SO4, filtering, and vacuum drying.

[0026] One of the technical solutions of the present invention is: a method of using the aforementioned test kit, comprising the following steps:

[0027] (1) Modification of gold electrodes

[0028] ① Add Apt1 solution dropwise onto the gold electrode, allow it to react, wash, and dry.

[0029] ②Immerse the electrode from step ① in MCH solution, react, wash, and dry;

[0030] ③ Add the sample to be tested onto the electrode surface from step ②, incubate, wash, and dry;

[0031] ④ Add the Apt2 solution dropwise to the electrode from step ③, allow it to react, wash, and dry.

[0032] ⑤ Immerse the electrode from step ④ in BIBB solution, allow it to react, wash, and dry.

[0033] ⑥ Immerse the electrode from step ⑤ in ARGET ATRP reaction solution, react, wash, and dry;

[0034] ⑦ Add the NCA-Fc solution dropwise onto the electrode surface from step ⑥, allow it to react, wash, and dry.

[0035] (2) Electrochemical detection

[0036] The modified electrode was immersed in LiClO4 solution, and the current response was recorded by square wave voltammetry. The cTnI content was analyzed based on the magnitude of the electrical signal.

[0037] Furthermore, the gold electrode undergoes pretreatment, the pretreatment method of which is as follows:

[0038] ① The gold electrode was ultrasonically washed with anhydrous ethanol and ultrapure water, respectively;

[0039] ② The surface of the gold electrode was polished and ground using 0.3μm and 0.05μm alumina powders, respectively;

[0040] ③ Wash with ultrasonic water, anhydrous ethanol and ultrapure water in sequence;

[0041] ④ Soak the gold electrode in freshly prepared piranha acid solution;

[0042] ⑤ Repeat step ③;

[0043] ⑥ Immerse the electrode in 0.5M H2SO4 solution, set the potential range to -0.3 to 1.5V, and the scan rate to 0.2V / s. Repeat the scan until overlapping cyclic voltammetry diagrams are obtained.

[0044] ⑦ Wash the electrode with ultrapure water and dry it with nitrogen.

[0045] Further, the reaction temperature of step ① is 35-40℃ and the time is 2.0h; the reaction temperature of step ② is 35-40℃ and the time is 0.5h; the incubation temperature of step ③ is 35-40℃ and the time is 1.0-2.0h; the reaction temperature of step ④ is 35-40℃ and the time is 1.0h; the reaction temperature of step ⑤ is 35-40℃ and the time is 0.5-1.0h; the reaction temperature of step ⑥ is 37℃ and the time is 0.5-1.0h; the reaction temperature of step ⑦ is room temperature and the time is 1.0-1.5h; the increase potential of the square wave voltammetry in step (2) is 4.0mV, the potential amplitude is 25mV, and the resting time is 30s.

[0046] One of the technical solutions of the present invention is the application of the aforementioned detection kit in the preparation of cTnI detection reagents.

[0047] The construction process of the detection method of the present invention is as follows: Figure 1 As shown.

[0048] The beneficial effects of this invention are:

[0049] 1. This invention presents a novel cTnI aptamer detection kit based on ARGET ATRP and ROP dual polymerization cascade amplification. It avoids the use of nanomaterials (complex operation, high cost) and biological enzymes (susceptible to external environmental factors such as pH and temperature) in commonly used signal amplification strategies, greatly improving sensitivity. Furthermore, this kit also offers advantages such as good selectivity and strong stability.

[0050] 2. Ferrocene and its derivatives possess excellent redox properties, stability, and low toxicity, and have been widely used as electroactive probes for electrochemical sensors. The monomer NCA-Fc synthesized in this invention can be readily obtained under mild reaction conditions at room temperature. When this monomer undergoes the ROP reaction, high-temperature and high-pressure reaction conditions are not required, making the operation relatively simple. Furthermore, it can be directly grafted onto the electrode surface, enabling its efficient application in cTnI detection.

[0051] 3. This invention successfully constructed a novel electrochemical aptamer detection kit based on an ARGET ATRP and ROP dual-polymerization amplification strategy. The kit includes: a gold electrode, Apt1, Apt2, MCH, BIBB, ATRP reaction solution, and NCA-Fc. The Apt1 probe self-assembles on the gold electrode surface via Au-S bonds, specifically recognizing and capturing cTnI. Apt2 is introduced to form a sandwich structure, and the initiator BIBB is linked to Apt2 via bromination. Simultaneously, the generated ATRP polymer serves as a macromolecular initiator for the ROP reaction. A large number of electroactive polymer chains are grafted onto the electrode surface via an amino-initiated ROP reaction, improving detection sensitivity. The detection linear range is 100 fg / mL to 100 ng / mL, with a detection limit as low as 32.24 fg / mL. After two weeks of continuous storage, the stability reaches 92.28% of the initial value, and the anti-interference ability reaches 95.2% to 98.7%. Meanwhile, this invention also has good accuracy and applicability when analyzing real serum samples, laying the foundation for its later application in the medical field for the detection of cTnI and the early diagnosis of AMI. Attached Figure Description

[0052] Figure 1 This is a schematic diagram illustrating the construction of a cTnI detection kit based on the ARGET ATRP and ROP dual-signal amplification strategy.

[0053] Figure 2 The image shows the infrared spectrum of the monomer NCA-Fc.

[0054] Figure 3 A shows the SWV signal diagrams of electrodes under different modification conditions; Figure 3 B represents the CV curves at different scan rates; Figure 3 C is the EIS curve of the electrode after each step of modification; Figure 3 D is the CV curve of the electrode after each step of modification.

[0055] Figure 4 Atomic force microscopy (AFM) characterization of the electrode surface before and after the ARGET ATRP and ROP reactions.

[0056] Figure 5 The water contact angle (WCA) of different modified electrode surfaces was characterized.

[0057] Figure 6 A represents the optimization of the BIBB reaction time; Figure 6 B represents the optimization of ARGET ATRP response time; Figure 6 C represents the optimization of the ROP reaction time.

[0058] Figure 7 A represents the SWV current intensity at different concentrations of cTnI; Figure 7 B is a graph showing the linear relationship between cTnI concentration and current intensity.

[0059] Figure 8 A represents a selectivity study of the kit of this invention; Figure 8 B represents the anti-interference study of the reagent kit of this invention. Detailed Implementation

[0060] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0061] cTnI specific antigen standard solution (L4C00102) was purchased from Shanghai Lingchao Biotechnology Co., Ltd.

[0062] Aptamers Apt1 and Apt2 were both synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their sequences are as follows:

[0063] Apt1:5'-SH-(CH2)6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'

[0064] (SEQ ID NO.1).

[0065] Apt2: 5'-NH2-(CH2)6-CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3' (SEQ ID NO. 2).

[0066] Example 1: Preparation of Materials

[0067] (1) Preparation of ARGET ATRP reaction solution:

[0068] ① Dissolve CuBr2 and tris(2-dimethylaminoethyl)amine (Me6TREN) in dimethyl sulfoxide (DMSO) to prepare a CuBr2 / Me6TREN solution with a concentration of 10 mM for both CuBr2 and Me6TREN.

[0069] ② Dissolve the monomer N-(3-aminopropyl)methacrylamide (NAMA) in PBS buffer to prepare a 40 mM monomer NAMA solution;

[0070] ③ Dissolve ascorbic acid (AA) in PBS buffer to prepare an AA solution with a concentration of 2 mM;

[0071] ④ Take 0.4 mL of each of the solutions from ①②③ above, add 2.8 mL of PBS buffer and mix well to obtain the ARGET ATRP reaction solution.

[0072] (2) Synthesis of NCA-Fc:

[0073] ① Weigh 3 mmol of ferrocene carboxylic acid and dissolve it in 60 mL of anhydrous dichloromethane (DCM). Slowly add 6 mmol of triethylamine at 0 °C, then add 3 mmol of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and 3 mmol of 1-hydroxybenzotriazole (HOBT) in sequence. After reacting at 0 °C for 1 h, evaporate to dryness by rotary evaporation to obtain the product Fc-OBT.

[0074] ② 1230 μL of trifluoroacetic acid (TFA) was slowly added dropwise to a solution of tert-butoxycarbonyl-L-lysine intracyclic anhydride (Boc-L-Lys NCA, CAS No.: 33043-60-6) at 0 °C (3 mmol of Boc-L-Lys NCA dissolved in 9 mL of DCM). After reacting at 0 °C for 1 h, the mixture was transferred to room temperature and stirred overnight to obtain the NCA product.

[0075] ③ 6 mmol of triethylamine was slowly added dropwise to the prepared Fc-OBT DCM solution (solvent: 20 mL DCM) at 0 °C. Then, the prepared NCA was added to the mixture. The reaction was carried out at room temperature, and the reaction process was monitored by thin-layer chromatography. After the reaction was completed, the mixture was washed successively with saturated NaHCO3 solution, 0.5 M hydrochloric acid solution, saturated NaHCO3 solution and ultrapure water to separate the DCM organic phase. The mixture was dried with anhydrous Na2SO4, filtered, and vacuum dried to obtain the monomer NCA-Fc.

[0076] To verify the successful synthesis of NCA-Fc, it was characterized using Fourier transform infrared spectroscopy. Figure 2 This is the infrared spectrum of the monomer NCA-Fc. Figure 2 It can be known that 3088cm -1 The peak at 1162 cm⁻¹ is attributed to the tensile vibration of the CH bond in the ferrocene ring. The characteristic peak of the tensile vibration of the C-C bond in the ferrocene ring is at 1162 cm⁻¹. -1 494cm -1 The peak at 3290 cm⁻¹ is a characteristic peak of the C-Fe bond stretching vibration. Additionally, the peak at 3290 cm⁻¹... -1 and 1665cm -1 The peak values ​​at 1256 cm⁻¹ are the characteristic peak values ​​of the NH bond tensile vibration and deformation vibration, respectively. -1 The peak at that point originates from the stretching vibration of the C=O bond. The stretching vibration of the C=O bond in the amide bond is at 1530 cm⁻¹. -1 The peak value appears at [a certain point]. Therefore, these results demonstrate the successful preparation of the monomer NCA-Fc.

[0077] Example 2: Construction of the reagent kit

[0078] A highly sensitive cTnI detection kit based on the ARGET ATRP and ROP dual-signal amplification strategy includes: gold electrode, Apt1, Apt2, 6-mercapto-1-hexanol (MCH), 2-bromoisobutyryl bromide (BIBB), ARGET ATRP reaction solution, NCA-Fc, PBS buffer, LiClO4, DMSO, DMF, ethanol, and ultrapure water.

[0079] When using this product, some of the ingredients need to be prepared into solutions. The concentrations of Apt1 solution are 1 μM, MCH solution is 2 mM, Apt2 solution is 1 μM, BIBB solution is 10 mM, NCA-Fc solution is 40 mM, PBS buffer is 0.1 M with pH 7.4, and LiClO4 solution is 1.0 M.

[0080] Example 3: Detection Method

[0081] A method for detecting cTnI includes the following steps:

[0082] (1) Electrode pretreatment

[0083] ① The gold electrode was ultrasonically washed for 30 seconds with anhydrous ethanol and ultrapure water, respectively;

[0084] ② Polish the surface of the gold electrode with 0.3μm and 0.05μm alumina powder respectively for 3-5 minutes;

[0085] ③ Wash with ultrapure water, anhydrous ethanol and ultrapure water in sequence for 30 seconds;

[0086] ④ Soak the gold electrode in freshly prepared piranha acid solution (30% H2O2 solution and 98% H2SO4 solution, v / v = 1:3) for 15 min;

[0087] ⑤ Repeat step ③;

[0088] ⑥ Immerse the electrode in 0.5M H2SO4 solution, set the potential range to -0.3 to 1.5V, and the scan rate to 0.2V / s. Repeat the scan until overlapping cyclic voltammetry diagrams are obtained.

[0089] ⑦ Wash the electrode with ultrapure water and dry it with nitrogen.

[0090] (2) Modification of gold electrodes

[0091] ① Add 10 μL of Apt1 solution (1 μM) dropwise to the gold electrode, react at 37 °C for 2 h, wash, and dry.

[0092] ②Immerse the electrode from step ① in 300μL MCH solution (2mM), react at 37℃ for 0.5h, wash and dry.

[0093] ③ Add 10 μL of the sample solution to be tested (containing cTnI specific antigen standard solution) to the electrode in step ②, incubate at 37℃ for 1 h, wash, and dry.

[0094] ④ Add 10 μL of Apt2 solution (1 μM) dropwise to the electrode from step ③, react at 37 °C for 1 h, wash, and dry.

[0095] ⑤ Immerse the electrode from step ④ in 200 μL of BIBB solution (10 mM), react at 37 °C for 40 min, wash, and dry.

[0096] ⑥ Immerse the electrode from step ⑤ in 200 μL of ARGET ATRP reaction solution, react at 37 °C for 50 min, wash, and dry.

[0097] ⑦ Add 10 μL of NCA-Fc solution (40 mM) to the electrode surface in step ⑥, react under vacuum at room temperature for 1 h (ROP reaction), wash, and dry.

[0098] Steps ①, ③, ④, and ⑥ involve gently washing the gold electrode surface with PBS buffer to remove unreacted substances and avoid nonspecific adsorption. Step ② involves washing with 70% (v / v) ethanol solution, step ⑤ involves washing with DMSO solution, and step ⑦ involves washing with 30% (v / v) DMF solution. Steps ①-⑦ involve drying with nitrogen gas.

[0099] (3) SWV detection

[0100] The electrode obtained in step (2) was immersed in 15 mL of LiClO4 solution (1.0 M), and the current response was recorded by square wave voltammetry (SWV) (potential increase: 4.0 mV; potential amplitude: 25 mV; rest time: 30 s). The content of cTnI was analyzed by the magnitude of the electrical signal.

[0101] Example 4: Feasibility Verification

[0102] To evaluate the feasibility of this invention for detecting cTnI, the redox current signals of a series of modified electrodes were measured using SWV and compared. The SWV curves of various modified electrodes are shown in [Figure number missing]. Figure 3A. Curves a–g in the figure reflect that no significant current signal was observed within the potential range of ferrocene when none of the following substances—Apt1, MCH, cTnI, Apt2, BIBB, ARGET ATRP, or ROP—were added during electrode construction. This indicates that ring-opening polymerization did not occur, and the non-specific adsorption of signal units on the electrode surface was negligible. When all the above components were continuously modified onto the electrode surface, a strong oxidizing current signal with a peak potential of ~0.41V was clearly observed (curve h). Therefore, the above experimental results demonstrate that the invented kit is feasible for cTnI detection.

[0103] Example 5: Electrochemical characterization of the modified electrode

[0104] To verify the actual occurrence of the polymerization reaction, the modified electrode was characterized using cyclic voltammetry (CV) in 1.0 M LiClO4 solution. The effect of different scan rates on the redox current intensity was investigated, with potentials ranging from 0 to 0.8 V. The results are as follows: Figure 3 As shown in B, in the range of 0.01–0.25 V s -1 At the scan rate, the peak current showed a good linear relationship with the scan rate, indicating that the electroactive polymer was fixed to the gold electrode by covalent bonds, which was independent of diffusion.

[0105] To demonstrate the success of the electrochemical aptamer kit construction process, a solution of 5 mM [Fe(CN)6] containing 0.1 M KNO3 was prepared. 3- / 4- Electrochemical impedance spectroscopy (EIS) spectra were collected stepwise at the same electrode during the continuous preparation process in an electrolyte solution. In the Nyquist plot, the diameter of the semicircle in the high-frequency region represents the charge transfer resistance (Ro). ct This is the resistance encountered by electrons during their transfer at the electrode surface. The result is as follows: Figure 3 As shown in C, the R of the bare gold electrode ct The Ω value is only 256 Ω (curve a), indicating that the electrode surface is clean and electrons can transfer rapidly between the electrode and solution interface. Modifying the electrode surface with Apt1, due to the formation of a self-assembled monolayer with [Fe(CN)6]... 3- / 4- Electrostatic repulsion exists between solutions, leading to R ct Increase (~597Ω, curve b). Subsequently, excess binding sites on the electrode surface are occupied by MCH, leading to R... ct (~810Ω, curve c) further increases. Next, cTnI specifically recognizes Apt1, forming a protein layer on the electrode surface, which hinders electron transfer efficiency, leading to R... ct Further increases (~1156Ω, curve d). Subsequently, the introduction of Apt2 forms a sandwich structure, leading to R ctContinue increasing (~1560Ω, curve e). After the initiator BIBB is fixed to the electrode, R ct (~2106Ω, curve f) increases again. After the ARGET ATRP reaction occurs, R ct The Ω value (~926 Ω, curve g) decreased significantly, which is attributed to the large number of amino groups on the ARGET ATRP polymer promoting electron transfer efficiency. Finally, after the ROP reaction, a large number of hydrophobic polymer links branched onto the electrode surface R ct A significant increase (~3903 Ω, curve h). The above EIS results indicate that the construction process of this invention is successful.

[0106] Furthermore, the characteristics of the electrode surface under different modification steps were evaluated using cyclic voltammetry. The results are as follows: Figure 3 As shown in Figure D, a pair of distinct redox peaks are observed at the interface of the bare gold electrode (curve a). When Apt1, MCH, cTnI, Apt2, and BIBB (curves b–f) are gradually added to the electrode surface, the peak current decreases sequentially with decreasing electron transfer rate, consistent with the EIS results. After the ARGET ATRP reaction occurs on the electrode surface, the peak current significantly increases (curve g). Finally, the peak current significantly decreases after the ROP reaction (curve h), which is attributed to the increased steric hindrance due to the large number of electroactive polymer chains. These results demonstrate the feasibility of the prepared cTnI detection kit.

[0107] Example 6: Characterization of electrode surface morphology

[0108] To further demonstrate the formation of the polymer, the surface morphology of the modified electrode was observed using atomic force microscopy (AFM). The gold electrode modified with Apt1 / MCH / cTnI / Apt2 / BIBB had a height of 17.0 nm. Figure 4 A) Following the ARGETATRP reaction, the gold electrode height increases to 26.8 nm due to polymer formation on the gold electrode. Figure 4 B). Furthermore, after further ROP reaction, its surface height continued to increase to 38.0 nm (B). Figure 4 (C) This is because a large number of electroactive polymer chains are attached to the electrode surface via an amino-initiated ROP reaction, indicating the feasibility of the prepared cTnI detection kit. Furthermore, due to the different hydrophilicity and hydrophobicity of the modified electrode surface groups, the water contact angle (WCA) can be used to characterize the changes in the electrode surface groups. Results are as follows... Figure 5 As shown, the electrode surfaces before and after the ARGET ATRP and ROP reactions were characterized. The WCA value of Au / Apt1 / MCH / cTnI / Apt2 / BIBB was 82.5°. Figure 5A) After the ARGET ATRP reaction occurs on the electrode surface, the WCA value decreases to 78.8° due to the presence of a large number of hydrophilic amino groups in the polymer. Figure 5 B). Finally, after the ROP reaction, WCA increased sharply to 87.1° ( Figure 5 (c) This significant difference is due to the extensive modification of the electrode surface with hydrophobic polymer chains. The change in WCA further confirms the successful modification of the electrode. The above results demonstrate the feasibility of using this invention for the detection of cTnI.

[0109] Example 7: Optimization of Detection Conditions

[0110] To optimize the detection performance of the kit, this invention optimizes key parameters in the electrode construction process, including BIBB reaction time, ARGET ATRP and ROP reaction time, to improve the analytical performance of the kit.

[0111] (1) BIBB reaction time

[0112] As an initiator in the ARGET ATRP reaction, the amount of BIBB attached to the electrode directly affects the polymerization reaction. With increasing reaction time, more and more initiator adheres to the electrode, providing more reaction sites for polymerization, and the current intensity gradually increases. The corresponding current intensity relationship is as follows: Figure 6 As shown in Figure A, the results indicate that the current increases with reaction time in the first 40 minutes, but remains almost constant after 40 minutes, because the reaction has reached equilibrium. Therefore, 40 minutes was chosen as the optimal reaction time for BIBB.

[0113] (2) ARGET ATRP reaction time

[0114] Similarly, the effect of ARGET ATRP reaction time on cTnI detection was investigated. Figure 6 As shown in Figure B, the current intensity gradually increases with the extension of polymerization time, reaching its maximum value at around 50 minutes. This is because a large amount of polymer is grafted onto the electrode, and the steric hindrance on the electrode surface gradually increases. Therefore, the optimal reaction time for ATRP is 50 minutes.

[0115] (3) ROP reaction time

[0116] The number of ferrocene electroactive molecules on the electrode surface significantly affects the analytical performance of the kit. To ensure the analytical sensitivity of the kit, the effect of ROP reaction time on the oxidation current value was investigated. The results are as follows: Figure 6As shown in Figure C, when the ROP reaction time is within the range of 30–60 min, the current intensity increases with increasing reaction time, and then the signal intensity tends to stabilize. This is because the increased steric hindrance limits the further growth of the polymer chain. Therefore, the optimal ROP reaction time is selected as 60 min.

[0117] Example 8: Performance Analysis

[0118] Under optimal experimental conditions, the current intensity generated by different concentrations of cTnI (100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL) was measured to study the detection performance of this invention for cTnI. The results are as follows: Figure 7 As shown in Figure A, within the concentration range of 100 fg / mL to 100 ng / mL, the current response gradually increases with increasing cTnI concentration. Figure 7 As shown in Figure B, a good linear relationship exists between the logarithm of cTnI concentration and the current response. The linear regression equation is I(μA) = 1.045log[C]. cTnI / ng mL -1 +5.362(R) 2 =0.998), detection limit (C) LOD The concentration was 32.24 fg / mL (C). LOD = 3σ / slope, where σ represents the standard deviation of the blank group). The results show that the cTnI detection kit prepared in this invention has a wide linear range and a low detection limit, demonstrating great application potential in detecting ultra-low levels of cTnI. The detection range and detection limit of this invention are compared with other existing detection methods in the table below.

[0119]

[0120] Example 9: Selective Experiment

[0121] To verify the selectivity of this signal amplification method, this invention compared the current intensities generated by the method under the same conditions in the blank group, bovine serum albumin (BSA), carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), and estrogen receptor (ERα). Specifically, the electrochemical signal intensities generated by 10 ng / mL cTnI and other interfering proteins at the same concentration were compared. The results are as follows: Figure 8 As shown in Figure A, the current signals generated by other interfering proteins are relatively smaller than those of cTnI and are basically equivalent to the signals of the blank group, proving that the method of the present invention has high selectivity.

[0122] Example 10: Reproducibility and Stability Study

[0123] To evaluate the stability of this invention, the signal strength of the newly constructed electrode and the electrode after a period of storage were compared. Two sets of modified electrodes were prepared under the same conditions. The signal strength of one set was detected by SWV immediately after construction, while the other set was stored in a refrigerator at 4°C. After two weeks of storage, the current signal of the modified electrode still remained at 92.28% of the initial current. Therefore, this demonstrates that the method of this invention has good stability.

[0124] The reproducibility of the method of the present invention was studied through intra-batch and inter-batch experiments. Intra-batch and inter-batch experiments (n=3) were conducted under the same experimental conditions. When the concentration of cTnI was 10 ng / mL, the relative standard deviations for intra-batch and inter-batch experiments were 4.06% and 3.56%, respectively, demonstrating that the method of the present invention has good reproducibility for the detection of cTnI.

[0125] Example 11: Anti-interference performance and analysis of actual samples

[0126] To evaluate the anti-interference capability of the method of the present invention, three different concentrations of cTnI (1 pg / mL, 100 pg / mL, and 10 ng / mL) were prepared using 5% (v / v) and 10% (v / v) human serum samples, and their current intensities were compared with the current signals of the same concentration of cTnI in PBS buffer. The results are as follows: Figure 8 As shown in Figure B, when the concentrations of cTnI are 1 pg / mL, 100 pg / mL, and 10 ng / mL, the current signals from 5% and 10% normal human serum (NHS) are 95.2% and 97.6%, 97.7% and 98.5%, and 97.7% and 98.7% of the peak current from the PBS buffer, respectively. This indicates that the method of the present invention has strong anti-interference ability for the detection of cTnI in normal human serum.

[0127] Finally, to verify the practical application value of the kit, six different clinical serum samples were tested. The use of clinical samples in this study was approved by the Ethics Committee of Henan Provincial Hospital of Traditional Chinese Medicine. The results obtained by this method are compared with those of commonly used clinical fluorescence immunoassay in the table below, with a relative error range of -4.52% to 3.26% and an RSD of less than 5%.

[0128] These results indicate that the kit is accurate in detecting cTnI in clinical samples and has certain clinical application value.

[0129]

Claims

1. A high-sensitivity cTnI detection kit based on ARGET ATRP and ROP dual polymerization cascade amplification, characterized in that, include: Gold electrode, Apt1, Apt2, 6-mercapto-1-hexanol (MCH), 2-bromoisobutyryl bromide (BIBB), ARGET ATRP reaction solution, NCA-Fc; The sequences of Apt1 and Apt2 are as follows: Apt1:5'-SH-(CH2)6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3' Apt2: 5'-NH2-(CH2)6-CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3'; The preparation method of ARGET ATRP reaction solution is as follows: ① Dissolve CuBr2 and tris(2-dimethylaminoethyl)amine (Me6TREN) in DMSO to prepare a CuBr2 / Me6TREN solution with a concentration of 10 mM for both CuBr2 and Me6TREN. ② Dissolve the monomer N-(3-aminopropyl)methacrylamide (NAMA) in PBS buffer to prepare a 40 mM monomer NAMA solution; ③ Dissolve ascorbic acid (AA) in PBS buffer to prepare an AA solution with a concentration of 2 mM; ④ Take 0.4 mL of each of the solutions from ①②③ above, add 2.8 mL of PBS buffer and mix well to obtain the ARGET ATRP reaction solution.

2. The detection kit according to claim 1, characterized in that, It also includes LiClO4, DMSO, DMF, ethanol, and PBS buffer.

3. The detection kit according to claim 1, characterized in that, The synthesis method of NCA-Fc is as follows: (1) Weigh ferrocene carboxylic acid and dissolve it in DCM, add triethylamine dropwise, and then add benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBT) in sequence. After the reaction, evaporate to dryness by rotation to obtain the product Fc-OBT. (2) Trifluoroacetic acid (TFA) was added dropwise to a DCM solution of tert-butoxycarbonyl-L-lysine intracyclic anhydride (Boc-L-Lys NCA). After the reaction, the solution was moved to room temperature and stirred overnight to obtain NCA. (3) Triethylamine was added dropwise to the prepared Fc-OBT, and then the prepared NCA was added. The reaction was carried out at room temperature, and the reaction process was monitored by thin-layer chromatography. After the reaction was completed, post-processing was performed to obtain the monomer NCA-Fc.

4. The detection kit according to claim 3, characterized in that, In steps (1) and (2), the molar ratio of ferroceneic acid:triethylamine:HBTU:HOBT:Boc-L-Lys NCA is 1:2:1:1:1; In step (2), the ratio of TFA:Boc-L-Lys NCA:DCM is 0.41 mL:1 mmol:3 mL; The amount of triethylamine used in steps (1) and (3) is the same; The dropping conditions in steps (1)-(3) are all 0℃; the reaction time in step (1) is 1 h, and the reaction time in step (2) is 1 h; In step (3), the post-treatment involves washing with saturated NaHCO3 solution, 0.5 M hydrochloric acid solution, saturated NaHCO3 solution and ultrapure water in sequence to separate the DCM organic phase, drying with anhydrous Na2SO4, filtering, and vacuum drying.

5. The detection kit according to any one of claims 1-4, characterized in that, The method of using the kit includes the following steps: (1) Modification of gold electrodes ① Add Apt1 solution dropwise onto the gold electrode, allow it to react, wash, and dry. ②Immerse the electrode from step ① in MCH solution, react, wash, and dry; ③ Add the sample to be tested onto the electrode surface from step ②, incubate, wash, and dry; ④ Add the Apt2 solution dropwise to the electrode from step ③, allow it to react, wash, and dry. ⑤ Immerse the electrode from step ④ in BIBB solution, allow it to react, wash, and dry. ⑥ Immerse the electrode from step ⑤ in ARGET ATRP reaction solution, react, wash, and dry; The NCA-Fc solution was added dropwise to the electrode surface in step ⑥, reacted, washed, and dried. (2) Electrochemical detection The modified electrode was immersed in LiClO4 solution, and the current response was recorded by square wave voltammetry. The cTnI content was analyzed based on the magnitude of the electrical signal.

6. The detection kit according to claim 5, characterized in that, The gold electrodes first undergo pretreatment, which is as follows: The gold electrode was ultrasonically washed with anhydrous ethanol and ultrapure water, respectively. The gold electrode surface was polished and ground using 0.3 μm and 0.05 μm alumina powder, respectively. The sample was ultrasonically washed sequentially with ultrapure water, anhydrous ethanol, and ultrapure water. The gold electrode was soaked in a freshly prepared piranha acid solution. Repeat steps ; The electrode was immersed in 0.5 M H2SO4 solution with a potential range of -0.3 to 1.5 V and a scan rate of 0.2 V / s. The scan was repeated until overlapping cyclic voltammetry plots were obtained. The electrode was washed with ultrapure water and dried with nitrogen.

7. The detection kit according to claim 5, characterized in that, The reaction temperature for step ① is 35-40℃, and the reaction time is 2.0 h; the reaction temperature for step ② is 35-40℃, and the reaction time is 0.5 h; the incubation temperature for step ③ is 35-40℃, and the incubation time is 1.0-2.0 h; the reaction temperature for step ④ is 35-40℃, and the reaction time is 1.0 h; the reaction temperature for step ⑤ is 35-40℃, and the reaction time is 0.5-1.0 h; the reaction temperature for step ⑥ is 37℃, and the reaction time is 0.5-1.0 h; The reaction temperature is room temperature and the time is 1.0-1.5 h; in step (2), the increase potential of the Chinese wave voltammetry is 4.0 mV, the potential amplitude is 25 mV, and the resting time is 30 s.

8. The use of the detection kit as described in any one of claims 1-4 in the preparation of cTnI detection reagents.

Images