Highly sensitive cTnI detection kit based on PEI-functionalized GO and ROP dual signal amplification strategy and its application

Abstract

The present invention discloses a highly sensitive cTnI detection kit and application based on a dual signal amplification strategy of PEI-functionalized GO and ROP. The kit includes: a gold electrode, Apt1, MCH, Apt2-GO-PEI bioconjugate, and NCA-Fc. The present invention successfully constructs a novel electrochemical aptamer detection kit based on a dual signal amplification strategy of PEI-functionalized GO and ROP. The Apt1 probe self-assembles on the surface of the gold electrode through Au-S bonds and captures cTnI through specific recognition. The Apt2-GO-PEI bioconjugate serves as a macromolecular initiator of the ROP reaction, and the NCA-Fc serves as a monomer of the ROP reaction. A large number of electroactive polymer chains are grafted to the electrode surface through an amino-triggered ROP reaction. The combined application of PEI-functionalized GO and NCA-Fc not only ensures that the reaction has abundant active sites, but also greatly improves the signal intensity and the sensitivity of detection. At the same time, the present invention has good selectivity, reproducibility, stability and anti-interference properties.

Description

Technical Field

[0001] The present invention relates to a highly sensitive cTnI detection kit and application based on a dual signal amplification strategy of polyethyleneimine (PEI) functionalized graphene oxide (GO) and ring-opening polymerization (ROP), belonging to the technical field of bioanalysis. Background Art

[0002] Acute myocardial infarction (AMI) is the leading cause of death in patients with cardiovascular disease, and early diagnosis is crucial for treating the irreversible damage caused by AMI. Conventional electrocardiograms (ECGs) are inadequate for specific detection of AMI. Cardiac troponin I (cTnI) is considered the gold standard for diagnosing AMI due to its high specificity and sensitivity. Generally, the cTnI value in healthy individuals is usually below 0.3 ng / mL, and a cTnI value of 1.0 ng / mL serves as the positive diagnostic threshold for AMI. Therefore, the development of a highly sensitive and specific cTnI electrochemical sensor is of great significance for the early diagnosis, disease assessment, and prognosis of AMI.

[0003] Currently, the main methods for detecting cTnI include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLI), fluorescence immunoassay (FLIA), surface plasmon resonance (SPR), colorimetry, and electrochemical sensing. Electrochemical sensing, due to its high sensitivity, rapid response, low cost, portability, and ease of miniaturization and automation, is playing an increasingly important role in the early diagnosis and prognosis of AMI. Aptamer-based electrochemical sensors, in particular, are gaining increasing favor among researchers due to their high specificity for antibodies, low immunogenicity, small size, excellent chemical stability, and ease of electrochemical unit modification.

[0004] To improve the analytical sensitivity of electrochemical sensors, researchers have developed numerous strategies for amplifying the output signal, including enzyme catalysis, nanomaterials, and polymer chains. Polymerization reactions achieve signal amplification by controlling the dynamic growth of polymer chains, effectively enhancing detection sensitivity. Among these, ring-opening polymerization (ROP) is a highly efficient living polymerization reaction with the advantages of atom economy and chemical specificity. The resulting polymers exhibit excellent biocompatibility and biodegradability. Ferrocene (Fc) and its derivatives are often used as electroactive probes in electrochemical sensors due to their excellent chemical stability, redox properties, and low toxicity. The most common method is the ring-opening polymerization of ferrocene oxirane, but this ROP reaction often requires high temperature and oxygen-free conditions. However, ROP, using α-amino acid-N-carboxylic anhydride (NCA) as a monomer, is a simple and effective method for preparing peptide polymers. It reacts at room temperature and enables the grafting of electroactive polymer chains onto biomolecules without affecting the protein structure, facilitating the widespread application of electrochemical biosensors in clinical testing. Therefore, Fc and NCA can be combined as monomers for the ROP reaction.

[0005] Graphene oxide (GO), a novel carbon nanomaterial, possesses exceptional properties, including large surface area, excellent dispersibility, and strong surface reactivity. The GO surface contains numerous oxygen-containing reactive groups, facilitating functional modification through covalent and coordination bonding with other materials or molecules. Polyethyleneimine (PEI), a water-soluble polymer containing numerous amino groups, can undergo ring-opening reactions with epoxy groups via covalent bonds. Therefore, PEI-functionalized GO (PEI-GO) can be prepared as a macromolecular initiator for ROP reactions.

[0006] In summary, the purpose of the present invention is to develop an electrochemical kit with NCA-Fc as the monomer, Apt2-GO-PEI as the initiator, and a dual signal amplification strategy based on PEI-GO nanomaterials and ROP polymerization reaction, so that it has the characteristics of high sensitivity and good selectivity, and can be used for the sensitive detection of cTnI. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a highly sensitive cTnI detection kit based on polyethyleneimine functionalized graphene oxide and ring-opening polymerization dual signal amplification and its use method, which has the characteristics of good selectivity and high sensitivity.

[0008] In order to achieve the above object, one of the technical solutions of the present invention is:

[0009] A highly sensitive cTnI detection kit based on a dual signal amplification strategy of PEI-functionalized GO and ROP, including: a gold electrode, Apt1, MCH, Apt2-GO-PEI bioconjugate, NCA-Fc, LiClO4, ultrapure water, and PBS buffer.

[0010] Furthermore, the sequence of Apt1 is:

[0011] 5'-SH-(CH2)6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'.

[0012] Furthermore, the synthesis method of NCA-Fc is:

[0013] (1) Ferrocenecarboxylic acid was weighed and dissolved in DCM. Triethylamine was added dropwise at 0°C, followed by HBTU and HOBT. After reacting for 1 h, the mixture was rotary evaporated to dryness to obtain the product Fc-OBT.

[0014] (2) TFA was added dropwise to the DCM solution of Boc-L-Lys NCA at 0°C. After reacting for 1 h, the mixture was moved to room temperature and stirred overnight to obtain the NCA product.

[0015] (3) Triethylamine was added dropwise to the prepared Fc-OBT at 0°C, and then the prepared NCA was added. The mixture was reacted at room temperature for 2 days. The reaction process was monitored by thin layer chromatography. After the reaction was completed, post-treatment was performed to obtain monomeric NCA-Fc.

[0016] in,

[0017] The molar ratio of ferrocenecarboxylic acid: triethylamine: HBTU: HOBT: Boc-L-Lys NCA in steps (1) and (2) is 1:2:1:1:1;

[0018] In step (2), the ratio of TFA:Boc-L-Lys NCA:DCM is 0.41 mL:1 mmol:3 mL;

[0019] The amount of triethylamine used in steps (1) and (3) is the same;

[0020] The post-treatment in step (3) is washing with saturated NaHCO3 solution, 0.5M hydrochloric acid solution, saturated NaHCO3 solution and ultrapure water in sequence, separating the DCM organic phase, drying with anhydrous Na2SO4, filtering and drying in vacuo.

[0021] Furthermore, the preparation method of Apt2-GO-PEI bioconjugate is as follows:

[0022] 4 mg of GO-PEI was dispersed in 2 mL of PBS buffer, and then 200 μL of 10 μM Apt2 solution was added to the dispersion and incubated at 37°C for 3 h. The product was centrifuged and washed, and finally the centrifuged product was redispersed with PBS buffer to obtain a uniform Apt2-GO-PEI bioconjugate.

[0023] The sequence of Apt2 is: 5'-CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3'.

[0024] One of the technical solutions of the present invention is: a method for using the detection kit, comprising the following steps:

[0025] (1) Electrode modification

[0026] ① Add Apt1 solution dropwise onto the gold electrode, react, wash, and blow dry;

[0027] ② Soak the electrode in step ① in MCH solution, react, wash, and blow dry;

[0028] ③ Add the sample to be tested to the electrode surface in step ②, react, wash, and blow dry;

[0029] ④ Add the Apt2-GO-PEI bioconjugate solution dropwise onto the electrode in step ③, react, wash, and blow dry;

[0030] ⑤ Add the NCA-Fc solution dropwise to the electrode surface in step ④, react, wash, and blow dry;

[0031] (2) Electrochemical detection

[0032] The modified electrode was immersed in LiClO4 solution, the current response was recorded by square wave voltammetry, and the cTnI content was analyzed based on the size of the electrical signal.

[0033] Furthermore, the gold electrode needs to be pre-treated, and the pre-treatment method is as follows:

[0034] ①Ultrasonic clean the gold electrode with anhydrous ethanol and ultrapure water respectively;

[0035] ② Polish the surface of the gold electrode with 0.3μm and 0.05μm alumina powder respectively;

[0036] ③Use ultrasonic washing with ultrapure water, anhydrous ethanol and ultrapure water in sequence;

[0037] ④ Soak the gold electrode in the freshly prepared piranha acid solution;

[0038] ⑤Repeat step ③;

[0039] ⑥ Immerse the electrode in 0.5M H2SO4 solution, set the potential range to -0.3~1.5V, scan rate to 0.2V / s, and repeat the scan until the overlapping cyclic voltammogram is obtained;

[0040] ⑦ Wash the electrode with ultrapure water and blow dry with nitrogen.

[0041] Furthermore, the reaction temperature of step ① is 35-40°C, and the time is 2 hours; the reaction temperature of step ② is 35-40°C, and the time is 0.5 hours; the incubation temperature of step ③ is 35-40°C, and the time is 1-2 hours; the reaction temperature of step ④ is 35-40°C, and the time is 1 hour; the reaction temperature of step ⑤ is room temperature, and the time is 1.0-1.5 hours; the increase potential of the square wave voltammetry in step (2) is: 4.0 mV, the potential amplitude: 25 mV, and the rest time: 30 seconds.

[0042] One of the technical solutions of the present invention is: use of the detection kit in preparing a cTnI detection reagent.

[0043] The construction process of the detection method of the present invention is as follows Figure 1 shown.

[0044] Beneficial effects of the present invention:

[0045] 1. This invention constructs a novel cTnI aptamer detection kit based on a dual signal amplification strategy using PEI-functionalized GO and ROP. This strategy avoids the use of enzymes (which are susceptible to environmental influences such as pH and temperature) in commonly used signal amplification strategies. The signal is amplified exponentially, resulting in high sensitivity, efficiency, and stability.

[0046] Ferrocene and its derivatives have excellent redox properties, stability, and low toxicity, and have been widely used as electroactive probes in electrochemical sensors. The monomer NCA-Fc synthesized in this invention can be successfully obtained under mild reaction conditions at room temperature. The monomer undergoes a relatively simple ROP reaction and can be directly grafted onto electrode surfaces, making it highly effective for cTnI detection.

[0047] 3. Based on a dual signal amplification strategy involving PEI-functionalized GO and ROP, this invention successfully constructed a novel electrochemical aptamer detection kit, achieving highly sensitive detection of cTnI. The Apt1 probe self-assembles on the gold electrode surface via Au-S bonds and specifically recognizes and captures cTnI. Subsequently, the Apt2-GO-PEI bioconjugate serves as a macromolecular initiator for the ROP reaction, and NCA-Fc serves as a monomer for the ROP reaction. A large number of electroactive polymer chains are grafted onto the electrode surface via an amino-triggered ROP reaction. The combined use of PEI-functionalized GO and NCA-Fc not only ensures abundant active sites for the reaction but also significantly enhances signal intensity and detection sensitivity. Under optimal conditions, this aptamer detection kit exhibits a wide linear range for cTnI detection (10 fg / mL to 10 ng / mL) with a detection limit as low as 3.78 fg / mL. Furthermore, the results demonstrate that the kit exhibits excellent selectivity, reproducibility, stability, and anti-interference properties. At the same time, the present invention has high sensitivity and high accuracy when analyzing real serum samples, and has great application potential in biomedical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the construction of a cTnI detection kit based on the dual signal amplification strategy of PEI-functionalized GO and ROP.

[0049] Figure 2 This is the infrared spectrum of monomer NCA-Fc.

[0050] Figure 3 A and 3B are transmission electron microscopy images of GO and Apt2-GO-PEI, respectively.

[0051] Figure 4 The zeta potential diagram of GO and Apt2-GO-PEI.

[0052] Figure 5 A is the SWV signal diagram of the electrode under different modification conditions; Figure 5 B is the CV curves at different scan rates; Figure 5 C is the EIS curve of the electrode modified in each step; Figure 5 D is the CV curve of the electrode after modification in each step.

[0053] Figure 6 Atomic force microscopy (AFM) characterization of the electrode surface before and after ROP reaction.

[0054] Figure 7 Water contact angle (WCA) characterization of different modified electrode surfaces.

[0055] Figure 8 A is the optimization of Apt2-GO-PEI reaction time; Figure 8 B is the optimization of monomer concentration (NCA-Fc); Figure 8 C is the optimization of ROP reaction time.

[0056] Figure 9 A is the SWV current intensity of different cTnI concentrations; Figure 9 B is the linear relationship between cTnI concentration and current intensity.

[0057] Figure 10 A is the selectivity study of the kit of the present invention; Figure 10 B is the anti-interference study of the kit of the present invention. DETAILED DESCRIPTION

[0058] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0059] cTnI-specific antigen standard solution (L4C00102) was purchased from Shanghai Lingchao Biotechnology Co., Ltd.

[0060] Aptamers Apt1 and Apt2 were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and their sequences are as follows:

[0061] Apt1: 5'-SH-(CH2)6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'(SEQ IDNO.1)

[0062] Apt2: 5'-CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3' (SEQ ID NO. 2).

[0063] Example 1: Synthesis of NCA-Fc

[0064] (1) Weigh 3 mmol of ferrocenecarboxylic acid and dissolve it in 60 mL of anhydrous dichloromethane (DCM). Slowly add 6 mmol of triethylamine dropwise at 0°C, then add 3 mmol of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and 3 mmol of 1-hydroxybenzotriazole (HOBT) in sequence. After reacting at 0°C for 1 h, the mixture was rotary evaporated to dryness to obtain the product Fc-OBT.

[0065] (2) 1230 μL of trifluoroacetic acid (TFA) was slowly added dropwise to a solution of tert-butyloxycarbonyl-L-lysine cyclic anhydride (Boc-L-Lys NCA, CAS No.: 33043-60-6) (3 mmol of Boc-L-Lys NCA dissolved in 9 mL of DCM) at 0°C. After reacting at 0°C for 1 h, the mixture was moved to room temperature and stirred overnight to obtain the NCA product (solution).

[0066] (3) 6 mmol of triethylamine was slowly added dropwise to the prepared Fc-OBT at 0°C, and then the prepared NCA solution was added to the mixture. The mixture was reacted at room temperature for 2 days. The reaction process was monitored by thin layer chromatography. After the reaction was completed, the mixture was washed with saturated NaHCO3 solution, 0.5 M hydrochloric acid solution, saturated NaHCO3 solution and ultrapure water in sequence. The DCM organic phase was separated, dried with anhydrous Na2SO4, filtered, and vacuum dried to obtain monomer NCA-Fc.

[0067] To verify the successful synthesis of NCA-Fc, it was characterized by Fourier transform infrared spectroscopy. Figure 2 is the infrared spectrum of monomer NCA-Fc. Figure 2 It can be seen that 3088cm -1 The peak at 1162 cm is attributed to the stretching vibration of the C-H bond of the ferrocene ring. -1 .494cm -1 The peak at 3290 cm is the characteristic peak of C-Fe bond stretching vibration. -1 and 1665cm -1 The peaks at 1256cm are the characteristic peaks of NH bond stretching vibration and deformation vibration. -1 The peak at 1530 cm comes from the stretching vibration of the C=O bond in the amide bond. -1 Therefore, these results demonstrate the successful preparation of monomeric NCA-Fc.

[0068] Example 2: Preparation of Apt2-GO-PEI Bioconjugate

[0069] (1) 20 mg of GO was added to 40 mL of ultrapure water and ultrasonically dispersed for 0.5 h. Then, 2 mL of PEI aqueous solution (2 mg / mL) was added dropwise to the above dispersion and stirred at room temperature for 24 h. After the reaction was completed, the product was purified by centrifugation and washed three times with ultrapure water. The GO-PEI solid product was obtained after vacuum drying.

[0070] (2) 4 mg of GO-PEI was dispersed in 2 mL of PBS buffer. 200 μL of Apt2 solution (10 μM) was then added to the dispersion. After incubation at 37°C for 3 h, the product was centrifuged and washed three times with PBS buffer. Finally, the centrifuged product was redispersed with PBS buffer to obtain a uniform Apt2-GO-PEI bioconjugate.

[0071] The morphology of GO and Apt2-GO-PEI bioconjugates was characterized by transmission electron microscopy. Figure 3A and 3B are transmission electron micrographs of GO and Apt2-GO-PEI, respectively. Figure 3 It can be seen that the morphology of GO reveals that the overall structure of GO is in the form of flakes with a rough and wrinkled surface ( Figure 3 A), when GO is covalently bound and electrostatically adsorbed, the specific surface area and interlayer spacing of Apt2-GO-PEI increase, and the surface becomes rougher and highly aggregated ( Figure 3 B).

[0072] Figure 4 The zeta potential diagram of GO and Apt2-GO-PEI. Figure 4 The results show that the zeta potential of GO dispersed in water is -41.5 mV, and the zeta potential of Apt2-GO-PEI is +30.5 mV, indicating that the functionalization of PEI increases the surface positive charge of GO.

[0073] Example 3: Construction of the kit

[0074] A highly sensitive cTnI detection kit based on PEI-functionalized GO and ROP dual signal amplification strategy includes: a gold electrode, Apt1, 6-mercapto-1-hexanol (MCH), Apt2-GO-PEI bioconjugate, NCA-Fc, PBS buffer, LiClO4, and N,N-dimethylformamide (DMF).

[0075] During use, some raw materials need to be prepared into solutions, among which the concentration of Apt1 solution is 1 μM, the concentration of MCH solution is 2 mM, the concentration of Apt2-GO-PEI bioconjugate is 2 mg / mL, the concentration of NCA-Fc solution is 100 mM, the concentration of LiClO4 solution is 1.0 M, the concentration of PBS buffer is 0.1 M, and the pH is 7.4.

[0076] Example 4: Detection method

[0077] A method for detecting cTnI, comprising the following steps:

[0078] (1) Electrode pretreatment

[0079] ① Ultrasonic wash the gold electrode with anhydrous ethanol and ultrapure water for 30 seconds respectively;

[0080] ② Polish the gold electrode surface with 0.3μm and 0.05μm alumina powder for 3-5 minutes respectively;

[0081] ③Use ultrasonic washing for 30 seconds in sequence with ultrapure water, anhydrous ethanol and ultrapure water;

[0082] ④ Soak the gold electrode in freshly prepared piranha acid solution (30% H2O2 solution and 98% H2SO4 solution, v / v = 1:3) for 15 minutes;

[0083] ⑤Repeat step ③;

[0084] ⑥ Immerse the electrode in 0.5M H2SO4 solution, set the potential range to -0.3~1.5V, scan rate to 0.2V / s, and repeat the scan until the overlapping cyclic voltammogram is obtained;

[0085] ⑦ Wash the electrode with ultrapure water and blow dry with nitrogen.

[0086] (2) Modification of gold electrode and ROP reaction

[0087] ① Add 10 μL of Apt1 solution (1 μM) onto the electrode, react at 37°C for 2 h, wash, and blow dry.

[0088] ② Soak the electrode prepared in step ① in 300 μL MCH solution (2 mM), react at 37°C for 0.5 h, wash, and blow dry.

[0089] ③ Add 10 μL of the test solution (cTnI-specific antigen standard solution) onto the electrode in step ②, incubate at 37°C for 1 hour, wash, and blow dry.

[0090] ④ Add 10 μL of the prepared Apt2-GO-PEI solution (2 mg / mL) dropwise to the electrode surface in step ③, react at 37°C for 1 h, wash, and blow dry.

[0091] ⑤ROP reaction: 10 μL of NCA-Fc solution (100 mM) was added dropwise to the electrode in step ④, and the reaction was carried out under vacuum at room temperature for 1.5 h, followed by washing and drying.

[0092] Wash the gold electrode surface with PBS buffer to remove unreacted substances and avoid nonspecific adsorption. Wash with 70% (v / v) ethanol solution in step 2 and 30% (v / v) DMF solution in step 5.

[0093] (3) SWV detection

[0094] The electrode obtained in step (2) was immersed in a LiClO4 solution (1.0 M), and the current response was recorded by square wave voltammetry (SWV) (increase potential: 4.0 mV; potential amplitude: 25 mV, rest time: 30 s). The cTnI content was analyzed by the size of the electrical signal.

[0095] Example 5: Feasibility Verification

[0096] In order to verify the feasibility of the proposed cTnI detection method, a series of blank control experiments were conducted. The SWV curves of the modified electrodes were compared by comparing the current intensity of the electrodes without different materials. Figure 5 A. In the absence of Apt1 (curve a), MCH (curve b), cTnI (curve c), Apt2-GO-PEI (curve d), and NCA-Fc (curve e), no obvious current signal was observed, indicating that ring-opening polymerization does not occur and the nonspecific adsorption of the signal unit on the electrode surface is negligible. When ROP / Apt2-GO-PEI / cTnI / MCH / Apt1 are modified on the electrode surface in sequence, a strong oxidation current signal with a peak potential of ~0.41V can be clearly observed (curve f). Therefore, the above experimental results show that the kit of the present invention is feasible for cTnI detection.

[0097] Example 6: Electrochemical Characterization of Modified Electrode

[0098] In order to prove the actual occurrence of ROP reaction, the modified electrode was characterized by CV in 1.0M LiClO4 solution to study the effect of different scan rates on the redox current intensity at a potential of 0 to 0.8 V. Figure 5 As shown in B, at 0.01~0.25V·s -1 At a scan rate of , there is a good linear relationship between the peak current and the scan rate, indicating that the redox process of ferrocene is independent of diffusion and the polymer is grafted onto the surface of the gold electrode.

[0099] To prove that the construction process of the electrochemical aptamer kit was successful, the step-by-step modified electrode was characterized by electrochemical impedance spectroscopy (EIS) in a solution containing potassium ferrocyanide and potassium ferrocyanide. In the Nyquist plot, the diameter of the semicircle in the high-frequency region is equal to the charge transfer resistance (R ct ). The result is as follows Figure 5 As shown in C, the bare gold electrode shows the smallest R ct (~259Ω, curve a), Apt1 is modified on the electrode surface, and the self-assembled monolayer formed by the [Fe(CN)6] 3- / 4- Electrostatic repulsion between solutions causes R ct After that, the occupation of the unbound active sites on the electrode surface by MCH leads to an increase in R ct Further increase (~1260Ω, curve c). Subsequently, Apt1 specifically recognizes cTnI to form a protein layer, and the difference in conductivity of cTnI biomolecules leads to R ct Apt2-GO-PEI was then added to the electrode surface. Due to the low conductivity of the Apt2-GO-PEI composite material, electron transfer was hindered, and R ct Finally, after the ROP reaction, a large number of hydrophobic polymer chains were grafted onto the electrode surface Rct The above EIS results prove that the fully modified electrode was successfully constructed.

[0100] In addition, the electrochemical interfacial properties of the gradually modified gold electrodes were evaluated by cyclic voltammetry. Figure 5 As shown in Figure 3D, the bare gold electrode exhibits a pair of distinct redox peaks at its interface (curve a). When Apt1, MCH, cTnI, and Apt2-GO-PEI (curves b–e) are gradually modified on the electrode surface, the peak current decreases sequentially with decreasing electron transfer rates, consistent with the EIS results. Notably, the peak current decreases significantly after the ROP reaction (22.00 A, curve f) due to the increased steric hindrance caused by the abundant electroactive polymer chains. These results demonstrate that the aptamer-based biorecognition interface is well assembled and that the prepared cTnI detection kit is feasible.

[0101] Example 7: Electrode surface morphology characterization

[0102] To further prove the formation of polymers, the surface morphology of the modified electrode was observed by atomic force microscopy (AFM). The formation of polymer chains was confirmed by comparing the changes in the electrode surface height before and after the polymerization reaction. Figure 6 A) The height of the modified electrode is 10.3 nm, and after ROP polymerization ( Figure 6 B) The height of the electrode is 32.8 nm, which is due to the fact that a large number of polymer chains are connected to the electrode surface through the ROP reaction initiated by the amino group, further proving the occurrence of the ROP reaction and indicating that the prepared cTnI detection kit is feasible.

[0103] In addition, the water contact angle (WCA) was used to characterize the step-by-step modified electrodes based on the differences in the hydrophilicity of the surface groups of the modified electrodes. Figure 7 It can be seen that due to the hydrophobicity of the gold electrode surface, its WCA value is 90.7° ( Figure 7 A). Due to the presence of hydrophilic phosphate groups in Apt1, the WCA decreased to 85.7° ( Figure 7 B). Next, due to the influence of the hydrophobic methylene and hydrophilic hydroxyl groups in the MCH molecule, the WCA of the electrode increased slightly (88.8°) ( Figure 7 C). cTnI ( Figure 7 D) and Apt2-GO-PEI( Figure 7 After E), the WCA decreased to 83.4° and 80.6°, respectively, due to the presence of amino and carboxyl groups in the molecule. Finally, after ROP reaction, the WCA increased sharply to 90.3° ( Figure 7F), which is due to the generation of a large number of hydrophobic polymer chains. The change in WCA further proves that the electrode has been successfully modified.

[0104] The above results demonstrate that the present invention is feasible for detecting cTnI.

[0105] Example 8: Optimization of detection conditions

[0106] In order to optimize the detection performance of the kit, the present invention optimizes important conditions in the electrode construction process, including Apt2-GO-PEI reaction time, monomer NCA-Fc concentration, and ROP reaction time, to improve the analytical performance of the kit.

[0107] (1) Apt2-GO-PEI reaction time

[0108] The specific binding between Apt2-GO-PEI bioconjugate and cTnI will directly affect the amount of primary amine attached to the electrode surface, and the amount of primary amine will provide sufficient active sites for the subsequent polymerization reaction. Therefore, the present invention optimizes the reaction time of Apt2-GO-PEI, and the corresponding current intensity relationship is as follows: Figure 8 As shown in Figure A, the results show that the peak current increases rapidly with the extension of the reaction time, reaching a maximum value at 60 minutes, after which the current no longer changes significantly. Therefore, 60 minutes was selected as the optimal reaction time for Apt2-GO-PEI.

[0109] (2) Monomer reaction concentration

[0110] In order to determine the optimal concentration of NCA-Fc, the present invention studied the peak current changes of the kit within the concentration range of 20 to 140 mM. Figure 8 As shown in Figure B, when the monomer concentration is between 20 and 100 mM, the oxidation current increases rapidly with increasing monomer concentration. Thereafter, increasing the monomer concentration does not significantly change the current intensity. Therefore, the optimal monomer concentration for the reaction is 100 mM.

[0111] (3) ROP reaction time

[0112] The number of ferrocene electroactive molecules on the electrode surface greatly affects the analytical performance of the kit. In order to improve the analytical sensitivity of the kit, the effect of ROP reaction time on the oxidation current value was studied. The results are shown in Figure 2. Figure 8 As shown in Figure C, when the ROP reaction time is within the range of 30 to 90 minutes, the current intensity increases with the reaction time, and then the signal intensity tends to stabilize. This is because the increase in steric resistance limits the further growth of the polymer chain. Therefore, the optimal ROP reaction time is selected as 90 minutes.

[0113] Example 9: Analytical Performance

[0114] Under the optimal experimental conditions, the current intensity generated by different concentrations of cTnI (10fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, 10ng / mL) was detected to study the detection performance of the present invention for cTnI. The results are as follows Figure 9 As shown in A, within the concentration range of 10fg / mL to 10ng / mL, the current response gradually increases with the increase of cTnI concentration. Figure 9 As shown in B, the cTnI current response and the logarithm of cTnI concentration showed a good linear relationship in the range of 10fg / mL to 10ng / mL. The linear regression equation was I(μA)=1.186log[C cTnI / pg mL -1 ]+3.590(R 2 =0.998), detection limit (C LOD ) was 3.78 fg / mL (C LOD = 3σ / slope, where σ represents the standard deviation of the blank group. The results demonstrate that the cTnI detection kit prepared by the present invention has a wide linear range and a low detection limit. The present method demonstrates great potential for detecting ultra-low levels of cTnI. A comparison of the detection range and detection limit of the present invention with other existing detection methods is shown in the table below.

[0115]

[0116] Example 10: Selectivity Experiment

[0117] To verify the selectivity of the signal amplification method, the present invention compared the current intensity generated by the blank group (Blank), bovine serum albumin (BSA), carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA 21-1) and estrogen receptor (ERα) under the same conditions. The concentrations of cTnI (1 ng / mL) and other interfering proteins were the same. Figure 10 As can be seen from Figure 4, the sample containing only cTnI (1 ng / mL) exhibited a significant oxidation current signal, while the current signals from BSA, CEA, CYFRA 21-1, and ERα were as low as 12.44%, 14.35%, 16.4%, and 12.08% of cTnI, respectively, which were comparable to the blank group signals, demonstrating that the method of the present invention has high selectivity.

[0118] Example 11: Reproducibility and stability studies

[0119] To evaluate the stability of the constructed fully modified electrodes, the present invention conducted storage experiments. Two sets of modified electrodes were prepared under identical conditions: one set was tested for signal intensity using SWV immediately after construction, while the other set was tested after being stored at 4°C for 14 days. The results showed that the modified electrodes retained 92.8% of the current signal after two weeks of storage compared to freshly prepared electrodes. This demonstrates the excellent stability of the present method.

[0120] The reproducibility of the method of the present invention was investigated through intra- and inter-batch experiments. Under identical experimental conditions, both intra- and inter-batch experiments (n = 3) were performed. At a cTnI concentration of 1 ng / mL, the intra- and inter-batch relative standard deviations were 1.97% and 3.31%, respectively, demonstrating the good reproducibility of the method for cTnI detection.

[0121] Example 12: Anti-interference and actual sample analysis

[0122] To evaluate the anti-interference ability of the method of the present invention, three different concentrations of cTnI (100 fg / mL, 10 pg / mL, and 1 ng / mL) were prepared using 5% (v / v) and 10% (v / v) human serum samples, and the current intensities were compared with the current signals of the same concentration of cTnI in PBS buffer. Figure 10 As shown in Figure B, when the concentrations of cTnI were 100 fg / mL, 10 pg / mL, and 1 ng / mL, respectively, the current signals from 5% and 10% normal human serum (NHS) were 97.8% and 95.9%, 98.7% and 96.7%, and 96.5% and 94.8% of the peak current from PBS buffer, respectively, indicating that the method of the present invention has strong anti-interference ability for the detection of cTnI in normal human serum.

[0123] The method's practical analytical capabilities were further evaluated by measuring serum samples from patients with acute myocardial infarction (AMI). Six clinical samples were provided by the First Affiliated Hospital of Henan University of Traditional Chinese Medicine. The use of clinical samples in this study was approved by the Ethics Committee of Henan Provincial Hospital of Traditional Chinese Medicine. The results of this method are compared with those of a commonly used clinical fluorescence immunoassay (FIA) in the table below. The relative errors ranged from -4.08% to 4.52%, with RSDs less than 5%. These results demonstrate that this kit accurately measures cTnI in clinical samples and has practical application value.

[0124]

Claims

1. A highly sensitive cTnI detection method for non-diagnostic and / or therapeutic purposes based on a PEI-functionalized GO and ROP dual signal amplification strategy, characterized in that: The materials used include: gold electrode, Apt1, MCH, Apt2-GO-PEI bioconjugate, NCA-Fc; The sequence of Apt1 is: 5'-SH-(CH2)6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'; The preparation method of Apt2-GO-PEI bioconjugate is as follows: 4 mg of GO-PEI was dispersed in 2 mL of PBS buffer, and then 200 μL of 10 μM Apt2 solution was added to the dispersion and incubated at 37°C for 3 h. The product was centrifuged and washed, and finally the centrifuged product was redispersed with PBS buffer to obtain a uniform Apt2-GO-PEI bioconjugate. The sequence of Apt2 is: 5'-CGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3' The synthesis method of NCA-Fc is: (1) Ferrocenecarboxylic acid was weighed and dissolved in DCM. Triethylamine was added dropwise at 0°C, followed by HBTU and HOBT. After reacting for 1 h, the mixture was rotary evaporated to dryness to obtain the product Fc-OBT. (2) TFA was added dropwise to the DCM solution of Boc-L-Lys NCA at 0°C. After 1 h of reaction, the mixture was moved to room temperature and stirred overnight to obtain the NCA product. (3) Triethylamine was added dropwise to the prepared Fc-OBT at 0°C, and then the prepared NCA was added. The reaction was allowed to react at room temperature for 2 days. The reaction process was monitored by thin layer chromatography. After the reaction was completed, post-treatment was performed to obtain monomeric NCA-Fc. The detection method comprises the following steps: (1) Modification of gold electrode and ROP reaction ① Add Apt1 solution dropwise onto the gold electrode, react, wash, and blow dry; ② Soak the electrode in step ① in MCH solution, react, wash, and blow dry; ③ Add the sample to be tested to the electrode surface in step ②, react, wash, and blow dry; ④ Add the Apt2-GO-PEI bioconjugate solution dropwise onto the electrode in step ③, react, wash, and blow dry; ⑤ Add the NCA-Fc solution dropwise to the electrode surface in step ④, react, wash, and blow dry; (2) Electrochemical detection The modified electrode was immersed in LiClO4 solution, the current response was recorded by square wave voltammetry, and the cTnI content was analyzed based on the size of the electrical signal.

2. The detection method according to claim 1, characterized in that The raw materials used also include LiClO4, ultrapure water, and PBS buffer.

3. The detection method according to claim 1, wherein In the synthesis method of NCA-Fc: The molar ratio of ferrocenecarboxylic acid: triethylamine: HBTU: HOBT: Boc-L-Lys NCA in steps (1) and (2) 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 post-treatment in step (3) is washing with saturated NaHCO3 solution, 0.5 M hydrochloric acid solution, saturated NaHCO3 solution and ultrapure water in sequence, separating the DCM organic phase, drying with anhydrous Na2SO4, filtering and vacuum drying.

4. The detection method according to claim 1, wherein Before use, the gold electrode needs to be pre-treated. The pre-treatment method is as follows: The gold electrode was ultrasonically cleaned with anhydrous ethanol and ultrapure water respectively; The gold electrode surface was polished with 0.3 μm and 0.05 μm alumina powder respectively; Ultrasonic washing was performed in sequence with ultrapure water, anhydrous ethanol, and ultrapure water; Soak the gold electrode in freshly prepared piranha acid solution; Repeat steps ; The electrode was immersed in 0.5 M H2SO4 solution, the potential range was set to -0.3~1.5 V, the scan rate was 0.2 V / s, and the scan was repeated until the overlapping cyclic voltammograms were obtained; Wash the electrode with ultrapure water and blow dry with nitrogen.

5. The detection method according to claim 1, wherein In the detection method, the reaction temperature of step ① is 35-40°C and the time is 2 h; the reaction temperature of step ② is 35-40°C and the time is 0.5 h; the incubation temperature of step ③ is 35-40°C and the time is 1-2 h; the reaction temperature of step ④ is 35-40°C and the time is 1 h; the reaction temperature of step ⑤ is room temperature and the time is 1.0-1.5 h; the increase potential of the square wave voltammetry in step (2) is: 4.0 mV, the potential amplitude is: 25 mV, and the rest time is: 30 s.

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