Highly sensitive cTnI detection kit and its application based on ROP signal amplification strategy induced by FFAPO

Abstract

The present invention discloses a highly sensitive cTnI detection kit and application based on the ROP signal amplification strategy triggered by FFAPO, and the kit includes an electrode, MPA, EDC, NHS, Ab1, BSA, Ab2, GA, potassium methoxide, FFAPO, and LiClO4. The present invention adopts a sandwich structure to introduce cTnI, and connects the initiator glycolic acid of the ROP reaction to Ab2 through an esterification reaction, so that a large amount of ferrocene electroactive polymer is grafted to the electrode surface through the ROP reaction, and successfully establishes a kit for detecting cTnI with high sensitivity and strong specificity. Experiments have shown that the linear range of the detection of the present invention is 1pg / mL to 1μg / mL, the correlation coefficient is 0.997, the detection limit is 57.14fg / mL, the stability reaches 94.8% of the initial value after continuous storage for two weeks, and the anti-interference reaches 96.2% to 103.1%. At the same time, it has good selectivity and reproducibility, which lays a foundation for the later application of cTnI detection in the medical field and early diagnosis of acute myocardial infarction.

Description

Technical Field

[0001] The invention relates to a highly sensitive cTnI detection kit and application thereof based on a ROP signal amplification strategy initiated by ferrocenylcarboxylic acid glycidyl ester (FFAPO), and belongs to the technical field of bioanalysis. Background Art

[0002] Acute myocardial infarction (AMI) is a disease that seriously threatens human life and health. It is the main cause of death from cardiovascular diseases and has a high mortality rate and morbidity. Cardiac troponin I (cTnI) has become the gold standard for diagnosing AMI because of its high myocardial specificity. Under normal circumstances, the cTnI concentration of normal healthy people is usually below 0.3ng / mL; a cTnI concentration higher than 2.0ng / mL means an increased risk of serious heart disease. Therefore, the development of a method with high sensitivity and high efficiency for detecting cTnI is of great significance in the fields of medicine and pathology.

[0003] At present, people have studied a variety of methods for detecting cTnI, such as enzyme-linked immunosorbent assay, chemiluminescent immunoassay, fluorescent immunoassay and electrochemical sensing. Among them, electrochemical methods have the advantages of high sensitivity, good selectivity, rapid analysis and simple operation compared with traditional technologies, and play an increasingly important role in the early diagnosis and prognosis of AMI. Therefore, it is of great significance to study a more sensitive cTnI detection method based on the existing electrochemical method. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a highly sensitive cTnI detection kit and application based on a ROP signal amplification strategy triggered by ferrocenylcarboxylic acid glycidyl ester (FFAPO).

[0005] In order to achieve the above object, one of the technical solutions of the present invention is:

[0006] A highly sensitive cTnI detection kit based on the ROP signal amplification strategy triggered by FFAPO includes the following raw materials: electrode, MPA, EDC, NHS, Ab1, BSA, Ab2, GA, potassium methoxide, FFAPO, LiClO4, ultrapure water, PBS buffer, and DMF.

[0007] Further, the preparation method of FFAPO is:

[0008] (1) Mix 0.69 g of ferrocenecarboxylic acid, 40 mL of DCM, 300 μL of glycidol, and 0.30 g of 4-dimethylaminopyridine, and stir evenly in an ice-water bath;

[0009] (2) 0.69 g of EDC was dissolved in 20 mL of DCM, and added dropwise to the mixture of step (1) in an ice-water bath under nitrogen protection, and reacted at room temperature for 24 h;

[0010] (3) The reaction solution was extracted with ultrapure water, and the DCM layer was retained, concentrated, and purified to obtain an orange-yellow solid, which was FFAPO.

[0011] Furthermore, the purification method in step (3) is silica gel column chromatography, and the developing solvent used is n-hexane / ethyl acetate (v / v) = 4:1.

[0012] Furthermore, when used, some raw materials need to be prepared into solutions, among which the concentration of MPA solution is 10mM, the concentration of EDC solution is 4mM, the concentration of NHS solution is 1mM, the concentration of EDC and NHS in the mixed solution is 0.4M, the concentration of NHS is 0.1M, the concentration of Ab1 solution is 1μg / mL, the concentration of Ab2 solution is 2μg / mL, the concentration of GA solution is 1mM, the concentration of potassium methoxide solution is 3.3mg / mL, the concentration of FFAPO solution is 100mM, the concentration of LiClO4 solution is 1M, the concentration of BSA solution is 1%, the concentration of PBS buffer is 0.1M, and the pH is 7.4.

[0013] One of the technical solutions of the present invention is: a method for using a high-sensitivity cTnI detection kit based on the ROP signal amplification strategy induced by FFAPO, comprising the following steps:

[0014] (1) MAP modification

[0015] Immerse the clean bare gold electrode in MPA solution, react, and wash;

[0016] (2) EDC and NHS activate the carboxyl group of MAP

[0017] The gold electrode in step (1) is immersed in a mixed solution of EDC and NHS for activation and washing;

[0018] (3) Ab1 modification

[0019] Add Ab1 solution dropwise onto the electrode surface in step (2), incubate, and wash;

[0020] (4) BSA modification

[0021] Dropping the BSA solution onto the electrode surface in step (3), reacting, and washing;

[0022] (5) cTnI modification

[0023] Dropping the sample solution to be detected onto the electrode surface in step (4), incubating, and washing;

[0024] (6) Antigen-antibody specific binding

[0025] Add the prepared GA-Ab2 solution dropwise to the electrode surface of step (5), incubate, and wash;

[0026] (7) ROP reaction

[0027] Immersing the electrode in step (6) in a potassium methoxide solution, reacting, then immersing the electrode in a FFAPO solution, reacting, and washing;

[0028] (8) Electrochemical detection

[0029] The electrode of step (7) was immersed in a LiClO4 solution and the current response was recorded by square wave voltammetry.

[0030] Further, the preparation method of the clean bare gold electrode is:

[0031] ① Polish the gold electrode with 0.3 and 0.05 μm alumina slurry respectively;

[0032] ② Use ultrasonic waves to wash the electrodes in anhydrous ethanol and ultrapure water respectively;

[0033] ③ Immerse in piranha acid solution for 15 minutes;

[0034] ④ Treat the gold electrode by cyclic voltammetry in H2SO4 solution, with a scanning potential of -0.3 to 1.5 V and a scanning rate of 0.2 V·s -1 , until a reproducible cyclic voltammogram is achieved;

[0035] ⑤ Wash the electrode in ultrapure water using ultrasound, dry with nitrogen and set aside.

[0036] Furthermore, the reaction temperature of step (1) is 37°C and the time is 2h; the activation temperature of step (2) is 35-40°C and the time is 0.5h; the incubation temperature of steps (3), (5) and (6) is 35-40°C and the time is 1h; the reaction temperature of step (4) is 35-40°C and the time is 0.5h; the reaction conditions of step (7) are: 45-55°C vacuum reaction for 1-2h.

[0037] Furthermore, the increasing potential of the square wave voltammetry was: 4.0 mV, the potential amplitude was: 25 mV, and the resting time was: 30 s.

[0038] Further, the preparation method of GA-Ab2 solution is:

[0039] (1) Mix 100 μL of EDC solution, 100 μL of NHS solution, and 100 μL of GA solution, and shake at 35-40°C until the carboxyl groups of GA in the mixture are completely activated;

[0040] (2) Add 200 μL of Ab2 solution to the mixture and continue the reaction for 2 h. The mixture is stored at 4°C for future use.

[0041] One of the technical solutions of the present invention is: an application of the above-mentioned kit in cTnI detection.

[0042] The construction process of the detection method of the present invention is as follows Figure 1 shown.

[0043] Beneficial effects of the present invention:

[0044] 1. Using ROP as a signal amplification strategy avoids the use of nanomaterials and biological enzymes (which are easily affected by the external environment such as pH and temperature) in the currently commonly used signal amplification strategies. The signal is amplified exponentially, and the sensitivity is greatly improved. At the same time, the kit has the advantages of good selectivity and strong stability.

[0045] 2. Ring-opening polymerization (ROP) of ferrocene oxiranes is a strategy for synthesizing electroactive polymers. ROP can synthesize polymers with clear structures, and ROP polymers have specific and controllable properties. In addition, ferrocene and its derivatives have good redox properties, good stability, and very low toxicity. Compared with existing ferrocene, the monomer ferrocene formate glycidyl ester (FFAPO) synthesized by the present invention does not require high temperature and high pressure reaction conditions, is more likely to react, and can be well applied to biological detection of target molecules. The present invention uses FFAPO for ROP reaction, which is simple to operate.

[0046] 3. The present invention uses a sandwich structure to introduce cTnI, and connects the initiator glycolic acid of the ROP reaction to Ab2 through an esterification reaction, thereby grafting a large amount of ferrocene electroactive polymer to the electrode surface through the ROP reaction, and successfully establishes a kit for detecting cTnI with high sensitivity and strong specificity. Experiments have shown that the linear range of the present invention is 1pg / mL to 1μg / mL, and the corresponding linear equation is I(μA)=0.629log[C cTnI / pg mL -1 ]+2.951, the correlation coefficient was 0.997, the detection limit was 57.14fg / mL, and the stability reached 94.8% of the initial value after continuous storage for two weeks. The anti-interference ability reached 96.2%~103.1%. It also had good selectivity and reproducibility, laying a foundation for the later application in the detection of cTnI in the medical field and the early diagnosis of acute myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the construction process of the detection method of the present invention.

[0048] Figure 2This is the H-NMR spectrum of monomer FFAPO.

[0049] Figure 3 To verify the feasibility.

[0050] Figure 4 and Figure 5 The redox properties of the grafted polymers on the electrode surface were studied for CV.

[0051] Figure 6 The electrochemical impedance spectroscopy (EIS) was used for characterization.

[0052] Figure 7 It is characterized by water contact angle (WCA).

[0053] Figure 8 Characterized by atomic force microscopy (AFM).

[0054] Fig. 9 is the relationship between oxidation current and cTnI incubation time.

[0055] Fig.10 is the relationship between oxidation current and ROP polymerization time.

[0056] Fig.11 is the relationship between oxidation current and monomer concentration (FFAPO).

[0057] Fig.12 To study the analytical performance of the kit.

[0058] Fig.13 This is the selectivity result of the kit.

[0059] Fig.14 This is the anti-interference result of the kit. DETAILED DESCRIPTION

[0060] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0061] cTnI specific antigen standard solution (L4C00102), cTnI polyclonal antibody Ab1 (Anti-cTnI, L3C00401) and cTnI polyclonal antibody Ab2 (Anti-cTnI, L3C00402) were all purchased from Shanghai Lingchao Biotechnology Co., Ltd.

[0062] Example 1: Synthesis of ferrocene formate glycidyl ester (FFAPO)

[0063] (1) 0.69 g of ferrocenecarboxylic acid (FCA), 300 μL of propylene oxide and 40 mL of dichloromethane (DCM) were added to a dry three-necked flask, and then 0.30 g of 4-dimethylaminopyridine (DMAP) was added as a superacylation catalyst. The mixture was placed in an ice-water bath and stirred for 10 min.

[0064] (2) 0.69 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was dissolved in 20 mL of DCM, and then added dropwise to the mixture of step (1) under an ice-water bath and nitrogen protection, and the reaction was stirred at room temperature for 24 h.

[0065] (3) The reaction solution was extracted three times with ultrapure water, and the DCM layer was retained and concentrated by a rotary evaporator. The crude product was purified by silica gel column chromatography (the developing solvent used was n-hexane / ethyl acetate = 4:1, v / v) to obtain an orange-yellow solid, which was the target compound FFAPO.

[0066] The structure of the orange-yellow solid was verified by H NMR. Figure 2 1H NMR (CDCl3, 400MHz) data are as follows: δ (ppm) = 4.84 (2H, m, cp), 4.58-4.61 and 4.03-4.08 (-CH2-), 4.42 (2H, m, cp), 4.22 (5H, m, cp), 3.33 (CH of ethylene oxide), 2.89-2.72 (CH2 of ethylene oxide), indicating that ferrocenecarboxylic acid glycidyl ester was successfully synthesized.

[0067] Example 2: Preparation of GA-Ab2

[0068] (1) Mix 100 μL of 4 mM EDC solution, 100 μL of 1 mM N-hydroxysuccinimide (NHS) solution, and 100 μL of 1 mM glycolic acid (GA) solution, and shake vigorously on a shaker until the carboxyl groups of GA in the mixture are fully activated (37°C, 2 h);

[0069] (2) Add 200 μL of 2 μg / mL Ab2 solution to the mixture and continue the reaction for 2 h. The resulting product is stored at 4°C for future use.

[0070] Example 3: Kit

[0071] A highly sensitive cTnI detection kit based on the ROP signal amplification strategy triggered by FFAPO includes the following raw materials: gold electrode, 3-mercaptopropionic acid (MPA), EDC, NHS, Ab1, bovine serum albumin (BSA), Ab2, GA, potassium methoxide, FFAPO, LiClO4, ultrapure water, PBS buffer, and N,N-dimethylformamide (DMF).

[0072] When in use, some raw materials need to be prepared into solutions, among which the concentration of MPA solution is 10mM, the concentration of EDC solution is 4mM, the concentration of NHS solution is 1mM, the concentration of EDC and NHS in the mixed solution is 0.4M, the concentration of NHS is 0.1M, the concentration of Ab1 solution is 1μg / mL, the concentration of Ab2 solution is 2μg / mL, the concentration of GA solution is 1mM, the concentration of potassium methoxide solution is 3.3mg / mL, the concentration of FFAPO solution is 100mM, the concentration of LiClO4 solution is 1M, the concentration of BSA solution is 1%, the concentration of PBS buffer is 0.1M, and the pH is 7.4.

[0073] Example 4: Detection method

[0074] (1) Electrode pretreatment

[0075] ① Polish the gold electrode with 0.3 and 0.05 μm alumina slurry respectively;

[0076] ② Use ultrasonic waves to wash the electrodes in anhydrous ethanol and ultrapure water respectively;

[0077] ③ Immerse the electrode in piranha acid solution (30% H2O2 and 98% H2SO4, v / v=1:3) for 15 min;

[0078] ④ Treat the gold electrode by cyclic voltammetry in H2SO4 solution (0.5M), with a scanning potential of -0.3 to 1.5 V and a scanning rate of 0.2 V·s -1 , until a reproducible cyclic voltammogram is achieved;

[0079] ⑤ Wash the electrode in ultrapure water with ultrasound to obtain a clean bare gold electrode. Dry it with nitrogen before use and set it aside.

[0080] (2) MAP modification

[0081] The clean bare gold electrode was immersed in 300 μL 10 mM MPA solution, reacted at 37 °C for 2 h, washed with ultrapure water, and dried with nitrogen gas;

[0082] (3) EDC and NHS activate the carboxyl group of MAP

[0083] The gold electrode in step (2) was immersed in 300 μL of a mixed solution of EDC and NHS (0.4 M EDC and 0.1 M NHS), activated at 37°C for 0.5 h, and washed with ultrapure water;

[0084] (4) cTnI primary antibody (Ab1) modification

[0085] 10 μL of 1 μg / mL Ab1 solution was dripped onto the electrode surface in step (3), incubated at 37°C for 1 h, and washed;

[0086] (5) BSA modification

[0087] 10 μL of 1% BSA solution was dripped onto the electrode surface in step (4), reacted at 37°C for 0.5 h, to block the sites on the gold electrode surface that were not bound by Ab1, and then washed;

[0088] (6) cTnI modification

[0089] 10 μL of the sample solution to be tested (containing cTnI-specific antigen) was dripped onto the electrode surface in step (5), incubated at 37° C. for 1 h, and washed;

[0090] (7) Antigen-antibody specific binding

[0091] 10 μL of the prepared GA-Ab2 solution was added dropwise to the electrode surface of step (6), incubated at 37°C for 1 h, and washed;

[0092] The washing in steps (4) to (7) is to rinse the electrode surface with phosphate buffered saline (PBS, 0.1 M, pH = 7.4) to remove unreacted substances and prevent specific adsorption;

[0093] (8) ROP reaction

[0094] The electrode in step (7) was immersed in 100 μL of 3.3 mg / mL potassium methoxide solution and reacted at 50°C in vacuum for 1 h. The electrode was then immersed in 100 μL of 100 mM FFAPO solution and reacted at 50°C in vacuum for 2 h. The electrode was rinsed with DMF and ultrapure water to remove unreacted monomers.

[0095] (9) Electrochemical detection

[0096] The electrode of step (8) was immersed in a 1.0 M lithium perchlorate (LiClO4) (10 mL) solution, and the square wave voltammetry (SWV) measurement value was recorded. The cTnI was detected according to the current response value (increase potential: 4.0 mV, potential amplitude: 25 mV, rest time: 30 s).

[0097] Example 5: Feasibility Verification

[0098] The present invention compares the current response of electrodes prepared differently by square wave voltammetry (SWV). Figure 3As shown, no obvious current signal was observed when MPA (curve a), Ab1 (curve b), cTnI (curve c), GA-Ab2 (curve d) or FFAPO (curve e) was added during the electrode modification process, indicating that the nonspecific adsorption of FFAPO monomer on the electrode surface was negligible. When the electrode surface was modified step by step with ROP / GA-Ab2 / cTnI / BSA / Ab1 / MPA, a strong oxidation current signal with a peak potential of ~0.41 V was clearly observed (curve f). These results clearly show that ROP can be used as a signal amplification strategy for cTnI electrochemical detection.

[0099] Example 6: Redox performance study

[0100] like Figure 4 and 5 As shown, in the range of 0.01 to 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 does not rely on independent diffusion and the polymer is fixed on the electrode surface.

[0101] Example 7: Characterization by electrochemical impedance spectroscopy (EIS)

[0102] EIS can characterize the modification of the electrode surface during the electrode construction process. In the presence of 5 mM [Fe(CN)6] 3- / 4- In the electrolyte solution, the EIS spectra of the same electrode in the continuous preparation process are gradually collected. Figure 6 As shown in the figure, due to the high electron transfer efficiency, the impedance of the bare gold electrode is the lowest (~268Ω, curve a). When MPA is fixed on the electrode surface, the electrostatic repulsion of the formed self-assembled monolayer hinders the transfer of [Fe(CN)6] 3- / 4Transfer on the electrode surface leads to a gradual increase in Rct (~695Ω, curve b). The carboxyl groups on MPA are activated with EDC / NHS, and the impedance is found to decrease (~300Ω, curve c). This is because the negatively charged carboxyl groups are replaced by succinimidyl esters, which eliminates electrostatic repulsion to a certain extent. When Ab1 is connected to the electrode surface through an amide bond, the protein reduces the available active sites during electron transfer, resulting in an increase in Rct (~878Ω, curve d). The use of 1% BSA to block the active sites on the electrode surface leads to a further increase in Rct (~1084Ω, curve e). In addition, due to the poor conductivity of biomolecules, Rct further increases after cTnI specifically recognizes Ab1 (~1475Ω, curve f). Similarly, the introduction of GA-Ab2 increases the number of biomolecules on the electrode surface, further increasing Rct (~1800Ω, curve g). Finally, due to the large number of high-density hydrophobic polymer chains connected to the electrode surface, Rct increases significantly after the ROP reaction is completed (~3800Ω, curve h). The above EIS results indicate that the cTnI detection electrode based on the ROP signal amplification strategy has been successfully prepared.

[0103] Example 8: Water Contact Angle (WCA) Characterization

[0104] The value of WCA is closely related to the hydrophilicity of the electrode surface and increases with decreasing hydrophilicity. Figure 7 As shown, due to the hydrophobicity of the bare gold electrode surface, its WCA value is 91.2° ( Figure 7 A). Due to the influence of the hydrophilic carboxyl groups in the MPA molecule, the WCA was significantly reduced to 87° ( Figure 7 B). After MPA modification, WCA increases continuously to 90.2° ( Figure 7 C). This is because the carboxyl groups in the MPA molecules on the electrode are activated into hydrophobic ester bonds by EDC / NHS. After Ab1, BSA and cTnI were incubated on the electrode surface in turn, the WCA dropped to 85.6° ( Figure 7 D), 79.2°( Figure 7 E) and 79°( Figure 7 F). After GA-Ab2 was incubated on the electrode surface, a sandwich structure was formed. Due to the presence of hydrophilic hydroxyl and carboxyl groups in GA-Ab2, WCA continued to decrease to 76.9° ( Figure 7 G). After ROP reaction, WCA increased sharply to 87.4°( Figure 7 H). This is because a large number of hydrophobic polymer chains are produced. These results confirm that the construction of the modified electrode is successful.

[0105] Example 9: Atomic Force Microscopy (AFM) Characterization

[0106] The electrode preparation process was further characterized by AFM. Figure 8 A) The height of the modified electrode is 17.1 nm, and after ROP polymerization ( Figure 8 B) The height of the electrode is 30.3 nm, which can be attributed to the large number of polymer chains attached to the electrode surface through the ROP reaction, further indicating that the preparation of a cTnI detection kit based on the ROP signal amplification strategy is feasible.

[0107] Example 10: Optimization of analytical conditions

[0108] (1) cTnI incubation time

[0109] like Fig. 9 As shown in the figure, the oxidation current increased significantly with the incubation time of cTnI from 15 to 60 minutes, reaching the maximum value at 60 minutes. When the incubation time was greater than 60 minutes, the peak current remained basically unchanged. Therefore, the optimal incubation time for cTnI was 60 minutes.

[0110] (2) ROP reaction time

[0111] The analytical performance of the kit based on the ROP signal amplification strategy depends largely on the number of ferrocene electroactive molecules on the electrode surface. In order to optimize the analytical performance, especially the sensitivity, the effect of polymerization time on the oxidation current value was studied. Fig.10 As shown in the figure, when the ROP reaction time is in the range of 0.5 to 2 h, the oxidation current increases significantly and then tends to be basically stable. This is due to the significant increase in steric hindrance and the dynamic growth of the polymer or the termination of free radicals. Therefore, the optimal ROP reaction time is selected as 2 h.

[0112] (3) Monomer reaction concentration

[0113] The effect of monomer concentration on the detection of cTnI by the kit was studied. Fig.11 As shown in the figure, when the monomer concentration is 20 to 100 mM, the oxidation current increases rapidly with the increase of monomer (FFAPO) concentration, and thereafter, the current intensity does not change significantly with the increase of monomer concentration. Therefore, in the following experiments, the optimal concentration of FFAPO was selected as 100 mM.

[0114] Example 11: Analytical Performance

[0115] According to the optimal experimental conditions obtained by optimizing the conditions in Example 10, the current intensity generated by different concentrations of cTnI (1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, 10ng / mL, 100ng / mL, 1μg / mL) was detected to study the detection performance of the method of the present invention for cTnI. Fig.12As shown in A, within the concentration range of 1pg / mL to 1μg / mL, the current response gradually increases with the increase of cTnI concentration. Fig.12 As shown in B, it can be found that the cTnI current response and the logarithm of the cTnI concentration show a good linear relationship in the range of 1pg / mL to 1μg / mL. The corresponding linear equation is I(μA)=0.629log[C cTnI / pg mL -1 ]+2.951(R 2 =0.997), LOD was calculated to be 57.14 fg / mL (C LOD =3σ / slope, σ represents the standard deviation of the blank group). Therefore, the kit prepared by the present invention exhibits a relatively wide linear range and a low LOD. Therefore, the method of the present invention shows great application potential in detecting ultra-low levels of cTnI.

[0116] Example 12: Selective Detection

[0117] According to the best experimental conditions obtained by optimizing the conditions in Example 10, the current intensity generated by the blank group (Blank), bovine serum albumin (BSA), carcinoembryonic antigen (CEA), and cytokeratin 19 fragment (CYFRA 21-1) using the method of the present invention was studied under the same conditions. Specifically, the electrochemical signal intensity generated by 100 ng / mL cTnI and other interfering proteins of the same concentration was compared. Fig.13 As shown, the current signal generated by other interfering proteins is smaller than that of cTnI, and is basically equivalent to the signal of the blank group, which proves that the method of the present invention has high selectivity. This can be attributed to the specific recognition of antigen and antibody, which enables it to have a high recognition ability for cTnI. Unpaired antigens will significantly reduce the recognition efficiency.

[0118] Example 13: Reproducibility test

[0119] According to the best experimental conditions obtained by the optimization of conditions in Example 10, intra-batch and inter-batch experiments (n=3) were performed under the same experimental conditions. When the concentration of cTnI was 10 ng / mL, the relative standard deviations within and between batches were 2.7% and 3.7%, respectively, proving that the method of the present invention has good reproducibility for the detection of cTnI.

[0120] Example 14: Stability test

[0121] According to the best experimental conditions obtained by the optimization of conditions in Example 10, under the same experimental conditions, the prepared electrodes were randomly divided into two groups (6 in each group), one group was used directly for SWV measurement after preparation, and the other group was stored in a refrigerator at 4°C. After two weeks of storage, the current signal of the modified electrode was still maintained at 94.8% of the initial current. Therefore, it is proved that the method of the present invention has good stability.

[0122] Example 15: Anti-interference analysis

[0123] Anti-interference performance is an important parameter reflecting the clinical application value of the kit. Three different concentrations of cTnI (10pg / mL, 1ng / mL and 100ng / 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. Fig.14 As shown, when the concentration of cTnI was 10pg / mL, 1ng / mL and 100ng / mL, respectively, the current signals from 5% and 10% normal human serum (NHS) were 96.2% and 102.5%, 98.6% and 103.1%, 99.2% and 101.6% of the peak current from PBS buffer, respectively, indicating that the kit has good anti-interference ability for the detection of cTnI in normal human serum and has certain practical application value in clinical detection.

Claims

1. A highly sensitive cTnI non-diagnostic and / or therapeutic detection method based on the ROP signal amplification strategy triggered by ferrocenylcarboxylic acid glycidyl ester FFAPO, characterized in that: The raw materials used include: electrode, 3-mercaptopropionic acid MPA, EDC, NHS, Ab1, BSA, Ab2, glycolic acid GA, potassium methoxide, FFAPO, LiClO4; wherein the preparation method of FFAPO is: (1) Mix 0.69 g of ferrocenecarboxylic acid (FCA), 40 mL of dichloromethane (DCM), 300 μL of propylene oxide, and 0.30 g of 4-dimethylaminopyridine (DMAP), and stir evenly in an ice-water bath; (2) Dissolve 0.69 g of EDC in 20 mL of DCM, add dropwise to the mixture of step (1) in an ice-water bath under nitrogen protection, and react at room temperature for 24 h; (3) Extract the reaction solution with ultrapure water, retain the DCM layer, concentrate and purify to obtain an orange-yellow solid, which is FFAPO; The detection method comprises the following steps: (1) MPA modification Immerse the clean bare gold electrode in MPA solution, react, and wash; (2) EDC and NHS activate the carboxyl end of MPA The gold electrode in step (1) is immersed in a mixed solution of EDC and NHS for activation and washing; (3) Ab1 modification Dropping Ab1 solution onto the electrode surface in step (2), incubating, and washing; (4) BSA modification Dropping the BSA solution onto the electrode surface in step (3), reacting, and washing; (5) cTnI modification Drop the sample solution to be tested onto the electrode surface in step (4), incubate the reaction for 60 minutes, and wash; (6) Antigen-antibody specific binding Add the prepared GA-Ab2 solution dropwise onto the electrode surface of step (5), incubate, and wash; (7) ROP reaction The electrode in step (6) was immersed in a potassium methoxide solution to react, and then the electrode was immersed in a 100 mM FFAPO solution to react for 2 hours and washed; (8) Electrochemical detection The electrode from step (7) was immersed in a LiClO4 solution and the current response was recorded by square wave voltammetry.

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

3. The detection method according to claim 1, characterized in that: In step (3) of the preparation method of FFAPO, the purification method is silica gel column chromatography, and the developing solvent used is n-hexane / ethyl acetate (v / v) = 4:

1.

4. The detection method according to any one of claims 1 to 3, characterized in that: When used, some raw materials need to be prepared into solutions, among which the concentration of MPA solution is 10 mM, the concentration of EDC solution is 4 mM, the concentration of NHS solution is 1 mM, the concentration of EDC and NHS in the mixed solution is 0.4 M EDC concentration and 0.1 M NHS concentration, the concentration of Ab1 solution is 1 μg / mL, the concentration of Ab2 solution is 2 μg / mL, the concentration of GA solution is 1 mM, the concentration of potassium methoxide solution is 3.3 mg / mL, the concentration of LiClO4 solution is 1 M, the concentration of BSA solution is 1%, the concentration of PBS buffer is 0.1 M, and the pH is 7.4.

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