An electrochemiluminescence detection kit based on GO-CDTPA-AuNPs and PET-RAFT

Abstract

This invention discloses an electrochemiluminescence detection kit based on GO-CDTPA-AuNPs and PET-RAFT, comprising a gold electrode, hDNA, MCH, MPA-NHS ester, GO-CDTPA-AuNPs, Me6TREN, EY, NAS, and luminol. This invention employs the GO-CDTPA-AuNPs composite material and the PET-RAFT multi-signal amplification strategy, avoiding the use of biological enzymes in commonly used signal amplification strategies. The signal is amplified many times over, improving detection sensitivity, stability, and reproducibility. Furthermore, it avoids the instability of luminol binding to GO through π-π conjugation, improving the surface utilization of GO, which will further enhance the electrochemiluminescence efficiency of luminol. Combining GO and AuNPs avoids the instability and easy aggregation of traditional nanomaterials. The photo-initiated RAFT polymerization reaction avoids the need for thermal initiation in traditional RAFT reactions, resulting in milder reaction conditions, faster reaction time, and "active" / controlled polymerization at room temperature; moreover, it does not produce biotoxicity and is more environmentally friendly.

Description

Technical Field

[0001] This invention relates to an electrochemiluminescence detection kit based on GO-CDTPA-AuNPs composite material and a photoinduced electron / energy transfer reversible addition-fragment chain transfer (PET-RAFT) multi-signal amplification strategy, as well as its usage and application, belonging to the field of bioanalytical technology. Background Technology

[0002] Non-small-cell lung cancer (NSCLC) accounts for 80%–85% of all lung cancers, and 20%–25% of NSCLC patients have mutations in the Kirsten rat sarcoma viral oncogene (KRAS). Among these, KRAS G12C (G→T) mutations account for 40% of KRAS mutations in NSCLC, and patients with KRAS G12C mutations have low survival rates. Early-stage NSCLC can be successfully resected surgically, while intermediate-to-late-stage NSCLC can be treated with radiotherapy and chemotherapy. However, in recent years, traditional chemotherapy for lung cancer has not seen any breakthrough progress and has serious adverse reactions. Therefore, developing a sensitive method for detecting KRAS G12 is of great significance for diagnosing the carcinogenic factors in NSCLC patients and for determining appropriate treatment options.

[0003] Several methods for detecting KRAS mutations have been reported, including liquid biopsy analysis, dideoxy sequencing, digital polymerase chain reaction (dPCR), quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS). However, these methods still suffer from drawbacks such as false positives, long processing times, and the need for specialized equipment and operators. Electrochemiluminescence immunoassay, on the other hand, combines the advantages of both chemiluminescence and electrochemical methods, such as high sensitivity, high accuracy, lower cost, and ease of operation, and is therefore widely used.

[0004] Furthermore, combining nanomaterials and signal amplification techniques such as polymerization reactions can improve the detection performance of sensors. Graphene oxide (GO), as a chemically modified graphene sheet nanomaterial, possesses a large specific surface area, good biocompatibility, and multiple oxygen-related functional groups, such as hydroxyl and epoxide groups on the basal surface and carboxyl groups on the planar edges. However, due to π-π bond interactions, it is prone to aggregation. Fortunately, this can be effectively addressed by introducing gold nanoparticles (AuNPs) between graphene layers. With these properties, GO possesses more active sites and unique catalytic activity, attracting widespread attention in many fields. In recent years, photo-initiated polymerization has garnered significant attention due to its good economic benefits and ecological prospects. Among these, PET-RAFT polymerization outperforms other photochemical technologies in terms of applicability, cost, and sustainability. The core advantages of PET-RAFT polymerization are its simplicity and energy efficiency, as it requires only a low-energy visible light source and can be carried out at room temperature. The polymer can be synthesized in the presence of oxygen and does not require heavy metal catalysts. Therefore, it is of great significance to study an electrochemiluminescence sensing platform based on GO-CDTPA-AuNPs composite materials and PET-RAFT multi-signal amplification strategy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a novel electrochemiluminescence assay kit, its usage method, and its application based on GO-CDTPA-AuNPs composite materials and a PET-RAFT multi-signal amplification strategy. This kit combines the advantages of nanomaterials with the multifunctionality and convenience of free radical polymerization, avoiding the use of heavy metal ion catalysts in traditional RAFT reactions. This results in signal amplification, improving detection sensitivity, stability, and reproducibility.

[0006] To achieve the above objectives, one of the technical solutions of the present invention is as follows:

[0007] An electrochemiluminescence detection kit based on GO-CDTPA-AuNPs and PET-RAFT comprises the following ingredients: gold electrode, hDNA, MCH, MPA-NHS ester, GO-CDTPA-AuNPs, Me6TREN, EY, NAS, luminol, PBS buffer, and H2O2.

[0008] Furthermore, the preparation method of MPA-NHS ester is to mix equimolar amounts of EDC, NHS and MPA evenly and react at 37°C for 3 hours to obtain an MPA-NHS ester solution with a concentration of 1 mM.

[0009] Furthermore, the preparation method of GO-CDTPA-AuNPs is as follows:

[0010] ① Dissolve 20 mg of GO in 10 mL of ultrapure water and sonicate to obtain a GO dispersion;

[0011] ② Disperse 20 mg ETA and 20 mg KOH in GO dispersion, react, centrifuge and wash, and vacuum dry to obtain ETA-modified GO, i.e. GO-ETA;

[0012] ③ Mix 2 mg CDTPA, 1.6 mg EDC, and 1 mg NHS evenly in 5 mL DMF and react to obtain a CDTPA-NHS ester solution;

[0013] ④ Dissolve 5 mg GO-ETA and 0.2 mg DMAP in 5 mL CDTPA-NHS ester solution, react, centrifuge, collect the precipitate and redisperse it with 1 mL ultrapure water to obtain GO-CDTPA dispersion;

[0014] ⑤ Mix 1 mM AuNPs solution and GO-CDTPA dispersion at a volume ratio of 2:3 until homogeneous, react, centrifuge, collect the precipitate and redisperse it with ultrapure water to obtain GO-CDTPA-AuNPs dispersion.

[0015] Furthermore, ① the ultrasonic treatment time is 30 min; ② the reaction condition is stirring at 80℃ for 24 h; ③ the reaction condition is reacting at 37℃ for 15–24 h; ④ the reaction condition is reacting at room temperature in darkness for 24 h; ⑤ the reaction condition is stirring at room temperature for 12 h.

[0016] Furthermore, some raw materials need to be prepared into solutions before use. The concentrations of the hDNA solution are as follows: 0.5 μM, MCH solution: 2 mM, MPA-NHS ester solution: 1 mM, Me6TREN solution: 1.2 mM, EY solution: 0.02 mM, NAS solution: 10 mM, luminol solution: 10 mM, PBS buffer solution: 0.1 M, pH = 7.4, and H2O2 solution: 10 mM.

[0017] One of the technical solutions of the present invention is: a method for using an electrochemiluminescence detection kit, comprising the following steps:

[0018] (1) Electrode pretreatment

[0019] Bare gold electrodes are polished to obtain a polished mirror finish;

[0020] (2) Electrode modification

[0021] ① Drop the hDNA solution onto the electrode surface and allow the reaction to proceed;

[0022] ② Immerse the electrode from step ① in the MCH solution and allow the reaction to proceed;

[0023] ③ The solution to be tested is dropped directly onto the electrode surface in step ②, and the reaction occurs;

[0024] ④ Immerse the electrode from step ③ in the MPA-NHS ester solution and allow the reaction to proceed;

[0025] ⑤ Drop the GO-CDTPA-AuNPs dispersion onto the electrode surface from step ④ and allow it to react;

[0026] ⑥ Place the electrode from step ⑤ in a mixed solution consisting of Me6TREN solution, EY solution, NAS solution, and PBS buffer for reaction;

[0027] ⑦ Place the electrode from step ⑥ in a luminol solution to allow the reaction to proceed;

[0028] ⑧ Place the electrode from step ⑦ in H2O2 to measure the luminescence intensity of luminol.

[0029] Furthermore, the reaction conditions are as follows: ① reaction temperature is 37℃, time is 2-8h; ② reaction temperature is 37℃, time is 0.5-1h; ③ reaction temperature is 37℃, time is 1.5-2h; ④ reaction temperature is 37℃, time is 1-2h; ⑤ reaction temperature is 37℃, time is 1-3h; ⑥ reaction conditions are room temperature, 470nm blue light irradiation, 2h; ⑦ reaction temperature is 37℃, time is 1-3h.

[0030] One of the technical solutions of the present invention is the application of the aforementioned kit in the preparation of reagents for detecting lung cancer.

[0031] Furthermore, the lung cancer in question is non-small cell lung cancer.

[0032] A schematic diagram of the detection method of this invention is shown below. Figure 1 As shown.

[0033] This invention employs a GO-CDTPA-AuNPs composite material and a PET-RAFT multi-signal amplification strategy. First, a hairpin DNA (hDNA) probe is attached to the electrode surface via self-assembled polar covalent bonds. After 6-mercaptohexanol (MCH) shuts off residual binding sites, target DNA (tDNA) is attached to the electrode surface through specific recognition. MPA and hDNA-NH2 are linked via an amide reaction. Then, the GO-CDTPA-AuNPs composite material is linked to MPA via Au-S bond self-assembly. Subsequently, under blue light irradiation, a large number of monomeric NAS molecules are linked to the chain transfer agent CDTPA via a PET-RAFT reaction, providing numerous binding sites for the luminol luminescent material, thereby significantly amplifying the ECL signal. Finally, many luminol molecules are tightly linked to a large number of NAS molecules through an amino condensation reaction.

[0034] The PET-RAFT process was used to polymerize NAS under 470 nm light irradiation, with CDTPA as the chain transfer agent, EY as the catalyst, and Me6TREN as the ligand. The reduction quenching pathway initiated upon blue light excitation at a specific wavelength; with photon absorption, electrons transitioned from the ground state (EY) to the excited state (EY*). Subsequently, EY* generated EY by stripping electron donors (NR3, Me6TREN). ·- and NR3 ·+ Based on three catalysts (EY, EY*, and EY) ·－ A reversible cycle was established to modulate the reduction and quenching pathway of photo-ATRP. The chain transfer agent (CDTPA) of PET-ATRP was activated, generating free radicals (Pn·), which subsequently interacted with the reduced RAFT reagent to produce dormant macromolecular RAFT substances, thereby shutting down the catalytic cycle. Through the above reduction-quenching reaction, a large amount of monomer was grafted onto the electrode surface. Then, the monomers were combined with luminol for ECL detection. Hydrogen peroxide is an effective co-reactant in the luminol ECL reaction, tending to decompose into superoxide radicals (OH·) and superoxide anion radicals (O2·). － Under a certain voltage, luminol and the co-reactant hydrogen peroxide are simultaneously oxidized. The hydrogen peroxide then rapidly decomposes to generate a high-energy free radical intermediate (which participates in the oxidation reaction of luminol, exciting it, and then returns it to the ground state, simultaneously producing light of the corresponding wavelength). It is noteworthy that GO and AuNPs play a crucial role in amplifying the ECL signal of luminol. GO increases the interfacial area of ​​the modified electrode, capturing more AuNPs and luminol through the interaction of positive and negative charges with PET-RAFT; while AuNPs promote electron transfer at the electrode interface, catalyzing the ECL process of luminol in the electrode and the chemical reaction products, thereby highly amplifying the ECL signal.

[0035] Beneficial effects of this invention:

[0036] This invention utilizes GO-CDTPA-AuNPs composite material and PET-RAFT multi-signal amplification strategy, avoiding the use of biological enzymes (which are easily affected by external environment and temperature) in commonly used signal amplification strategies. The signal is amplified many times over, improving the sensitivity, stability and reproducibility of detection.

[0037] This invention employs a GO-CDTPA-AuNPs composite material and a PET-RAFT multi-signal amplification strategy, which not only avoids the instability of luminol binding with GO through π-π conjugation but also improves the surface utilization of GO, further enhancing the electrochemiluminescence efficiency of luminol. Combining GO and AuNPs avoids the instability and easy aggregation inherent in traditional nanomaterials. The photo-initiated RAFT polymerization reaction eliminates the need for thermal initiation in traditional RAFT reactions, resulting in milder reaction conditions, faster reaction time, and "living" / controlled polymerization at room temperature; moreover, it does not produce biotoxicity, making it more environmentally friendly. Attached Figure Description

[0038] Figure 1 This is a schematic diagram illustrating the principle of the detection method of the present invention.

[0039] Figure 2 (A) represents the electrochemiluminescence intensity under different conditions. Among them, curve a represents the absence of hDNA, curve b represents the absence of tDNA, curve c represents the absence of MPA, curve d represents the absence of GO-CDTPA-AuNPs, curve e represents the absence of PET-RAFT reaction solution, curve f represents the absence of luminol, and curve g represents luminol / NAS / GO-CDTPA-AuNPs / MPA / tDNA / MCH / hDNA / Au.

[0040] Figure 2 (B) shows the evolution of the impedance curves of the electrode (curve a→h) after each step of modification from the bare gold electrode.

[0041] Figure 2 (C) shows the CV curves of the electrode after each step of modification from the bare gold electrode (curve a→h).

[0042] Figure 3 The image shows the UV-Vis spectrum (A) and particle size distribution (B) of AuNPs.

[0043] Figure 4 Scanning electron microscopy characterization (A) and energy-dispersive spectrum (B) of GO-CDTPA-AuNPs.

[0044] Figure 5 The morphology of the electrode surfaces under different modification states is shown in the figures. (A) is the scanning electron microscope (SEM) characterization of the bare gold electrode, (B) is the SEM characterization of the electrode before PET-RAFT reaction modification, (C) is the SEM characterization of the electrode after PET-RAFT reaction modification, (D) is the atomic force microscope (AFM) characterization of the bare gold electrode, (E) is the AFM characterization of the electrode before PET-RAFT reaction modification, and (F) is the AFM characterization of the electrode after PET-RAFT reaction modification.

[0045] Figure 6 Optimization of GO concentration (A), optimization of GO to AuNPs volume ratio (B), and optimization of PET-RAFT reaction time (C).

[0046] Figure 7 The graph shows the relationship between electrochemiluminescence (ECL) intensity and tDNA concentration (A) and the corresponding linear correlation curve (B).

[0047] Figure 8 (A) is a comparison of the electroluminescence intensity of 10 pM tDNA and the same concentrations of single-base mismatched DNA (SBM), double-base mismatched DNA (DBM), fully mismatched DNA (NC), and no DNA (blank) under the same detection conditions.

[0048] Figure 8 (B) Signal intensity of tDNA at different concentrations in TE buffer and 10% (v / v) serum samples. Detailed Implementation

[0049] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0050] hDNA, tDNA (KRAS G12C), SBM, DBM, and NC were all purchased from Sangon Biotech (Shanghai) Co., Ltd., and their sequences are as follows:

[0051]

[0052] Example 1: Reagent Kit

[0053] An electrochemiluminescence detection kit based on GO-CDTPA-AuNPs and PET-RAFT comprises the following raw materials: gold electrode, hDNA, 6-mercaptohexanol (MCH), MPA-NHS ester, GO-CDTPA-AuNPs, tris(2-dimethylaminoethyl)amine (Me6TREN), eosin Y (EY), N-acryloyloxysuccinimide (NAS), luminol, PBS buffer, and H2O2.

[0054] Some raw materials need to be prepared into solutions before use. The concentrations of the following solutions are as follows: hDNA solution 0.5 μM, MCH solution 2 mM, MPA-NHS ester solution 1 mM, Me6TREN solution 1.2 mM, EY solution 0.02 mM, NAS solution 10 mM, luminol solution 10 mM, PBS buffer 0.1 M, pH 7.4, and H2O2 solution 10 mM.

[0055] The preparation method of GO-CDTPA-AuNPs is as follows:

[0056] ① Dissolve 20 mg of graphene oxide (GO) in 10 mL of ultrapure water and sonicate for 30 min to obtain a GO dispersion;

[0057] ② Disperse 20 mg ethanolamine (ETA) and 20 mg KOH in GO dispersion, stir at 80 °C for 24 h, centrifuge and wash several times with anhydrous ethanol and ultrapure water, and vacuum dry the crude product to obtain ETA-modified GO (GO-ETA);

[0058] ③ Mix 2 mg of 4-cyano-4-[(dodecylthioalkylthiocarbonyl)thioalkyl]valeric acid (CDTPA), 1.6 mg of carbodiimide hydrochloride (EDC), and 1 mg of N-hydroxysuccinimide (NHS) uniformly in 5 mL of N,N-dimethylformamide (DMF) and react on a shaker at 37 °C for 20 h to obtain a CDTPA-NHS ester solution with a concentration of 1 mM;

[0059] ④ Dissolve 5 mg GO-ETA and 0.2 mg 4-dimethylaminopyridine (DMAP) in 5 mL CDTPA-NHS ester solution, continue the reaction for 24 h in the dark at room temperature, then centrifuge multiple times to remove free CDTPA molecules, collect the precipitate and redisperse it with 1 mL of ultrapure water to obtain GO-CDTPA dispersion.

[0060] ⑤ Add 666 μL of gold nanoparticle (AuNPs) solution (1 mM) to the GO-CDTPA dispersion and stir vigorously at room temperature for 12 h; then centrifuge multiple times to remove free AuNPs, collect the precipitate and redisperse it with 1 mL of ultrapure water to obtain the GO-CDTPA-AuNPs dispersion.

[0061] The preparation method of gold nanoparticles (AuNPs) is as follows:

[0062] ① Add 2.06 g of polyethyleneimine (PEI) to 20 mL of 1 mM gold chloride trihydrate (HAuCl4·3H2O) aqueous solution;

[0063] ② After mixing evenly, heat the solution from ① in a water bath and stir with a magnetic bead. Increase the temperature to 65℃ at 5℃ / min, then increase it to 80℃ at 1℃ / min. Maintain the temperature and heat until the solution turns ruby ​​red. Stop heating and stirring until room temperature is reached to obtain AuNPs solution.

[0064] The preparation method of MPA-NHS ester is to mix equimolar amounts of EDC, NHS and MPA (3-mercaptopropionic acid) evenly and react at 37℃ for 3h to obtain an MPA-NHS ester solution with a concentration of 1mM.

[0065] Example 2: How to use the reagent kit

[0066] (1) Electrode pretreatment

[0067] Bare gold electrodes are polished to obtain a polished mirror finish;

[0068] (2) Electrode modification

[0069] ① Drop 5 μL of hDNA solution (0.5 μM) onto the electrode surface, react at 37 °C for 2 h, wash with ultrapure water, and dry with nitrogen gas;

[0070] ②Immerse the electrode (hDNA / Au) from step ① in 150μL MCH solution (2mM), react at 37℃ for 0.5h, wash with ultrapure water, and dry with nitrogen gas;

[0071] ③ Drop 10 μL of the test solution (containing tDNA) directly onto the electrode (MCH / hDNA / Au) surface from step ②, react at 37°C for 1.5 h, wash with ultrapure water, and dry with nitrogen.

[0072] ④ Immerse the electrode (tDNA / MCH / hDNA / Au) from step ③ in 150 μL of MPA-NHS ester solution (1 mM), react at 37 °C for 1 h, wash with ultrapure water, and dry with nitrogen.

[0073] ⑤ Drop 10 μL of GO-CDTPA-AuNPs dispersion onto the electrode surface (MPA / tDNA / MCH / hDNA / Au) from step ④, react at 37 °C for 2 h, wash with ultrapure water, and dry with nitrogen gas;

[0074] ⑥ Place the electrode from step ⑤ in a mixed solution (PET-RAFT solution) consisting of 5 μL of tris(2-dimethylaminoethyl)amine (Me6TREN) solution (1.2 mM), 5 μL of eosin Y (EY) solution (0.02 mM), 60 μL of N-acryloyloxysuccinimide (NAS) solution (10 mM) and 1930 μL of PBS buffer (0.1 M, pH=4), and react for 2 h at room temperature under 470 nm blue light irradiation (PET-RAFT). Wash with ultrapure water and dry with nitrogen gas.

[0075] ⑦ Place the electrode (NAS / GO-CDTPA-AuNPs / MPA / tDNA / MCH / hDNA / Au) from step ⑥ in 150 μL of luminol solution (10 mM), react at 37 °C for 2 h, wash with ultrapure water, and dry with nitrogen.

[0076] ⑧ Place the electrode from step ⑦ (luminol / NAS / GO-CDTPA-AuNPs / MPA / tDNA / MCH / hDNA / Au) in 6 mL of H2O2 solution (10 mM, PBS buffer). Measure the luminescence intensity of luminol using an MPI-E electrochemiluminescence detector. Calculate the tDNA concentration based on the luminescence intensity.

[0077] Example 3: Feasibility Verification

[0078] To demonstrate the feasibility of establishing the detection method of this invention, six sets of blank control experiments were conducted and the ECL (enhanced chemiluminescence) reaction was recorded. The results are as follows: Figure 2 As shown in Figure A, the ECL signal was very weak in the absence of hDNA (curve a), tDNA (curve b), MPA (curve c), GO-CDTPA-AuNPs (curve d), PET-RAFT solution (curve e), or luminol (curve f), while a significant ECL response was clearly observed on the fully modified electrode (curve g). In summary, the above experimental results demonstrate the necessity of each modification step and the feasibility of this method for detecting KRAS G12C mutations.

[0079] Example 4: Characterization Analysis

[0080] To verify the successful preparation of AuNPs, their surface morphology was characterized using ultraviolet-visible spectroscopy (UV-Vis) and dynamic light scattering (DLS). Figure 3 As shown in Figure A, AuNPs exhibit a characteristic UV absorption peak at 520 nm, typical of gold nanoparticles, as observed in UV-Vis. Figure 3 The particle size distribution diagram (B-type) shows that the AuNPs are uniformly distributed with an average particle size of 18 nm, indicating that the preparation of AuNPs was successful.

[0081] To definitively confirm the successful synthesis of the GO-CDTPA-AuNPs composite material, its surface morphology was characterized using scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS). The SEM results are shown below. Figure 4 As shown in Figure A, gold nanoparticles are densely loaded on the GO sheet. EDS results are as follows: Figure 4 As shown in Figure B, the composite material is composed of five elements: C, N, O, S, and Au. This further confirms that AuNPs and CDTPA are attached to the GO surface. These results indicate that the composite material was successfully synthesized.

[0082] To demonstrate the successful fabrication of PET-RAFT, the morphology of different modified electrode surfaces was characterized. For example... Figure 5As shown in the SEM image of A, the surface of the bare gold electrode is relatively smooth. When the composite material is attached to the electrode surface, the electrode morphology changes significantly, and a large amount of particulate matter is generated. Figure 5 B), this should be AuNPs. After culturing in PET-RAFT solution for 2 hours, SEM imaging clearly showed a large amount of polymer distributed on the electrode surface (B). Figure 5 C), which also confirms the introduction of CDTPA and the growth of the polymer. Similarly, atomic force microscopy (AFM) characterization of the electrode shows a surface height of 9.9 nm for the bare gold electrode. Figure 5 D). A higher image was obtained after the GO-CDTPA-AuNPs composite material was attached to the electrode surface, with a surface height of 25.4 nm. Figure 5 E). When the electrode was polymerized in a 10 mM NAS mixture, its surface height further increased to 46.1 nm. Figure 5 F). The aggregation process was observed to occur normally in both SEM and AFM, which strongly supports the success of PET-RAFT.

[0083] To verify the successful construction of the modified electrode, the construction process was characterized using EIS. The results are as follows: Figure 2 As shown in Figure B, the impedance spectrum of the bare gold electrode exhibits a relatively small semicircle (~0.19 kΩ, curve a). The assembly of hDNA (~0.73 kΩ, curve b) and MCH (~1.01 kΩ, curve c) leads to a gradual increase in Rct, which is attributed to the interaction between the phosphorylation sites of DNA and the carboxyl sites of the protein with [Fe(CN)6]. 3- / 4- Electrostatic repulsion. Similarly, after tDNA hybridization, due to DNA phosphorylation sites and [Fe(CN)6]... 3- / 4- Electrostatic repulsion between them further increases Rct (~1.41 kΩ, curve d). With the binding of small molecule MPA to the amino groups of hDNA, the impedance further increases (~2.01 kΩ, curve e), possibly due to the ionization of thiol groups in water by hydrogen ions, making the electrode surface negatively charged and thus hindering electron transfer. However, the self-assembly of GO-CDTPA-AuNPs with MPA causes a sharp decrease in impedance (~0.27 kΩ, curve f), as the crystal structure and large specific surface area of ​​Au enhance the electrocatalytic activity of the electrode. Subsequently, the formation of NAS chains (~2.32 kΩ, curve g) increases Rct because the modified highly hydrophobic polymer chains reduce surface hydrophilicity and prevent redox probes from entering the electrode surface. Finally, the attachment of luminol, which is also highly hydrophobic, leads to a significant increase in Rct (~3.37 kΩ, curve h). Furthermore, as... Figure 2 As shown in Figure C, the results of cyclic voltammetry (CV) are negatively correlated with EIS. These results clearly demonstrate the successful manufacture of the test kit.

[0084] Example 5: Condition Optimization

[0085] To improve detection sensitivity, this invention investigated the effects of GO concentration, the volume ratio of GO to AuNPs, and the reaction time of PET-RAFT on ECL signal intensity.

[0086] 1. GO concentration optimization

[0087] The results are as follows Figure 6 As shown in Figure A, the intensity of the ECL signal initially increases with increasing GO dosage, then tends to stabilize. The inflection point for the ECL signal intensity is a GO dispersion concentration of 2 mM. Therefore, in subsequent experiments, the optimal concentration of the GO dispersion was set at 2 mM.

[0088] 2. Optimization of the volume ratio of GO-CDTPA dispersion to AuNPs solution

[0089] The results are as follows Figure 6 As shown in Figure B, when the volume ratio of GO-CDTPA dispersion to AuNPs solution increased from 6:1 to 3:2, the ECL intensity increased with the increase in the proportion of AuNPs solution. This is because with the increase in the proportion of AuNPs solution, the loading of AuNPs on GO also increases, thus producing a stronger catalytic effect on the ECL process of luminol. However, further increases in the proportion of AuNPs solution lead to a decrease in current value. This may be due to the limited number of active sites on the GO flakes and the intense competition among AuNPs, resulting in a large number of AuNPs being in a free state, which prevents them from recombining well with GO. Therefore, the optimal mixing volume ratio of GO-CDTPA dispersion to AuNPs solution is set at 3:2.

[0090] 3. Optimization of PET-RAFT reaction time

[0091] The results are as follows Figure 6 As shown in Figure C, the ECL signal increases continuously with the reaction time of PET-RAFT until it plateaus at 120 min, indicating that the reaction time of PET-RAFT reaches saturation at 120 min. Therefore, 120 min was selected as the optimal reaction time for PET-RAFT.

[0092] Example 6: Performance Analysis

[0093] Under optimal conditions, the limit of detection (LOD) and linear response range of the KRAS G12C tDNA detection kit were evaluated using ECL. Results are as follows: Figure 7 As shown in Figure A, when the concentration of KRAS G12C tDNA is in the range of 1 fM to 10 nM, the ECL signal increases linearly. Figure 7B further showed a positive correlation between the logarithm of I(au) and the concentration of KRAS G12C tDNA. The corresponding linear equation is I(au) = 1438log[C tDNA / pM]+6224(R 2 =0.997), and the calculated LOD is 0.12fM (S / N = 3). Compared with other methods, the method proposed in this invention for detecting KRAS G12C mutations has a relatively low detection limit and a wide detection range (see table below) due to the combination of the sensitivity of ECL, the GO-CDTPA-AuNPs composite material, and the advantages of the in-situ induced PET-RAFT signal amplification strategy. This indicates that the kit of this invention has potential application value in the early detection of lung cancer.

[0094]

[0095] Example 7: Analysis of selectivity, reproducibility, stability and anti-interference ability

[0096] Under the same experimental conditions, the present invention tested the electrochemiluminescence (ECL) signals of tDNA, SBM, DBM, NC, and blank to demonstrate the selectivity of the proposed method. Figure 8 As shown in Figure A, the electrochemiluminescence signal of SBM is 30.17% of that of tDNA, while the ECL signals of DBM, NC, and blank are 78.16%, 94.93%, and 95.6% lower than that of tDNA, respectively. These results indicate that the prepared kit has high selectivity for the detection of tDNA.

[0097] Furthermore, the reproducibility of the kit was investigated based on intra- and inter-batch tests (n=5). The relative standard deviations (RSDs) between and within batches were 2.46% and 2.27%, respectively, indicating high reproducibility of the fabricated modified electrode. To evaluate the stability of the modified electrode, five identical modified electrodes were prepared and stored in a 4°C refrigerator. After three weeks, up to 91.51% of the ECL signal was observed to be retained. Therefore, the stability of the modified electrode during storage is satisfactory.

[0098] To evaluate the interference resistance of this kit, the ECL signal of tDNA in 10% (v / v) human serum samples was investigated. A certain amount of tDNA was injected into 10% (v / v) serum to prepare a serum sample. The corresponding current signal in the 10% (v / v) serum sample was compared with the ECL signal in TE buffer. The results are as follows: Figure 8As shown in Figure B, the current signals of 10% (v / v) serum samples were 101.9%, 95.9%, and 91.1% of those of 10 fM, 1 pM, and 100 pM tDNA in TE buffer, respectively. Therefore, the kit of the present invention exhibits good anti-interference ability in serum samples, indicating its significant potential for clinical application.

Claims

1. A GO-CDTPA-AuNPs and PET-RAFT based electrochemiluminescence detection kit, characterized in that, The raw materials include: gold electrode, hDNA, MCH, MPA-NHS ester, GO-CDTPA-AuNPs, Me6TREN, EY, NAS, luminol; the sequence of hDNA is shown as SEQ ID NO. 1; the preparation method of GO-CDTPA-AuNPs is: ① 20 mg of GO is dissolved in 10 mL of ultrapure water and ultrasonically treated to obtain a GO dispersion; ② 20 mg of ETA and 20 mg of KOH are dispersed in the GO dispersion, reacted, centrifuged and washed, and vacuum dried to obtain ETA-modified GO, i.e. GO-ETA; ③ 2 mg of CDTPA, 1.6 mg of EDC and 1 mg of NHS are uniformly mixed in 5 mL of DMF, reacted to obtain a CDTPA-NHS ester solution; ④ 5 mg of GO-ETA and 0.2 mg of DMAP are dissolved in 5 mL of the CDTPA-NHS ester solution, reacted, centrifuged, and the precipitate is collected and re-dispersed with 1 mL of ultrapure water to obtain a GO-CDTPA dispersion; ⑤ 1 mM of AuNPs solution and the GO-CDTPA dispersion are uniformly mixed in a volume ratio of 2:3, reacted, centrifuged, and the precipitate is collected and re-dispersed with ultrapure water to obtain a GO-CDTPA-AuNPs dispersion.

2. The electrochemiluminescent test kit according to claim 1, characterized in that, PBS buffer and H2O2 are also included.

3. The electrochemiluminescent detection kit according to claim 1, wherein The preparation method of MPA-NHS ester is to uniformly mix equal moles of EDC, NHS and MPA and react at 37°C for 3 h to obtain a MPA-NHS ester solution with a concentration of 1 mM.

4. The electrochemiluminescent detection kit according to claim 1, wherein The ultrasonic treatment time of ① is 30 min; the reaction condition of ② is stirring at 80°C for 24 h; the reaction condition of ③ is reaction at 37°C for 15-24 h; the reaction condition of ④ is reaction at room temperature in the dark for 24 h; and the reaction condition of ⑤ is stirring at room temperature for 12 h.

5. The electrochemiluminescent detection kit according to claim 1 or 2, characterized in that, Some raw materials need to be prepared into solutions when used, wherein the concentration of the hDNA solution is 0.5 μM, the concentration of the MCH solution is 2 mM, the concentration of the MPA-NHS ester solution is 1 mM, the concentration of the Me6TREN solution is 1.2 mM, the concentration of the EY solution is 0.02 mM, the concentration of the NAS solution is 10 mM, the concentration of the luminol solution is 10 mM, the concentration of the PBS buffer is 0.1 M, pH=7.4, and the concentration of the H2O2 solution is 10 mM.

6. A method of using the electrochemiluminescent test kit of claim 1, wherein, The following steps are included: (1) electrode pretreatment The bare gold electrode is polished to obtain a polished mirror surface; (2) electrode modification ① The hDNA solution is dropped onto the surface of the electrode and reacted; ② The electrode of step ① is immersed in the MCH solution and reacted; ③ The solution to be detected is directly dropped onto the surface of the electrode of step ② and reacted; ④ The electrode of step ③ is soaked in the MPA-NHS ester solution and reacted; ⑤ The GO-CDTPA-AuNPs dispersion is dropped onto the surface of the electrode of step ④ and reacted; ⑥ The electrode of step ⑤ is placed in a mixed solution composed of the Me6TREN solution, the EY solution, the NAS solution and the PBS buffer and reacted; Reaction temperature of ① is 37℃, time is 2-8 h; reaction temperature of ② is 37℃, time is 0.5-1 h; reaction temperature of ③ is 37℃, time is 1.5-2 h; reaction temperature of ④ is 37℃, time is 1-2 h; reaction temperature of ⑤ is 37℃, time is 1-3 h; reaction condition of ⑥ is room temperature, 470 nm blue light irradiation, 2 h; reaction temperature of ⑦ is 37℃, time is 1-3 h.

8. Use of the kit of any one of claims 1-5 in the preparation of a reagent for detecting lung cancer.

7. The electrochemiluminescent detection kit according to claim 6, wherein The lung cancer is non-small cell lung cancer. ​ 9. Use according to claim 8, characterized in that, ​

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