An electrochemiluminescence detection kit based on PET-RAFT signal amplification strategy and its use method and application

Abstract

The present invention discloses an electrochemiluminescence detection kit based on a PET-RAFT signal amplification strategy and a method for use and application. The kit includes: a gold electrode, hDNA, MCH, CDTPA-NHS ester solution, Me6TREN, EY, APMA, NGQDs, K2S2O8. The present invention adopts NGQDs as a luminophore and a PET-RAFT signal amplification strategy. First, a hairpin DNA (hDNA) probe is self-assembled and connected to the electrode surface through Au-S bonds, and after the residual binding site is blocked with 6-mercaptohexanol (MCH), the target DNA (tDNA) is connected to the electrode surface, and the hDNA is opened and the amino group is exposed. Subsequently, the carboxyl group of CDTPA is connected to the hDNA through an amide reaction. Then, under the irradiation of blue light, a PET-RAFT reaction is triggered, and an APMA polymer chain is formed on CDTPA, which provides a large number of binding sites for the signal unit NGQDs, thereby further significantly amplifying the ECL signal. The tDNA concentration is analyzed by observing the strength of the ECL signal.

Description

Technical Field

[0001] The present invention relates to an electrochemiluminescence detection kit based on NGQDs luminophore and photoinduced electron / energy transfer reversible addition-fragment chain transfer (PET-RAFT) multi-signal amplification strategy, as well as a use method and application, belonging to the field of bioanalysis technology. Background Art

[0002] With the advancement of molecular biotechnology, the detection of driver mutations and appropriate targeted drug therapy in patients with non-small cell lung cancer (NSCLC) have been gradually incorporated into medical practice, offering hope for long-term survival. Kirsten rat sarcoma viral oncogene (KRAS), the most common driver oncogene in NSCLC, accounts for approximately 15%–20% of all NSCLC mutations. Notably, the KRAS G12C mutation (G→T) accounts for 40% of KRAS mutations. Therefore, mutant gene sequences associated with KRAS G12C are often used as targeted DNA (tDNA) for NSCLC diagnosis. The gold standard for mutation detection is DNA sequencing, which offers advantages such as high reproducibility for known genes and the ability to detect unknown mutations. However, due to its low sensitivity, time-consuming nature, and high cost, it is not widely used in clinical practice. Notably, the low mutation levels in early-stage NSCLC patients result in a significant number of patients being diagnosed at advanced stages. Therefore, developing a method for sensitive detection of KRAS G12C is of great significance for early diagnosis of NSCLC and drug development.

[0003] Electrochemiluminescence (ECL) biosensors have both the sensitivity of luminescence technology and the simplicity of sensing technology. Compared with chemiluminescence, ECL is highly controllable, and the excited state of the luminophore can be adjusted by changing the applied potential. Compared with fluorescence analysis, ECL does not require an external light source, avoiding problems such as light scattering and background interference from luminescent impurities. Therefore, it is widely used in ultrasensitive nucleic acid detection. ECL luminophores are of great significance to improving the performance of sensing systems. ECL luminophores are divided into three categories: (1) Inorganic systems, mainly composed of organometallic complexes; (2) Organic systems, covering polycyclic aromatic hydrocarbons; (3) Nanomaterial systems, especially quantum dots (QDs). Compared with traditional molecular ECL emitters (such as Ru(bpy)3 2+Compared with luminol, quantum dots can effectively improve the reactivity of interfacial electrochemistry and thus improve the intensity of ECL due to their advantages such as small size effect, multiple surface effects, large specific surface area and multiple surface active sites. As a new type of luminescent nanoparticles, graphene quantum dots (GQDs) have the advantages of low cytotoxicity, good water solubility and excellent biocompatibility, quantum confinement effect and simple preparation method, which has attracted extensive research by scientists in various fields. By doping GQDs with heteroatoms, their local electronic configuration, polarizability, defect level and band structure are changed, thereby effectively improving the photoelectric properties of GQDs. In order to further improve the sensitivity of ECL sensors constructed with NGQDs, people have been seeking various signal amplification strategies to expand their application in biological detection.

[0004] Photoinduced electron / energy transfer reversible addition fragment chain transfer (PET-RAFT) polymerization has superior performance in terms of applicability, cost and sustainability. (1) It can provide a suitable temperature for the polymerization of monofunctional monomers, such as room temperature or lower, to reduce the occurrence of side reactions. (2) It has a high degree of temporal and spatial control, that is, the removal of the light source will cause the polymerization to stop. (3) The all-organic system greatly reduces the amount of metal catalyst used. Based on its excellent performance, the present invention aims to use PET-RAFT polymerization reaction based on eosin Y (EY) excitation for signal amplification to study an electrochemiluminescence sensing system for highly sensitive detection of KRAS G12C based on NGQDs luminophore and PET-RAFT signal amplification strategy, which is of great significance for the early diagnosis of NSCLC. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide an electrochemiluminescence kit based on NGQDs luminophore and PET-RAFT signal amplification strategy, as well as a usage method and application. Different raw material ratios are used to synthesize NGQDs with the strongest electroluminescence signal to construct an electrochemiluminescence sensing system. On the one hand, it combines the advantages of nanomaterials and the versatility and convenience of photoinitiated free radical polymerization, avoiding the use of heavy metal ion catalysts in traditional RAFT reactions, and grafting a large number of signal unit NGQDs into the sensing system to reduce the detection limit and improve the sensitivity, stability and reproducibility of detection.

[0006] In order to achieve the above object, one of the technical solutions of the present invention is:

[0007] An electrochemiluminescence detection kit based on a PET-RAFT signal amplification strategy includes: a gold electrode, hDNA, MCH, a CDTPA-NHS ester solution, Me6TREN, EY, APMA, NGQDs, and K2S2O8.

[0008] Furthermore, the sequence of hDNA is shown as SEQ ID NO.1.

[0009] Furthermore, synthesis of NGQDs: citric acid and urea were added into a crucible at a molar ratio of 1:4 and reacted at 200 °C for 8 h to obtain NGQDs.

[0010] Furthermore, the CDTPA-NHS ester solution was prepared by mixing an EDC solution, an NHS solution, and a CDTPA solution of equal molar concentration and volume, and reacting the mixture at 37° C. for 3 h.

[0011] Furthermore, the novel electrochemiluminescence detection kit further includes PBS buffer.

[0012] Furthermore, some raw materials need to be prepared into solutions when used, among which the concentration of hDNA solution is 0.5 μM, the concentration of MCH solution is 2 mM, the concentration of CDTPA-NHS ester solution is 2 mM, the concentration of Me6TREN solution is 1%, and the concentration of EY solution is 0.5 mg mL -1 , the concentration of APMA solution was 1 M, and the concentration of NGQDs solution was 3 mg mL -1 , the concentration of K2S2O8 solution is 10mM.

[0013] One of the technical solutions of the present invention is: a method for using a novel electrochemiluminescence detection kit, comprising the following steps:

[0014] (1) Add hDNA solution dropwise to the surface of the gold electrode for reaction;

[0015] (2) immersing the electrode of step (1) in MCH solution for reaction;

[0016] (3) directly adding the sample solution to be tested to the electrode surface in step (2) for reaction;

[0017] (4) soaking the electrode in step (3) in a CDTPA-NHS ester solution for reaction;

[0018] (5) placing the electrode in step (4) in a mixed solution consisting of Me6TREN solution, EY solution, APMA solution and PBS buffer solution for reaction;

[0019] (6) placing the electrode from step (5) in the NGQDs solution for reaction;

[0020] (7) Place the electrode in step (6) in a K2S2O8 solution and measure the luminescence intensity of NGQDs.

[0021] Furthermore, the bare gold electrode was pretreated by grinding the gold electrode to obtain a polished mirror surface. Then, the electrode was placed in Piranha solution for 15 minutes and ultrasonically rinsed with ultrapure water, anhydrous ethanol, and ultrapure water for 30 seconds. Finally, the gold electrode was placed in 0.1M H2SO4 solution and scanned by cyclic voltammetry. When two completely consistent reduction peaks were obtained, it indicated that the gold electrode had been cleaned. It was then ultrasonically washed with ultrapure water and dried with N2. The cyclic voltammetry scan potential range was -0.3 to 1.5 V, and the scan rate was 0.2 V s. -1 .

[0022] Furthermore, the reaction temperature of step (1) is 37°C and the time is 2 to 8 hours; the reaction temperature of step (2) is 37°C and the time is 0.5 to 1 hour; the reaction temperature of step (3) is 37°C and the time is 1.5 to 2 hours; the reaction temperature of step (4) is 37°C and the time is 1 to 2 hours; the reaction conditions of step (5) are room temperature, 470nm light irradiation, 75 minutes; the reaction temperature of step (6) is 37°C and the reaction time is 1 to 3 hours.

[0023] One of the technical solutions of the present invention is: use of a novel electrochemiluminescence detection kit in the preparation of a KRAS G12C detection reagent.

[0024] The schematic diagram of the detection method of the present invention is as follows Figure 1 shown.

[0025] The present invention uses NGQDs as a luminophore and a PET-RAFT signal amplification strategy. First, a hairpin DNA (hDNA) probe is self-assembled and attached to the electrode surface via Au-S bonds. After the residual binding sites are blocked with 6-mercaptohexanol (MCH), the target DNA (tDNA) is attached to the electrode surface. At the same time, the hDNA is opened and the amino groups are exposed. Subsequently, the carboxyl groups of CDTPA are connected to the hDNA through an amide reaction. Then, under the irradiation of blue light, a PET-RAFT reaction is triggered, forming APMA polymer chains on CDTPA, providing a large number of binding sites for the signal unit NGQDs, thereby further significantly amplifying the ECL signal. The tDNA concentration is analyzed by observing the strength of the ECL signal.

[0026] The PET-RAFT reaction principle is as follows Figure 1 As shown in the figure, the monomer APMA is polymerized under 470nm blue light irradiation with CDTPA as chain transfer agent, EY as catalyst, and Me6TREN as ligand. Under the excitation of blue light of a specific wavelength, the ground state EY is converted to the excited state EY* by absorbing photons. After that, electrons are transferred from the donor (NR3, Me6TREN) to EY*, and EY is generated at the same time. ·- and NR3 ·+Based on three catalysts (EY, EY* and EY ·－ ), a reversible cyclic pathway was established to regulate the reductive quenching pathway of PET-RAFT and graft a large amount of monomer APMA onto the electrode surface.

[0027] The present invention uses NGQDs as a luminophore to cooperate with the PET-RAFT signal amplification strategy. NGQDs avoid the shortcomings of traditional GQDs such as unstable luminescence signals and low quantum yield, as well as the need for thermal initiation in traditional RAFT reactions. This strategy has mild reaction conditions and can achieve "active" / controllable polymerization at room temperature; it does not produce biological toxicity and is more environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the principle of the detection method of the present invention.

[0029] Figure 2 X-ray photoelectron spectra (A) and high-resolution C1s XPS spectra (B) of NGQDs synthesized with different ratios of raw materials.

[0030] Figure 3 UV-visible spectra (A), Raman spectra (B), and electrochemiluminescence spectra (C) of NGQDs synthesized with different ratios of raw materials.

[0031] Figure 4 A represents the ECL signal intensity under different electrode surface modification conditions. Curve a represents the absence of hDNA, curve b represents the absence of tDNA, curve c represents the absence of CDTPA, curve d represents the absence of PET-RAFT reaction solution, curve e represents the absence of NGQDs, and curve f represents NGQDs / APMA / CDTPA / tDNA / MCH / hDNA / Au.

[0032] Figure 4 B is the evolution of the impedance curve of the electrode after each step of modification from the bare gold electrode (curve a→g).

[0033] Figure 4 C is the CV curve of the electrode after each step of modification from the bare gold electrode (curve a→g).

[0034] Figure 5The morphological characterizations of the electrode surfaces at different modification states are shown in Figure 1. (A) is the scanning electron microscopy (SEM) characterization of the electrode before PET-RAFT modification, (B) is the SEM characterization of the electrode after PET-RAFT modification, and (C) is the SEM characterization of the electrode after NGQDs modification. (D) is the AFM characterization of the electrode before PET-RAFT modification, (E) is the AFM characterization of the electrode after PET-RAFT modification, and (F) is the AFM characterization of the electrode after NGQDs modification.

[0035] Figure 6 Condition optimization. Relationship between ECL signal intensity and CDTPA concentration (A), PET-RAFT reaction time (B), and NGQDs concentration (C).

[0036] Figure 7 Figure 2 is the relationship between ECL signal intensity and tDNA concentration (A) and the corresponding linear correlation curve (B).

[0037] Figure 8 A is a comparison of the electroluminescent signal intensities of 1 nM tDNA (Target) and the same concentrations of single-base mismatch DNA (SBM), double-base mismatch DNA (DBM), and fully mismatched DNA (NC) under the same detection conditions.

[0038] Figure 8 B is a comparison of the electroluminescence signal intensities of tDNA at different concentrations in TE buffer and 10% serum samples. DETAILED DESCRIPTION

[0039] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0040] hDNA, tDNA, SBM, DBM, and NC were purchased from Sangon Biotech (Shanghai) Co., Ltd., and their sequences are as follows:

[0041]

[0042] Example 1, kit

[0043] An electrochemiluminescence detection kit based on NGQDs luminophore and PET-RAFT multi-signal amplification strategy includes: a gold electrode, hDNA, 6-mercaptohexanol (MCH), CDTPA-NHS ester solution, tris(2-dimethylaminoethyl)amine (Me6TREN), eosin Y (EY), N-(3-aminopropyl) methacrylamide (APMA), nitrogen-doped graphene quantum dots (NGQDs), and K2S2O8.

[0044] Some raw materials need to be prepared into solutions when used. Among them, the concentration of hDNA solution is 0.5μM, the concentration of MCH solution is 2mM, the concentration of CDTPA-NHS ester solution is 2mM, the concentration of Me6TREN solution is 1% v / v, and the concentration of EY solution is 0.5mg mL -1 , the concentration of APMA solution is 1 M, and the concentration of NGQDs solution is 3 mg mL -1 , the concentration of K2S2O8 solution is 10mM.

[0045] Preparation of CDTPA-NHS ester solution:

[0046] Equal volumes of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution (6 mM), N-hydroxysuccinimide (NHS) solution (6 mM), and 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA) solution (6 mM) were mixed and reacted at 37°C for 3 h.

[0047] Synthesis of nitrogen-doped graphene quantum dots (NGQDs):

[0048] (1) Citric acid and urea were added into a crucible at a molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6 and reacted at 200 °C for 8 h to obtain NGQDs;

[0049] (2) NGQDs synthesized in different proportions were ground, dispersed in water, and ultrasonicated to obtain 3 mg mL -1 NGQDs solution;

[0050] (3) Select the NGQDs with the strongest electrochemiluminescence intensity and set aside.

[0051] To verify whether the NGQDs were successfully prepared, the NGQDs were characterized by X-ray photoelectron spectroscopy (XPS), high-resolution C1s XPS spectrum, ultraviolet-visible spectrum (UV-Vis), Raman spectrum and electrochemiluminescence (ECL) spectrum. Figure 2 A shows the XPS of NGQDs at different ratios, showing three elements, including C1s, N1s, and O1s, representing nitrogen-rich and oxygen-rich groups, respectively. Figure 2 B shows the high-resolution C1s XPS spectra of NGQDs at different ratios, and four peaks can be observed at around 284, 285, 287 and 288 eV. The peaks at 284 and 285 eV are sp 2 -sp 2 C(CC) and N-sp 2The binding energy of C(CN) bonds is 287 and 288 eV, respectively, for CO-type bonds (CO and OC=O). Figure 3 Figure A shows the UV-Vis spectra of NGQDs prepared at different ratios. Two characteristic UV absorption peaks are observed: one at 340 nm, attributed to the π→π* transition of the conjugated graphene network, and another at 400 nm, attributed to the n-π* electron transition associated with nitrogen and oxygen free electron pairs. Notably, the NGQDs prepared with a 1:1 ratio of citric acid and urea lack an absorption peak at 400 nm, due to the relatively low nitrogen doping. As the ratio of citric acid to urea increases, the UV absorption of the NGQDs undergoes a significant red shift, indicating an increase in the number of cochromatic groups in the NGQDs and an increase in the mobility of the electron cloud in the conjugated system. Figure 3 B shows the Raman spectra of NGQDs at different ratios. At approximately 1360 cm -1 The D peak (C atom crystal defect) and the G peak (C atom sp 2 Hybrid in-plane stretching vibration) appears at 1570 cm -1 With the increase of urea content, a gradual increase in the ID / IG ratio was observed, indicating an increase in the degree of defects in NGQDs, and the ID / IG ratio stabilized when the ratio of citric acid to urea content reached approximately 1:4. Figure 3 Figure C shows the ECL spectra of NGQDs at different urea ratios. The ECL intensity of NGQDs gradually increases with increasing urea ratio. When the ratio increases to 1:4, NGQDs exhibit excellent ECL intensity. This is due to the increase in N and O doping content, which increases the degree of defects and facilitates electron conduction. Therefore, the optimal molar ratio of citric acid to urea selected in this invention is 1:4.

[0052] Example 2: Method of using the kit

[0053] (1) Electrode pretreatment

[0054] The bare gold electrode was polished to obtain a mirror-like surface. Then, the electrode was placed in piranha solution for 15 min and ultrasonically rinsed with water, anhydrous ethanol, and water for 30 s. Finally, the gold electrode was placed in 0.1 M H2SO4 solution and scanned by cyclic voltammetry (CV) (potential range: -0.3 to 1.5 V, scan rate: 0.2 V s -1 ), when two completely consistent reduction peaks are obtained, it indicates that the gold electrode has been cleaned. It is then ultrasonically washed with ultrapure water and dried with N2.

[0055] (2) Electrode modification

[0056] ① Add 5 μL hDNA solution (0.5 μM) dropwise to the electrode surface, react at 37°C for 2-8 h, wash with ultrapure water, and blow dry with N2;

[0057] ② Immerse the electrode (hDNA / Au) from step ① in 150 μL of MCH solution (2 mM) at 37°C for 0.5–1 h, then wash with ultrapure water and blow dry with N2;

[0058] ③ Add 10 μL of the sample solution to be tested (containing tDNA) dropwise to the surface of the electrode (MCH / hDNA / Au) prepared in step ②, incubate at 37°C for 1.5-2 hours, wash with ultrapure water, and blow dry with N2;

[0059] ④ Immerse the electrode (tDNA / MCH / hDNA / Au) prepared in step ③ in 150 μL of CDTPA-NHS ester solution (2 mM) at 37°C for 1–2 h, then wash with ultrapure water and blow dry with N2.

[0060] ⑤ Place the electrode (CDTPA / tDNA / MCH / hDNA / Au) prepared in step ④ in 136 μL Me6TREN solution (1% v / v), 200 μL EY solution (0.5 mg mL -1 ), 800 μL APMA solution (1 M) and 864 μL PBS buffer (0.1 M), reacted at room temperature under 470 nm light irradiation for 75 min (PET-RAFT reaction), washed with ultrapure water, and dried with N2;

[0061] ⑥ Place the electrode (APMA / CDTPA / tDNA / MCH / hDNA / Au) prepared in step ⑤ in 150 μL of NGQDs solution (3 mg mL -1 ), react for 1 to 3 hours, wash with ultrapure water, and blow dry with N2;

[0062] ⑦ Place the electrode (NGQDs / APMA / CDTPA / tDNA / MCH / hDNA / Au) prepared in step ⑥ in 6 mL of K2S2O8 solution (10 mM, solvent is PBS buffer, pH = 7.4) and measure its luminescence intensity using an MPI-E electrochemiluminescence detector.

[0063] Example 3: Feasibility Verification

[0064] In order to prove the feasibility of establishing the detection method of the present invention, 6 groups of blank control experiments were carried out. Figure 4As shown in Figure A, in the absence of hDNA (curve a), tDNA (curve b), CDTPA (curve c), PET-RAFT solution (curve d), or NGQDs (curve e), the ECL signals are very weak, while a significant ECL response is clearly observed on the fully modified electrode (curve f). In summary, the above experimental results demonstrate the feasibility of this detection method.

[0065] Example 4: Characterization Analysis

[0066] In order to prove the successful preparation of PET-RAFT and the successful connection of NGQDs, the present invention used scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize the morphology of the electrode surfaces with different modifications. Figure 5 A SEM image of the electrode before PET-RAFT reaction modification shows a relatively smooth surface. After the PET-RAFT reaction, the electrode morphology changes significantly, with a large amount of polymer distributed on the electrode surface ( Figure 5 B). When NGQDs were attached to the electrode surface, the electrode morphology changed significantly and a large amount of particulate matter was generated ( Figure 5 C), which indicates that the NGQDs are successfully connected. Similarly, the surface height of the electrode before PET-RAFT reaction modification is 10.0 nm ( Figure 5 D). The surface height of the electrode modified by PET-RAFT reaction is 22.9 nm ( Figure 5 E). When NGQDs are attached to the electrode surface, their surface height further increases to 33.5 nm ( Figure 5 F). The normal connection process was observed by both SEM and AFM, which strongly indicates the successful PET-RAFT polymerization and the successful connection of NGQDs.

[0067] In order to verify whether the sensing system is successfully constructed, the present invention characterized its construction process by electrochemical impedance spectroscopy (EIS). The measurement solution is 5 mM [Fe(CN)6] 3- / 4- Electrolyte solution. Figure 4 As shown in B, the impedance spectrum of the bare gold electrode has a relatively small semicircle (~0.23 kΩ, curve a). Incubation with hDNA (~0.45 kΩ, curve b) and MCH (~0.63 kΩ, curve c) both resulted in a gradual increase in Rct, which is due to the interaction between the phosphorylation sites of DNA and the carboxyl sites of proteins with [Fe(CN)6] 3- / 4- Similarly, after tDNA hybridization, due to the electrostatic repulsion between DNA phosphorylation sites and [Fe(CN)6] 3- / 4-Due to the electrostatic repulsion between the two groups, Rct further increases (~0.91 kΩ, curve d). As the small molecule CDTPA binds to the amino groups of hDNA, the impedance value further increases (~1.26 kΩ, curve e), because the highly hydrophobic groups after modification reduce the surface hydrophilicity, moving the redox probe away from the electrode surface. Interestingly, the formation of APMA chains causes a sharp decrease in impedance (~0.54 kΩ, curve f). This is because the large number of amino groups gives the electrode surface a positive charge, which promotes the [Fe(CN)6] 3- / 4 The opposite attraction between them promotes the transfer of charge. Subsequently, the attachment of NGQDs leads to a significant decrease in Rct (~0.35kΩ, curve g). This is because the N doping of GQDs increases the carrier concentration of quantum dots and accelerates the mobility, thereby promoting electron transfer. In addition, Figure 4 The results of cyclic voltammetry (CV) were negatively correlated with those of EIS, as shown in Figure C. These results clearly demonstrated the successful construction of the DNA biosensing system.

[0068] Example 5: Condition Optimization

[0069] In order to improve the sensitivity of detection, the present invention studied the effects of CDTPA concentration, PET-RAFT reaction time, and NGQDs concentration on the ECL signal intensity.

[0070] 1. CDTPA concentration

[0071] like Figure 6 As shown in Figure A, as the concentration of CDTPA solution increases, the ECL signal intensity first increases slowly and then stabilizes. A CDTPA concentration of 2 mM is the turning point of the ECL signal intensity. Therefore, in subsequent experiments, the optimal concentration of CDTPA solution was set at 2 mM.

[0072] 2. Optimization of PET-RAFT reaction time

[0073] like Figure 6 As shown in Figure B, the ECL signal intensity increases with the extension of reaction time until it reaches a plateau at 75 min, indicating that the polymer chain stops growing after 75 min. Therefore, 75 min was selected as the ideal reaction time for PET-RAFT.

[0074] 3. NGQDs concentration optimization

[0075] like Figure 6 As shown in C, when the concentration of NGQDs solution increased from 0.5 mg mL -1 Increase to 3 mg mL -1, the ECL intensity gradually increased. However, as the concentration of NGQDs further increased, the ECL signal intensity further decreased. This is due to the aggregation effect that occurs when the concentration of NGQDs is too high, which prevents the quantum dots from attaching to the monomers. Therefore, the optimal concentration of NGQDs is 3 mg mL -1 .

[0076] Example 6: Performance Analysis

[0077] Under optimal conditions, the limit of detection (LOD) and linear response range of the KRAS G12C DNA detection kit were evaluated by ECL. Figure 7 As shown in A, when the concentration of KRAS G12C DNA is in the range of 10 fM to 10 nM, the ECL signal intensity increases linearly with the concentration of the target. Figure 7 B further shows that there is a positive correlation between I(au) and the logarithmic value of KRAS G12C DNA concentration. The corresponding linear equation is I(au) = 1090log[C tDNA / pM ]+2876(R 2 =0.9991), with a limit of detection (LOD) of 8 fM (S / N = 3). Compared with other methods for detecting KRAS mutations, the proposed method, based on the NGQDs and PET-RAFT biosensor system, has a wider detection range and a relatively low limit of detection (Table below). This demonstrates the potential application of the present kit in the early detection of NSCLC.

[0078]

[0079] Example 7: Analysis of selectivity, reproducibility, stability and anti-interference ability

[0080] Under the same experimental conditions, the present invention tested the ECL signal intensity of tDNA, SBM, DBM, and NC at the same concentration (1 nM) to prove the good selectivity of the proposed method. Figure 8 As shown in Figure A, the ECL signal intensities of SBM, DBM, and NC are 25.3%, 15.97%, and 6.78% of the tDNA ECL signal intensity, respectively, indicating that the prepared biosensor system has high selectivity for the detection of tDNA.

[0081] In addition, the reproducibility of the kit was investigated based on intra-batch and inter-batch tests (n=5). The inter-batch and intra-batch relative standard deviations (RSDs) were 2.1% and 2.4%, respectively, indicating that the electrochemiluminescence detection kit has high reproducibility. To evaluate the stability of the electrochemiluminescence detection kit, five identical modified electrodes were prepared and stored in a 4°C refrigerator. After three weeks of storage, the ECL signal intensity reached 93.67% of the initial signal intensity. This result indicates that the electrochemiluminescence detection kit has good stability.

[0082] In order to evaluate the anti-interference ability of the kit, the present invention uses the kit to compare the ECL signal intensity of tDNA in 10% (v / v) human serum samples and tDNA in TE buffer. A certain amount of tDNA is injected into 10% (v / v) serum to form serum samples with tDNA concentrations of 10fM, 1pM and 100pM respectively. The ECL signal intensity of the 10% (v / v) serum sample is compared with the ECL signal intensity of the corresponding concentration of tDNA in TE buffer. The results are shown in FIG. Figure 8 As shown in Figure B, the ECL signal intensities in TE buffer were 105.5%, 97.8%, and 105.1% of the ECL signal intensities of 100 fM, 10 pM, and 1 nM tDNA in 10% (v / v) serum samples, respectively. Therefore, the electrochemiluminescence detection kit has good anti-interference ability in serum samples, indicating that it has significant potential for clinical application.

Claims

1. An electrochemiluminescence detection kit based on PET-RAFT signal amplification strategy, characterized in that: include: Gold electrode, hDNA, 6-mercaptohexanol MCH, CDTPA-NHS ester solution, tris(2-dimethylaminoethyl)amine Me6TREN, eosin Y EY, N-(3-aminopropyl)methacrylamide APMA, nitrogen-doped graphene quantum dots NGQDs, K2S2O8; The sequence of hDNA is shown in SEQ ID NO. 1; The CDTPA-NHS ester solution is prepared by mixing equal volumes of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution, N-hydroxysuccinimide (NHS) solution, and 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA) solution at equimolar concentrations and reacting the mixture.

2. The electrochemiluminescence detection kit according to claim 1, characterized in that Synthesis of NGQDs: Citric acid and urea were added into a crucible at a molar ratio of 1:4 and reacted at 200 °C for 8 h to obtain NGQDs.

3. The electrochemiluminescence detection kit according to claim 1, characterized in that The reaction temperature was 37°C and the reaction time was 3 h.

4. The electrochemiluminescence detection kit according to claim 1, characterized in that PBS buffer is also included.

5. The electrochemiluminescence detection kit according to any one of claims 1 to 4, characterized in that Some raw materials need to be prepared into solutions when used. The concentration of hDNA solution is 0.5 μM, the concentration of MCH solution is 2 mM, the concentration of CDTPA-NHS ester solution is 2 mM, the concentration of Me6TREN solution is 1%, and the concentration of EY solution is 0.5 mg mL -1 , the concentration of APMA solution was 1 M, and the concentration of NGQDs solution was 3 mg mL -1 , the concentration of K2S2O8 solution is 10 mM.

6. Use of the electrochemiluminescence detection kit according to claim 1 in preparing a KRAS G12C detection reagent.

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