Isothermal amplification method and kit for detecting EGFR-L858R and EGFR-T790M point mutation

Abstract

The invention belongs to the technical field of gene mutation detection, and particularly relates to an isothermal amplification method and a kit for detecting EGFR-L858R and EGFR-T790M point mutation. One purpose of the invention is to provide a kit for detecting EGFR-L858R and EGFR-T790M point mutation. The kit comprises a capture probe sequence as shown in SEQ ID NO.5 and a signal probe sequence as shown in SEQ ID NO.6, wherein the capture probe sequence is used for detecting L858R point mutation; a capture probe sequence as shown in SEQ ID NO.10 and a signal probe sequence as shown in SEQ ID NO.11, which are used for detecting T790M point mutation. According to the technical scheme, PdAgBP is taken as a solid-phase immobilized substrate, EXPAR isothermal amplification and nucleic acid site LNA padlock are taken as a dual signal amplification mechanism, and biosensors based on BPE-ECL are respectively constructed for detection of point mutation EGFR L858R and EGFR T790M.

Description

Technical Field

[0001] This invention belongs to the field of gene mutation detection technology, and specifically relates to an isothermal amplification method and kit for detecting EGFR-L858R and EGFR-T790M point mutations. Background Technology

[0002] EGFR gene mutations play a crucial role in the pathological process of lung cancer. The L858R point mutation is one of the common activating mutations in the EGFR kinase domain, leading to persistent activation of the EGFR signaling pathway and promoting tumor cell proliferation, survival, and metastasis. The T790M point mutation is one of the main mechanisms leading to resistance to EGFR tyrosine kinase inhibitors. This mutation alters the structure of the EGFR ATP binding site, weakening the binding and inhibitory effects of tyrosine kinase inhibitors, ultimately resulting in treatment failure. Therefore, accurate detection of EGFR L858R and T790M mutations is of great significance for the formulation of treatment strategies and prognostic assessment in lung cancer patients.

[0003] In recent years, to improve detection performance, new technologies such as LAN and exponential amplification reaction (AAR) have been gradually applied to the field of gene mutation detection. LAN is a class of chemically modified nucleic acid analogs whose ribose rings are connected by a methylene bridge between the 2'-O and 4'-C molecules, forming a rigid double-loop structure. This structure significantly enhances the binding stability between LAN and complementary nucleic acids, increases the melting temperature of the hybrid double strand, and thus exhibits stronger sequence discrimination ability when identifying single-base mutations. Even if the target sequence differs by only a single base, LAN-modified probes can achieve highly sensitive identification of mutations through significant changes in hybridization characteristics, reducing interference from wild-type background and improving detection accuracy. Meanwhile, AAR, as a highly efficient isothermal nucleic acid amplification technology, can achieve exponential amplification of the target sequence under isothermal conditions through the synergistic action of DNA polymerase and endonuclease, significantly improving detection sensitivity. Compared with polymerase chain reaction (PCR), this technology does not require temperature cycling equipment, is rapid, and easy to operate, making it more suitable for rapid detection in clinical settings. Its amplification product is single-stranded DNA, facilitating integration with downstream detection methods.

[0004] While techniques such as LAN and exponential amplification reactions have provided new avenues for mutation detection, the detection of single-base mutations still faces multiple challenges in practical applications. Because single-base mutation sequences are short and carry limited genetic information, the number of hydrogen bonds formed during hybridization with probes is small and the interactions are weak, resulting in weak signal changes and making high-sensitivity detection difficult. Furthermore, the difference in a single base limits the ability of conventional methods to accurately distinguish between mutant and wild-type sequences, easily leading to false positives or false negatives and affecting detection specificity. In addition, the content of mutated genes in clinical samples is often extremely low, placing higher demands on the sensitivity of detection methods. In particular, when attempting to combine the high specificity of LAN with the high efficiency of exponential amplification to construct an integrated detection system capable of simultaneously detecting L858R and T790M dual-site mutations, a more complex design challenge arises: LAN-modified probes need to ensure high discrimination against single-base mismatches, while their sequences and structures must also be highly compatible with the primer design and enzyme kinetics of the exponential amplification reaction. Any incompatibility may inhibit amplification efficiency or introduce non-specific amplification. Furthermore, detection components (e.g., probes and primers) targeting two different mutation sites may interfere with each other in the same reaction system, requiring precise sequence design and reaction condition optimization to achieve a balance between specificity and sensitivity in dual-channel detection.

[0005] Therefore, there is an urgent need to develop a highly sensitive detection strategy that can effectively amplify single-base difference signals and accurately distinguish between wild-type and point mutant genes, so as to meet the clinical need for reliable detection of low-abundance point mutations.

[0006] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0007] This invention belongs to the field of gene mutation detection technology, and specifically relates to an isothermal amplification method and kit for detecting EGFR-L858R and EGFR-T790M point mutations.

[0008] One objective of this invention is to provide a kit for detecting EGFR-L858R and EGFR-T790M point mutations, comprising... The capture probe sequence shown in SEQ ID NO.5 and the signal probe sequence shown in SEQ ID NO.6 are used to detect L858R point mutations. The capture probe sequence shown in SEQ ID NO. 10 and the signal probe sequence shown in SEQ ID NO. 11 are used to detect the T790M point mutation.

[0009] According to a preferred embodiment, the kit comprises Nt.AlwI polymerase, KF restriction enzyme, target L858R and / or T790M, and hairpin template primer. Preferably, it contains 0.5–1.0 μL of Nt.AlwI restriction enzyme (10 U / μL), 0.5–1.0 μL of Klenow polymerase (5 U / μL), 1–2 μL of target fragment (1 μM), and 1–2 μL of hairpin template (1 μM).

[0010] Specifically, solutions A and B were prepared on ice baths. 3–5 μL of solution A consisted of 0.5–0.7 μL of 10×NEBBuffer 2, 1–1.5 μL of hairpin template (1 μM), 0.8–1.0 μL of RNase inhibitor (10 U / μL), 1.5–2.0 μL of dNTPs (2.5 mM), 0.2–0.5 μL of DEPC water, and 1–2 μL of target fragment (1 μM). 5–7 μL of solution B consisted of 0.5 μL of restriction enzyme Nt. AlwI (10 U / μL), 0.5–0.7 μL of polymerase Klenow (5 U / μL), 0.5–0.7 μL of Smart 2 Buffer, and 3.5–4.5 μL of DEPC water. Subsequently, 10 μL of solutions A and B were mixed thoroughly and incubated at 37 °C for 40 min for EXPAR amplification. Then, the enzyme activity was inactivated at 95 °C for 5 min, and the product was placed on ice.

[0011] One objective of this invention is to provide a method for preparing a nucleic acid sensor, which includes the following steps: PdAgBP bimetallic nanosol was dropped onto the ITO-BPE array cathode to obtain a PdAgBP / ITO electrode. After annealing and looping, the Capture probe was drop-coated onto the surface of the PdAgBP / ITO electrode and incubated at room temperature to obtain the Capture probe / PdAgBP / ITO electrode. HT was drop-coated onto the cleaned electrode surface, incubated at room temperature, and then washed away with PBS buffer to remove non-specific adsorption, thus obtaining the HT / Capture probe / PdAgBP / ITO electrode. The signal probe was dropped onto the surface of the HT / Capture probe / PdAgBP / ITO electrode, hybridized, and then rinsed with PBS buffer to terminate the reaction.

[0012] According to a preferred embodiment, the preparation method of PdAgBP bimetallic nanosol includes the following steps: NH4F solution, boric acid solution, K2PdCl4 solution, and AgNO3 solution were sequentially added to a 4.5 mg / mL DDAC suspension, and the mixture was heated and stirred. NH3 was then added... H2O was quickly added to the mixture above and stirred further until colorless to obtain an emulsion; Add NaH2PO2 dropwise to the emulsion and stir for 20 min in a water bath at a temperature not lower than 95°C. Inject DMAB into the solution, stir vigorously, and continue stirring at a temperature not lower than 95 °C until the solution turns dark brown.

[0013] The optimal ratio of the above-mentioned preparation components is: =3.5 ~5.0 mg / mL, =3~5 wt%, =4.0~5.0 (molar ratio, the total volume of both added is 2 mL,) =10 mM, =10 mM), =1.0~2.0 (volume ratio, the total volume of both added is 3 mL), =0.034 M, =0.1 M).

[0014] According to a preferred embodiment, the preparation method of PdAgBP bimetallic nanosol includes the following steps: A 4.5 mg / mL DDAC suspension was prepared using ultrapure water; NH4F solution (2-3 mL, 0.337 M), boric acid solution (2-3 mL, 0.101 M), K2PdCl4 solution (1.63-1.67 mL, 10 mM), and AgNO3 solution (0.37-0.39 mL, 10 mM) were sequentially added to 20 mL of DDAC suspension, and the mixture was heated and stirred. NH3 was then added... H2O (0.8~1.0 mL, 4 wt%) was rapidly added to the above mixture and stirred further until colorless to obtain an emulsion; Add NaH2PO2 (1.8~2.0 mL, 0.034 M) dropwise to the emulsion and stir for 20 min in a water bath at a temperature not lower than 95 °C. Add DMAB (1.2~1.5 mL, 0.1 M) to the solution, stir vigorously, and continue stirring at a temperature not lower than 95 °C until the solution turns dark brown.

[0015] According to a preferred embodiment, the concentration of the capture probe is 0.5-3 μM.

[0016] According to a preferred embodiment, the concentration of the signal probe is 0.5-2 μM.

[0017] One of the objectives of this invention is to provide a nucleic acid sensor, which is prepared based on the above-described method for preparing a nucleic acid sensor.

[0018] According to a preferred embodiment, the nucleic acid sensor can confirm the target concentration by acquiring luminescent images.

[0019] A method for detecting EGFR-L858R and EGFR-T790M point mutations, comprising the following steps: The target or target amplicon is dropped into the above-mentioned nucleic acid sensor and incubated. The target amplicon was prepared by mixing Nt.AlwI polymerase, KF endonuclease, target L858R and / or T790M, hairpin template primer, incubating at 37 ℃ for 40 min, and then heating at 90 ℃ for no more than 10 min.

[0020] According to a preferred embodiment, the incubation time of the target amplicon or the target on the nucleic acid sensor is 20-60 min.

[0021] One of the objectives of this invention is to provide the application of the above-mentioned kit for detecting EGFR-L858R and EGFR-T790M point mutations in the preparation of drugs for detecting EGFR gene-mediated diseases.

[0022] One of the objectives of this invention is to provide the application of the above-mentioned sensor in the preparation of drugs for detecting EGFR gene-mediated diseases.

[0023] This invention aims to utilize PdAgBP mesoporous bimetallic nanospheres as the substrate-supported electrode material. By designing corresponding LNA latch positions on the capture probe and signal probe, specific identification of point mutation sites in EGFR L858R and EGFR T790M fragments can be achieved. Then, based on EXPAR isothermal amplification, the nucleic acid concentration of the two target fragments is amplified. Furthermore, the special "Y-shaped" structure formed by the single-stranded target amplicon, LNA-modified capture probe, and signal probe can be stably maintained at room temperature to enhance the ECL signal of anolyl luminol. Thus, an ECL sensing and detection strategy for EGFR L858R and EGFR T790M is constructed.

[0024] This technical solution uses PdAgBP as a solid-phase substrate and employs EXPAR isothermal amplification and LNA locking at nucleic acid sites as dual signal amplification mechanisms to construct BPE-ECL-based biosensors for the detection of point mutations EGFR L858R and EGFR T790M. On one hand, the integration of a portable BPE electrode driving device with smartphone imaging significantly reduces the sensor's dependence on traditional laboratory equipment, providing a feasible technical path for point-of-care testing. On the other hand, the nucleic acid modified with LNA can distinguish specific Y-shaped structures formed by nucleic acid sequences at different mutation sites through a precise molecular recognition mechanism, further improving the sensitivity and accuracy of detection. This sensing interface exhibits good linear response over a wide concentration range from 100 aM to 1 μM: the linear regression equation for L858R is Y = 629.7013X + 10570.7570, with a correlation coefficient R0. 2 =0.9893, LOD as low as 14.58 aM; the calibration curve equation for T790M is Y=600.9911X +10691.6382, with a correlation coefficient R. 2 =0.9941, with a LOD as low as 15.23 aM. Performance evaluation and preliminary clinical sample testing results further confirm that the sensor can accurately distinguish between L858R and T790M mutation-positive and negative samples, demonstrating excellent analytical performance. Although this Y-structure-based single-base recognition strategy shows promising application prospects in preliminary studies, the clinical applicability of the sensor still needs to be further validated through large-scale, multi-center clinical studies to comprehensively evaluate its stability and reliability in different clinical scenarios. From a clinical application perspective, this sensor can not only be used for the detection of EGFR gene mutations, but can also be extended to the detection of other disease-related single nucleotide polymorphisms (SNPs), providing important technical support for the development of personalized medicine plans and precision medicine. Attached Figure Description

[0025] Figure 1 The effects of pairwise interactions of various factors on PdAgBP ECL; Figure 2 The structure of the surfactant DDAC is shown, where red spheres represent nitrogen atoms, blue spheres represent carbon atoms, and white spheres represent hydrogen atoms. Figure 3 The diagram shows the synthesis of PdAgBP. (A) The synthesis process of PdAgBP; (B) HRTEM image of PdAgBP and (C, D) IFFT and FTT diffraction patterns of selected regions (inset in C); (E1, E2) HRTEM and HAADF-STEM images of single and multiple PdAgBP nanospheres and their HAADF-STEM-EDS spectra, including full spectrum (Ea, Fa), palladium (Eb, Fb), silver (Ec, Fc), palladium-silver composite (Ed, Fd), boron (Ee, Fe), and phosphorus (Ef, Ff); Figure 4 XPS characterization of the electronic states of the surface of PdAgBP alloy mesoporous nanospheres: (A) Pd 3d spectrum, (B) Ag 3d spectrum, (C) B 1s spectrum, and (D) P 2p spectrum; Figure 5 The diagram shows the sensor signal acquisition process. (A) Electrochemiluminescence analyzer and its testing interface; (B) A mobile phone screen with a portable device driven in dark conditions. Figure 6 The following is a schematic diagram of the sensor construction strategy and working principle: (A) Structural domain of the EXPAR hairpin template sequence; (B) Single base mutation of the target EGFR L858R and EGFR T790M; (C) Portable DC drive device; (D) Assembly of ITO-BPE; (E) Actual photo taken with a mobile phone showing anodic emission; (F) EXPAR amplification reaction; (G) Cathode reaction interface modification process. Figure 7 For the electrode interface modification and assembly process, (A) ECL signal response, (B) electrochemical impedance value (the inset in B is its equivalent circuit diagram), where a is bare ITO, b is PdAgBP / ITO, c is Capture probe / PdAgBP / ITO, d is HT / Capture probe / PdAgBP / ITO, e is L858R+Signal probe / HT / Capture probe / PdAgBP / ITO, and f is T790M+Signal probe / HT / Capture probe / PdAgBP / ITO; Figure 8The images are PAGE electrophoresis verification diagrams. (A) EXPAR amplification products, (B) formation of the Y-shaped structure. Figure 9 The results of the optimized experimental conditions are shown in the figure. (A) Capture probe incubation concentration, (B) Signal probe incubation concentration, (C) EXPAR amplification time, and (D) Y-type structure hybridization time. Figure 10 The following are the ECL response curves at different concentrations: (A) L858R, (B) T790M and their corresponding linear regression equations (C) L858R, (D) T790M. Figure 11 Images of the anodic emission of L858R and T790M at different gradient concentrations: (A) ECL signal of L858R captured by a mobile phone; (B) ECL signal of T790M captured by a mobile phone. Figure 12 Colorimetric values I Relationship between L858R and T790M concentrations, (A) L858R, (B) T790M; Figure 13 The diagram shows the stability of the biosensor's response to different concentrations of L858R and T790M ECL. (A) L858R, (B) T790M. Figure 14 The specificity of the sensing strategy for detecting targets L858R (red) and T790M (green); Figure 15 For biosensors to perform L858R and T790M detection on real samples. ΔI' ; Figure 16 The image shows a comparison of the test results of the target biosensor and gene detection for L858R and T790M in NSCLC samples. Green represents negative and red represents positive. Detailed Implementation

[0026] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0028] Example 1 1. Experimental Materials 1.1 Instruments The main instruments used in the experiment are shown in Table 1.

[0029] Table 1

[0030] 1.2 Reagents The main reagents used in this experiment are shown in Table 2.

[0031] Table 2

[0032] All nucleic acid sequences used in the experiment were synthesized by Shanghai Sangon Biotech Co., Ltd., as detailed in Table 3.

[0033] Table 3

[0034] Note: [base] refers to the base labeled with LNA; "base" refers to a single base mutation site, which can specifically complement [base] on the Capture probe; (base) refers to the base mismatch site relative to the Mutant DNA.

[0035] 2 Experimental Methods 2.1 Design of Y-shaped structural coupling For the detection of target fragments with high homology in nucleic acid sequences in complex biological samples, especially the detection of single-base mutations relative to wild-type fragments, it is necessary to design and synthesize DNA probes with high specificity and hybridization efficiency. In this study, based on the sequences of the target point-mutated genes L858R and T790M, a set of DNA probes (L858R group and T790M group, see Table 3) were designed, including: a hairpin capture probe with an amino group at both the 5' and 3' ends and an LNA site specifically complementary to the target point-mutated base; and a signal probe containing two LNA sites and a ferrocene (Fc) modification at the 3' end. The purpose of modifying the LNA sites in the capture and signal probes is to reduce the conformational flexibility of ribose, improve selectivity for complementary sequences to reduce non-specific binding, and further weaken the background signal. The double-amino-modified capture probe can be firmly coupled to the PdAgBP / ITO surface via Ag-NH2 and Pd-N bonds. The stem of the capture probe consists of 5 base pairs (bp) and serves as a rigid support to keep the probe upright. The capture probe loop provides a platform for partial hybridization with the target fragment and the signal probe, respectively. The signal probe cannot bind to the electrode surface before the target fragment (L858R or T790M) binds to the capture probe because the melting temperatures Tm are 25 °C (L858R group) and 23 °C (T790M group), respectively, which are much lower than the experimental operating temperature of 37 °C; the Tm values ​​were calculated using relevant calculation websites. However, when the target is present, the Tm values ​​are 48.8 °C (L858R group) and 44.8 °C (T790M group), which are higher than the operating temperature, and a stable Y-shaped connection structure can be formed between the target, capture probe, and signal probe.

[0036] 2.2 Synthesis of PdAgBP Based on the optimal synthesis conditions for ECL observed in the BBD experiment, the PdAgBP alloy with the best performance was prepared. 90 mg of DDAC was uniformly dispersed in 20 mL of ultrapure water under ultrasonic stirring, and the solution changed from turbid to transparent. Then, NH4F solution (2 mL, 0.337 M), boric acid solution (2 mL, 0.101 M), K2PdCl4 solution (1.63 mL, 10 mM), and AgNO3 solution (0.37 mL, 10 mM) were sequentially added to the suspension. After gentle stirring and heating at 35 °C for 5 min, NH3... H₂O (0.8 mL, 4 wt%) was rapidly added to the above mixture, and the mixture was stirred until colorless. NaH₂PO₂ (1.8 mL, 0.034 M) was added dropwise to the emulsion, and the mixture was stirred in a 95 °C water bath for 20 min. Finally, freshly prepared DMAB (1.2 mL, 0.1 M) was injected into the solution, and the mixture was stirred vigorously and continued to be stirred at 95 °C for 30 min. After DMAB reduction, the color changed from light yellow to dark brown, indicating that the PdAgBP alloy had formed. After centrifugation at 12000 rpm, the mixture was washed three times repeatedly with 70% ethanol to remove DDAC. Finally, the product was ultrasonically dispersed in 6 mL of ultrapure water and stored at 4 °C for later use.

[0037] 2.2.1 Statistical Analysis of BBD Design Based on DDAC concentration ( a ), NH3 H2O concentration ( b ), Pd 2+ / Ag + Add molar ratio ( c ) and the volume ratio of NaH2PO2 / DMAB added ( d The BBD model was designed with 29 experimental runs based on the selected range of variation for each factor. The ECL test data corresponding to these 29 experimental runs are shown in Table 4.

[0038] Table 4

[0039] The results of the analysis of variance for the ECL quadratic model of PdAgBP are shown in Table 5 (Results of the analysis of variance for the particle size response surface model of PdAgBP). As can be seen from the table, the regression model is statistically significant. p <0.0001). DDAC concentration and Pd 2+ / Ag + Add molar ratio (ac) and four-factor DDAC concentration ( a ), NH3 H2O concentration ( b ), Pd 2+ / Ag + Add molar ratio ( c ), NaH2PO2 / DMAB addition volume ratio ( d Its interaction with itself a 2 , b , 2 c , 2 d 2 The effect on the ECL of PdAgBP was statistically significant.p <0.05, while other factors were not statistically significant ( p >0.05).

[0040] Using software simulation analysis, the functional relationships of the interactions of various synthesis conditions on particle size during the synthesis of PdAgBP were clarified as follows:

[0041] Furthermore, the R of this model 2 The value is 0.9544, which is close to 1, indicating that the consistency between the actual value and the predicted value is very good. The PdAgBP model "Adeq.Precision" value obtained in this study is 16.766, indicating that the noise signal has little impact on the model. Therefore, the influence of the noise signal can be ignored when analyzing the experimental results.

[0042] Table 5

[0043] (R) 2 =0.9544, R 2 Adj =0.9188,Adeq. Precision: 16.766) 2.2.2 Response Surface Analysis Based on the function model constructed above, a 3D surface response plot of the ECL intensity of PdAgBP was plotted, and the DDAC concentration was further analyzed. a ), NH3 H2O concentration ( b ), Pd 2+ / Ag + Add molar ratio ( c ), NaH2PO2 / DMAB addition volume ratio ( d The interaction between them.

[0044] like Figure 1 As shown, the ECL response surface of the pairwise interactions of the four parameters reveals that any combination of interactions significantly affects the ECL surface response intensity of PdAgBP, indicating a substantial influence on the curvature change and the corresponding ECL variation range. This suggests that these four variables are three key parameters in the PdAgBP preparation process. The ECL intensity increases with the concentration or value of these factors, reaching a peak after the parameters reach a moderate value, and then decreasing with further increases. This demonstrates that precise control of the ratios of these factors plays a decisive role in regulating the ECL properties of the material and is crucial for preparing high-performance PdAgBP. The optimal synthesis formulation after BBD optimization is as follows: =4.5 mg / mL, =4 wt%, =9 : 2 = 4.5 (molar ratio, the total volume of both added is 2 mL) =10 mM, =10 mM), =3:2 =1.5 (volume ratio, the total volume of both added is 3 mL). =0.034 M, =0.1 M).

[0045] 2.2.3 Morphology and property characterization of PdAgBP DODAC, as an amphiphilic surfactant, has two hydrophobic long-chain tails in its structure (C... 18 ) and a hydrophilic quaternary ammonium head, which can drive self-assembly into rod-shaped cylindrical micelles in aqueous solution, especially in the presence of a negatively charged metal precursor ( Figure 2 After mixing with the metal precursors K₂PdCl₄ and AgNO₃ solution, the electrostatic and coordination interactions stabilize the mesoporous micelles, thereby inhibiting the PdCl₄ oxidation. 2 and Ag + The mobility was then utilized to directly assemble and grow PdAgBP nanospheres with continuous mesopores along the cylindrical micelles of DODAC using a reducing agent in a co-synergistic manner.

[0046] Figure 3 A illustrates the synthesis process of mesoporous PdAgBP nanospheres. To demonstrate the successful preparation of bimetallic PdAgBP, TEM and HRTEM techniques were first used to characterize the size, shape, physical structure, and chemical composition of the PdAgBP nanospheres. In a single nanosphere ( Figure 3 E1, 3E2) and multiple nanospheres ( Figure 3 Under F1 and 3F2 fields of view, the prepared PdAgBP alloy exhibits strong dispersion, with a diameter of approximately 100-130 nm, resembling a hydrangea. Higher magnification electron microscopy images further reveal its mesoporous structure; under HRTEM, the internal mesoporous network displays a three-dimensional dendritic structure with a central radiating channel morphology. Figure 3 B); The scanned lattice fringes were analyzed using software ( Figure 3 B); The selected region is processed by Inverse Fast Fourier Transform (IFFT) image as follows Figure 3 As shown in Figure C, the inset is the corresponding FTT diffraction pattern; after selecting the range in the IFFT image, the interplanar spacing of the eight spacer lines is 1.824 nm. Figure 3 D), the lattice fringe spacing of the PdAgBP alloy was calculated to be 0.228 nm. Figure 3B). To further verify that the quaternary bimetallic PdAgBP has been successfully prepared, elemental spectrum analysis was performed using HAADF-STEM-EDS, such as... Figure 3 As shown in Ea-Ef and Fa-Ff, whether it is the full spectrum, the Pd-Ag binary spectrum, or the individual single-element spectra of Pd, Ag, B, and P, all show that each element is uniformly distributed throughout the entire nanocluster.

[0047] To further characterize the chemical composition and surface state of PdAgBP nanomaterials, XPS technology was used to systematically analyze the chemical energy level valence states of Pd, Ag, B, and P elements in the material. Figure 4 As shown in Figure A, two sets of characteristic peaks were observed in the Pd 3d spectrum: peaks with binding energies in the ranges of 343-338 eV and 337-333 eV correspond to the Pd 3d3 / 2 and Pd 3d5 / 2 orbitals, respectively, indicating that Pd exists in an elemental state. Similarly, the Ag 3d spectrum ( Figure 4 B) Two pairs of characteristic peaks are observed in the ranges of 375-372 eV and 369-366 eV, which are attributed to Ag 3d. 3 / 2 and Ag 3d 5 / 2 The orbitals confirmed that Ag also exists in its elemental state. Furthermore, analysis of the high-resolution XPS spectra of B 1s and P 2p revealed that B and P exist simultaneously in both elemental and oxidized states. Figure 4 These analytical results not only confirm the successful integration of the four elements Pd, Ag, B, and P into the material, but also provide conclusive experimental evidence for the successful synthesis of quaternary PdAgBP alloy materials. This unique elemental composition and chemical state distribution may be one of the important reasons why the material exhibits excellent electrochemical performance.

[0048] 2.3 Preparation of EXPAR amplification products The EXPAR amplification system based on targets L858R and T790M is prepared using two premixed solutions, A and B, primarily to prevent the formation of byproducts. Solutions A and B are prepared on ice. 5 μL of solution A consists of 0.5 μL 10×NEB Buffer 2, 1 μL hairpin template (1 μM), 0.8 μL RNase inhibitor (10 U / μL), 1.5 μL dNTPs (2.5 mM), 0.2 μL DEPC water, and 1 μL target fragment (1 μM). 5 μL of solution B consists of 0.5 μL restriction enzyme Nt. AlwI (10 U / μL), 0.5 μL polymerase Klenow (5 U / μL), 0.5 μL Smart 2 Buffer, and 3.5 μL DEPC water. Subsequently, 10 μL of solutions A and B were mixed thoroughly and incubated at 37 °C for 40 min for EXPAR amplification. Then, the enzyme activity was inactivated at 95 °C for 5 min, and the product was placed on ice.

[0049] 2.4 Assembly of Nucleic Acid Sensors Before sensor fabrication, ITO-BPE was sequentially immersed in beakers containing anhydrous ethanol, 50% anhydrous ethanol, and deionized water, and ultrasonically cleaned for 20 min in an ultrasonic cleaner. The cleaned bipolar electrodes were then dried in a 37 ℃ constant temperature drying oven. After drying, PDMS was attached for later use. During biosensor modification, 20 μL of the PdAgBP bimetallic nanosol synthesized in section 2.2 was first added to the cathode side of the smooth ITO-BPE array and dried at 37 ℃ to obtain a PdAgBP / ITO electrode. Then, 20 μL of 1 μM annealed and looped Capture probe was drop-coated onto the surface of the PdAgBP / ITO electrode and incubated at room temperature for 2 h to obtain a Capture probe / PdAgBP / ITO electrode. Unimmobilized probes were washed away with PBS buffer (pH 7.4). Next, 10 μL of 10 mM HT was drop-coated onto the cleaned electrode surface to passivate it and block non-specific sites. After incubation at room temperature for 1 h, the non-specific adsorption was washed away with PBS buffer (pH 7.4) to obtain the HT / Capture probe / PdAgBP / ITO electrode. Finally, 20 μL of a mixture containing 10 μL of 1 μM signal probe and 10 μL of 1 μM target amplicon was drop-coated onto the surface of the HT / Capture probe / PdAgBP / ITO electrode and hybridized at 37 °C for 50 min. This allowed the Mut DNA amplicon, Capture probe, and signal probe to form a Y-shaped structure through complementary base pairing. The electrode was then rinsed with PBS buffer (pH 7.4) to terminate the reaction, dried, and stored briefly at 4 °C for subsequent ECL detection. The sensor interface construction was thus completed.

[0050] 2.5 Sensor Signal Acquisition Method Photomultiplier tube counting (electrochemiluminescence analyzer): The assembled ITO-BPE array electrode plate was placed in the photomultiplier tube box. Alligator clips were used to connect the pre-installed ITO driving electrodes on both sides of the BPE. The driving electrode on the reaction port (cathode) side was connected to the working electrode wire, while the driving electrode on the opposite side (reporter port (anode)) was connected to the counter electrode and reference electrode. 200 μL of PBS buffer (pH 7.4) was added to each reaction cell, and 200 μL of luminol solution (10 μM) was added to the reporter cell. A voltage of 0-4 V (photomultiplier tube high voltage PMT = 300 V) was applied using cyclic voltammetry. The ECL signal of each channel's anode port was measured sequentially and independently. E pa The ECL peak appears at +4.0 V; the instrument and its test interface are as follows. Figure 5 As shown in Figure A.

[0051] Color Depth Counting (Portable Device-Driven Mobile Phone Camera Acquisition): Shooting was conducted in a completely dark room with opaque walls. A DC-DC regulator module stably outputs a +4.0V drive voltage across the ITO-BPE sensor. Using the phone's rear camera (night mode, 10s exposure time, ISO 3200), the ECL signal of the anode luminol was captured. The actual shooting interface of the portable device-driven mobile phone is shown below. Figure 5 As shown in B, the light emission signal of the aperture can not only be qualitatively distinguished by the naked eye in terms of its brightness and chromaticity, but also the specific values ​​of the three primary colors in the red, green, blue (RGB) color mode can be analyzed using image analysis software. Based on the average value of the three, I = (R+G+B) / 3, the color depth can be quantitatively counted.

[0052] 2.6 Extraction of cfDNA from blood samples The equipment required for the experiment was soaked in 0.1% DEPC water overnight to inactivate the RNA-degrading enzymes on the equipment. After soaking, it was autoclaved at 120 °C for 25 min and then placed in a drying oven to dry, so that the residual DPEC on the equipment could be decomposed.

[0053] This experiment was approved by the Ethics Committee of the Western Theater General Hospital of the Chinese People's Liberation Army (2025EC11-ky051) and Chengdu Medical College (Chengdu Medical College Ethics Review 2022 NO.18, 2025CYFYIRB-BA-118). A total of 15 blood samples were collected from the Department of Oncology of the First Affiliated Hospital of Chengdu Medical College and the Department of Nuclear Medicine of the Western Theater General Hospital of the Chinese People's Liberation Army. Among them, 13 were NSCLC pathological samples. The inclusion criteria were: (1) clinically diagnosed with NSCLC; (2) first diagnosis and treatment, and no prior treatment; (3) age ≥ 18 years; (4) complete clinical data; (5) signed informed consent; and the exclusion criteria were also met: (1) patients with malignant tumors in other parts of the body; (2) patients with mental illness. The other 2 samples were negative control samples from healthy individuals. Inclusion criteria: (1) No abnormalities were found in the comprehensive physical examination, routine laboratory tests, and cardiopulmonary function tests; (2) No history of drug abuse; (3) Age ≥ 18 years; (4) No history of hereditary major diseases in immediate family members; (5) Signed informed consent; and simultaneously met the exclusion criteria: (1) Use of drugs that may affect the study indicators within the past month; (2) Acute infectious disease outbreak or chronic infection within the past 3 months; (3) History of serious organic disease. All samples underwent serum separation after collection for cfDNA, as cfDNA fragments in peripheral blood originate from the metabolites of tumor cells and can reflect the tumor burden in real time for early diagnosis of lung cancer. The separated serum was stored in an ultra-low temperature freezer at -80°C.

[0054] The serum / plasma cell-free DNA extraction kit is used to extract cfDNA from the serum isolated above. The specific steps are as follows: ① Take 100-200 μL of serum / plasma into a 2 mL centrifuge tube. If the volume is less than 100 μL, add buffer GA to bring the final volume to 100 μL. ② Add 20 μL of Proteinase K solution and vortex to mix. ③ Add 200 μL of buffer GB, gently invert to mix, and incubate at 56°C for 10 min, shaking the sample occasionally. Briefly centrifuge to remove droplets from the inner wall of the tube cap. ④ Add 200 μL of anhydrous ethanol. If the room temperature exceeds 25°C, pre-cool the ethanol on ice. Gently invert to mix the sample, incubate at room temperature for 5 min, and briefly centrifuge to remove droplets from the inner wall of the tube cap. ⑤ Add the solution obtained in the previous step to an adsorption column CR2 (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 30 s, discard the waste liquid, and return the adsorption column CR2 to the collection tube. ⑥ Add 500 μL of buffer GD to the adsorption column CR2, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and return the adsorption column CR2 to the collection tube. ⑦ Add 600 μL of wash buffer PW to the adsorption column CR2, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and return the adsorption column CR2 to the collection tube. ⑧ Repeat step ⑦. ⑨ Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column CR2 at room temperature for 2-5 min to thoroughly dry any residual wash liquid in the adsorption material. ⑩ Transfer the adsorption column CR2 to a clean centrifuge tube, add 30-50 μL of elution buffer TB dropwise to the center of the adsorption membrane, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 2 min, and collect the solution in the centrifuge tube. Store the extracted nucleic acid in an ultra-low temperature freezer at -80 ℃. Aliquot in advance to avoid repeated freeze-thaw cycles that could degrade the DNA.

[0055] 2.7 Determination of actual samples Samples containing 10% normal human serum (A, B) were prepared using Tris-HCl buffer (10 mM). Synthesized L858R and T790M gene fragments at fixed concentrations were added, and nucleic acids were extracted using a nucleic acid extraction kit. The ECL intensity obtained from each spiked sample test was substituted into the standard curve to calculate the actual detected mutant gene concentration. The detected concentration was then compared with the spiked concentration to verify the spiked recovery rate. Each sample was tested three times to calculate the RSD.

[0056] Furthermore, this experiment still uses gene testing results as the gold standard to compare the accuracy of this sensor in determining the genotype of NSCLC patient samples. The ECL background signal value when the target is blank, i.e., the concentration is 0, is defined as... I 0, the value measured for each actual sample is 0.I i Subtracting the two gives Δ I i '= I i - I 0, meaning the change in ECL signal value Δ for each sample. I i To minimize experimental error and false positive rate, the ΔE ratio was adjusted for NSCLC pathological serum samples (experimental group) and normal human serum samples (control group). I i Statistical analysis was performed to determine the significance of differences, thereby verifying whether the sensor effectively detected L858R and T790M mutations in NSCLC pathological samples compared to the negative control sample. Finally, by comparing the sensor detection results with the gene detection results, the performance of this biosensor in actual sample detection can be qualitatively evaluated. Data processing used statistical software; one-way ANOVA was used for inter-group comparisons, and multiple comparison tests were used to identify statistical differences between the pathological experimental group and the normal control group. p A value <0.05 is considered statistically significant.

[0057] 3 Results 3.1 Working principle of the sensor Figure 6 This paper presents the main components of the ECL sensor construction system used in this study. The nucleic acid sequence structures of the hairpin initiation template used for target EXPAR amplification are shown below. Figure 6 As shown in Figure A, its 3' end contains a self-primer domain, the loop region contains the antisense sequence of the self-primer and the ENase recognition site, and the 5' end contains a target recognition domain. The single-base mutations in the target EGFR L858R and EGFR T790M sequences are shown below. Figure 6 As shown in B. In addition to traditional electrochemiluminescence assays, this study will construct a sensing interface based on an ITO-BPE array (…). Figure 6 D), innovatively introducing a portable DC power supply device ( Figure 6 C) Drive luminol emission at the anode, and measure the target concentration by visualizing the image using a mobile phone. Figure 6 E). Successful EXPAR amplification targeting the target is crucial for signal amplification in the sensing platform, such as... Figure 6F. Once the hairpin template encounters the target single-stranded nucleic acid, it hybridizes with the target recognition domain of the hairpin template and releases its own primers via toe-point exchange, forcing the hairpin template to transform into an "activated" conformation. During this process, the self-primer hybridizes with its antisense sequence in preparation for initiation. In the presence of Nt.AlwI polymerase, the self-primer extends along the hairpin template, at which point the target nucleic acid is freely displaced, producing dsDNA containing a nick endonuclease recognition site. This dsDNA can be cleaved by KF endonuclease, and the cleaved dsDNA is then extended by Nt.AlwI polymerase, producing single-stranded amplicon that serves as a target analog. The regenerated double-stranded DNA is ready for the next nick extension reaction, and the released target amplicon can serve as a new target to initiate another round of EXPAR. After the above amplification cycle, the concentration of the target fragment in the reaction system is significantly increased. When mixed with a signal probe and incubated on an HT / Capture probe / PdAgBP / ITO surface, the number of Y-shaped structures that can be formed increases under the driving force of the target, and correspondingly, the ECL signal intensity also increases. Conversely, if there is no target, Y-shaped structures cannot be formed, and the ECL signal intensity is low (e.g., ...). Figure 6 As shown in G).

[0058] 3.2 Characterization of the sensor assembly process Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry-electrochemiluminescence (CV-ECL) were used to characterize the stepwise fabrication process of the proposed ECL biosensor. The electrode connections were consistent with the synthesis method of PdAgBP described above. Figure 7 As shown in A and B, the bare ITO electrode exhibits a low ECL intensity and a high EIS impedance (curve a). Subsequently, when PdCuBP nanomaterials are modified onto the ITO electrode, the ECL intensity increases significantly, while the corresponding resistance decreases markedly (curve b), indicating that the highly conductive PdCuBP nanomaterials can effectively accelerate electron transfer. Next, when the capture probe is modified onto the electrode surface, the ECL value decreases and the impedance increases due to the steric hindrance effect of DNA (curve c). Furthermore, when HT blocks the non-specific binding site, the ECL intensity further decreases, and the impedance continues to increase, mainly due to the obstruction of electron transfer between the anode and cathode (curve d). After the signal probe and the mutant target are incubated on the electrode, a Y-shaped structure is formed between the capture probe, signal probe, and target, and a large amount of ferrocene is immobilized on the electrode, resulting in a significant increase in the ECL signal value and a significant decrease in the impedance (curves e and f; where e corresponds to the L858R group and f corresponds to the T790M group).

[0059] 3.3 Electrophoresis verification of EXPAR amplification products and Y-type structure assembly status First, PAGE electrophoresis was used to verify the successful amplification of single-stranded target fragments by EXPAR (each nucleoplast used a concentration of 1 µM). Figure 8 As shown in Figure A, Nt.AlwI polymerase and KF endonuclease, the target L858R or T790M, and the hairpin template primer are all indispensable for initiating isothermal EXPAR amplification. Only when all three are present can the target be successfully amplified, forming a high concentration of amplicon (circled in red), and the bp number of the amplified band matches the theoretical value. The amplification results show that this EXPAR amplification method has a low non-specific amplification rate, high sensitivity, and good reproducibility. It requires only one nucleic acid template, avoiding complex and multiplexed amplification templates and exogenous primers and probes, which helps to suppress non-specific amplification reactions and reduce amplification variation. This method is a powerful tool for amplifying target short-chain sequences.

[0060] Subsequently, the specific differences in the formation of Y-shaped structures with Capture probe and Signal probe between single-base mutant DNA and wild-type DNA were investigated. Figure 8 As shown in Figure B, gel electrophoresis results indicate that only DNA fragments containing specific point mutations can successfully hybridize with the Capture probe and Signal probe, forming Y-shaped complexes of the expected size (lanes 4 and 9). In contrast, unmutated wild-type DNA fragments cannot effectively bind to the probes (lanes 5 and 10). This result fully confirms that the LNA-modified probes designed in this study can significantly enhance the specificity of base recognition, successfully achieving the goal of single-base resolution. However, it is worth noting that some non-specific byproducts were observed in the lanes where the Y-shaped complexes were formed. This suggests that there is still room for optimization in the current probe design, and future research needs to further improve the probe sequence or optimize the hybridization conditions to reduce the generation of non-specific binding fragments, thereby improving the accuracy and reliability of detection.

[0061] 3.4 Optimization of Experimental Conditions 3.4.1 Capture probe incubation concentration First, the concentration of the capture probe immobilized on the electrode surface was optimized. For example... Figure 9As shown in Figure A, the anodic luminol ECL intensity decreased sharply with the increase of the capture probe concentration. When the concentration of the capture probe reached 3 μM and continued to increase, the anodic ECL signal intensity showed a slow decrease and reached a plateau. This indicates that when the capture probe concentration is 3 μM, its bonding with the bimetallic nanosubstrate PdAgBP is basically saturated. Therefore, 3 μM was selected as the incubation concentration of the capture probe in this experiment.

[0062] 3.4.2 Signal probe incubation concentration The more Y-shaped structures formed on the electrode surface, the higher the variable threshold of the ECL signal response. Figure 9 As shown in Figure B, when the concentration of the signal probe increases from 0.5 µM to 2 µM, the anolyte ECL signal intensity increases rapidly. However, when the concentration exceeds 2 µM, the growth rate of the ECL signal value gradually slows down significantly and reaches a relatively stable stage. This indicates that when the signal probe concentration is 2 µM, the Y-shaped structure formed on the electrode interface has basically reached saturation. Therefore, based on these experimental results, this study selects 2 µM as the optimal concentration for the signal probe.

[0063] 3.4.3 EXPAR amplification time The concentration of the target is crucial in influencing the number of Y-shaped structures formed and the magnitude of the ECL signal response threshold; therefore, the amplification time of the target EXPAR was optimized. Figure 9 As shown in Figure C, when the isothermal amplification time is between 10 and 30 minutes, the ECL signal intensity increases rapidly, reaching its peak at 30 minutes. Furthermore, the ECL signal value does not increase significantly with further extension of the amplification time. Therefore, 30 minutes was selected as the amplification time for EXPAR in this experiment.

[0064] 3.4.4 Y-shaped structure assembly incubation time The hybridization time among the target, capture probe, and signal probe is also crucial for the formation of the Y-shaped structure. For example... Figure 9 As shown in Figure D, when the incubation time of the three components on the electrode was extended from 20 min to 60 min, the ECL signal intensity significantly increased, reaching a peak at 60 min. Thereafter, even with further extension of the incubation time, the ECL signal value remained stable without significant change. Based on this experimental result, this study selected 60 min as the optimal hybridization time for the Y-shaped structure.

[0065] 3.5 ECL Response Standard Curves for EGFR L858R and EGFR T790M Based on the above-mentioned optimization of experimental parameters, this study constructed BPE-ECL sensors targeting the EGFR L858R and EGFR T790M mutation sites, respectively, and systematically evaluated their ECL response characteristics in the concentration range of 100 aM to 1 μM. Figure 10 As shown in Figures A and B, the ECL response curves of both L858R and T790M exhibit significant concentration dependence. To establish a quantitative detection model, the logarithm of the target concentration (lg...) is used. C With as the independent variable and the anodic ECL intensity as the dependent variable, the corresponding standard curve was plotted. Figure 10 (C, D). Through linear regression analysis, the calibration curve equation for L858R is Y = 629.7013X + 10570.7570 (R²). 2 =0.9893), the calibration curve equation for T790M is Y=600.9911X+10691.6382 (R = 0.9893). 2 =0.9941), indicating that the constructed sensor has excellent quantitative detection performance.

[0066] To verify the practical application potential of the visualization detection method, this study used the night scene mode of a smartphone to record the anodic emission images of L858R and T790M under different concentration gradients in dark conditions. Figure 11 (A, B). Experimental results show that the luminescence intensity of the anode aperture is significantly positively correlated with the target concentration; that is, the brightness of the anode aperture increases significantly with increasing target concentration. Notably, when the target concentration differs by 3-4 orders of magnitude, this brightness difference can be directly identified by the naked eye, providing an intuitive visual interpretation method for rapid semi-quantitative assessment of target concentration. This finding not only confirms the practical application value of the developed ECL sensor but also provides important experimental evidence for the development of portable bedside detection devices.

[0067] Furthermore, the RGB values ​​of the actual images of the BPE anode ECL were analyzed using Photoshop, and the results are shown in Table 6 (RGB count values ​​of the actual images corresponding to different target concentrations), where A represents L858R (red) and B represents T790M (green). A linear fit was performed with the logarithm of the target DNA concentration as the x-axis and the average RGB value I as the y-axis. The results show that the intensity of the BPE anode ECL is directly proportional to the logarithm of the target concentration and has a good linear relationship, such as... Figure 12 As shown.

[0068] Table 6

[0069] The target BPE-ECL sensor constructed in this study demonstrated a good linear response from 100 aM to 1 μM for the detection of L858R and T790M, with detection limits of 14.58 aM and 15.23 aM, respectively (S / N=3, where S and N are the standard deviation of the analyte blank sample and the slope of the calibration curve, respectively). Compared with existing technologies (Table 7, comparison of detection limits of the target ECL biosensor with other methods), the sensing system developed in this study exhibits a superior lower detection limit.

[0070] Table 7

[0071] 3.6 Sensor Performance Evaluation To evaluate the stability of the constructed sensor in detecting L858R and T790M at different concentrations, the synthesized L858R and T790M fragments were prepared into solutions with three concentration gradients of 1 fM, 1 pM, and 1 nM for systematic testing. Figure 13 As shown, the experimental results demonstrate a strong positive correlation between ECL signal intensity and target concentration; that is, the ECL signal intensity increases with increasing target concentration. Peak values ​​were calculated in four independent experiments, and the RSDs for both targets at all concentrations were below 3.00%, indicating that the developed biosensor possesses excellent detection stability. This high stability ensures reliable quantitative analysis of target genes in practical applications, providing crucial technical support for the accurate detection of clinical samples.

[0072] To systematically verify the specific recognition ability of the constructed sensing method for base mutations, this study designed a rigorous control experimental system: wild-type wt DNA, single-base mismatched fragments, and three-base mismatched fragments were used as interfering agents, forming five experimental systems together with the target mutDNA and mixed samples. The ECL response intensity of each system was measured. The net signal intensity change was obtained by subtracting the background signal from the blank control. ΔI' ).like Figure 14 As shown, the detection results for L858R (red) and T790M (green) indicate that wtDNA, single-base mismatches, and triple-base mismatches in the samples produced... ΔI'The values ​​were not significantly different from the blank group and were significantly lower than those of the experimental group containing the target mutDNA. This phenomenon can be attributed to the fact that only when the target mutDNA is present can it specifically bind to both the capture probe and the signal probe simultaneously, forming a stable Y-shaped structure on the electrode surface, thereby immobilizing a large number of ferrocene molecules on the cathode pore surface. This structure significantly improves the conductivity of the electrode surface, effectively promotes electron transfer efficiency, and ultimately leads to a significant enhancement of the ECL signal of luminol in the anode pore. These experimental results not only confirm that the prepared biosensor has excellent selective recognition ability for single-base mutations, but also demonstrate good anti-interference performance.

[0073] 3.7 Actual Sample Measurement 3.7.1 Spike Recovery Experiment As shown in Table 8 (Spiked Recovery Rates of L858R and T790M Measured by the Target Biosensor), spiked recovery experiments were conducted on two targets using serum samples A and B from two healthy individuals. The spiked recovery rate for L858R ranged from 99.43% to 103.13%, with an RSD < 6.67%; the spiked recovery rate for T790M ranged from 98.93% to 105.18%, with an RSD < 6.58%. The RSD values ​​for both targets were below 7%, further confirming the high accuracy and reproducibility of the constructed biosensor for detecting L858R and T790M. This indicates that the detection method proposed in this study has significant clinical application potential and is expected to be used in the detection of EGFR gene mutations in clinical samples.

[0074] Table 8

[0075] 3.7.2 Methodological Comparison of ECL Sensors and Gene Detection like Figure 15 As shown, the biosensor was used to perform ECL measurements on cfDNA extracted from serum samples of healthy individuals (Control) and serum samples of 13 NSCLS patients (Samples 1-13). The ECL signal change value was obtained after subtracting the background signal from the blank sample. ΔI' Experimental results showed that significant differences were observed between different samples when detecting the L858R and T790M mutation sites. ΔI'The difference in values ​​stems from the varying specific molecular recognition capabilities of the sensing systems for mutated nucleic acids. Specifically, this biosensor achieves single-base mutation detection based on a specific molecular recognition mechanism: when the single-base mutation site of the target DNA is perfectly complementary to the LNA-modified base of the capture probe, a stable double-stranded structure with a high melting temperature (Tm value higher than the operating temperature) is formed; simultaneously, the target DNA binds to the LNA-modified region of the signal probe, ultimately constructing and immobilizing a stable Y-type complex on the electrode surface, thereby generating significant... ΔI' Signal response. Conversely, if there is a mismatch in the target sequence, the Y-shaped structure cannot be formed due to insufficient hybridization stability (at which point the Tm value is lower than the operating temperature), resulting in a weak change in the ECL signal.

[0076] The Dunnett test was used to detect and calculate the Δ value of cfDNA in serum samples from healthy individuals and NSCLC patients. I' Statistical difference analysis was performed on the values, with normal samples as negative controls. Values ​​showing statistically significant differences were considered. p Pathological samples with a concentration <0.05 were considered positive. Table 9 shows the results obtained from the sensor measurement of L858R. ΔI' Based on the statistical analysis of L858R detection results using the Dunnett test, samples 2, 4, 5, 9, and 12 were confirmed as EGFR L858R mutation-positive samples, while the mutation was not detected in the remaining samples. Similarly, based on the statistical analysis of T790M detection results (Table 10, T790M measurements obtained by the sensor),... ΔI' Based on the results of the Dunnett test, sample 7 was determined to be a blood sample from a patient with the EGFR T790M mutation, while the remaining samples were non-T790M mutation samples.

[0077] Table 9

[0078] Table 10

[0079] Note: ns indicates no statistical significance. for p <0.0001.

[0080] Table 11 Gene detection results of actual samples

[0081] like Figure 16As shown, the sensor's detection results are highly consistent with the gene detection results in Table 11. In the existing sample set, the sensor's detection results for L858R and T790M mutations are consistent with the gold standard method. This finding preliminarily confirms that the sensor has excellent detection performance: its high sensitivity ensures the effective detection of low-abundance mutations, while its high specificity effectively avoids false positive results, thus ensuring the reliability of the diagnostic results.

[0082] Example 2 Synthesis of PdAgBP This embodiment is basically the same as Embodiment 1, except for the composition ratio of the synthesized PdAgBP.

[0083] The proportions of the above-mentioned preparation components are as follows: =5.0 mg / mL, =5 wt%, =5.0 (molar ratio, the total volume of both added is 2 mL,) =10 mM, =10 mM), =2.0 (volume ratio, the total volume of both added is 3 mL), =0.034 M, =0.1M).

[0084] Example 2 Synthesis of PdAgBP This embodiment is basically the same as Embodiment 1, except for the composition ratio of the synthesized PdAgBP.

[0085] The preparation method of PdAgBP bimetallic nanosol includes the following steps: A 4.5 mg / mL DDAC suspension was prepared using ultrapure water; NH4F solution (3 mL, 0.337 M), boric acid solution (3 mL, 0.101 M), K2PdCl4 solution (1.67 mL, 10 mM), and AgNO3 solution (0.39 mL, 10 mM) were sequentially added to 20 mL of DDAC suspension, heated, and stirred. NH3 was then added... H2O (1.0 mL, 4 wt%) was rapidly added to the above mixture and stirred further until colorless to obtain an emulsion; Add 2.0 mL of NaH2PO2 (0.034 M) dropwise to the emulsion and stir for 20 min in a water bath at a temperature not lower than 95 °C. Inject DMAB (1.5 mL, 0.1 M) into the solution, stir vigorously, and continue stirring at a temperature not lower than 95 °C until the solution turns dark brown.

[0086] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a nucleic acid sensor, characterized in that, Includes the following steps: PdAgBP bimetallic nanosol was dropped onto the ITO-BPE array cathode to obtain a PdAgBP / ITO electrode. After annealing and loop-back, the capture probe was drop-coated onto the surface of a PdAgBP / ITO electrode and incubated at room temperature to obtain a capture probe / PdAgBP / ITO electrode. The capture probe includes the capture probe sequence shown in SEQ ID NO.5 for detecting the L858R point mutation and the capture probe sequence shown in SEQ ID NO.10 for detecting the T790M point mutation. HT was drop-coated onto the cleaned electrode surface, incubated at room temperature, and then washed away with PBS buffer to remove non-specific adsorption, thus obtaining the HT / capture probe / PdAgBP / ITO electrode. A mixture of signal probe and target amplicon was dropped onto the surface of an HT / capture probe / PdAgBP / ITO electrode for hybridization, and then rinsed with PBS buffer to terminate the reaction. The signal probe contained the signal probe sequence shown in SEQ ID NO.11 for detecting the T790M point mutation and the signal probe sequence shown in SEQ ID NO.6 for detecting the L858R point mutation.

2. The preparation method according to claim 1, characterized in that, The preparation method of the PdAgBP bimetallic nanosol includes the following steps: NH4F solution, boric acid solution, K2PdCl4 solution, and AgNO3 solution were sequentially added to the DDAC suspension, and the mixture was heated and stirred. NH3 was then added. H2O was quickly added to the mixture above and stirred further until colorless to obtain an emulsion; Add NaH2PO2 dropwise to the emulsion and stir for 20 min in a water bath at a temperature not lower than 95°C. Inject DMAB into the solution, stir vigorously, and continue stirring at a temperature not lower than 95 °C until the solution turns dark brown.

3. The preparation method according to claim 1, characterized in that, The concentration of the capture probe is 0.5-3 μM; the concentration of the signal probe is 0.5-2 μM.

4. A nucleic acid sensor for detecting point mutations in EGFR-L858R and EGFR-T790M, characterized in that, The nucleic acid sensor is prepared based on the preparation method described in any one of claims 1-3.

5. The nucleic acid sensor according to claim 4, characterized in that, The nucleic acid sensor comprises Nt.AlwI polymerase, KF endonuclease, target L858R and / or T790M, and hairpin template primers.

6. The use of the nucleic acid sensor for detecting EGFR-L858R and EGFR-T790M point mutations as described in claim 4 or 5 in the preparation of a medicament for detecting EGFR gene-mediated diseases.

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