EGFR-TKI drug-resistant biomarker detection kit for screening aptamer based on non-immobilized GO-SELEX and preparation method of EGFR-TKI drug-resistant biomarker detection kit

Abstract

The invention provides an EGFR-TKI drug-resistant biomarker detection method based on a non-immobilized GO-SELEX screening aptamer and a kit. In the preparation process of the kit, a nucleic acid aptamer Osi-1 capable of being specifically combined with characteristic protein (Calnexin) in EGFR-TKI drug-resistant cell supernatant is obtained through a GO-SELEX technology. Furthermore, an unmarked optical sensing platform is constructed in combination with gold nanoparticles, so that rapid and sensitive detection of Calnexin is realized. The kit is suitable for early recognition of EGFR-TKI drug resistance, and can be used for clinical liquid biopsy and precise treatment monitoring.

Description

Technical Field

[0001] This invention relates to a biological detection method and device, and more specifically to an EGFR-TKI resistance biomarker detection kit based on non-immobilized GO-SELEX screening aptamers and its preparation method. Background Technology

[0002] Lung cancer is one of the leading malignant tumors threatening human health, with its mortality rate consistently ranking first among all malignant tumors, posing a serious threat to human life and health. Clinically, lung cancer is mainly divided into two categories based on pathological characteristics: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Among them, NSCLC is the most common, accounting for more than 80% of all lung cancer cases. Due to the insidious nature of early symptoms, more than 70% of patients are already in locally advanced stages or have distant metastases at the time of diagnosis, which limits the effectiveness of traditional treatments such as chemotherapy and radiotherapy in controlling disease progression. Studies have shown that approximately 40% to 50% of East Asian NSCLC patients have EGFR-sensitive mutations. Targeted application of EGFR TKIs can significantly prolong progression-free survival (PFS), improve response rates, and enhance quality of life. However, regardless of whether first-, second-, or third-generation EGFR-TKIs are used, acquired resistance inevitably develops, eventually leading to tumor recurrence or progression. The emergence of resistance has become a core challenge limiting the long-term efficacy of EGFR-targeted therapy. Clinically, management strategies for drug-resistant patients mainly focus on targeted therapy or chemotherapy; however, overall efficacy remains limited. Current clinical diagnosis primarily relies on imaging assessments, such as tumor enlargement or the presence of new metastatic lesions. Serum biomarkers, including carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE), lack sufficient sensitivity and specificity to identify drug resistance. A significant time lag exists between the actual onset of resistance and clinical diagnosis, potentially leading to missed opportunities for early intervention. Therefore, screening for highly sensitive and specific biomarkers closely related to EGFR-TKI resistance, and establishing a dynamic monitoring and early detection system for TKI resistance in NSCLC patients, is of significant scientific and clinical value for further improving the survival outcomes of these patients.

[0003] Accurate quantification of biomarkers relies heavily on the recognition and binding capabilities of probes. Nucleic acid aptamers are oligonucleotide molecules composed of short single-stranded DNA or RNA, selected from random libraries using Systematic Evolutionary Exponential Enrichment (SELEX) technology. Due to their ability to spontaneously fold into unique three-dimensional structures, aptamers can recognize their targets with high affinity and specificity, and are therefore often considered "chemical antibodies." With the development of nanotechnology, aptamers combined with various nanomaterials are widely used in the field of biomarker detection. In particular, nanomaterials such as gold nanoparticles (AuNPs), due to their good stability, modifiability, and biocompatibility, have been extensively studied in cell imaging, targeted therapy, gene regulation, and disease diagnosis. Modifying these nanocarriers with aptamers not only enhances the recognition ability of probes but also significantly improves the detection sensitivity of target molecules.

[0004] Although existing studies have demonstrated the potential of aptamers in peripheral blood biomarker detection, specific molecular biomarkers for EGFR-TKI resistance in non-small cell lung cancer (NSCLC) remain relatively scarce, and there is also a lack of mature aptamer sensing systems for real-time monitoring of resistance progression. Therefore, constructing a highly sensitive aptamer-based detection platform and combining it with resistance mechanisms to uncover key molecular targets holds promise for providing new technical approaches and research foundations for early warning and precise intervention of EGFR-TKI resistance. Summary of the Invention

[0005] To address the problems of existing technologies described in the background section, the applicant established a new research model based on existing technologies, specifically including model development, aptamer screening, target identification, and sensor development. A graphene oxide-based SELEX (GO-SELEX) strategy was employed to screen for high-affinity single-stranded DNA aptamers targeting resistant cell supernatants, while avoiding chemical fixation of the target to maintain its native structure. After multiple rounds of screening, the candidate sequence Osi-1 exhibited excellent binding ability and high specificity. Notably, calnexin was identified for the first time as a specific molecular target of Osi-1. Based on this discovery, we transformed Osi-1 into a practical diagnostic tool by developing a label-free aptamer sensor platform based on gold nanoparticles (AuNPs). System optimization of the sensing system, including NaCl concentration, aptamer loading, and incubation time, ensured reliable and reproducible detection performance. This sensing platform demonstrated exceptional specificity and sensitivity in detecting cadherin, reaching the nanomolar detection limit. The materials used included the nucleic acid aptamer Osi-1, gold nanoparticles, and PBS buffer.

[0006] The preparation method of the biomarker detection kit includes the following steps:

[0007] 1) PC9 and HCC827 cells were induced by a stepwise increase in drug concentration to construct osimertinib-resistant cell lines, and their cell supernatants were collected as aptamer screening targets.

[0008] 3) The non-fixed target GO-SELEX strategy is adopted. By adsorbing unbound sequences through π-π stacking of GO, the target binding sequence is efficiently enriched. Combined with reverse screening to remove non-specific binding fragments to parental cells, the highly specific sequence Osi-1 is obtained through multiple rounds of screening as SEQ ID NO.1: (5´-TTGGTGCGGGGGGGTGGAACTGGTTGCTGCGTCGTCATGC-3´).

[0009] 4) Biotinylated Osi-1 was linked to streptavidin magnetic beads, and the binding protein was analyzed by pull-down analysis. SDS-PAGE and mass spectrometry confirmed for the first time that the target specifically bound by Osi-1 is Calnexin.

[0010] 5) A sensor was constructed using the electrostatic adsorption between the aptamer and the AuNP surface. By adjusting the aptamer concentration, NaCl-induced aggregation conditions, and incubation time, the color change of AuNP from red to blue reflected the target concentration, thus achieving spectroscopic detection of Calnexin.

[0011] 6) The sensing platform of the present invention exhibits good linearity in the range of 10–500 nM, with a detection limit of 7.89 nM, and maintains a recovery rate of 97.43%–107.12% in serum samples, making it suitable for early diagnosis and clinical monitoring of drug resistance processes.

[0012] The beneficial technical effects of this invention are as follows:

[0013] ① This invention adopts the "non-fixed GO-SELEX" strategy to avoid conformational changes caused by target fixation, thereby improving the efficiency of target screening;

[0014] ②The technical solution of the present invention enables aptamer identification for osimertinib resistance biomarkers;

[0015] ③ The label-free AuNP aptamer sensing platform constructed by the technical solution described in this invention is simple to operate and has low cost;

[0016] ④ The technical solution described in this invention has stable detection performance and is suitable for complex biological samples;

[0017] ⑤ This invention represents a major breakthrough in existing detection technologies for the early monitoring of tumor drug resistance development;

[0018] ⑥ The technical solution, method and platform described in this invention can be extended to the screening and detection of aptamers for other drug resistance-related proteins, tumor markers, inflammatory factors, etc., providing a general technical foundation for building a multi-dimensional and rapid clinical diagnosis and treatment monitoring system. Attached Figure Description

[0019] Figure 1 : A schematic diagram of the EGFR-TKI resistance biomarker detection method based on non-immobilized GO-SELEX screening aptamers described in this invention;

[0020] Figure 2 Characterization diagram of drug-resistant cell construction;

[0021] Figure 3 : The GO-SELEX screening and monitoring process diagram described in this invention;

[0022] Figure 4 Characterization of Osi-1 aptamer affinity and specificity;

[0023] Figure 5 : Identification of Osi-1 binding targets;

[0024] Figure 6 : Sensor platform system optimization;

[0025] Figure 7 Quantitative detection based on Osi-1 aptamer sensing platform. Detailed Implementation

[0026] Example 1 Preparation of the reagent kit

[0027] 1. The method for detecting EGFR-TKI resistance biomarkers based on non-immobilized GO-SELEX aptamers described in this invention is as follows: Figure 1 As shown. This includes the following specific steps:

[0028] This study established a complete technical system encompassing drug resistance model establishment, aptamer screening, target analysis, and sensor construction. First, an osimertinib-resistant NSCLC cell line was successfully obtained through an escalation dosing induction strategy. Figure 1 Step I (A) was then used to screen single-stranded DNA aptamers capable of efficiently binding to the target molecule from the culture supernatant of drug-resistant cells using graphene oxide-based SELEX technology (GO-SELEX). Figure 1 A, Step II–VII). After multiple rounds of enrichment and selection, the candidate sequence Osi-1 showed the most outstanding binding ability and high specificity.

[0029] Further pull-down and mass spectrometry analysis for the first time clearly identified Calnexin as a specific recognition target of Osi-1. Based on this, Osi-1 was used to construct a label-free gold nanoparticle (AuNP) aptamer sensing platform. Figure 1 (B) This system enables the quantitative detection of target molecules. Through optimization of key parameters such as NaCl concentration, aptamer dosage, and incubation time, the sensing system achieved stable and reproducible detection performance. Results show that the platform can quantitatively detect Calnexin with nanomolar sensitivity and excellent selectivity. Furthermore, it maintains good stability and recovery rate in serum samples, further enhancing the application potential of this strategy in early clinical drug resistance monitoring.

[0030] 1.1 Construction of an osimertinib-resistant NSCLC cell model:

[0031] Non-small cell lung cancer cell lines PC9 and HCC827 carrying EGFR activating mutations were selected and routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum. To establish an osimertinib resistance model, 20–50 nM osimertinib was used as the initial stimulation concentration, followed by a gradient increase in drug dose, maintaining each concentration for approximately 7 days to allow cells to gradually adapt to drug stress. After continuous induction, cells could stably grow under 1.5 μM osimertinib conditions, successfully obtaining the resistant lines PC9·Osi and HCC827·Osi. The IC50 of osimertinib in the two cell lines was evaluated using the CCK-8 assay. 50 The results showed that the drug resistance index exceeded 100, further proving the reliability of the drug resistance model (see...). Figure 2 In the subsequent supernatant collection step, the drug-resistant cells were cultured to approximately 80% confluence, the serum-containing medium was discarded, and the cells were cultured in serum-free medium for another 24 h. The cell supernatant was then collected and stored at 4°C for use in subsequent experiments.

[0032] 2 Aptamer Screening and Target Identification Based on GO-SELEX

[0033] 2.1 Optimization of filtering criteria:

[0034] First, the ratio of ssDNA library to GO was optimized, and a 1:30 ssDNA / GO quality ratio was finally determined as the best condition for subsequent screening. Figure 3 A). The effects of different parameters on amplification efficiency during PCR were then optimized, including seven annealing temperatures (52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃) and multiple cycle numbers (12, 14, 16, 18, 20, 22). The results showed that the strongest fluorescence signal was obtained at an annealing temperature of 58℃, and the bands obtained after 20 cycles were the clearest and had the highest intensity in gel electrophoresis. Figure 3 (B–C), therefore this parameter was selected as the optimized condition for subsequent PCR. In the early stages of GO-SELEX, the high-affinity sequences were positively enriched by incubating the random ssDNA library with PC9 Osi cell supernatant in a binding buffer system (20 mM Tris-HCl, 100 mM NaCl, 2 mM MgCl2, 5 mM KCl, 1 mM CaCl2, pH 7.4). The PCR amplification products were further bound to streptavidin magnetic beads to prepare secondary libraries for the next round of screening. To improve specificity, PC9 par cell supernatant was introduced in some rounds for reverse screening, thereby removing non-specific binding sequences to proteins derived from the parent cell.

[0035] The efficiency of each round of screening was evaluated using the recovery rate of ssDNA. For example... Figure 3 As shown in Figure D, the recovery rate stabilized after the 6th round and remained at approximately 59%, indicating that high-affinity sequences in the library had been largely enriched. Subsequently, high-throughput sequencing was performed on the ssDNA library obtained in the 8th round, yielding a total of 40,661 valid sequences. The top 12 sequences were selected by abundance and named Osi-1 to Osi-12, with Osi-1, Osi-2, and Osi-3 having the highest proportions, at 35.52%, 27.89%, and 25.31%, respectively. Figure 3 E). Clustal multiple sequence alignment further revealed a clearly conserved base repeat structure among Osi-1, Osi-2, and Osi-3. Figure 3 F), suggesting that they may share key binding site features.

[0036] 2.2 Analysis of candidate aptamer binding ability

[0037] Osi 1-12 aptamers were dissolved separately in binding buffer and incubated with the supernatant of PC9-Osi cells. Unbound single-stranded DNA (ssDNA) was removed by adding graphene oxide (GO). The supernatant was collected and its fluorescence intensity was measured. Figure 4 As shown in Figure A, Osi-1, Osi-2, and Osi-3 exhibited significantly higher fluorescence intensity compared to other sequences, indicating the highest binding affinity for PC9-Osi cell supernatant. Serial dilutions of Osi-1, Osi-2, and Osi-3 were prepared and labeled with FAM fluorescent tags. Results ( Figure 4B) showed that the dissociation constants (Kd) of Osi-1, Osi-2, and Osi-3 with PC9-Osi cell supernatant were 71.93 nM, 97.78 nM, and 113.67 nM, respectively, while they showed no affinity for PC9-par cell supernatant. Given that Osi-1 had the highest affinity, it was selected for further experiments. To further investigate the binding specificity of the Osi-1 aptamer, FAM-labeled Osi-1 nucleic acid aptamer (500 nM) was incubated with different cell supernatants. Notably, Osi-1 showed superior binding specificity to PC9-Osi and HCC827-Osi supernatants compared to other supernatants, indicating that Osi-1 has high specificity. Figure 4 C).

[0038] 2.3 Identification of Osi-1 specific targets

[0039] The Osi-1 aptamer, used as a target recognition element, had its specific binding ability verified by a protein capture experiment. First, biotinylated Osi-1 was co-incubated with PC9-Osi cell supernatant to allow the aptamer to form a complex with the potential target protein. This complex was then enriched using streptavidin magnetic beads, and the resulting protein components were separated and analyzed using 10% SDS-PAGE. Several controls were set up in the experimental system: (1) whole-cell lysate and supernatant protein as a positive control; (2) a biotinylated randomized library as a blank control; and (3) biotin-Osi-1 as the experimental group. Electrophoresis results (Figure 5A) showed a distinct band at approximately 90 kDa in the experimental group, while no corresponding protein was observed in the blank control. After cutting and mass spectrometry identification of this characteristic band, its main protein component was confirmed to be Calnexin, whose molecular weight was highly consistent with its electrophoretic migration position, and its reliability score was also at a high level. To further verify whether Osi-1 can specifically capture Calnexin, pull-down assays were performed using biotin-Osi-1 and biotin randomized libraries, and the enriched proteins were detected by Western blot. The results (Figure 5B) showed that only the biotin-Osi-1 group detected a strong positive Calnexin signal, while the randomized library group showed virtually no detectable bands. These results collectively demonstrate that Osi-1 can efficiently and specifically enrich Calnexin, thus establishing Calnexin as the target recognition molecule of Osi-1.

[0040] Example 2 Activity assessment of the kit

[0041] 1. Preparation of reagents

[0042] Add 500 μL of 1% HAuCl4·3H2O solution to 49.5 mL of deionized water and heat to boiling under continuous vigorous stirring. After the system has completely boiled, quickly add 1 mL of 1% trisodium citrate as a reducing agent. Continue stirring for about 5 minutes. During this process, the solution color can be observed to gradually change to a typical wine-red color, indicating that the formation reaction of gold nanoparticles has been completed. After the reaction is complete, allow the system to cool naturally to room temperature, and transfer the prepared gold nanoparticle solution to a glass container for storage at 4°C for later use.

[0043] 2. Detection Principle

[0044] In the absence of external interference, gold nanoparticles (AuNPs) can be stably dispersed in the system, exhibiting a typical wine-red appearance and a characteristic absorption peak at approximately 520 nm. Upon the addition of sodium chloride, the ionic strength of the solution increases, partially shielding the surface charge of the AuNPs and weakening their electrostatic repulsion. This promotes aggregation between the particles, resulting in a change in solution color from red to blue.

[0045] Under undisturbed conditions, gold nanoparticles (AuNPs) can be stably dispersed in solution, exhibiting a typical wine-red color and a characteristic absorption peak at approximately 520 nm. Upon addition of sodium chloride, the ionic strength of the system increases, partially shielding the surface charge of the AuNPs and weakening the electrostatic repulsion between particles, leading to particle aggregation and a change in solution color from red to blue.

[0046] When the Osi-1 aptamer is present in the solution system, it adsorbs onto the AuNP surface. The single-stranded nucleic acid backbone provides the particles with additional spatial and charge stability, thereby significantly improving the colloidal stability of AuNP, effectively inhibiting NaCl-induced aggregation, and maintaining the solution color.

[0047] Upon the addition of Calnexin, due to the higher affinity of Osi-1 for Calnexin, the aptamer undergoes conformational folding and desorbs from the AuNP surface, instead specifically binding to Calnexin and forming a stable G-quadruplex structure. The AuNP, now without its aptamer protective layer, rapidly aggregates in a high-salt environment, and the system color changes back from wine-red to blue.

[0048] 3. Optimize the testing platform

[0049] The concentration of sodium chloride (NaCl) has a decisive influence on the aggregation state of gold nanoparticles (AuNPs) in aptamer sensing systems. For example... Figure 6 As shown in Figure A, when the NaCl concentration is 15–45 mM, A 695 / A 520The ratio remained at a low level, indicating that the AuNPs were still well dispersed. When the NaCl concentration increased to 55 mM, the ratio rose significantly, indicating significant particle aggregation. Therefore, 55 mM was determined to be the optimal salt concentration for subsequent experiments.

[0050] After optimizing the salt concentration, the time required for NaCl and AuNPs to reach equilibrium was further investigated. Figure 6 B). The results showed that, within the range of 2–18 min, A 695 / A 520 The ratio change was not significant, indicating that the aggregation process could reach equilibrium within about 2 minutes, so 2 minutes was set as the optimal reaction time.

[0051] The protective effect of aptamer Osi-1 on AuNPs was optimized under the determined salt conditions (55 mM, 2 min). As the aptamer concentration increased from 20 nM to 260 nM, the protective effect of A... 695 / A 520 The ratio gradually decreases ( Figure 6 C) indicates that its stabilizing effect on AuNPs is enhanced. When the aptamer concentration reaches 310–350 nM, this ratio tends to stabilize, suggesting that the AuNP surface is sufficiently covered by the aptamer. Therefore, 310 nM was selected as the optimal aptamer concentration.

[0052] Finally, the incubation time between the aptamers and AuNPs was evaluated. Figure 6 As shown in D, within the first 10 minutes, A 695 / A 520 The ratio decreased rapidly and then remained relatively stable within 10–45 min, indicating that the adsorption process of the aptamer on the AuNP surface could be completed within 10 min. Therefore, 10 min was determined as the optimal incubation time.

[0053] 4. Specificity and quantitative detection based on the Aptamer sensing platform

[0054] To verify the specificity of the constructed aptamer sensing platform, the applicant tested the culture supernatants of various cell lines (PC9 Osi, HCC827 Osi, PC9 par, HCC827 par, A549 WT, HepG2 WT, and Beas-2b). Figure 7 As shown in Figure A, the supernatant of drug-resistant cells PC9 Osi and HCC827 Osi generated a significantly enhanced signal on the sensing platform, which was significantly higher than that of non-drug-resistant cells and the blank control group, while the signals of other cell lines were weak and close to the baseline level, indicating that the sensor can effectively distinguish between samples from drug-resistant and non-drug-resistant sources.

[0055] Further investigation was conducted into the sensor's response characteristics at different calnexin concentrations. Figure 7 B), the result shows A 695 / A 520 The ratio gradually increases with increasing calnexin concentration. Within the 1–5000 nM range, the ratio continues to rise; however, it plateaus in the 5000–30000 nM range, indicating that the system has reached saturation at high concentrations. Notably, within the 10–500 nM calnexin concentration range, A… 695 / A 520 The ratio showed a good linear relationship with the calnexin concentration. Figure 7 C), the regression equation is y = 0.001x + 0.2826, and the correlation coefficient R² = 0.9912. Based on 3 times the standard deviation and the slope of the calibration curve, the limit of detection (LOD) of the platform is calculated to be 7.89 nM, indicating that it has high sensitivity and good specificity.

[0056] To evaluate the platform's applicability in complex biological samples, we added different concentrations of calnexin to fresh serum from healthy donors for testing. Figure 7 (D) The results showed that there was no significant signal difference between PBS and serum samples, indicating that the serum matrix had a limited impact on detection performance. Meanwhile, the spiked recoveries were 97.43%–107.12%, with an average RSD < 5%, demonstrating that the platform has good accuracy and stability in a serum environment.

[0057] In summary, the constructed aptamer sensing platform not only achieves highly sensitive detection of calnexin, a specific biomarker for drug-resistant cells, but also maintains excellent analytical performance in complex biological samples.

Claims

1. An EGFR-TKI drug resistance biomarker detection kit for screening aptamer based on non-immobilized GO-SELEX, characterized in that, The kit comprises nucleic acid aptamer Osi-1, gold nanoparticles and PBS buffer.

2. The nucleotide sequence of the aptamer Osi-1 according to claim 1 is SEQ ID NO.

1.

3. The method for preparing the biomarker detection kit according to claim 1, characterized in that, Based on non-immobilized GO-SELEX screening of high-affinity single-chain Osi-1 aptamer for drug-resistant cell supernatant, the affinity of Osi-1 to drug-resistant cell supernatant is determined; the target of Osi-1 is identified as calnexin protein; a sensing platform for detecting calnexin is constructed using Osi-1; and the sensing platform is optimized.

4. The method for preparing the biomarker detection kit according to claim 3, characterized in that, The ratio of ssDNA / GO in the non-immobilized GO-SELEX screening is 1:

30.

5. The method for preparing the biomarker detection kit according to claim 3, characterized in that, The reliability screening of the high-affinity single-chain Osi-1 aptamer for drug-resistant cell supernatant is that the enriched proteins are analyzed by pull down experiment of Osi-1 combined with SDS-PAGE electrophoresis, and calnexin is identified by mass spectrometry to verify the reliability.

6. The method for preparing the biomarker detection kit according to claim 3, characterized in that, The optimization of the sensing platform includes optimization of NaCl concentration, aptamer loading and incubation time.

7. Application of the biomarker detection kit in detection of EGFR-TKI drug resistance biomarkers.

8. Application of the preparation method of the biomarker detection kit according to claim 7 in detection of EGFR-TKI biomarkers, specifically in detection of lung cancer drug resistance diseases.

9. The application according to claim 8, the linear equation of detecting fluorescence signal and calnexin cell is: y = 0.001x + 0.2826, R 2 = 0.9912.

10. Application of the biomarker detection kit according to claim 7 in detection of EGFR-TKI biomarkers, specifically in detection of lung cancer drug resistance diseases.

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