A multiplexed signal amplification electrochemical sensor for hotalr detection

Abstract

The application discloses a multiplex signal amplification electrochemical sensor for HOTAIR detection. A specific DNA fragment in a HOTAIR reverse transcription product is taken as a target, and a FAM / biotin double-labeled dsDNA is generated through a ligase chain reaction; the product is sequentially combined with an anti-FAM antibody modified magnetic microbead and SA-PolyHRP to form a complex and is magnetically fixed on the surface of a magnetic glassy carbon electrode. A reduction current is generated by SA-PolyHRP catalyzing a TMB-H2O2 system, and quantitative detection of HOTAIR is realized, which can be used for detecting the expression level of HOTAIR in cell, lung tissue and blood samples, and provides a detection means for assisting in judging the benignity and malignancy of a lung ground glass nodule.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical biosensing and detection technology, specifically relating to a HOTAIR electrochemical sensor based on ligase chain reaction, antibody-magnetic microbead enrichment, and multi-signal amplification catalyzed by polyhortradiction peroxidase, and the application of this sensor in the auxiliary judgment of the nature of lung ground-glass nodules. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is one of the most common pathological types of lung cancer. Early-stage NSCLC often lacks specific clinical symptoms, and clinical diagnosis relies mainly on a comprehensive approach combining imaging examinations, serum tumor marker detection, and histopathological examination. For early lung lesions presenting as ground-glass opacities (GGOs), imaging follow-up and pathological diagnosis are crucial in determining malignancy and in clinical decision-making. However, imaging follow-up alone has a long cycle and cannot promptly reflect changes at the molecular level in lesions; while tissue biopsy or surgical sampling can provide clear pathological evidence, it is invasive and unsuitable for frequent, dynamic monitoring. Therefore, developing a non-invasive, highly sensitive, specific, and repeatable molecular detection method is of significant application value in the auxiliary assessment of the malignancy of ground-glass opacities.

[0003] Long non-coding RNAs (lncRNAs) are a class of RNA molecules longer than 200 nucleotides that typically do not encode proteins. They participate in various biological processes such as tumor cell proliferation, apoptosis, migration, invasion, angiogenesis, and treatment resistance. HOX transcript antisense RNA (HOTAIR) is one of the important lncRNAs closely related to the development, prognosis, and drug resistance of NSCLC. It can be found in tumor tissues, cells, and blood samples, and has potential value as a molecular biomarker for liquid biopsy. Especially in the auxiliary assessment of benign and malignant lung nodules, HOTAIR-related molecular signals are expected to provide supplementary information to traditional imaging evaluation. However, lncRNAs are usually less abundant in clinical samples and are easily interfered with by background nucleic acids, proteins, and complex matrix components. Direct detection suffers from insufficient sensitivity, difficulty in specific identification, and poor result stability.

[0004] Current HOTAIR or lncRNA detection technologies mainly include Northern blotting, microarrays, reverse transcription quantitative PCR (RT-qPCR), fluorescence detection, colorimetric detection, chemiluminescence detection, and some electrochemical detection methods. Among these, Northern blotting is complex to operate and has limited sensitivity; microarrays are suitable for high-throughput screening but have limited ability to quantitatively detect low-abundance targets; while RT-qPCR is widely used and has high sensitivity, it typically requires reverse transcription, thermal cycling amplification, specialized instruments, and standardized procedures, making the detection process relatively cumbersome. Although some signal amplification-based detection methods can improve detection sensitivity, there is still room for improvement in areas such as tolerability in complex clinical samples, target molecule enrichment efficiency, low-abundance target recognition ability, and detection signal stability.

[0005] Ligase chain reaction (LCR) is a nucleic acid amplification technique based on the specific hybridization and ligation reaction of adjacent oligonucleotide probes. Compared with conventional amplification methods, LCR relies on the precise pairing of probe ends with the target sequence, offering advantages such as high specificity, low background signal, and the ability to amplify target recognition events, making it suitable for high-specificity detection of low-abundance nucleic acid targets. Addressing the challenge of the long full-length HOTAIR sequence and the difficulty of direct detection, short fragments can be selected as detection target regions based on the conservation and specificity of the HOTAIR sequence, and LCR probes can be designed around these target regions. Furthermore, magnetic microbeads possess characteristics such as large specific surface area, easy surface modification, convenient separation, high enrichment efficiency, and the ability to capture target molecules in homogeneous solutions, making them suitable for the separation and enrichment of target nucleic acids or amplification products in complex samples. Polyhorseradish peroxidase (PolyHRP) exhibits stronger enzyme catalytic signal amplification capabilities compared to monomeric horseradish peroxidase, significantly enhancing electrochemical detection signals. Combining LCR-specific amplification, magnetic microbead enrichment and separation, and PolyHRP-catalyzed electrochemical signal amplification could potentially establish a highly sensitive and specific electrochemical sensing method for HOTAIR detection in complex clinical samples, providing a new molecular detection tool for the auxiliary determination of benign and malignant pulmonary ground-glass nodules. Summary of the Invention

[0006] One of the objectives of this invention is to provide a multi-signal amplification electrochemical sensor for HOTAIR detection, in order to solve the problem of low-abundance HOTAIR being difficult to detect sensitively and specifically in complex samples.

[0007] The second objective of this invention is to provide a preparation method and a detection method that combine LCR amplification, anti-FAM antibody-magnetic microbead enrichment and SA-PolyHRP catalysis, to achieve HOTAIR quantification through electrochemical chronocurrent signal and to use it as an auxiliary method for determining the benign or malignant nature of pulmonary ground-glass nodules.

[0008] To achieve the above objectives, the present invention employs the following technical solution: a detection method for a multiplex signal amplification electrochemical sensor for HOTAIR detection and auxiliary determination of the nature of pulmonary ground-glass nodules, comprising: obtaining total RNA containing HOTAIR from a sample to be tested and reverse transcribing it into cDNA; using a specific DNA fragment in the HOTAIR reverse transcription product as a template, adding probe A1, probe A2, CP-FAM, and Biotin-SP, and performing a ligase chain reaction under the action of a thermostable DNA ligase to obtain a dsDNA amplification product with FAM at one end and biotin at the other end; and combining the dsDNA amplification product with an epoxy resin functionalized magnetic material modified with an anti-FAM antibody. Incubation with magnetic microbeads allows dsDNA to assemble onto the surface of magnetic microbeads via the specific binding of FAM and anti-FAM antibodies. Streptavidin-labeled horseradish peroxidase is added, allowing it to bind to the dsDNA / Antibody / MMBs surface via biotin-streptavidin interaction, forming a PolyHRP / dsDNA / Antibody / MMBs complex. This complex is magnetically enriched on the surface of a magnetic glassy carbon electrode. Electrochemical reduction current signals are collected in a TMB-H2O2 substrate system, and the current signals are used to quantify HOTAIR or assist in determining the nature of pulmonary ground-glass nodules. The specific DNA fragment in the HOTAIR reverse transcription product is the sequence shown in SEQ ID NO.1, probe A1 is the sequence shown in SEQ ID NO.2, probe A2 is the sequence shown in SEQ ID NO.3, CP-FAM is the sequence shown in SEQ ID NO.4 with FAM modified at the 3' end, and Biotin-SP is the sequence shown in SEQ ID NO.5 with biotin modified at the 5' end.

[0009] Specifically, the reaction system of the ligase chain reaction includes target cDNA, probe A1, probe A2, CP-FAM, Biotin-SP, 10× ligase reaction buffer, Tris-HCl buffer, and thermostable DNA ligase; wherein the final concentration of probe A1, probe A2, CP-FAM, and Biotin-SP is 1-50 nM, preferably 10 nM.

[0010] Specifically, the thermal cycling conditions for the ligase chain reaction are: denaturation at 95°C for 1 min, hybridization / ligation at 60°C for 2 min, repeated for 30 cycles; preferably, denaturation at 95°C for 1 min, hybridization / ligation at 60°C for 2 min, repeated for 30 cycles.

[0011] Specifically, the anti-FAM antibody-modified epoxy resin functionalized magnetic microbeads are prepared by mixing epoxy resin functionalized magnetic microbeads, anti-FAM monoclonal antibody, high-salt PBS buffer, and ammonium sulfate buffer, followed by rotation incubation.

[0012] Specifically, the dsDNA amplification product is incubated with magnetic microbeads modified with anti-FAM antibody at room temperature for 20-60 min; then diluted SA-PolyHRP is added and incubated at room temperature for 10-30 min, preferably 15 min.

[0013] Specifically, the PolyHRP / dsDNA / Antibody / MMBs complex was washed, resuspended in PBS, dropped onto the surface of a magnetic glassy carbon electrode and magnetically fixed. The three-electrode system was then placed in a TMB substrate solution, and the current signal was recorded using chronoamperometry.

[0014] Specifically, when the sample to be tested is a lung tissue sample, the volume of tissue reverse transcription product added to the ligase chain reaction system is 1-5 μL, preferably 3 μL; when the sample to be tested is a blood sample, the volume of blood reverse transcription product added to the ligase chain reaction system is 0.4-1.2 μL, preferably 1 μL.

[0015] A multi-signal amplification electrochemical sensor for HOTAIR detection is prepared by the above method. The sensor includes a HOTAIR-specific LCR probe set, epoxy resin functionalized magnetic microbeads modified with anti-FAM antibody, SA-PolyHRP, a magnetic glassy carbon electrode, and a TMB-H2O2 electrochemical substrate system.

[0016] The preparation method described above or the multi-signal amplified electrochemical sensor can be used to prepare a kit for auxiliary determination of benign and malignant pulmonary ground-glass nodules.

[0017] Compared with existing technologies, this invention has the following advantages: First, it utilizes LCR to achieve specific amplification of nucleic acid targets, improving the detection sensitivity of low-abundance HOTAIR; second, it uses magnetic microbeads to increase the reaction interface and facilitate separation and enrichment, improving target capture efficiency in complex samples and reducing matrix interference; third, it uses SA-PolyHRP to replace monomeric HRP to achieve stronger enzyme catalytic signal amplification; fourth, it uses electrochemical chronoamperometry for reading, resulting in lower equipment cost, faster response speed, and ease of clinical sample detection; fifth, this method can detect HOTAIR expression in various samples such as cells, tissues, and blood, providing a new method for auxiliary judgment of benign and malignant pulmonary ground-glass nodules. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the design principle of the HOTAIR electrochemical sensor with multiple signal amplification according to the present invention.

[0019] Figure 2 Non-denaturing PAGE characterization of the feasibility of ligase chain reaction (LCR) amplification.

[0020] Figure 3 A diagram validating the assembly feasibility of FAM-dsDNA / Antibody / MMBs and PolyHRP / dsDNA / Antibody / MMBs.

[0021] Figure 4 This is a scanning electron microscope (SEM) characterization of the stepwise assembly process on the surface of magnetic microspheres.

[0022] Figure 5 The images show the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) characterizations of different assembly stages on the surface of the magnetic glassy carbon electrode.

[0023] Figure 6 The graph shows the optimization of LCR probe concentration, ligase dosage, ligation temperature, and number of cycles.

[0024] Figure 7 The graph shows the effect of the amount of magnetic microbeads on the electrochemical response signal of the sensor of this invention.

[0025] Figure 8 The diagram shows the effect of the amount of anti-FAM antibody on the electrochemical response signal of the sensor of this invention.

[0026] Figure 9 The graph shows the effect of SA-PolyHRP concentration on the electrochemical response signal of the sensor in this invention.

[0027] Figure 10 The graph shows the electrochemical response, linear range, and detection limit of the sensor of this invention to different concentrations of HOTAIR target chains.

[0028] Figure 11 This is a diagram showing the verification results of the specificity of the sensor of the present invention for the HOTAIR target chain and different mismatch sequences.

[0029] Figure 12 A diagram illustrating the storage stability evaluation of pre-prepared anti-FAM antibody-modified magnetic microbead components.

[0030] Figure 13 The image shows the results of HOTAIR detection in different cell samples by the sensor of this invention.

[0031] Figure 14 This is a graph showing the results of the sensor of the present invention detecting HOTAIR in lung tissue samples and helping to distinguish between SPN and NSCLC.

[0032] Figure 15 Figure 1 shows the results of HOTAIR detection in lung tissue samples by optimizing the volume of reverse transcription products added to lung tissue and the sensor of the present invention.

[0033] Figure 16 The figure shows the RT-qPCR validation and intergroup comparison results of the relative expression level of HOTAIR in blood samples.

[0034] Figure 17 Figure 1 shows the results of optimizing the addition volume of blood reverse transcription products and the detection of HOTAIR in blood samples by the sensor of this invention, which helps distinguish between NSCLC and SPN / healthy donors. Detailed Implementation

[0035] The present invention will be further illustrated below with reference to the embodiments. It should be understood that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Without departing from the spirit and essence of the present invention, those skilled in the art can make equivalent substitutions or conventional optimizations to the reagent concentration, incubation time, reaction temperature, sample type, and electrochemical detection parameters.

[0036] The reagents and materials used in this invention are all from the following companies:

[0037]

[0038]

[0039] The solution used in this invention is prepared as follows:

[0040] (1) Phosphate buffer (PB): NaH2PO4·2H2O and Na2HPO4·12H2O are mixed in a volume ratio of 19:81 to prepare a 10.0 mM phosphate buffer with a pH of 7.4.

[0041] (2) Phosphate buffer solution (PBS): NaH2PO4·2H2O and Na2HPO4·12H2O were mixed in a volume ratio of 19:81 to prepare a 10.0 mM phosphate buffer solution with a pH of 7.4, and then 0.1 M NaCl was added to prepare PBS.

[0042] (3) High concentration phosphate buffer solution (high salt PBS): NaH2PO4·2H2O and Na2HPO4·12H2O are mixed in a volume ratio of 19:81 to prepare a 10.0 mM pH 7.4 phosphate buffer, and then 1 M NaCl is added to prepare PBS.

[0043] (4) Tris-HCl buffer: First prepare a certain volume of 10 mM Tris solution, then adjust the pH value to 8.0 with a small amount of concentrated hydrochloric acid.

[0044] (5) Ammonium sulfate buffer: Prepare ammonium sulfate buffer by dissolving (NH4)2SO4 in 10.0 mM pH 7.4 phosphate buffer to 3 M (NH4)2SO4.

[0045] (6) TE buffer: Dissolve EDTA and NaCl in Tris-HCl buffer at pH 8.0 to prepare TE buffer containing 1.0 mM EDTA and 1.0 M NaCl.

[0046] (7) Magnetic bead cleaning buffer: First, prepare a 10.0 mM pH 7.4 phosphate buffer by dissolving EDTA and NaCl and adding an appropriate amount of Tween-20 to prepare a magnetic bead cleaning buffer containing 10 mM PB, 0.5 mM EDTA, 1 M NaCl and 0.01 wt% Tween-20.

[0047] (8) SA-HRP enzyme solution: The SA-HRP mother solution was diluted stepwise with magnetic bead washing buffer to a 1:1000 enzyme solution for use and stored temporarily at 4°C in the dark.

[0048] (9) SA-PolyHRP enzyme solution: The SA-PolyHRP stock solution was gradually diluted to 1:1000 with magnetic bead washing buffer and stored temporarily at 4°C in the dark.

[0049] (10) DNA stock solution: The synthesized DNA is a sample that has been identified by the manufacturer through HPLC-MS. It is a very light dry film attached to the wall of the container tube and is very easy to dissipate when opened. Therefore, before dissolving, the centrifuge tube containing DNA is centrifuged at 3000 rpm for 10 min. Then, the cap is slowly opened and a certain volume of TE buffer is added according to the instructions to prepare a 100 µM stock solution. In order to avoid repeated freeze-thaw cycles, the stock solution is aliquoted and stored at -20℃.

[0050] (11) DMEM cell culture medium: Dissolve one packet of DMEM powder in 800 mL of ultrapure water, adjust the pH to 7.2 with sodium hydroxide or hydrochloric acid, and then verify with pH test paper (to rule out inaccurate pH meters). Then bring the volume to 1 L. Next, filter through a 0.22 μm filter membrane for sterilization, and then add 100 mL of fetal bovine blood and 10 mL of (100×) penicillin-streptomycin solution. The working concentration of streptomycin is 0.1 mg / mL, and the working concentration of penicillin is 100 U / mL.

[0051] (12) Trypsin: Weigh 12.5 g of trypsin, dissolve it in 800 mL of 1 × PBS, adjust the pH to 7.2 with sodium hydroxide or hydrochloric acid, and then verify it with pH test paper (to exclude the case of inaccurate pH meter). Then make up to 1 L, filter it through a 0.22 μm filter membrane for sterilization, aliquot it and store it in a 4°C refrigerator.

[0052] The clinical blood samples used in this experiment were obtained from the First Affiliated Hospital of Fujian Medical University and were approved ethically and with informed consent.

[0053] Example 1: Nucleic Acid Probe Sequence Design

[0054] This invention selects a 43 nt conserved target sequence from HOTAIR as the probe recognition region and uses the specific DNA fragment obtained by reverse transcription of this sequence as the detection target. Based on the base pairing principle, four DNA probes required for the ligase chain reaction (LCR) are designed, including probe A1, probe A2, CP-FAM, and Biotin-SP, to construct a multiplexed electrochemical sensor for HOTAIR detection. The detection principle is as follows: Figure 1As shown. In the presence of the target strand, probes A1, A2, CP-FAM, and Biotin-SP hybridize adjacently on the target strand template and undergo a ligation amplification reaction catalyzed by a thermostable DNA ligase, forming a double-labeled dsDNA product with FAM at one end and biotin at the other end. In this invention, SEQ ID NO.1 is the target recognition fragment taken from the HOTAIR reverse transcription product; SEQ ID NO.2 to SEQ ID NO.5 are LCR probe sequences designed according to SEQ ID NO.1; SEQ ID NO.6 and SEQ ID NO.7 are RT-qPCR primer sequences used for HOTAIR expression level verification. SEQ ID NO.1 to SEQ ID NO.7 can all be artificially synthesized by commercial nucleic acid synthesis institutions according to the nucleotide sequences listed in Table 1. In this embodiment, the oligonucleotide sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; wherein, the 3' end of CP-FAM is modified with FAM, and the 5' end of Biotin-SP is modified with biotin. After the synthesized oligonucleotides were identified as qualified by HPLC-MS, they were dissolved in TE buffer to prepare 100 μM stock solutions, aliquoted, and stored at -20℃ to avoid repeated freeze-thaw cycles. The primer sequences are shown in Table 1.

[0055] Table 1. Primer sequences

[0056] In the above sequence, FAM and Biotin are functional modification groups. The FAM terminus of CP-FAM is used to bind to anti-FAM antibodies, and the biotin terminus of Biotin-SP is used to bind SA-PolyHRP.

[0057] Example 2: Solution and Reagent Preparation

[0058] Phosphate-buffered saline (PB): Prepare a 10 mM phosphate-buffered saline solution (PB) with NaH₂PO₄·2H₂O and Na₂HPO₄·12H₂O in a specific ratio, with a pH of 7.4. Phosphate-buffered saline (PBS): Prepare PBS containing 0.1 M NaCl by adding NaCl to 10 mM PB; high-salt PBS can be prepared as PBS containing 1 M NaCl. Tris-HCl buffer: Prepare a 10 mM Tris solution and adjust the pH to 8.0 with hydrochloric acid. Magnetic bead washing buffer: Includes 10 mM PB, 0.5 mM EDTA, 1 M NaCl, and 0.01 wt% Tween-20. SA-PolyHRP enzyme solution: Dilute the SA-PolyHRP stock solution with magnetic bead washing buffer, preferably at a dilution ratio of 1:1000, and store temporarily at 4°C protected from light. DNA stock solution: After the synthesized DNA was verified by HPLC-MS, it was centrifuged and dissolved in TE buffer to prepare a 100 μM stock solution. After aliquoting, it was stored at -20℃ to avoid repeated freeze-thaw cycles.

[0059] Example 3: Ligase chain reaction (LCR)

[0060] Prepare a 100 μL LCR reaction system according to Table 2. For detection of artificial target strands, the target strand or blank control can be added; for detection of actual samples, cDNA obtained by reverse transcription of total RNA from cells, lung tissue or blood can be added.

[0061] Table 2. LCR Reaction System

[0062]

[0063] In Table 2, “Tris-HCl” is prepared in the same way as in Example 2; “ampligase® DNA ligase” is a thermostable DNA ligase.

[0064] The optimal LCR reaction parameters are: denaturation at 95℃ for 1 min, hybridization / ligation at 60℃ for 2 min, constituting one cycle, for a total of 30 cycles. After the reaction, the product is stored at 4℃. Through LCR amplification, the target sequence triggers the rapid consumption of the four substrate strands, forming a FAM / biotin-labeled dsDNA product.

[0065] Example 4: Preparation of epoxy resin functionalized magnetic microspheres modified with anti-FAM antibody

[0066] Epoxy resin functionalized magnetic microbeads (MMBs) were mixed by pipetting with magnetic bead washing buffer (prepared as in Example 2) and enriched on a magnetic rack. The supernatant was discarded, and the washing was repeated twice. Subsequently, the anti-FAM-antibody and epoxy resin functionalized magnetic beads (hereinafter referred to as magnetic beads) binding reaction system was prepared according to Table 3, and incubated by rotation for 12 h to obtain Antibody / MMBs, which were stored at 4°C for later use. Table 3. Solution preparation for the reaction of anti-FAM-antibody with magnetic beads

[0067]

[0068] The "high-salt PBS buffer" in Table 3 is prepared in the same way as in Example 2.

[0069] The resulting Antibody / MMBs can specifically recognize the FAM group on the LCR product through anti-FAM antibody, thereby immobilizing the dsDNA amplification product on the surface of magnetic microbeads, and obtaining epoxy resin functionalized magnetic microbeads modified with anti-FAM antibody.

[0070] Example 5: Preparation of PolyHRP / dsDNA / Antibody / MMBs complex

[0071] The LCR reaction product dsDNA was added to a centrifuge tube containing Antibody / MMBs. After assembly at room temperature for 30 min, the dsDNA / Antibody / MMBs were washed twice with magnetic bead washing buffer. Then, 100 μL of SA-PolyHRP (preferably 1:1000) enzyme solution was added, and the tube was incubated at room temperature for 15 min by rotation, allowing SA-PolyHRP to bind to the other end of the LCR product dsDNA via biotin-streptavidin interaction. After incubation, the tube was washed twice more with magnetic bead washing buffer and resuspended in 10 μL PBS to obtain the PolyHRP / dsDNA / Antibody / MMBs complex.

[0072] Example 6: Pretreatment and electrochemical detection of magnetic glassy carbon electrode

[0073] Before each test, the magnetic glassy carbon electrode is polished with a 0.05 μm Al2O3 powder suspension, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water in sequence to remove residual Al2O3 powder and other impurities. The magnetic glassy carbon electrode is then dried with nitrogen gas for later use.

[0074] The PolyHRP / dsDNA / Antibody / MMBs complex was dropped onto the surface of a magnetic glassy carbon electrode treated in the above steps. After magnetic fixation for 1 min, electrochemical detection was performed. A three-electrode system was used: the magnetic glassy carbon electrode as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode; the detection solution was a TMB-H2O2 substrate system. The reduction current was recorded by chronoamperometry, with a sampling interval of 0.1 s and a sampling time of 100 s. The higher the content of the target HOTAIR, the more LCR products were produced, the more SA-PolyHRP was bound, and the stronger the corresponding reduction current signal.

[0075] Example 7: Method Feasibility and Interface Characterization

[0076] The feasibility of LCR was verified using non-denaturing polyacrylamide gel electrophoresis. The results are as follows: Figure 2 As shown, no obvious long double-stranded DNA product is formed when there is no target strand, no ligase, or no partial primer strand; only when the target strand, four substrate probes, and thermostable DNA ligase are present simultaneously is an obvious dsDNA product band formed, indicating that the HOTAIR target fragment can specifically trigger LCR amplification.

[0077] The feasibility of the sensor construction was verified by FAM fluorescence observation, TMB color development, and electrochemical detection. The results are as follows: Figure 3 As shown in AD. Samples containing the target sequence can form FAM-dsDNA / Antibody / MMBs and exhibit significant fluorescence ( ). Figure 3 (A in the text); upon further integration with SA-PolyHRP, the TMB substrate exhibited a significant color change ( Figure 3 In sample B), the electrochemical reduction peak or chronocurrent signal is significantly enhanced, while the signal is lower in samples without the target sequence. Figure 3 (CD in the middle).

[0078] The surface morphology of MMBs, Antibody / MMBs, dsDNA / Antibody / MMBs, and PolyHRP / dsDNA / Antibody / MMBs was observed using scanning electron microscopy. The results are as follows: Figure 4 As shown in Figures A-D, the surface morphology and particle size of the magnetic microbeads change accordingly as the antibody, dsDNA, and PolyHRP are gradually assembled. Figure 4 Obvious voids can be observed on the surface of bare MMBs A in this case, where the diameter of the MMBs is 2.40 µm. Figure 4 In the sample B, small white, poorly defined granular clumps were observed on its surface, with significantly reduced porosity and a diameter of 2.44 µm, indicating that Anti-FAM-Antibody was successfully assembled on MMBs. Figure 4 In the C sample, numerous small, white, clearly defined granular clumps with a diameter of 2.47 µm were observed on its surface, indicating that the dsDNA was successfully assembled on Antibody / MMBs. Figure 4 In D, it can be observed that the entire surface of MMBs has similar characteristics. Figure 4 The presence of small white particles (B) and a few newly formed, more aggregated clusters with a diameter of 2.51 µm indicates that HRP was successfully assembled onto MMBs. As self-assembly progressed, the surface morphology of the MMBs changed, and their diameter gradually increased. The electrochemical properties of the magnetic glassy carbon electrode surface at different assembly stages were characterized using CV and EIS, and the results are as follows: Figure 5 As shown, Figure 5 In the figure, A represents the CV curves of different fabrication stages scanned within a potential range of -0.1 to 0.6 V. Compared to bare glassy carbon electrodes, after MGCE / MMBs assembly, [Fe(CN)6] 3− The peak current gradually decreases after MGCE / Antibody / MMBs assembly, [Fe(CN)6] 3− The peak current increased after MGCE / dsDNA / Antibody / MMBs assembly, [Fe(CN)6] 3− The peak current decreased after MGCE / HRP / dsDNA / Antibody / MMBs assembly, [Fe(CN)6] 3− The peak current increased again. Figure 5 The Nyquist plot of EIS shown in Figure B indicates that the resistance of the bare electrode is extremely weak. Assembly of MMBs on the electrode slightly increases the semicircle diameter. When Antibody / MMBs are assembled onto the electrode, the semicircle radius decreases significantly. This is because antibodies, as proteins, carry a large amount of positive charge, resulting in lower electronegativity. The proximity of potassium ferricyanide further reduces the impedance. Assembly of dsDNA / Antibody / MMBs further increases the semicircle diameter. However, assembly of HRP / dsDNA / Antibody / MMBs significantly reduces the semicircle diameter, likely because SA-PolyHRP carries a large amount of positive charge, leading to lower impedance. Both EIS and CV results show that the surface properties of MGCE change accordingly with gradual assembly, indicating successful assembly.

[0079] Example 8: Optimization of Experimental Conditions

[0080] The concentrations of LCR probes, Ampligase enzyme, ligation temperature, number of cycles, amount of magnetic beads, amount of anti-FAM antibody, and concentration of SA-PolyHRP were optimized. The results are as follows: Figure 6As shown in Figure A, when the final concentration of the four substrate probes in the LCR is below 10 nM, the target signal increases with increasing probe concentration while the background remains relatively stable; above 10 nM, the background signal increases significantly, therefore 10 nM is preferred as the LCR probe concentration. Figure 6 B- Figure 6 As shown in D, the preferred dosage of Ampligase enzyme is 1.0 U; the preferred ligation temperature is 60℃; and the preferred number of thermal cycles is 30. The dosage of magnetic microbeads (…) Figure 7 ), dosage of anti-FAM antibody ( Figure 8 ) and SA-PolyHRP concentration ( Figure 9 Both have optimal ranges; too low a range can lead to insufficient capture or catalytic signal, while too high a range may result in a reduced signal-to-noise ratio due to steric hindrance or nonspecific adsorption. Under optimal conditions, SA-PolyHRP achieves a higher electrochemical signal response than SA-HRP without a significant increase in background signal.

[0081] Example 9: Sensitivity, Specificity, and Stability

[0082] Under optimized experimental conditions, different concentrations of the HOTAIR target chain were used for detection. The results are as follows: Figure 10 As shown in Figure 10, A-C, it can be seen through colorimetry that as the concentration of the target chain increases, a clear color change in the solution is observed to the naked eye, and the current signal gradually increases as well. Plotting the logarithm of the target concentration (lgCTarget) on the x-axis and the final current signal recorded (Current / I) on the y-axis yields the following result: Figure 10 The linear relationship of B in the graph is good, and the linear equation is: I = 2972.737lgCTarget + 48814.624 (R² = 0.991), with a linear range from 100 aM to 100 pM. According to the calculation method of the detection limit (i.e., the blank sample value plus the sample concentration corresponding to 3 times the standard deviation of the blank sample signal), the following is obtained: Figure 10 The bar chart for C shows the system without any target concentration added (Blank). In this case, the average blank signal is 44.33 nA. After adding three times the standard deviation, the result is 53.48 nA, which is less than the signal of 191.33 nA at the target concentration of 10 aM. According to the LOD (Limit of Detection) three-times-blank standard deviation method, the minimum concentration of the sample to be detected relative to the blank value needs to be above three times the blank standard deviation, i.e., 3σ blank (or 3S blank). Therefore, we can conclude that the final detection limit obtained by this experimental method is 10 aM.

[0083] Specificity was verified using 1-base mismatch, 3-base mismatch, and 5-base mismatch sequences as interfering agents. The results are as follows: Figure 11 As shown in AB, Figure 11As shown in Figure A, the solution obtained from the reaction of 10 fM target DNA is a deep blue, while the solution of 100 fM containing four mismatched base sequences is close to the color of the blank solution, and they can be clearly distinguished by the naked eye. Figure 11 As shown in B, the current intensity corresponding to the non-mismatched target strand is significantly higher than that corresponding to other mismatched sequences, consistent with the phenomenon obtained by colorimetric methods. The current signal of the target DNA is significantly higher than the signal value generated by the base mismatched sequence, and can be completely distinguished. The experiment shows that the sensor has very high specificity.

[0084] The sensor response was detected after pre-prepared antibody / MMBs were stored at 4°C. Results are as follows: Figure 12 As shown, the signal retention rate is high within a certain storage time, indicating that the pre-prepared magnetic bead components have good short-term stability and can be used to reduce the pretreatment time for detection.

[0085] Example 10: Detection of cell, lung tissue and blood samples

[0086] The sensor of the present invention obtained in Example 6 was used for HOTAIR detection in normal alveolar / bronchial epithelial cells and non-small cell lung cancer cells. The results are as follows: Figure 13 As shown in AC, the it current signals measured in human non-small cell lung cancer strains were all higher than those measured in normal human alveolar epithelial cells. Figure 13 The AB in the figure indicates that the expression level of HOTAIR in non-small cell lung cancer cells is higher than that in normal alveolar epithelial cells. Furthermore, the results were compared with those from RT-qPCR detection. Figure 13 Comparing C) with those in the previous study, the trends were similar. Since the magnitude of the electrochemical signal can significantly distinguish between normal alveolar epithelial cells and non-small cell lung cancer cells, the experiment verified the accuracy of the electrochemical sensor in real samples. RT-qPCR results also demonstrated that the expression level of HOTAIR in normal cells was significantly lower than that in non-small cell lung cancer cells. Notably, the A549DDP cell line exhibited cisplatin resistance, and its signal response in both electrochemical and RT-qPCR detection was greater than that of other non-small cell lung cancer cell lines. This is attributed to the high expression of HOTAIR and its association with tumor drug resistance.

[0087] The sensor of the present invention obtained in Example 6 was used for the detection of lung tissue samples from patients with pulmonary ground-glass nodules. Due to the complexity of the tissue reverse transcription product system, the amount of tissue cDNA added to the LCR system affects the amplification efficiency. Optimization showed that the preferred amount of tissue reverse transcription product added was 3 μL. Detection results are as follows... Figure 14 AB and Figure 15As shown in Figure 14, A represents the expression level analysis of HOTAIR extracted from clinical tissue samples (SPN and NSCLC patients) using RT-qPCR. Figure 14 The statistical significance of sensor B in the figure was analyzed using a t-test. Error bars represent the standard deviation of the measured values ​​(n = 3). Figure 15 In the diagram, A represents the optimization of the amount of tissue reverse transcription product added to LCR; Figure 15 In the figure, B represents the expression level analysis of HOTAIR extracted from clinical tissue samples (SPN and NSCLC patients) using the electrochemical sensor constructed in this paper. Figure 15 In the figure, C represents the statistical significance of the sensor readings, analyzed using a t-test. Error bars represent the standard deviation of the measurements (n = 3). The experimental results showed that the overall HOTAIR electrochemical signal in the lung tissue of NSCLC patients was higher than that in SPN patients, and this was consistent with RT-qPCR and pathological results, which can help differentiate between benign and malignant GGOs.

[0088] The sensor of the present invention obtained in Example 6 was used for blood sample detection. Since the components of blood reverse transcription products are complex, the preferred amount added is 1 μL. The detection results are as follows: Figure 16 AC and Figure 17 As shown in AD. Where, Figure 16 In the figure, A represents the expression level analysis of HOTAIR extracted from clinical blood samples (SPN patients, NSCLC patients, and healthy donors) by RT-qPCR. Figure 16 B- Figure 16 The statistical significance of C was analyzed using a t-test. Error bars represent the standard deviation of the measurements (n = 3). Results showed that the HOTAIR electrochemical signal in blood samples from NSCLC patients was higher than that from SPN patients and healthy donors, consistent with the trend observed in RT-qPCR. These results indicate that the method of this invention can detect HOTAIR in blood samples, providing a liquid biopsy protocol for the auxiliary assessment of GGO properties.

[0089] This invention constructs a multi-signal amplified electrochemical sensor based on HOTAIR-specific LCR amplification, enrichment of magnetic microbeads modified with anti-FAM antibody, and SA-PolyHRP catalytic signal amplification. This sensor combines the advantages of high sensitivity, high specificity, low cost, and adaptability to complex samples. It can be used for quantitative detection of HOTAIR and can be further used to assist in the determination of benign or malignant pulmonary ground-glass nodules.

[0090] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, combinations, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a multi-signal amplified electrochemical sensor for HOTAIR detection, characterized in that, Includes the following steps: (1) Obtain total RNA containing HOTAIR from the sample to be tested, and reverse transcribe the obtained total RNA into cDNA; (2) Using the specific DNA fragment in the cDNA of the HOTAIR reverse transcription product as a template, probe A1, probe A2, CP-FAM and Biotin-SP were added, and a ligase chain reaction was carried out under the action of thermostable DNA ligase to obtain a dsDNA amplification product with FAM at one end and biotin at the other end. (3) The dsDNA amplification product obtained in step (2) is incubated with epoxy resin functionalized magnetic microbeads modified with anti-FAM antibody, so that the dsDNA is assembled on the surface of the magnetic microbeads by the specific binding of the anti-FAM antibody in the epoxy resin functionalized magnetic microbeads modified with anti-FAM antibody through the FAM at one end of the dsDNA, thus obtaining dsDNA / Antibody / MMBs. (4) Add streptavidin-labeled polyhortradiction peroxidase SA-PolyHRP, so that it binds to the surface of dsDNA / Antibody / MMBs through biotin-streptavidin action to form a PolyHRP / dsDNA / Antibody / MMBs complex. (5) In the TMB-H2O2 substrate system, the PolyHRP / dsDNA / Antibody / MMBs complex is magnetically enriched on the surface of a magnetic glassy carbon electrode, and an electrochemical reduction current signal is collected in the TMB-H2O2 substrate system. The current signal can be used to quantify HOTAIR or assist in judging the nature of lung ground-glass nodules. The specific DNA fragment in the cDNA of the HOTAIR reverse transcription product is the sequence shown in SEQ ID NO.1, the probe A1 is the sequence shown in SEQ ID NO.2, the probe A2 is the sequence shown in SEQ ID NO.3, the CP-FAM is the sequence shown in SEQ ID NO.4 with FAM modified at the 3' end, and the Biotin-SP is the sequence shown in SEQ ID NO.5 with biotin modified at the 5' end.

2. The preparation method according to claim 1, characterized in that, The reaction system for the ligase chain reaction includes target cDNA, probe A1, probe A2, CP-FAM, Biotin-SP, 10× ligase reaction buffer, Tris-HCl buffer, and thermostable DNA ligase; wherein the final concentrations of probe A1, probe A2, CP-FAM, and Biotin-SP are 1-50 nM.

3. The preparation method according to any one of claims 1-2, characterized in that, The thermal cycling conditions for the ligase chain reaction were: denaturation at 95°C for 1 min, hybridization / ligation at 60°C for 2 min, repeated for 30 cycles.

4. The preparation method according to claim 1, characterized in that, The anti-FAM antibody-modified epoxy resin functionalized magnetic microbeads were prepared by mixing epoxy resin functionalized magnetic microbeads, anti-FAM monoclonal antibody, high-salt PBS buffer, and ammonium sulfate buffer, followed by rotation incubation.

5. The preparation method according to claim 1 or 4, characterized in that, The dsDNA amplification product described in step (2) is incubated with epoxy resin functionalized magnetic microbeads modified with anti-FAM antibody at room temperature for 20-60 min; then diluted SA-PolyHRP is added and incubated at room temperature for 10-30 min.

6. The preparation method according to claim 1, characterized in that, The PolyHRP / dsDNA / Antibody / MMBs complex was washed, resuspended in PBS, dropped onto the surface of a magnetic glassy carbon electrode and magnetically fixed. The three-electrode system was then placed in a TMB substrate solution, and the current signal was recorded using chronoamperometry.

7. The preparation method according to claim 1, characterized in that, When the sample to be tested is lung tissue, the volume of tissue reverse transcription product added to the ligase chain reaction system is 1-5 μL; when the sample to be tested is blood, the volume of blood reverse transcription product added to the ligase chain reaction system is 0.4-1.2 μL.

8. A multi-signal amplified electrochemical sensor for HOTAIR detection, characterized in that, Prepared by the preparation method according to any one of claims 1-7, the invention comprises a HOTAIR-specific LCR probe set, epoxy resin functionalized magnetic microbeads modified with anti-FAM antibody, SA-PolyHRP, a magnetic glassy carbon electrode, and a TMB-H2O2 electrochemical substrate system. The HOTAIR-specific LCR probe set consists of probe A1, probe A2, CP-FAM, and Biotin-SP. The target of the HOTAIR-specific LCR probe set is a specific DNA fragment in the cDNA of the HOTAIR reverse transcription product. The specific DNA fragment is the sequence shown in SEQ ID NO.

1. Probe A1 is the sequence shown in SEQ ID NO.

2. Probe A2 is the sequence shown in SEQ ID NO.

3. CP-FAM is the sequence shown in SEQ ID NO.4 and modified with FAM at its 3' end. Biotin-SP is the sequence shown in SEQ ID NO.5 and modified with biotin at its 5' end.

9. The multi-signal amplified electrochemical sensor for HOTAIR detection obtained by the preparation method according to any one of claims 1-7 is used in the preparation of a kit for auxiliary determination of benign and malignant pulmonary ground-glass nodules.

10. The application of the multi-signal amplification electrochemical sensor according to claim 8 in the preparation of a kit for auxiliary determination of benign and malignant pulmonary ground-glass nodules.

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