Biosensor based on proximity hybridization for single lncrna-initiated multiple rolling circle amplification and its application in breast cancer lncrna ratio fluorescent detection

Abstract

The application belongs to the technical field of fluorescence detection, and particularly relates to a single lncRNA starting multiple rolling circle amplification biosensor based on proximity hybridization and application thereof in breast cancer long-chain non-coding RNA (lncRNA) one-step ratio fluorescence detection. The application utilizes the proximity hybridization effect of target lncRNA to assist the hybridization of primers and circular templates, realizes single lncRNA molecule starting multiple rolling circle amplification, and combines fluorescence resonance energy transfer (FRET) to construct a ratio fluorescence detection system, develops a template-dependent one-step lncRNA detection biosensor and a corresponding detection method, effectively improves the amplification efficiency and sensitivity of detection, reduces the detection cost, simultaneously utilizes the self-calibration function of ratio fluorescence to reduce the detection error, realizes sensitive and accurate detection of lncRNA at a single cell level and in a clinical tissue sample, and has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, specifically relating to a biosensor based on proximity hybridization-initiated single lncRNA multiple rolling circle amplification and its application in one-step ratio fluorescence detection of long non-coding RNA (lncRNA) in breast cancer. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Long non-coding RNAs (lncRNAs) are a class of non-coding ribonucleic acid molecules exceeding 200 nucleotides in length, widely distributed throughout the human genome, and exhibiting high evolutionary conservation and tissue specificity. Although lncRNAs do not participate in protein coding, they play crucial roles in various important physiological activities such as epigenetic regulation, post-transcriptional regulation, and cell cycle regulation. Abnormal expression of lncRNAs is a significant driving factor in the occurrence and development of malignant tumors. Among them, lung adenocarcinoma metastasis-associated transcript 1 (lncRNA MALAT1) is dysregulated in solid tumors such as breast cancer, lung cancer, and liver cancer, as well as hematologic malignancies, and is closely related to tumor occurrence and metastasis. It is an important biomarker for early tumor screening and prognostic assessment, and also a potential target for targeted tumor therapy. Therefore, sensitive, rapid, and accurate detection of lncRNA expression levels is of great significance for biomedical research, clinical diagnosis, and targeted therapy of tumors.

[0004] Currently, methods for detecting lncRNA mainly include classic molecular biology techniques such as RNA sequencing (RNA-seq), gene chips, and quantitative real-time polymerase chain reaction (qRT-PCR), as well as novel detection methods based on electrochemistry, photoelectrochemistry, and localized surface plasmon resonance. However, these methods all have many limitations: RNA-seq detection is costly and data analysis is complex; gene chips are prone to false positive signals due to cross-hybridization; qRT-PCR is cumbersome to operate and relies on sophisticated thermal cycling equipment; and electrochemical and photoelectrochemical methods suffer from complex electrode modification, cumbersome operation steps, and long detection times.

[0005] To address the aforementioned issues, fluorescence detection methods based on isothermal nucleic acid amplification strategies have been applied to lncRNA detection, such as strand displacement amplification, hybridization chain reaction, and TtAgo-mediated exponential amplification. However, most of these methods rely on multiple polymerases or carefully designed nucleic acid probes, significantly increasing detection costs. Furthermore, most methods employ multi-step reaction procedures or use a single fluorescence signal as the readout signal, making the liquid-phase transfer process susceptible to aerosol contamination. The fluorescence signal is highly sensitive to minute fluctuations in the reaction system, easily generating false positive signals, resulting in poor stability of the detection results. In addition, rolling circle amplification (RCA), as a highly efficient isothermal nucleic acid amplification technique, has been widely used in nucleic acid detection, but due to the excessive length of lncRNA chains, it cannot be directly used as an RCA primer, making it difficult to directly apply to lncRNA detection.

[0006] Therefore, developing an analytical method that is simple to operate, fast to detect, highly sensitive, stable, and capable of one-step detection of lncRNA has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a biosensor for single-lncRNA-initiated multiple rolling circle amplification based on proximity hybridization and its application in ratiometric fluorescence detection of lncRNAs in breast cancer. This invention utilizes the proximity hybridization effect of the target lncRNA to assist primers in hybridizing with a circular template, enabling single-lncRNA molecule-initiated multiple rolling circle amplification. Combined with fluorescence resonance energy transfer (FRET), a ratiometric fluorescence detection system is constructed, developing a template-dependent one-step lncRNA detection biosensor and corresponding detection method. This effectively improves the amplification efficiency and sensitivity of detection, reduces detection costs, and utilizes the self-calibration function of ratiometric fluorescence to reduce detection errors. It achieves sensitive and accurate detection of lncRNAs at the single-cell level and in clinical tissue samples, demonstrating significant practical application value.

[0008] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a biosensor for multiple rolling circle amplification initiated by a single lncRNA based on proximity hybridization, the biosensor comprising at least specific primers, a circular template, Bst DNA polymerase, a Cy3-modified donor probe, a Cy5-modified acceptor probe, and a dNTPs mixture. The specific primer is a single-stranded DNA with only 7 complementary base pairs to the circular template, and cannot stably hybridize with the circular template under normal conditions; the specific primer can hybridize with a specific sequence of the target lncRNA MALAT1, and form a stable primer-template hybridization structure with the circular template through the proximity hybridization effect mediated by the target lncRNA. The circular template is a single-stranded DNA containing specific sequences complementary to the target lncRNA MALAT1, specific primers, donor probes, and acceptor probes. It can be amplified by rolling circles starting with specific primers under the action of Bst DNA polymerase. The Cy3-modified donor probe and the Cy5-modified acceptor probe are single-stranded DNA, which can specifically hybridize with specific sequences of rolling circle amplification products. After hybridization, Cy3 and Cy5 are spatially close, which can produce efficient fluorescence resonance energy transfer (FRET).

[0009] The Bst DNA polymerase is Bst DNA 2.0 polymerase, which has strand displacement activity. During rolling circle amplification, it can remove lncRNA that has hybridized with the circular template from the template, so that the released lncRNA can mediate new primer-template hybridization and initiate a new round of rolling circle amplification.

[0010] In a second aspect, the present invention provides a method for initiating multiple rounds of isothermal amplification based on a single target. This method uses a single lncRNA as the trigger molecule. Through gradient molar ratio verification, it is found that as the molar ratio of primer, template and lncRNA increases, the fluorescence intensity of the system increases in a dose-dependent manner, demonstrating that a single lncRNA can simultaneously bind multiple primer and template complexes, initiating multiple rounds of rolling circle isothermal amplification and achieving signal amplification.

[0011] A third aspect of the present invention provides the application of the above-described biosensor in the detection of lncRNA, preferably in the detection of lncRNA MALAT1 in breast cancer samples.

[0012] A fourth aspect of the present invention provides a method for detecting lncRNA using the above-described biosensor, wherein the method is a one-step isothermal amplification detection method.

[0013] A fifth aspect of the present invention provides the application of the above-described biosensors and / or detection methods in the diagnosis of lncRNA-related tumors, the detection and analysis of lncRNA in tumor-related biological samples, and the screening of lncRNA-targeted drugs.

[0014] Beneficial technical effects of one or more of the above technical solutions (1) Achieve single-molecule-initiated multiplex amplification, significantly improving detection sensitivity. This invention utilizes the proximity hybridization effect of target lncRNA to mediate the hybridization of primers and circular templates, initiating rolling circle amplification. Furthermore, the strand displacement activity of Bst DNA polymerase removes the lncRNA from the template during amplification, allowing the released lncRNA to continue mediating new primer-template hybridization. This enables single-lncRNA molecule-initiated multiplex rolling circle amplification, significantly improving amplification efficiency compared to traditional single-target initiation single-amplification methods. The detection limit for lncRNA MALAT1 is as low as 0.283 aM, and it can achieve single-cell-level lncRNA detection, with sensitivity far superior to existing detection methods.

[0015] (2) One-step isothermal reaction, simple operation and rapid detection The detection method of this invention is a one-step isothermal amplification reaction, which does not require a complicated thermal cycling procedure or a multi-step liquid phase transfer operation. After mixing all reagents with the sample to be tested, amplification can be completed by incubating at a constant temperature of 65 °C for 20 min. This greatly simplifies the operation steps, shortens the detection time, and is suitable for point-of-care testing (POCT).

[0016] (3) Ratio fluorescence readout is used, which has high detection stability and accuracy. This invention combines the fluorescence resonance energy transfer of Cy3-Cy5 to construct a ratiometric fluorescence detection system, using the fluorescence intensity ratio of Cy5 / Cy3 as the detection signal. Compared with a single fluorescence signal, it has a built-in self-calibration function, which can effectively eliminate detection errors caused by factors such as small fluctuations in the reaction system and differences in probe concentration, significantly improve the stability and accuracy of detection results, and reduce the generation of false positive signals.

[0017] (4) Low testing cost and wide applicability The biosensor of this invention requires only one conventional Bst DNA polymerase for amplification, eliminating the need for multiple enzymes or expensive precision instruments, thus significantly reducing detection costs. Furthermore, by rationally designing the primer and circular template sequences, this method can be extended to the detection of other long nucleic acids (such as mRNA and other lncRNAs), showing broad application prospects in biomedical research and clinical diagnosis of tumors and other diseases.

[0018] (5) High specificity, enabling accurate detection of clinical samples. The specific primers of this invention cannot stably hybridize with the circular template under normal conditions. The amplification reaction can only be initiated under the mediation of the target lncRNA. Moreover, the donor probe and the recipient probe only specifically hybridize with the rolling circle amplification product, effectively avoiding interference caused by non-specific amplification and hybridization. This method can accurately distinguish the expression differences of lncRNA between cancer cells and normal cells, and can effectively identify the lncRNA expression levels between clinical breast cancer tissues and normal tissues. The area under the receiver operating characteristic (ROC) curve (AUC) is 1.0, which has excellent clinical diagnostic value.

[0019] In summary, compared with existing methods, the above-mentioned technical solution has advantages such as simple operation, rapid detection, high sensitivity, strong specificity, good stability, and low cost. The one-step isothermal amplification binding ratio fluorescence detection enables sensitive and accurate detection of lncRNA in single-cell and clinical tissue samples, and has great application potential in the fields of early clinical diagnosis of tumors, prognostic assessment, targeted drug screening, and biomedical research. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The present invention provides a schematic diagram of the mechanism of lncRNA ratio fluorescence detection for single lncRNA-induced multiple rolling circle amplification based on proximity hybridization; wherein (A) is the overall process of lncRNA detection, and (B) is the detailed process of lncRNA-induced proximity hybridization driving multiple rolling circle amplification. Figure 2 The feasibility verification diagram of rolling circle amplification products in this embodiment of the invention is shown in (A) for 4% denaturing polyacrylamide gel electrophoresis (PAGE) analysis, where lane 1 is the lncRNA-primer-template complex, lane 2 is the control group without lncRNA, and lane 3 is the amplification product group with lncRNA; (B) for SYBR Gold staining fluorescence spectroscopy analysis; (C) for Cy3-Cy5 FRET fluorescence spectroscopy analysis; (D) for ratiometric fluorescence detection normalized fluorescence spectroscopy analysis; and (E) for normalized Cy5 fluorescence intensity comparison. Figure 3 The amplification efficiency evaluation results in the embodiments of the present invention; wherein (A) is the fluorescence spectral analysis result under different lncRNA:primer-template ratios; (B) is the fluorescence intensity change result under different lncRNA:primer-template ratios; Figure 4The detection performance evaluation results in the embodiments of the present invention include: (A) normalized fluorescence spectra of lncRNA at different concentrations; (B) normalized Cy5 fluorescence intensity of lncRNA at different concentrations; (C) linear relationship between normalized Cy5 fluorescence intensity and the logarithm of lncRNA concentration; and (D) specificity verification results of the method. Figure 5 The results of intracellular lncRNA detection in this embodiment of the invention; wherein (A) is a heatmap of normalized Cy5 fluorescence intensity of different cell lines; (B) is a comparison of normalized Cy5 fluorescence intensity of different cell lines; (C) is a normalized fluorescence spectrum of different numbers of MCF-7 cells; (D) is a linear relationship between normalized Cy5 fluorescence intensity and the logarithm of the number of MCF-7 cells; Figure 6 The detection results of clinical breast cancer tissue samples in this embodiment of the invention; wherein (A) is a normalized Cy5 fluorescence intensity heatmap of cancer tissue and normal tissue; (B) is a comparison of normalized Cy5 fluorescence intensity of cancer tissue and normal tissue; (C) is the distribution of lncRNA expression levels of cancer tissue and normal tissue; (D) is the ROC curve for the method to distinguish cancer tissue from normal tissue; Figure 7 The results of optimized reaction conditions in the embodiments of the present invention; wherein (A) is the change of F / F0 value at different reaction times; (B) is the change of F / F0 value at different Bst polymerase concentrations; and (C) is the change of normalized Cy5 fluorescence intensity at different receptor probe concentrations. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0025] As mentioned earlier, existing lncRNA detection methods suffer from problems such as complex operation, time-consuming detection, low sensitivity, high cost, and poor stability of detection results. Furthermore, rolling circle amplification (RoBA) technology is difficult to directly apply to lncRNA detection due to lncRNA chain length limitations. In view of this, this invention constructs a single-lncRNA-initiated multiplex RoBA system based on the proximity hybridization effect, and establishes a ratiometric fluorescence detection method using Cy3-Cy5 fluorescence resonance energy transfer. This results in a one-step, isothermal, and ultrasensitive lncRNA detection biosensor, enabling rapid and accurate detection of lncRNA MALAT1 in breast cancer samples. The detection limit is as low as 0.283 aM, allowing for single-cell level detection and effectively differentiating lncRNA expression differences between clinical breast cancer tissue and normal tissue. This method has significant application value in biomedical research and clinical diagnosis. A schematic diagram of the specific mechanism is shown below. Figure 1 As shown.

[0026] In a typical embodiment of the present invention, the provided biosensor for single lncRNA-initiated multiple rolling circle amplification based on proximity hybridization includes at least specific primers, a circular template, Bst DNA 2.0 polymerase, a Cy3-modified donor probe, a Cy5-modified acceptor probe, and a dNTPs mixture. The specific primer is a single-stranded DNA with a nucleotide sequence of 5'-CGG AGC AGC ACG AAG ACA A-3'. It can only form 7 complementary base pairs with the circular template. It cannot stably hybridize with the circular template under constant temperature of 65 °C, but can specifically hybridize with the specific sequence of the target lncRNA MALAT1. The circular template is a single-stranded DNA with the nucleotide sequence 5'-TCC TCT GAA GAC CCG ACG TG TTTTACC AGC ACT ACT CCA TCG AC TTTT AGC TTC TCC ATC ACA TGT AG TTG TCT T GGC TGTCTG CTT GGG AAA TCT TA-3', containing specific sequences complementary to the target lncRNA MALAT1, specific primers, donor probes, and acceptor probes; The nucleotide sequence of the Cy3-modified donor probe is 5'-TCC TCT GAA GAC CCG ACG TG-Cy3-3', and the nucleotide sequence of the Cy5-modified acceptor probe is 5'-Cy5-ACC AGC ACT ACT CCA TCG AC-3'. Both can specifically hybridize with specific sequences of rolling circle amplification products. The Bst DNA 2.0 polymerase has strand displacement activity, which can remove and release lncRNA that has hybridized with the circular template during rolling circle amplification, so that the released lncRNA can mediate new primer-template hybridization and initiate a new round of rolling circle amplification.

[0027] In another specific embodiment of the present invention, the target lncRNA is lung adenocarcinoma metastasis-associated transcript 1 (lncRNA MALAT1), whose nucleotide sequence is 5'-UAA GAU UUC CCA AGC AGA CAG CCC GUG CUG CUCCG-3'. This lncRNA is an important biomarker for malignant tumors such as breast cancer.

[0028] In another specific embodiment of the present invention, a one-step isothermal amplification method for detecting lncRNA using the above-mentioned biosensor is provided, specifically including the following steps: S1. Preparation of the sample to be tested Total RNA was extracted from biological samples such as breast cancer cells and clinical breast tissue. The specific procedures are as follows: Cell samples: Breast cancer cells (MCF-7), lung cancer cells (A549), and normal breast epithelial cells (MCF-10A) cultured to the exponential growth phase were digested with trypsin, washed with PBS buffer, and centrifuged at 4°C to collect the cell pellet. Total RNA was extracted from the cells using the miRNeasy Mini Kit according to the manufacturer's instructions. The extracted RNA was dissolved in enzyme-free water and stored at -80°C for later use. Tissue samples: After dewaxing and hydrating the formaldehyde-fixed paraffin-embedded (FFPE) breast tissue samples, total RNA was extracted from the tissues using the same total RNA extraction kit described above, and stored at -80 ℃ for later use. The extracted RNA extract was used directly as the sample for subsequent testing.

[0029] S2. Preparation of a one-step isothermal amplification reaction system With a total reaction volume of 20 μL, the components and their final concentrations are as follows: Specific primers 20 pM, circular template 20 pM, Bst DNA 2.0 polymerase 0.4 U / μL, dNTPs mixture 200 μM, Cy3-modified donor probe 200 nM, Cy5-modified acceptor probe 200 nM, 1×ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 1% Triton X-100, pH 8.8), and 1–5 μL of RNA sample to be tested. The remaining volume was made up with enzyme-free water. A blank control group without test samples was also set up, and the remaining components were the same as those in the above reaction system.

[0030] S3, isothermal rolling-ring amplification reaction After thoroughly vortexing the prepared reaction system, incubate it in a 65 ℃ constant temperature metal bath for 20 min to perform rolling circle amplification. After the amplification reaction is completed, heat the reaction system at 95 ℃ for 5 min to inactivate Bst DNA 2.0 polymerase and terminate the amplification reaction.

[0031] S4. Ratio fluorescence detection and data analysis The terminated reaction system was placed in a fluorescence spectrophotometer, and the fluorescence emission intensity (F) of Cy3 at 566 nm was scanned and recorded using 520 nm as the excitation wavelength. D ) and the fluorescence emission intensity of Cy5 at 668 nm (F A ); Calculate the fluorescence intensity ratio of Cy5 / Cy3 (F). A / F D The Cy5 fluorescence intensity was normalized (normalized Cy5 intensity = F). A / F D ); Based on the linear regression equation of normalized Cy5 intensity and logarithm of lncRNA concentration, the target lncRNA MALAT1 in the sample can be quantitatively detected. At the same time, the rolling circle amplification products can be verified by gel electrophoresis. Specifically, the amplification reaction products without fluorescent probe are stained with SYBR Gold fluorescent dye and then subjected to 4% denaturing polyacrylamide gel electrophoresis (110V constant voltage electrophoresis for 40 min). The amplification product bands are observed by gel imaging analysis system.

[0032] In another specific embodiment of the present invention, the above-mentioned biosensor and detection method are provided for the detection of breast cancer lncRNA MALAT1, analysis of tumor-related biological samples lncRNA, clinical diagnosis of breast cancer, and screening of lncRNA-targeted anticancer drugs. The biological samples include isolated breast cancer cells, breast tissue, blood, body fluids, etc. The detection method of the present invention can realize the detection of lncRNA at the single-cell level and can effectively distinguish the lncRNA expression differences between breast cancer tissue and normal breast tissue. The area under the receiver operating characteristic (ROC) curve (AUC) reaches 1.0, which has excellent clinical diagnostic value.

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0034] Example 1 (I) Experimental methods and procedures 1. Experimental Materials and Reagents Oligonucleotides: Specific primers, circular templates, Cy3-modified donor probes, and Cy5-modified acceptor probes were all purified by HPLC and purchased from Changsha Aikerui Biotechnology Co., Ltd. Enzymes and buffers: Bst DNA 2.0 polymerase, 10×ThermoPol reaction buffer, and dNTPs mixture were all purchased from New England Biolabs, USA. Cell lines: Human breast cancer adenocarcinoma cell line (MCF-7), human lung cancer cell line (A549), and human normal breast epithelial cell line (MCF-10A), purchased from the Cell Bank of the Chinese Academy of Sciences; Clinical samples: Cancer tissue samples from 5 breast cancer patients and normal breast tissue samples from 5 healthy individuals, all of which were formaldehyde-fixed paraffin-embedded sections obtained from the Affiliated Hospital of Guangdong Medical University; Reagents and kits: miRNeasy Mini Kit total RNA extraction kit (Qiagen, Germany), SYBR Gold fluorescent dye (Thermo Fisher Scientific), DMEM medium, fetal bovine serum (FBS), penicillin-streptomycin mixture (Gibco, USA). Experimental instruments: Techcomp FS5C fluorescence spectrophotometer (Edinburgh Instruments, UK), WD-9413C gel imaging analysis system (Beijing Liuyi Biotechnology), constant temperature metal bath, high-speed refrigerated centrifuge, vertical electrophoresis apparatus, clean bench, etc.

[0035] 2. Experimental methods and procedures (1) Cell culture MCF-7, A549, and MCF-10A cells were seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture and cultured in a constant temperature incubator at 37 ℃ and 5% CO2. The medium was changed every 2-3 days. Subsequent experiments were carried out when the cells grew to the exponential growth phase.

[0036] (2) Total RNA extraction Cell samples: Cells in the exponential growth phase were digested with 0.25% trypsin, washed twice with pre-cooled PBS buffer, and centrifuged at 800×g for 5 min at 4 ℃ to collect the cell pellet. Cells were lysed with lysis buffer according to the miRNeasy Mini Kit instructions. Total RNA was extracted by RNA binding, washing, and elution. After dissolving in RNase-free water, RNA concentration and purity were detected using a nucleic acid quantification instrument. The samples were stored at -80 ℃ for later use. Tissue samples: After dewaxing (xylene soaking) and hydration (gradient ethanol solution) of FFPE breast tissue sections, digestion with proteinase K was performed. Subsequently, total RNA was extracted according to the above kit instructions. After detecting the concentration and purity, the samples were stored at -80 °C for later use.

[0037] (3) Amplification efficiency assessment To verify that a single long non-coding RNA (lncRNA) can trigger multiple rounds of rolling circle amplification, this embodiment constructed a reaction system with gradient ratios: lncRNA and primer-template complex were prepared into reaction systems at molar ratios of 1:0, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12, respectively. After the isothermal rolling circle amplification reaction was completed, fluorescent nucleic acid dye was added for staining, and the fluorescence emission spectrum in the range of 500-640 nm was detected by a fluorescence spectrophotometer. The fluorescence intensity at the maximum emission wavelength (approximately 538 nm) was extracted for quantitative analysis.

[0038] (4) Optimization of reaction conditions To obtain the best lncRNA detection results, three key reaction conditions—reaction time, Bst DNA 2.0 polymerase concentration, and Cy5 receptor probe concentration—were optimized. The F / F0 value (F being the SYBR Gold fluorescence intensity with lncRNA and F0 being the SYBR Gold fluorescence intensity without lncRNA) or normalized Cy5 fluorescence intensity was used as the evaluation index. Three parallel experiments were set up for each condition. Reaction time optimization: Set reaction times to 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, and detect the F / F0 value at different times; Polymerase concentration optimization: Bst polymerase concentrations were set to 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, and 0.5 U / μL, and the F / F0 values ​​at different concentrations were measured. Receptor probe concentration optimization: The receptor probe concentrations were set to 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, with the donor probe concentration being consistent with the receptor probe concentration. The normalized Cy5 fluorescence intensity at different concentrations was then detected.

[0039] (5) Validation of rolling circle amplification products An amplification reaction system without Cy3 donor probe and Cy5 receptor probe was prepared, with a lncRNA concentration of 1 pM. Amplification was performed under the optimized conditions described above, and the reaction was terminated. 1×SYBR Gold fluorescent dye was added to the amplification product, and the mixture was incubated at room temperature in the dark for 10 min. Then, 4% denaturing polyacrylamide gel was added for electrophoresis, and the electrophoresis was performed at a constant voltage of 110 V for 40 min. The electrophoretic bands were observed and recorded using a gel imaging analysis system. (6) Performance evaluation of the detection method Sensitivity detection: A series of lncRNA MALAT1 standards with gradient concentrations of 0 M, 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, and 1 pM were prepared. The optimized reaction system and method were used for detection. The fluorescence intensities of Cy3 and Cy5 at each concentration were recorded, and the normalized Cy5 fluorescence intensity was calculated. A standard curve was plotted with the logarithm of lncRNA concentration on the x-axis and the normalized Cy5 fluorescence intensity on the y-axis. A linear regression equation was established, and the limit of detection (LOD) was calculated by adding three times the standard deviation (3×SD) to the blank value. Specificity detection: Non-coding RNAs (miR-21, miR-221, let7-a, let7-c, lncRNA HOTAIR) highly expressed in cancer were selected as interferants. A blank control group, each interferant group (concentration of 1 pM) and the target lncRNA MALAT1 group (concentration of 1 pM) were set up. The detection was performed according to the above method, and the normalized Cy5 fluorescence intensity of each group was recorded to evaluate the specificity of the method.

[0040] (7) Detection of intracellular lncRNA lncRNA detection in different cell lines: Total RNA was extracted from MCF-7, A549, and MCF-10A cells, and the results were analyzed according to the optimized reaction system. Three parallel experiments were set up for each cell line. The normalized Cy5 fluorescence intensity of each group was recorded, and the expression level of lncRNA MALAT1 in different cell lines was compared. Single-cell level detection: Total RNA was extracted from 1, 10, 100, 300, and 1000 MCF-7 cells, respectively, and detected using the method described above. The normalized Cy5 fluorescence intensity was recorded at different cell numbers. A linear regression equation was established with the logarithm of the cell number as the x-axis and the normalized Cy5 fluorescence intensity as the y-axis to evaluate the detection capability of the method at the single-cell level.

[0041] (8) Detection of clinical breast tissue samples Total RNA was extracted from cancerous tissues of 5 breast cancer patients and normal breast tissues of 5 healthy individuals. The RNA was detected according to the optimized reaction system and method. Three parallel experiments were set up for each sample, and the normalized Cy5 fluorescence intensity of each group was recorded. The receiver operating characteristic (ROC) curve analysis method was used to distinguish the efficacy of breast cancer tissues from normal tissues, and the area under the curve (AUC) was calculated.

[0042] (II) Results Analysis and Discussion 1. Results of Optimization of Reaction Conditions The results of the reaction condition optimization are as follows Figure 7 As shown: Reaction time ( Figure 7 A): When the reaction time is in the range of 5 to 20 min, the F / F0 value increases significantly with the extension of time; when the reaction time exceeds 20 min, the F / F0 value shows a decreasing trend. This is because excessive incubation time will lead to an increase in non-specific amplification. Therefore, 20 min is selected as the optimal reaction time. Bst polymerase concentration ( Figure 7 B): When the polymerase concentration is in the range of 0.1~0.4 U / μL, the F / F0 value increases significantly with increasing concentration; when the concentration exceeds 0.4 U / μL, the F / F0 value decreases. This is because excessively high enzyme concentrations can increase non-specific reactions. Therefore, 0.4 U / μL is selected as the optimal enzyme concentration. Receptor probe concentration ( Figure 7 C): When the acceptor probe concentration is in the range of 50~200 nM, the normalized Cy5 fluorescence intensity increases significantly with increasing concentration; when the concentration exceeds 200 nM, the fluorescence intensity decreases because excessively high probe concentration will lead to an increase in background fluorescence. Therefore, 200 nM is selected as the optimal acceptor probe concentration, and the donor probe concentration is simultaneously set to 200 nM.

[0043] 2. Validation results of rolling circle amplification products Gel electrophoresis results showed ( Figure 2In the control group without lncRNA, only a 98 nt circular template band was observed, with no amplification product band. In the experimental group with added lncRNA MALAT1, a 152 nt lncRNA-primer-template complex band was observed, and a clear rolling circle amplification product diffuse band appeared. This indicates that the rolling circle amplification reaction can only be initiated by the binding of primers to the circular template through the proximity hybridization effect when the target lncRNA is present, thus proving the feasibility of the method of the present invention.

[0044] 3. Performance evaluation results of the detection method Performance evaluation results as follows Figure 4 As shown: Sensitivity: When the concentration of lncRNA MALAT1 was in the range of 1 aM to 1 pM, the normalized Cy5 fluorescence intensity showed a good linear relationship with the logarithm of the lncRNA concentration. The linear regression equation was: I = 0.31 lgC + 5.86 (R 2 =0.997), where I is the normalized Cy5 fluorescence intensity and C is the lncRNA concentration; calculated based on the blank value plus 3 times the standard deviation, the limit of detection (LOD) of this method is 0.283 aM, which is far superior to existing reported lncRNA detection methods, such as the globular nucleic acid nanomachine method (51 pM), the dual HCR fluorescence method (1 pM), and the CRISPR / Cas13a photoelectrochemical method (53.1 aM). Specificity: The normalized Cy5 fluorescence intensity of the blank control group and the interfering groups such as miR-21, miR-221, let7-a, let7-c, and lncRNA HOTAIR remained at a very low level, with no significant difference from the blank control group; however, the normalized Cy5 fluorescence intensity of the target lncRNA MALAT1 group was significantly increased, indicating that the biosensor and detection method of the present invention have excellent specificity for lncRNA MALAT1 and no cross-reactivity.

[0045] 4. Detection results of intracellular lncRNA The results of intracellular lncRNA detection are as follows: Figure 5 As shown.

[0046] Different cell lines: The normalized Cy5 fluorescence intensity of MCF-7 breast cancer cells and A549 lung cancer cells was significantly higher than that of MCF-10A normal breast epithelial cells, indicating that lncRNA MALAT1 was highly expressed in cancer cells, which is consistent with the results of existing studies. This proves that this method can accurately detect intracellular lncRNA and distinguish the lncRNA expression differences between cancer cells and normal cells. At the single-cell level: When the MCF-7 cell count was in the range of 1–1000, the normalized Cy5 fluorescence intensity showed a good linear relationship with the logarithm of the cell count, and the linear regression equation was: I = 0.34 lgN + 0.28 (R²). 2 =0.996), where N is the number of cells; and the normalized Cy5 fluorescence intensity of one MCF-7 cell was significantly different from that of the blank control group, proving that this method can achieve single-cell level lncRNA detection with ultra-high detection sensitivity.

[0047] 5. Detection results of clinical breast tissue samples Test results as follows Figure 6 As shown.

[0048] The normalized Cy5 fluorescence intensity of 5 breast cancer tissue samples was significantly higher than that of 5 normal breast tissue samples, and the fluorescence intensity distributions of the two groups did not overlap, indicating that lncRNA MALAT1 was significantly highly expressed in breast cancer tissue. ROC curve analysis showed that the area under the curve (AUC) for this method to distinguish between breast cancer tissue and normal tissue was 1.0, indicating that this method has a 100% accuracy rate and can be effectively applied to the clinical auxiliary diagnosis of breast cancer.

[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A biosensor for single-lncRNA-initiated multiple rolling circle amplification based on proximity hybridization, characterized in that, The biosensor includes at least specific primers, a circular template, Bst DNA polymerase, a Cy3-modified donor probe, a Cy5-modified acceptor probe, and a dNTPs mixture.

2. The biosensor according to claim 1, characterized in that, The specific primer is a single-stranded DNA that hybridizes to a specific sequence of the target lncRNA MALAT1; the sequence of the specific primer is shown in SEQ ID NO.1: 5'-CGG AGCAGC ACG AAG ACA A-3'.

3. The biosensor according to claim 1 or 2, characterized in that, The circular template is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID NO.2: 5'-TCC TCT GAA GAC CCG ACG TG TTTT ACC AGC ACTACT CCA TCG AC TTTT AGC TTC TCC ATC ACA TGT AG TTG TCT T GGC TGT CTG CTT GGGAAA TCT TA-3'.

4. The biosensor according to any one of claims 1-3, characterized in that, The nucleotide sequence of the Cy3-modified donor probe is shown in SEQ ID NO.3: 5'-TCC TCT GAA GAC CCG ACG TG-Cy3-3'; the nucleotide sequence of the Cy5-modified acceptor probe is shown in SEQ ID NO.4: 5'-Cy5-ACC AGC ACT ACT CCA TCG AC-3'.

5. A method for multi-round isothermal amplification based on a single target, characterized in that, Using lncRNA as a trigger molecule, multiple rounds of rolling circle isothermal amplification are initiated to achieve signal amplification.

6. The application of a biosensor as described in any one of claims 1-4 in the detection of lncRNA ratio fluorescence.

7. The application according to claim 6, characterized in that, The application of the biosensor in detecting lncRNA MALAT1 in breast cancer samples and in the screening of lncRNA-targeted drugs.

8. A method for detecting lncRNA using a biosensor as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of the sample to be tested: Extract total RNA from the biological sample; S2. Prepare a one-step isothermal amplification reaction system; S3, isothermal rolling ring amplification reaction; S4, Ratio fluorescence detection.

9. The method according to claim 7, characterized in that, The isothermal rolling ring amplification reaction is carried out by incubating the reaction system at a constant temperature of 65°C for 20 min.

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