DNA sensor based on endogenous glutathione activation for logic cascade signal amplification in miRNA imaging applications

Abstract

The application provides a miRNA imaging application method based on an endogenous glutathione activated DNA sensor for logic cascade signal amplification. The technology constructs a "local catalytic hairpin assembly DNA nanomachine", and triggers a cascade reaction through endogenous glutathione (GSH) and target miRNA-21: GSH cleaves the disulfide bond on the GH1 hairpin, so that the conformation is changed into a metastable state H1, and then hybridizes with miRNA-21 to start branch migration, releases miRNA-21 to circulate and catalyze H2 hairpin assembly, and realizes exponential amplification of signals. FAM and BHQ1 on the H2 hairpin are spatially separated due to double-stranded formation, and the fluorescence resonance energy transfer is released, so that a strong fluorescence signal positively related to the concentration of miRNA-21 is generated. The method has high sensitivity (the detection limit is 3.92 pM), high specificity and excellent stability, is suitable for high-fidelity imaging detection of miRNA in living cells, and provides an efficient tool for cancer diagnosis and treatment integration.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry detection technology, specifically relating to a DNA sensor based on endogenous glutathione activation for miRNA imaging applications using logic cascade signal amplification. Background Technology

[0002] MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 18-24 nucleotides in length, playing a crucial role in gene expression regulation and disease progression in eukaryotes. Aberrant miRNA expression has been identified as a biomarker and potential therapeutic target for various cancers. Therefore, developing sensitive and accurate miRNA detection methods is urgently needed. To achieve sensitive detection, various signal amplification strategies have been employed. Among them, polymerase chain reaction (PCR) and rolling loop amplification (RCA) are widely used for enzyme-assisted signal amplification, but the high sensitivity of enzymes to temperature and pH significantly limits their widespread application in complex systems. Therefore, to further improve detection sensitivity, enzyme-free signal amplification systems have been developed, such as hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA). Of these methods, CHA is a typical isothermal free energy-driven reaction, offering advantages such as high amplification efficiency, good biocompatibility, simple operation, and low cost. However, traditional enzyme-free signal amplification systems have certain limitations, including off-target signal leakage and instability in living cells—the amplification products are passively triggered before reaching the target region, generating background interference. Therefore, there is an urgent need to develop a novel and controllable sensing platform to achieve high-fidelity imaging of miRNAs in living cells.

[0003] To address the aforementioned technical bottlenecks, this study constructed a "locally catalytic hairpin assembly DNA nanomachine (S-CHA)" for rapid, efficient, and reliable fluorescence resonance energy transfer (FRET) imaging of miRNA-21 in living cells. When endogenous glutathione (GSH) coexists with the target miRNA-21, a cascade reaction mechanism is triggered: GSH specifically cleaves the disulfide bonds on the GH1 hairpin, exposing the shielded toe region, causing the GH1 conformation to transform into the metastable H1 hairpin; the H1 hairpin hybridizes with miRNA-21, initiating branching migration and opening the stem region to generate the H1-miRNA21 intermediate; this intermediate undergoes a catalytic hairpin assembly (CHA) reaction with the H2 hairpin through the exposed binding site, forming the H1-H2 double-stranded complex and releasing miRNA-21 to participate in cyclic amplification, achieving exponential signal amplification; the formation of the H1-H2 double-stranded complex forces the FAM labeled on the H2 hairpin to spatially separate from BHQ1, disrupting the FRET effect, resulting in a significant enhancement of the FAM fluorescence signal. The signal intensity is positively correlated with the concentration of miRNA-21, thus achieving high sensitivity and high specificity of target detection. Summary of the Invention

[0004] The purpose of this invention is to design a DNA sensor based on endogenous glutathione activation for miRNA imaging with logic cascade signal amplification.

[0005] The objective of this invention is achieved through the following technical solution.

[0006] A DNA sensor based on endogenous glutathione activation is used for miRNA imaging with logic cascade signal amplification, characterized by the following steps:

[0007] (1) Preparation of DNA Sensor: All oligonucleotide chains were prepared in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to a final concentration of 100 μM. Reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.5) was also used. Before assembling the CHA-based sensor scaffold (S-CHA), hairpin structures GH1 and H2 were annealed at 95°C for 5 minutes and then cooled at 4°C for 2 hours to form hairpin structures (GH1 and H2 probes). The assembly procedure for the CHA-based sensor scaffold was as follows: 2 μL of GH1 probe, 2 μL of H2 probe, and 2 μL of S-chain were mixed in a centrifuge tube and incubated at 37°C for 2 hours. After cooling to room temperature, the CHA-based sensor scaffold was constructed and stored at 4°C for subsequent studies.

[0008] (2) Non-denaturing polyacrylamide gel electrophoresis (PAGE) experiment: A 12% non-denaturing polyacrylamide gel was prepared by mixing 4 mL of 30% acrylamide, 4 mL of ultrapure water, and 2 mL of 5×TBE buffer. After thorough mixing, 70 μL of ammonium persulfate (APS) and 7 μL of N,N,N',N'-tetramethylethylenediamine (TEMED) were added, mixed well, and allowed to polymerize at 37°C. Subsequently, the mixture was transferred to a glass plate and reacted for 60 minutes to form a 12% polyacrylamide gel. DNA loading buffer was mixed with the DNA sample at a volume ratio of 1:5. Polyacrylamide gel electrophoresis (PAGE) was performed in 1×TBE buffer at 110 V for 60 minutes. After electrophoresis, the polyacrylamide gel was stained with a mild nucleic acid dye, and the gel image was recorded. The DNA strand concentration was 2 μM.

[0009] (3) In vitro fluorescence measurement experiment: The prepared DNA sensor (10 μL, 2 μM) was mixed with different concentrations of miRNA-21. GSH (20 μL, 100 μM) was added, and the reaction volume was adjusted to 200 μL with Tris-HCl buffer (50 mM Tris-HCl, 100 mM NaCl and 10 mM MgCl2, pH 8.5). The above mixture was incubated at 37°C for 4 hours before fluorescence intensity testing. The excitation wavelength of the fluorescent group (FAM) was set to 488 nm, and its fluorescence emission was collected on an FL-7000 spectrometer, with the acquisition range controlled between 508-650 nm.

[0010] (4) Selectivity and Stability Tests: Selectivity analysis was performed using single-base, double-base, and triple-base mismatched sequences of four additional miRNAs (miRNA-155, miRNA-141, let-7a, and miRNA-429) and miRNA-21. The DNA sensor (100 nM) was incubated with different miRNAs (10 nM) at 37°C for 4 hours in the presence of glutathione (GSH, 10 nM), followed by fluorescence detection. To assess stability to nucleases and serum, the DNA sensor (100 nM) was incubated with deoxyribonuclease I (2 U / mL) and human serum (10%, v / v) at 37°C for 0, 2, 4, 6, 8, and 10 hours, followed by fluorescence detection. The DNA sensor was stored at 4°C, and fluorescence measurements were performed for 7 consecutive days to assess its temporal stability. Furthermore, the integrity of the sensor structure was investigated by PAGE experiments after incubation with 10% (v / v) fetal bovine serum (FBS) for different times. Attached Figure Description

[0011] [ Figure 1 A schematic diagram of endogenous glutathione activation in the S-CHA system for the detection of specific miRNAs.

[0012] [ Figure 2 Self-assembly of S-CHA nanomachines.

[0013] [ Figure 3 (a) Non-denaturing polyacrylamide gel electrophoresis analysis of glutathione (GSH) activated free CHA (catalytic hairpin assembly) circuit. (b) Non-denaturing polyacrylamide gel electrophoresis analysis of glutathione activated CHA sensing scaffold (S-CHA) after treatment at 37°C for 4 hours.

[0014] [ Figure 4(a) Schematic diagram of the feasibility study of endogenous glutathione (GSH) regulation in the C-CHA circuit. (b) Schematic diagram of the feasibility study of endogenous glutathione (GSH) regulation in the S-CHA circuit. (c) Fluorescence response of S-CHA to miRNA-21 (10 nM) after incubation at 37°C for 4 hours, with and without glutathione (GSH). (d) Fluorescence spectra of C-CHA and S-CHA systems after incubation for 4 hours under different treatment combinations: (a') C-CHA; (b') C-CHA + miRNA-21; (c') C-CHA + GSH; (d') C-CHA + GSH + miRNA-21; (a) S-CHA; (b) S-CHA + miRNA-21; (c) S-CHA + GSH; and (d) S-CHA + GSH + miRNA-21. Error bars represent the standard deviation of three parallel experiments.

[0015] [ Figure 5 (a) Fluorescence spectra of the GSH-activated S-CHA system at different GSH concentrations. (b) Fluorescence intensity changes in the S-CHA system at different GSH concentrations. Error bars represent the standard deviation of three parallel experiments.

[0016] [ Figure 6 Time-dependent fluorescence response of S-CHA nanomachines under different treatment conditions. Error bars represent the standard deviation of three parallel experiments.

[0017] [ Figure 7 (a) Fluorescence emission spectrum response of the S-CHA system to different concentrations of miRNA-21. (b) Fluorescence intensity changes induced by the S-CHA system at different concentrations of miRNA-21. Inset: Linear calibration curve showing the correlation between fluorescence intensity changes and miRNA-21 concentration. Concentration range: 0.01–1 nM. Error bars represent the standard deviation of three parallel experiments.

[0018] [ Figure 8 (a) The glutathione-activated S-CHA system was used to analyze the fluorescence spectra of miRNA-21 (10 nM) and various miRNA-21 mutants. (b) The glutathione-activated S-CHA system was used to analyze the fluorescence spectra of miRNA-21 and several interfering miRNAs. Error bars represent the standard deviation of three parallel experiments.

[0019] [ Figure 9(a) Fluorescence intensity of the GSH-activated S-CHA system after incubation with 10 mM GSH or various amino acids in the presence of 10 nM miRNA-21. (b) Fluorescence spectrum of the GSH-regulated S-CHA system treated with GSH or various amino acids (10 mM) in the presence of 10 nM miRNA-21. Error bars represent the standard deviation of three sets of parallel experiments.

[0020] [ Figure 10 (a) Stability analysis of the DNA sensor in 10% human serum. (b) Stability analysis of the DNA sensor in DNase I. (c) Stability of the DNA sensor in 10% fetal bovine serum (FBS). Error bars represent the standard deviation of three parallel experiments.

[0021] [ Figure 11 The time stability of the DNA sensor. The error bars represent the standard deviation of three parallel experiments.

[0022] [ Figure 12 The sequences of all DNA oligonucleotides in this study. Detailed Implementation Plan

[0023] Here, specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention, but do not limit the scope of application or extension of the present invention.

[0024] Example 1: The principle of the S-CHA system activated by endogenous glutathione.

[0025] like Figure 1 As shown, this study developed a locally catalytic hairpin assembly DNA nanomachine (S-CHA) for the specific detection of miRNA-21. This mechanism integrates a GSH activation module for spatially selective circuit activation and a catalytic hairpin assembly (CHA) module for signal amplification.

[0026] The endogenous GSH activation module consists of two key components: a disulfide-protected catalytic probe GH1 hairpin and a capture probe H2, both of which self-assemble onto the stator chain S, forming the S-CHA nanomachine. In the absence of target miRNA-21, the GH1 hairpin essentially prevents target-induced conformational transitions, thereby inhibiting spontaneous CHA responses and maintaining a low background signal. The H2 hairpin is doubly modified, carrying 6-carboxyfluorescein (FAM) as a donor fluorophore and BHQ1 as an acceptor quencher, forming a fluorescence resonance energy transfer (FRET) pair. In the quiescent state, FAM and BHQ1 are closely adjacent, enabling efficient FRET and significantly quenching FAM fluorescence, thus keeping the biosensor in a "shutdown" state.

[0027] When the endogenous activator GSH and the target miRNA-21 coexist, GSH first specifically cleaves the disulfide bonds on the GH1 hairpin, exposing the previously shielded toe region. Next, the activated GH1 hairpin undergoes a conformational change, forming a metastable H1 hairpin. This hairpin hybridizes with the target miRNA-21, initiating branching migration and opening the stem region, generating the H1-miRNA-21 intermediate. Subsequently, the newly exposed binding site on the H1-miRNA-21 intermediate undergoes a CHA reaction with the H2 hairpin, causing the H2 hairpin to open and form a stable H1-H2 double-stranded complex. This further releases miRNA-21, which participates in the next round of the CHA cycle, thereby promoting exponential signal amplification. The formation of the H1-H2 double-stranded complex spatially separates the FAM and BHQ1 moieties on the H2 hairpin. This separation disrupts the FRET effect, leading to a significant increase in FAM fluorescence output, proportional to the concentration of the target miRNA-21. Therefore, this study achieved highly sensitive and specific detection of miRNA-21.

[0028] Example 2: Gel validation analysis of the S-CHA system activated by endogenous glutathione.

[0029] First, the assembly of the S-CHA nanomachines was verified by polyacrylamide gel electrophoresis (PAGE). Figure 2 As shown, in lane 4, the addition of the H2 probe significantly reduced the migration rate of the DNA band, confirming the successful binding of the capture probe H2 to the stator strand S1. In lane 5, the addition of the GH1 probe also reduced the band migration rate, indicating that the GH1 hairpin structure effectively binds to S1. Lane 6 shows that free GH1 and H2 probes do not react. Lane 7 shows that when all components (S1, GH1, and H2) coexist, one-pot self-assembly forms a composite band with lower migration rate. This indicates that both the GH1 and H2 hairpin structures can self-assemble with the stator strand S to form stable nanostructures, confirming the successful assembly of the S-CHA nanomachine. This lays the foundation for subsequent glutathione (GSH) activation of the S-CHA system signal amplification.

[0030] Subsequently, PAGE verified the detailed process by which GSH activates free GH1 and H2 hairpin structures. For example... Figure 3As shown in lane a, lane 3 indicates that without GSH, the target miRNA-21 cannot open the GH1 hairpin structure to initiate branching migration. Compared to lane 3, lane 4 produces a band with reduced migration, indicating that GSH successfully activates a conformational change in the GH1 hairpin structure, forming a metastable H1 hairpin structure. This H1 then interacts with the target miRNA-21 to generate the H1-miRNA21 intermediate. Lane 8 shows a band with even lower migration, indicating that in the free system, a CHA reaction occurs in the co-present presence of GSH and the target miRNA-21, forming a stable H1-H2 double-stranded complex.

[0031] Similarly, as Figure 3 As shown in b, lane 4 reveals that when GSH and target miRNA-21 were added to the S-CHA nanomachine system, a band with lower migration was generated compared to lanes 1-3. This indicates that GSH and target miRNA-21 can trigger the CHA reaction and alter the intramolecular conformation of the S-CHA nanomachines.

[0032] Example 3: Feasibility analysis of the S-CHA system activated by endogenous glutathione.

[0033] This study further explored the fluorescence feasibility of the glutathione (GSH)-activated S-CHA system. Figure 4 As shown in b, GSH and miRNA-21 are cleverly configured as dual-input conditions, meaning that significant fluorescence signal recovery can only be observed when both input conditions are present simultaneously. For other input combinations, the fluorescence signal either fails to recover or remains at background levels. This fully validates the specific dual-gate activation mechanism of the S-CHA nanomachines for GSH and miRNA-21, demonstrating excellent spatial selectivity and specificity. It provides a powerful platform for the precise detection of microRNAs in complex biological environments.

[0034] Subsequently, the GSH-activated S-CHA system was verified through fluorescence experiments. Figure 4 c) No significant fluorescence signal was captured when miRNA-21 was added alone. GSH stimulation alone produced a slight change in signal, while a significant FAM fluorescence signal was produced when GSH and miRNA-21 were present simultaneously. These fluorescence results were highly correlated with previous polyacrylamide gel electrophoresis (PAGE) analyses, indicating that GSH and miRNA-21, as dual inputs, can activate the S-CHA system for efficient signal transduction.

[0035] We also compared the fluorescence intensity of the C-CHA and S-CHA systems, which produced similar background signals ( Figure 4 a, Figure 4d). However, after the addition of GSH and miRNA-21, the fluorescence signal of the S-CHA system was significantly higher than that of the C-CHA system. This difference is because the C-CHA system contains a control hairpin structure (NH1), which has the same sequence structure as GH1 but lacks disulfide bond modification. Under the same reaction conditions, no GSH-triggered CHA reaction was observed in the C-CHA system. This demonstrates the indispensable role of disulfide bonds in the GSH activation process.

[0036] Example 4: Time optimization of the S-CHA system activated by endogenous glutathione.

[0037] To improve sensing performance and achieve efficient signal output, the GSH concentration was optimized. Figure 5 Subsequently, 10 mM GSH was selected. Fluorescence kinetic analysis was performed under optimal conditions. Figure 6 As shown, the fluorescence resonance energy transfer (FRET) signals generated by GSH and miRNA-21 increased continuously with incubation time, reaching a plateau at 4 hours. These results demonstrate the sequential activation process of the S-CHA system: GSH activates the probe, enabling miRNA-21 to trigger the CHA reaction and generate an amplified fluorescence signal.

[0038] Example 5: Sensitivity analysis of the S-CHA system activated by endogenous glutathione.

[0039] After confirming the feasibility of using the S-CHA system for detecting miRNA-21, under optimal incubation conditions, the fluorescence signal gradually increased with increasing miRNA-21 concentration. Figure 7 a). Calibration curve ( Figure 7 b) showed that there was a strong linear correlation between fluorescence intensity and miRNA-21 concentration in the range of 10 pM to 1 nM (R² = 0.9999). Figure 7 (b. Illustration). The limit of detection (LOD) calculated using the 3σ / k method was 3.92 pM. This indicates that the glutathione (GSH) activated S-CHA system has high sensitivity for detecting low-abundance miRNA-21.

[0040] Example 6: Performance analysis of the S-CHA system activated by endogenous glutathione.

[0041] The performance of the S-CHA system hinges on the specific activation of glutathione (GSH) and accurate recognition of miRNA-21. Given the complexity of the intracellular environment, potential interference from other biomolecules must be considered. Therefore, a series of single-base (1-Mut), double-base (2-Mut), and triple-base (3-Mut) mutants of miRNA-21 (10 nM) were used. Figure 8a) and other miRNA analogs (miRNA-141, miRNA-155, miRNA-429, and let-7a) Figure 8 b) This was added to S-CHA to investigate its selectivity and specificity. Only the intact miRNA-21 induced significant signal amplification, indicating that glutathione-activated S-CHA has high specificity and selectivity. Subsequently, by introducing various amino acids to replace glutathione (GSH) input, the selective activation of thiol-modified catalytic hairpin assembly (S-CHA) by GSH was further verified. The carboxyfluorescein (FAM) fluorescence signal of GSH-activated S-CHA was observed to be significantly higher than that of other amino acid groups (…). Figure 9 Therefore, GSH can specifically cleave disulfide-modified GH1 probes, thereby triggering subsequent catalytic hairpin assembly (CHA) reactions. Based on these results, the GSH-activated S-CHA system exhibits excellent selectivity.

[0042] Example 7: Stability and biocompatibility of the endogenous glutathione-activated S-CHA system.

[0043] Given the complexity of the intracellular environment, we investigated the stability of the S-CHA system in 10% human serum. Figure 10 a) The background signal remained stable over 10 hours, indicating reliable detection performance and strong anti-interference ability. Given that DNA endonuclease I can recognize and cleave specific DNA sites, we further tested the system's resistance to DNase I digestion. Figure 10 As shown in b, after 10 hours of DNase I treatment (2 U / mL), the background signal level of S-CHA fluctuated relatively little, indicating its reliable resistance to enzymatic degradation. Furthermore, fetal bovine serum (FBS) is essential for cell culture. Therefore, as... Figure 10 As shown in Figure c, we verified the stability of the S-CHA system in 10% (v / v) FBS medium using polyacrylamide gel electrophoresis (PAGE). After incubating the S-CHA system with 10% FBS for 10 hours, no diffuse bands were observed, indicating that the nanosensor can maintain its integrity for an extended period in the 10% FBS environment. Furthermore, the fluorescence intensity of S-CHA remained almost unchanged over 7 days. Figure 11 It exhibits excellent time stability.

Claims

1. A DNA sensor based on endogenous glutathione activation for miRNA imaging with logic cascade signal amplification, characterized in that... The sensor includes: A DNA nanomachine system for high-fidelity imaging detection of microRNA (miRNA) in living cells is characterized in that: the system is a locally catalytic hairpin assembly DNA nanomachine (S-CHA), which is formed by the self-assembly of a stator chain S, a hairpin probe GH1 modified with disulfide bonds, and a fluorescently labeled hairpin probe H2. In this process, GH1 and H2 bind to the S chain through complementary pairing, forming a stable ternary complex—the S-CHA nanomachine. Only when endogenous glutathione (GSH) and the target miRNA-21 are present simultaneously, GSH breaks the disulfide bonds on GH1, exposing the toe region and causing GH1 to conformate into a metastable hairpin H1. H1 hybridizes with miRNA-21 and induces branching migration, generating the H1-miRNA21 intermediate. This intermediate further undergoes a catalytic hairpin assembly (CHA) reaction with H2, opening the H2 hairpin to form the H1-H2 double-stranded complex and releasing miRNA-21 into cyclic amplification. The unfolding of the H2 structure leads to the spatial separation of FAM and BHQ1, disrupting the FRET effect and generating a significantly enhanced FAM fluorescence signal, achieving exponential signal amplification and specific recognition of miRNA-21.

2. The DNA nanomachine system as described in claim 1, characterized in that: The hairpin probe GH1 is modified with disulfide bonds, which are complementary to the S portion of the fixed strand. In the presence of glutathione, it undergoes a conformational change, transforming from a disulfide-protected catalytic probe into a metastable H1 hairpin structure, exposing the binding site to the target miRNA-21.

3. The DNA nanomachine system as described in claim 1, characterized in that: The hairpin probe H2 is labeled with a fluorescent donor group and a fluorescent quencher group at its two ends, forming a fluorescent resonance energy transfer pair. In the inactive state, the fluorescent donor group and the fluorescent quencher group are spatially adjacent, and the fluorescence is in a quenched state. When a double-stranded complex is formed, the fluorescent donor group and the fluorescent quencher group are spatially separated, the fluorescent resonance energy transfer effect is destroyed, and the fluorescence signal is restored.

4. The DNA nanomachine system as described in claim 1, characterized in that: The activation of the S-CHA system has a dual-input logic gating characteristic, meaning that a significant fluorescence response can only be triggered when GSH and miRNA-21 coexist; a single input cannot initiate an effective signal output.

5. The DNA nanomachine system as described in claim 1, characterized in that: The system is applied to in situ imaging detection of miRNA-21 in human cancer cells, utilizing the high concentration of GSH in tumor cells as an endogenous activator to achieve spatially selective activation and background noise suppression.

6. The DNA nanomachine system as described in claim 1, characterized in that: The system is resistant to nuclease degradation and maintains structural integrity and low background signal level after 10 hours of incubation in an environment containing 10% human serum or 2 U / mL DNase I.

7. The DNA nanomachine system as described in claim 1, characterized in that: The system showed no significant change in fluorescence background after being stored at 4°C for 7 days, demonstrating good time stability.

8. A method for preparing a DNA nanomachine system as described in any one of claims 1 to 7, characterized in that... Includes the following steps: GH1 hairpin, H2 hairpin, and S chain were each dissolved in TE buffer to prepare 100 μM stock solutions. GH1 and H2 probes were subjected to thermal annealing: heated at 95°C for 5 minutes, then slowly cooled to 4°C for 2 hours to ensure complete formation of the hairpin secondary structure. GH1 probe, H2 probe, and S chain were mixed in the reaction system and incubated at 37°C for 2 hours to allow the three to self-assemble into S-CHA nanomachines. After the reaction was completed, the samples were stored at 4°C for later use.

9. A method for detecting miRNA-21, using the S-CHA system as described in any one of claims 1 to 7, characterized in that... include: The sample to be tested was mixed with the S-CHA nanomachine and incubated at 37°C for 4 hours in a Tris-HCl buffer system (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.5). The fluorescence intensity of FAM was detected by fluorescence spectroscopy (excitation wavelength 488 nm, emission wavelength 508–650 nm). The content of miRNA-21 was quantitatively analyzed based on the linear relationship between fluorescence intensity and miRNA-21 concentration (R² = 0.9999 in the range of 0.01–1 nM). The limit of detection (LOD) reached 3.92 pM (calculated based on the 3σ / k method).

10. Use of the S-CHA system as described in any one of claims 1 to 7 in the preparation of molecular diagnostic reagents for early cancer diagnosis, prognostic monitoring, or efficacy assessment, particularly for the detection of lung cancer, breast cancer, or colon cancer-related diseases based on the aberrant expression characteristics of miRNA-21.

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