A microRNA-responsive 1,2-dioxetane chemiluminescent compound, a dual-probe system, a kit, a synthesis method and applications thereof

Abstract

This invention discloses a microRNA-responsive 1,2-dioxane-based chemiluminescent compound, its dual-probe system, kit, synthesis method, and applications. The compound's core is an o-acrylate-substituted phenoxyadamantane-1,2-dioxane, with the phenolic hydroxyl group protected by an azide benzyl group. The two-component probe system constructed based on this compound comprises an azide-modified first probe and a phosphine-modified second probe, each complementary to different segments of the target microRNA. Hybridization brings the two probes closer, triggering a Staudinger reaction to remove the protecting group and activate the chemiluminescent signal. This probe system requires no enzyme amplification or nucleic acid extraction, achieves a detection limit of 1.37 fM, and a signal-to-noise ratio as high as 157-fold. It can be successfully used for real-time imaging of endogenous microRNA-21 in live cells, showing broad application prospects in early tumor screening, auxiliary diagnosis, and prognostic assessment.

Description

Technical Field

[0001] This invention belongs to the field of fine chemical technology, specifically relating to a microRNA-responsive 1,2-dioxane chemiluminescent compound, its dual-probe system, kit, synthesis method and application, which are suitable for serum and live cells, and can be used for early lung cancer screening. Background Technology

[0002] MicroRNAs (miRNAs) are a class of non-coding RNA molecules approximately 18–25 nucleotides in length, playing crucial roles in biological processes such as gene expression regulation, cell proliferation, differentiation, and apoptosis. Current clinical methods for miRNA detection primarily include quantitative real-time polymerase chain reaction (qRT-PCR), gene chips, and Northern blotting. However, these traditional methods have significant limitations: First, they typically require complex sample pretreatment steps, including lysis and RNA extraction, which not only increases operational complexity but may also lead to sample loss or contamination. Second, due to the extremely low concentration of miRNAs in blood (often at the femtomolar level) and the presence of significant homologous sequence interference, existing technologies struggle to achieve ultrasensitive and highly specific detection of specific miRNAs. Furthermore, these methods cannot perform in-situ real-time monitoring in a living cellular environment, limiting their application in dynamic biological process research.

[0003] Chemiluminescence, also known as cold light, is the light radiation produced by a chemical reaction without any excitation from light, heat, or electric fields. Advantages include: no need for an external excitation source, avoidance of background and stray light interference, reduced noise, and improved signal-to-noise ratio. It features high sensitivity, a wide linear range, simple equipment, convenient operation, easy automation, and rapid analysis, making it ideal for point-of-care testing. Despite the excellent detection performance of chemiluminescence technology, its application to miRNA detection still faces challenges. In summary, there is currently no mature chemiluminescent miRNA detection platform that requires no enzyme amplification and can work directly in clinical serum or live cells. Existing probe systems generally suffer from insufficient sensitivity, weak anti-interference capabilities, and reliance on complex sample pretreatment, making it difficult to meet the practical needs of early cancer screening for highly sensitive, highly specific, rapid, and extraction-free detection. Summary of the Invention

[0004] This invention aims to overcome the long-standing bottlenecks of existing microRNA detection technologies, such as insufficient sensitivity, poor specificity, reliance on enzyme amplification, difficulty in applying to live cells or clinical samples, and cumbersome sample processing. It provides a 1,2-dioxane-based chemiluminescent compound and, based on this compound, establishes a novel chemiluminescent dual-probe system, kit, and application that combines an azide-phosphine-based Staudinger reaction with chemiluminescent signal output for highly sensitive and specific detection of tumor marker microRNA, more preferably microRNA-21 (miR-21).

[0005] The compound is designed based on bioorthogonal reactions. Its activated form can be coupled separately to antisense short RNAs complementary to the target microRNA region, forming a two-component recognition probe. In the presence of the target microRNA, the proximity-driven effect induced by the target microRNA pulls the bioorthogonal reaction substrates at both ends of the probe closer to the effective reaction distance, triggering the removal of the phenol protecting group. This, in turn, activates the o-acrylate-substituted phenoxyadamantane-1,2-dioxane chemiluminescent core, achieving precise coupling between the luminescent signal and target microRNA recognition (mechanism as follows: ...). Figure 1 (As shown). This probe system requires no complex sample pretreatment or excitation light, and can work stably in complex biological environments (such as serum and live cells). It has a detection limit of 1.37 fM and a signal-to-noise ratio of 157-fold. It also supports the direct detection of target microRNAs in live cells and clinical serum samples, providing a new strategy with high sensitivity, high specificity, and clinical translational potential for early screening, auxiliary diagnosis, and prognostic assessment of lung cancer.

[0006] This invention provides a microRNA-responsive 1,2-dioxane chemiluminescent compound, the general structural formula of which is shown in Figure I:

[0007]

[0008] I

[0009] R1 is selected from 2-methyl-4-azidobenzyl, 3-methyl-4-azidobenzyl, and p-azidobenzyl; p-azidobenzyl is most preferably selected from R1.

[0010] R2 is selected from carboxyl or aryl carboxylic acid groups, preferably from carboxyl or 4-carboxyphenyl.

[0011] II. On the other hand, the present invention provides a two-component probe system for microRNA-responsive 1,2-dioxane-based chemiluminescent compounds, comprising a first probe component and a second probe component:

[0012] The first probe component has the following general formula II:

[0013]

[0014] II

[0015] The first probe component is formed by covalently linking a chemiluminescent compound and a first oligonucleotide sequence through a linker group; the structure of the chemiluminescent compound is shown in general formula I, which includes a chemiluminescent core that can be activated by a bioorthogonal reaction, and a protecting group R1 containing a bioorthogonal substrate vinyl or azide group connected to the phenolic oxygen group of the core by an ester bond;

[0016] The second probe component has the following general formula III;

[0017]

[0018] III

[0019] The second probe component comprises a covalent linker of an active group and a second oligonucleotide sequence; wherein the active group in the general formula III is selected from diphenylphosphine or its salt; it is linked to the second oligonucleotide sequence via a carboxyl or benzoic acid group; preferably selected from 3-(diphenylphosphine)propionic acid.

[0020] The first and second oligonucleotide sequences respectively hybridize complementaryly with two adjacent but different segments of the same target microRNA molecule;

[0021] The first and second probe components pair via a Staudinger bioorthogonal reaction. When the target microRNA is present, it simultaneously hybridizes with the first and second oligonucleotide sequences, forming a ternary hybridization complex through base complementarity pairing. This complex brings the bioorthogonal substrate in the first and second probe components closer, triggering a bioorthogonal reaction, removing the protecting group R1, and thereby activating the chemiluminescent properties of the 1,2-dioxane-based chemiluminescent compound.

[0022] In a preferred embodiment, the target microRNA is microRNA-21.

[0023] More preferably, the first oligonucleotide sequence is the sequence shown in SEQ ID NO.1: 5′-UGAUAAGCUA-3′; which targets the 5′ end region of microRNA-21; the second oligonucleotide sequence is the sequence shown in SEQ ID NO.2: 5′-UCAACAUCAGU-3′, which targets the 3′ end region of microRNA-21.

[0024] The protecting group R1 is selected from 2-methyl-4-azidobenzyl, 3-methyl-4-azidobenzyl, p-azidobenzyl, with p-azidobenzyl being the most preferred; R2 is selected from carboxyl or arylcarboxylic acid, with carboxyl or 4-carboxyphenyl being the most preferred.

[0025] For the technical solution described above, preferably, in the most preferred embodiment, the first probe component is a compound (RNA-N3CL) having the following structure:

[0026]

[0027] The second probe component is a compound (RNA-PPh2) having the following structure:

[0028]

[0029] Any of the probe components described above can serve as a novel nucleic acid labeling tool (chemiluminescent probe monomer) based on the Staudinger reaction (one of the biological orthogonal reactions) to construct a proximity-triggered detection system (dual probe system). Those skilled in the art will understand that by replacing the oligonucleotide sequence with a sequence complementary to other target single-stranded RNAs (especially other microRNAs) and following the same probe construction principles, the detection platform of this invention can be applied to a wider range of target detection scenarios.

[0030] III. Test Kit

[0031] This invention also provides a chemiluminescent detection kit for in vitro detection of target microRNA, comprising:

[0032] The above two-component probe system (i.e. RNA-N3CL and RNA-PPh2).

[0033] Buffer systems suitable for nucleic acid hybridization and Staudinger reactions; for example: buffer system 100 mM Tris·HCl, pH 7.4, containing 150 mM NaCl;

[0034] The kit allows for the direct addition of the two-component probe system to the biological sample to be tested (such as human serum, plasma, or cell culture medium). After incubation at 25–42°C for 3–15 minutes, the chemiluminescent signal is detected without the need for RNA extraction, reverse transcription, or PCR amplification. This achieves "extraction-free, one-step" detection.

[0035] IV. Synthesis Method

[0036] This invention also provides a method for synthesizing 1,2-dioxane-based chemiluminescent compounds:

[0037]

[0038] Includes the following steps:

[0039]

[0040] (1) At 0~30℃, the raw material is dissolved in an organic solvent, and an organic base, magnesium chloride and paraformaldehyde are added in sequence. The molar ratio of the raw material, weak base, paraformaldehyde and magnesium chloride is 1:2~4:2~4:2~5. The reaction mixture is added to the solvent. The reflux temperature is 12~24 h. After the reaction is completed, excess dichloromethane is added for dilution and the mixture is washed with dilute hydrochloric acid. The volume ratio of the reaction organic solvent to dichloromethane / dilute hydrochloric acid is 1:5~8. The organic phase is collected and the solvent is evaporated to obtain the intermediate product S-1.

[0041] (2) In an organic solvent, S-1 and a triphenylphosphine compound containing an R2 group were added. After reacting for 0.5 to 3 h, the mixture was extracted, concentrated, and purified to obtain an intermediate product S-2 containing an R2 substitution, wherein the molar ratio of S-1 to the triphenylphosphine compound containing an R2 group was 1:1.2 to 3.

[0042] (3) Dissolve S-2 in an organic solvent, slowly add an inorganic base solution, stir at room temperature for 2-5 h, add ethyl acetate and dilute hydrochloric acid to wash, combine the organic phases, and evaporate the solvent to obtain S-3; wherein, the molar ratio of compound S-2 to strong base solution is 1:1-10, and the volume ratio of reaction organic solvent to ethyl acetate / dilute hydrochloric acid is 1:5-8.

[0043] (4) Under the protection of an inert gas, in an organic solvent at 20~30℃, S-3 and an organic base are dissolved in the organic solvent, and a bromide containing an R1 group is added. A condensation reaction occurs under the catalysis of the organic base. After recrystallization and purification, S-4 is obtained, wherein the molar ratio of S-3 to the bromide containing an R1 group is 1:1~5.

[0044] (5) In an organic solvent, at 0°C, S-4 and methylene blue are dissolved in the organic solvent, and S-4 is oxidized under laser catalysis. After HPLC purification, 1,2-dioxane chemiluminescent compounds of general formula I are obtained, wherein the molar ratio of S-4 to methylene blue is 1:0.01~0.05.

[0045] For the technical solution described above, it is further preferred that, in step (1), the organic solvent is selected from one of dichloromethane, chloroform, acetone, tetrahydrofuran, and acetonitrile;

[0046] In step (2), the organic solvent is selected from any one or a mixture of several of benzene, toluene, o-dichlorobenzene, DMF, and dichloromethane;

[0047] In step (3), the inorganic base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium acetate, sodium ethoxide, and lithium hydroxide;

[0048] In step (4), the organic solvent is selected from any one or a mixture of several of ethanol, acetic acid, acetic anhydride, and DMF; the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, and propanol; and the organic base is selected from any one of triethylamine, pyridine, and DIPEA.

[0049] V. Testing Methods

[0050] The present invention also provides a method for detecting target microRNA in in vitro samples, comprising the following steps: mixing a reaction system containing the above-mentioned two-component probe system with the sample to be tested, incubating under conditions suitable for hybridization and Staudinger reaction, and detecting chemiluminescent signals.

[0051] For the technical solution described above, a further preferred embodiment includes the following steps:

[0052] (a) Provide a biological sample to be tested, the sample being selected from serum, plasma, cell lysate or cell culture supernatant; more preferably, a human serum sample that has not undergone RNA extraction;

[0053] (b) The sample is mixed with the two-component probe system. More preferably, the two-component probe system is added to the serum sample to form a reaction system with a total volume of 100 μL, wherein the final concentrations of the first probe component (RNA-N3CL) and the second probe component (RNA-PPh2) are both 400 nM.

[0054] (c) Incubate at 25-42°C for 3-15 minutes; more preferably, incubate at 37°C for 5 minutes;

[0055] (d) Detect the intensity of the chemiluminescence signal generated by the reaction system; more preferably, use a multi-functional microplate reader to detect the intensity of the chemiluminescence signal (unit: RLU).

[0056] (e) Quantitative analysis of the chemiluminescence signal based on a standard curve to determine the concentration of the target microRNA.

[0057] In a further preferred embodiment, the target microRNA is microRNA-21, and step (e) specifically involves: quantifying the concentration of microRNA-21 in serum based on a calibration curve plotted with microRNA-21 concentration on the x-axis and luminescence intensity on the y-axis. This method is particularly suitable for detecting microRNA-21.

[0058] This method has been successfully applied to 30 clinical serum samples (including 22 lung cancer patients). The signal in the lung cancer group was significantly higher than that in the healthy control group (p<0.001), and it was highly consistent with the results of qRT-PCR.

[0059] VI. Pharmaceutical Uses

[0060] The present invention further provides the application of the two-component probe system or detection kit in the preparation of in vitro diagnostic reagents for detecting target microRNAs.

[0061] For the applications described above, a further preferred application is the use of the two-component probe system or detection kit in the preparation of in vitro diagnostic reagents for early screening, auxiliary diagnosis, or prognostic assessment of non-small cell lung cancer; wherein the use is for in vitro detection of the expression level of target microRNA in human serum or plasma, and does not involve direct treatment methods for disease diagnosis.

[0062] The in vitro diagnostic reagent is used to achieve the application by detecting the expression level of microRNA-21 in human serum or plasma.

[0063] More preferably, the method of using the in vitro diagnostic reagent includes: directly adding the two-component probe system to a human serum sample that has not undergone RNA extraction, reacting at 37°C for 5 minutes, detecting the chemiluminescence signal intensity, and setting X = [according to...] Figure 13 The statistically determined threshold (signal value ≥ 1500 RLU) is used as the standard for determining lung cancer positivity.

[0064] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0065] This invention provides a two-component probe system (NPP probe) based on Staudinger reaction and chemiluminescence signal amplification, which achieves highly sensitive and specific detection of target miRNAs through the proximity-driven effect induced by microRNA-21.

[0066] Ultra-high sensitivity: Utilizing a target cyclic amplification mechanism, the detection limit can reach the femtomolar (fM) level (e.g., 1.37 × 10⁻⁶). -15 M); far superior to conventional chemiluminescent or fluorescent probes;

[0067] It exhibits high specificity: it can accurately distinguish miR-21 from its homologous miRNAs (such as miR-21-5p, miR-143, etc.), and even when faced with interfering RNAs with single-base mismatches, the signal-to-noise ratio remains above 10:1 (see...). Figure 9 Excellent anti-interference ability: In environments containing 5 mM reducing agents (cysteine, TCEP) or oxidizing agents (H2O2), the probe signal shows no significant attenuation (see...). Figure 7This indicates that its Staudinger response mechanism has strong tolerance to the complex redox environment inside cells;

[0068] Fast response dynamics: Signal output completed within 15 minutes (see...) Figure 2 This is significantly faster than traditional methods that rely on hybridization chain reaction (HCR) or enzyme-catalyzed amplification;

[0069] Excellent biocompatibility and applicability: Suitable for quantitative analysis across a wide concentration range from picomoles to hundreds of nanomoles, and supports real-time live-cell imaging (see...). Figure 11 Furthermore, it exhibits extremely low cytotoxicity (cell viability >90%, see [reference]). Figure 10 );

[0070] Simple to operate: No complex sample pretreatment is required, enabling "one-stop" detection and imaging. This method has been successfully applied to unpurified clinical serum samples (see...). Figure 13 In 30 real-world samples (including 22 lung cancer patients and 8 healthy controls), direct detection of microRNA-21 was achieved, and the signal in the lung cancer patient group was significantly higher than that in the healthy group (p<0.001), which was highly consistent with the pathological diagnosis results (see...). Figure 12 ).

[0071] Therefore, this invention not only solves the bottlenecks of existing technologies in terms of sensitivity, enzyme-dependent amplification, specificity, in vivo applicability, and cumbersome sample processing, but also exhibits comprehensive advantages such as speed, stability, enzyme-free, interference resistance, and direct use in clinical samples, and has strong prospects for clinical translation. Attached Figure Description

[0072] Figure 1 This is a diagram illustrating the detection mechanism of the NPP probe.

[0073] Figure 2 To compare the chemiluminescence kinetics detection of the probe TCT;

[0074] Figure 3 The image shows the chemiluminescence kinetics of the NPP probe.

[0075] Figure 4 The absorption spectrum of RNA-N3CL (gray) and the absorption spectrum of RNA-N3CL after the Staudinger reaction (orange) are shown.

[0076] Figure 5 The images show the chemiluminescence spectrum (blue) of RNA-N3CL and the absorption spectrum (green and red) of RNA-N3CL after undergoing the Staudinger reaction.

[0077] Figure 6The graph shows the performance analysis of the probe NPP in detecting microRNA; where 6A is the chemiluminescence response intensity of the probe NPP to different concentrations of microRNA substrate; and 6B is the linear fitting curve between the chemiluminescence intensity and the logarithm of the target microRNA concentration.

[0078] Figure 7 This is a test diagram of the anti-interference test of the NPP probe;

[0079] Figure 8 This is a selectivity test plot for the NPP probe;

[0080] Figure 9 This is a specificity test diagram of the NPP probe;

[0081] Figure 10 This is a graph showing the cell toxicity assay of the NPP probe.

[0082] Figure 11 Chemiluminescence imaging of the NPP probe on A549 and 3T3 cells;

[0083] Figure 12 This image shows a qRT-PCR assay for microRNA-21 in serum samples from lung cancer patients and healthy individuals.

[0084] Figure 13 This is a clinical detection diagram of the NPP probe on serum samples from lung cancer patients and healthy individuals. Detailed Implementation

[0085] The structural formula of the probe molecule is shown in general formula I below:

[0086]

[0087] I

[0088] Wherein, R1 is preferably p-azidobenzyl, and R2 is preferably carboxyl, with the following structural formula:

[0089]

[0090] The chemiluminescence detection system of this invention consists of two oligonucleotide probes:

[0091] ,

[0092] (1) RNA-N3CL (i.e., compound r): a chemiluminescent core containing an azide group (-N3) coupled to the 5′ end (phenoxyadamantane-1,2-dioxane derivative).

[0093] (2) RNA-PPh2: A recognition sequence containing a diphenylphosphine group (-PPh2) is coupled to the 5′ or 3′ end.

[0094] The nucleic acid portions of the two probes were designed to be complementary to adjacent regions of the target microRNA-21 (miR-21, sequence: 5′-UAGCUUAUCAGACUGAUGUUGA-3′):

[0095] The RNA-N3CL sequence (5′-UGAUAAGCUA-3′) is complementary to positions 1-10 of miR-21;

[0096] The RNA-PPh2 sequence (5′-UCAACAUCAGU-3′) is complementary to positions 11-22 of miR-21 (ensuring that the two ends are close together after hybridization).

[0097] When miR-21 is absent, the two probes are free in the solution. The azide and phosphine groups are far apart and cannot react effectively, resulting in a very low background signal.

[0098] When miR-21 is present, both probes bind simultaneously to the same miR-21 molecule via base pairing, forming a ternary complex (miR-21:RNA-N3CL:RNA-PPh2). This forces the 5′ azide group and phosphine group to be brought closer to an effective distance, thereby efficiently triggering a biological reaction. This process removes the phenol protecting group, generating highly reactive phenoxy radicals in situ, which in turn initiates the cleavage of the adamantane-1,2-dioxane-butane structure, releasing a large number of chemiluminescent photons (λ). max ≈450 nm).

[0099] This "miRNA-induced proximity-driven effect" achieves a coupling of highly specific recognition and signal amplification: only perfectly matched target miR-21 can stably bridge the two probes; non-target miRNAs, due to mismatch, cause the complex to be unstable and cannot effectively trigger luminescence. Therefore, RNA-N3CL and RNA-PPh2 must be used in pairs; neither component alone can produce a significant chemiluminescent signal. Together, they constitute the core detection unit of this invention.

[0100] The synthesis steps are as follows:

[0101]

[0102] The synthesis steps of Example 1 are as follows:

[0103]

[0104] Example 1

[0105] Synthesis of Compound 1.1

[0106]

[0107] Under nitrogen protection, compound f (3.38 g, 11.08 mmol) was dissolved in 15 mL of anhydrous acetonitrile, followed by the addition of ultra-dry anhydrous magnesium chloride (2.62 g, 30.3 mmol) and anhydrous triethylamine (2.41 g, 3.33 mL, 23.82 mmol), and then paraformaldehyde (1.22 g, 40.3 mmol). The reaction mixture was heated to reflux and reacted overnight. The reaction system changed from white to yellow. Thin-layer chromatography monitored the reaction; under UV light at 365 nm, the product spots showed pale blue fluorescence. After the reaction was complete, the mixture was diluted with 50 mL of dichloromethane and washed with 50 mL of dilute hydrochloric acid (0.5 mol / L). The organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered, and excess solvent was removed by evaporation under reduced pressure. The crude product was purified by silica gel column chromatography (V... 石油醚 V 乙酸乙酯 = 50:1), to give compound 1.1 as a pale yellow solid (3.4 g, 10.23 mmol), yield 92.33%.

[0108] Synthesis of Compound 1.2

[0109]

[0110] Compound 1.1 (1 g, 3 mmol) was dissolved in 15 mL of anhydrous dichloromethane, and triphenylphosphine-methyl acetate (1.17 g, 3.3 mmol) was added in portions. After stirring at room temperature for about 1 h, the reaction was monitored by thin-layer chromatography, and the product spots showed bright yellow fluorescence. After the reaction was complete, the excess solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography (V... 石油醚 V 乙酸乙酯 = 40:1), to give compound 1.2 as a white solid (0.66 g, 1.7 mmol), yield 56.6%.

[0111] Synthesis of Compound 1.3

[0112]

[0113] Compound 1.2 (0.66 g, 1.7 mmol) was dissolved in 10 mL of tetrahydrofuran, and 10 mL of 1 M lithium hydroxide solution (0.24 g, 10 mmol) was slowly added. After stirring at room temperature for about 3 h, the reaction was monitored by thin-layer chromatography, and the reaction was basically completed. The solution was diluted with 50 mL of ethyl acetate and washed with 50 mL of dilute hydrochloric acid (0.5 mol / L). The organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and the excess solvent was removed by evaporation under reduced pressure to give compound 1.3 as a pale yellow solid (0.6 g, 1.58 mmol), with a yield of 93.4%.

[0114] Synthesis of compound 1.4

[0115]

[0116] Under nitrogen protection, compound 1.3 (50 mg, 0.13 mmol) was dissolved in 10 mL of anhydrous N,N-dimethylformamide, and anhydrous potassium carbonate (45.8 mg, 0.33 mmol) was added. The mixture was stirred at room temperature for 20 min. Then, p-azidobenzyl alcohol (34.9 mg, 0.17 mmol) was added. After approximately 2 h of reaction, the reaction was monitored by thin-layer chromatography. Upon completion, 50 mL of ethyl acetate was added to the reaction mixture, followed by washing with 50 mL of water and 50 mL of saturated sodium chloride solution. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The crude product was subjected to silica gel column chromatography (V... 石油醚 V 乙酸乙酯 After purification with a ratio of 50:1, compound 1.4 was obtained as a white solid (51 mg, 0.10 mmol), with a yield of 75.9%.

[0117] Synthesis of Compound 1

[0118]

[0119] In a clear quartz test tube, compound 1.4 (51 mg, 0.1 mmol) was dissolved in 10 mL of dichloromethane. Separately, the photosensitizer methylene blue (3.2 mg, 0.01 mmol) was dissolved in 1 mL of methanol and added to the reaction system. The reaction was cooled and maintained at 0°C, and then subjected to a 660 nm laser (0.2 W / cm²). 2 The reaction system was irradiated and oxygen was continuously bubbled in. Real-time monitoring was performed using high-performance liquid chromatography (HPLC). After the reaction was complete, the reaction system was subjected to low-temperature, light-protected conditions, and excess solvent was removed under reduced pressure. Purification was performed using preparative-grade high-performance liquid chromatography (RP-HPLC) with a mobile phase of water (A) and acetonitrile (B), using a gradient of 0–15 min, 40%–100% B, 15–30 min, and 100% B. The product retention time was 20–22 min, and the final probe molecule 1 was a colorless solid (20 mg, 0.037 mmol), with a yield of 37.2%.

[0120] Synthesis of activated compound 1

[0121]

[0122] At room temperature, N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (0.013 g, 0.037 mmol) was added to 3 mL of a DMF solution of compound q (20 mg, 0.037 mmol), followed by the addition of triethylamine (0.1 mL). The reaction was allowed to proceed for 2 h, and then the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The precipitate was purified by HPLC to obtain activated compound 1 (0.021 g, 0.033 mmol, Y = 89.7%).

[0123] Synthesis of compound RNA-N3CL

[0124] In this invention, compound 1, which is activated by compound N-hydroxysuccinimide (NHS), is an N-hydroxybenzyl azidoluminescent core derivative. Its active ester group can undergo a highly efficient amide condensation reaction with the primary amino group to form a stable amide bond.

[0125] The oligonucleotide sequence 5′-NH2-UGAUAAGCUA-3′ is a partially complementary sequence to microRNA-21 (corresponding to positions 1-10 of miR-21). A primary amino group (-NH2) is introduced at its 5′ end via a C6 or C3 spacer arm for covalent coupling to activated compound 1. This sequence is designed to specifically hybridize with the target miR-21 (5′-UAGCUUAUCAGACUGAUGUUGA-3′) to form a local double-stranded structure, thereby bringing the chemiluminescent core (via compound 1) closer to the phosphine-modified probe (RNA-PPh2) to an effective reaction distance, triggering subsequent chemiluminescence.

[0126] The structure of the obtained compound RNA-N3CL is shown below:

[0127]

[0128] The terminal amino-modified oligonucleotide sequence 5'-NH2-UGAUAAGCUA-3' (1 eq) was dissolved in 0.1 mM sodium bicarbonate buffer (pH = 8.45). Compound 1 (3 mM, 30 eq) dissolved in DMSO was added. The reaction was carried out at room temperature for 3 hours, and the reaction process was monitored by high performance liquid chromatography (HPLC). The major product was formed, with no obvious side reactions. The modified oligonucleotide was purified by HPLC, and the sample was immediately lyophilized to remove the solvent. The purity of all compounds was verified by LC-MS before activation experiments. Separation was performed using a Phenomenex Clarity Oligo-MS column (2.1 mm × 150 mm, 2.6 μm). The mobile phase A was TEA:HFIP:H2O (0.4:30:1000 v / v), and the mobile phase B was MeOH. The LC gradient was as follows: elution with 5% MeOH for 1 minute, followed by gradient elution with 5% MeOH for 15 minutes to 50% MeOH. The final product was compound r, which is an oligonucleotide probe with a 5′-terminal chemiluminescent core coupled to the core, named RNA-N3CL (where “N3” represents the azide functional group and “CL” represents the chemiluminescent unit).

[0129] Synthesis of compound RNA-PPh2

[0130]

[0131] The terminal amino-modified oligonucleotide sequence 5'-UCAACAUCAGU-NH2-3' (1 eq) was dissolved in 0.1 mM sodium bicarbonate buffer (pH = 8.45). Compound 2,5-dioxopyrrolidone-1-yl-3-(diphenylphosphine)propionic acid (PPh2, 3 mM, 30 eq) dissolved in DMSO was added. The reaction was carried out at room temperature for 3 hours, and the reaction process was monitored by high-performance liquid chromatography (HPLC). The major product was formed, with no significant side reactions. The modified oligonucleotide was purified by HPLC, and the sample was immediately lyophilized to remove the solvent, finally yielding compound RNA-PPh2.

[0132] The two components, RNA-N3CL and RNA-PPh2, are called NPP.

[0133] Comparative compound 2

[0134]

[0135] Synthetic route

[0136]

[0137] Synthesis of Compound 2.1

[0138]

[0139] Compound i (100 mg, 0.26 mmol) and pyridine (100 μL, catalytic amount) were dissolved in anhydrous DCM (20 mL). Under a nitrogen atmosphere at 0 °C, a DCM solution of benzyl p-nitrochloroformate (70.7 mg, 0.4 mmol) was added dropwise to the reaction solution while stirring. The reaction was continued at 0 °C for 2 h, then heated to room temperature and stirred overnight. After the reaction was complete, the solvent was removed under reduced pressure to obtain a yellow viscous liquid, which could be used as the crude product of compound 2.1 (approximately 200 mg). The crude product could be rapidly added to subsequent reactions without further purification.

[0140] Synthesis of Compound 2.2

[0141]

[0142] In a clear quartz test tube, compound 2.1 (150 mg, 0.28 mmol) was dissolved in 10 mL of dichloromethane. Separately, the photosensitizer methylene blue (9.6 mg, 0.03 mmol) was dissolved in 1 mL of methanol and added to the reaction system. The reaction was cooled and maintained at 0°C, and then subjected to a 660 nm laser (0.2 W / cm²). 2 The reaction system was irradiated and oxygen was continuously bubbled in. Real-time monitoring was performed using high-performance liquid chromatography (HPLC). After the reaction was complete, the reaction system was subjected to low-temperature, light-protected conditions, and excess solvent was removed under reduced pressure. Purification was performed using preparative-grade high-performance liquid chromatography (RP-HPLC), and compound 2.2 was finally obtained as a colorless solid (80 mg, 0.14 mmol), with a yield of 50%.

[0143] Synthesis of Compound 2

[0144]

[0145] The crude product of compound 2.2 (80 mg, 0.14 mmol) and N,N-diisopropylethylamine (DIPEA, 50 μL) were dissolved in a 20 mL mixture of anhydrous DCM and DMF in equal proportions, and stirred for 30 min at 0 °C under a nitrogen atmosphere. Then, a DCM solution containing (4E)-trans-cyclooctenol (25 mg, 0.2 mmol) was added dropwise to the above reaction solution, and the mixture was heated to 40 °C and reacted overnight. After the reaction was complete, the reaction solution was treated with water and extracted with DCM. The organic phase was washed three times with saturated brine, and the solvent was removed under reduced pressure. The residue was purified by HPLC to give a white solid as compound 2 (26.0 mg, 0.047 mmol), with a yield of 33.3%.

[0146] Synthesis of activated compound 2

[0147]

[0148] At room temperature, N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (0.017 g, 0.047 mmol) was added to 3 mL of a DMF solution of compound 2 (26.0 mg, 0.047 mmol), followed by the addition of triethylamine (0.1 mL). The reaction was allowed to proceed for 2 h, and then the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The precipitate was purified by HPLC to obtain activated compound 2 (0.026 g, 0.039 mmol, Y = 82.9%).

[0149] Synthesis of compound RNA-TCO

[0150] In this invention, the activated compound 2 is an N-hydroxysuccinimide (NHS) activated trans-cyclooctene carbonate-based chemiluminescent core derivative, whose active ester group can undergo a highly efficient amide condensation reaction with the primary amino group to form a stable amide bond.

[0151] The oligonucleotide sequence 5′-NH2-UGAUAAGCUA-3′ is a partially complementary sequence to microRNA-21 (corresponding to positions 1-10 of miR-21). A primary amino group (-NH2) is introduced at its 5′ end via a C6 or C3 spacer arm for covalent coupling to activated compound 2. This sequence is designed to specifically hybridize with the target miR-21 (5′-UAGCUUAUCAGACUGAUGUUGA-3′) to form a local double-stranded structure, thereby bringing the chemiluminescent core (via compound 2) closer to the tetrazine-modified probe (RNA-Tz) to an effective reaction distance, triggering subsequent chemiluminescence.

[0152] The structure of the obtained compound RNA-TCO is shown below:

[0153]

[0154] The terminal amino-modified oligonucleotide sequence 5'-NH2-UGAUAAGCUA-3' (1 eq) was dissolved in 0.1 mM sodium bicarbonate buffer (pH = 8.45). Compound 2,5-dioxopyrrolidone-1-yl-2-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)acetate (abbreviated as Tz, 3 mM, 30 eq) dissolved in DMSO was added. The reaction was carried out at room temperature for 3 hours, and the reaction process was monitored by high-performance liquid chromatography (HPLC). The major product was formed, with no significant side reactions. The modified oligonucleotide was purified by HPLC, and the sample was immediately lyophilized to remove the solvent, finally yielding the compound RNA-TCO.

[0155]

[0156] The terminal amino-modified oligonucleotide sequence 5'-UCAACAUCAGU-NH2-3' (1 eq) was dissolved in 0.1 mM sodium bicarbonate buffer (pH = 8.45). Compound 2,5-dioxopyrrolidone-1-yl-2-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)acetate (abbreviated as Tz, 3 mM, 30 eq) dissolved in DMSO was added. The reaction was carried out at room temperature for 3 hours, and the reaction process was monitored by high-performance liquid chromatography (HPLC). The major product was formed, with no significant side reactions. The modified oligonucleotide was purified by HPLC, and the sample was immediately lyophilized to remove the solvent, finally yielding compound RNA-Tz.

[0157] The two components, RNA-TCO and RNA-Tz, are collectively referred to as TCT.

[0158] Reaction kinetics testing of NPP probe and contrast probe TCT

[0159] To evaluate the difference in kinetic performance between the probe of this invention and the traditional chemiluminescent probe (TCT) in miRNA detection, a comparative experiment was conducted. For example... Figures 2-3 As shown, in this experiment, the two components of the NPP probe, RNA-N3CL and RNA-PPh2, or the two components of the control probe, RNA-TCO and RNA-Tz, were both kept at a concentration of 300 nM. These components were mixed with the substrate microRNA-21 in a buffer solution (100 mM Tris·HCl, pH = 7.4, 150 mM NaCl) (100 μL) as the blank experimental group, with a substrate concentration of 300 nM. Both the experimental and standard control groups were reacted at 37°C, and the chemical reaction kinetics were measured using a microplate reader. It should be noted that the blank group did not contain microRNA-21 and was used to assess the probe's own background signal; the experimental group contained 300 nM of synthetic microRNA-21 mimicry to assess the probe's response ability in the presence of the target.

[0160] The test results are as follows Figures 2-3 As shown, the horizontal axis represents time (minutes), and the vertical axis represents relative luminescence intensity (RLU). The red line represents the response curve of the TCT probe after adding different concentrations of microRNA-21, while the gray line represents the control group without microRNA-21. The results show that the NPP probe exhibits significantly better detection performance than the control probe TCT at 37°C and in buffer solution (100 mM Tris). In a system of HCl (pH = 7.4, 150 mM NaCl), when the concentration of microRNA-21 was 300 nM, the maximum chemiluminescent response value of the NPP probe reached 25230, with a signal-to-noise ratio (SBR) as high as 157; while the maximum chemiluminescent response value of the contrast probe TCT was only 900, with an SBR of only about 1. These results demonstrate that the azide-phosphine Staudinger triggering system constructed in this invention can achieve extremely low background and high-intensity signal output under the same conditions, significantly outperforming the traditional tetrazine-trans-cyclooctene (Tz-TCO) system.

[0161] It is noteworthy that this significant difference reveals the core innovation of this invention: by designing a two-component probe system and utilizing the miRNA-induced proximity effect to trigger the Staudinger reaction, high specificity and rapid response to the target miRNA are achieved. In contrast, traditional TCT probes suffer from significantly lower sensitivity and response speed due to higher background signals. This result not only demonstrates the technical superiority of this invention but also provides a novel solution to the inefficiency problem in existing miRNA detection methods.

[0162] Based on key indicators such as comprehensive reaction kinetics curves, maximum luminescence intensity, and signal-to-noise ratio, the NPP probe exhibits significant advantages in detection sensitivity, signal response intensity, and specificity, better meeting the needs of ultrasensitive detection of microRNA-21 and applications involving complex biological samples. Therefore, all subsequent in vitro quantitative assays, live-cell imaging, and clinical serum sample validation experiments were performed using the NPP probe.

[0163] Basic spectral testing of NPP probes

[0164] To verify the basic spectral characteristics of the NPP probe before and after detection, UV-Vis and chemiluminescence spectroscopy were used to systematically detect the spectral changes of the NPP probe before and after the reaction. The test system contained 20 μM RNA-N3CL and different concentrations of RNA-PPh2, and was carried out at 37°C in 100 mM Tris·HCl (pH 7.4) and 150 mM NaCl buffer.

[0165] Figure 4The graph shows the UV absorption spectrum (gray) of the NPP probe and the absorption spectrum (orange) of RNA-N3CL after the Staudinger reaction; the x-axis represents wavelength (nm) and the y-axis represents absorbance (au). The gray line represents a test system containing 20 μM RNA-N3CL in PBS buffer. The results show that the maximum absorption wavelength of RNA-N3CL is at 260 nm. The orange line represents a test system containing 20 μM RNA-N3CL with 200 μM (10 equivalents of RNA-N3CL) of RNA-PPh2 added to PBS buffer, incubated at 37°C for 60 minutes. The results show that RNA-N3CL undergoes a Staudinger reaction with RNA-PPh2, and the product exhibits a red shift of approximately 30 nm compared to the RNA-N3CL wavelength, confirming the effective occurrence of the reaction.

[0166] Figure 5 The graph shows the chemiluminescence spectrum (blue) of the NPP probe and the absorption spectra (green and red) of RNA-N3CL after the Staudinger reaction; the x-axis represents wavelength (nm) and the y-axis represents fluorescence intensity (au). The test system contained 20 μM RNA-N3CL and different concentrations (0 μM, 20 μM, 200 μM) of RNA-PPh2. Chemiluminescence spectra were measured after incubation at 37°C in PBS buffer for 60 minutes. The results show that the blue line represents the solution containing only RNA-N3CL, with a very weak background chemiluminescence intensity of approximately 1. The green line represents the chemiluminescence spectrum after adding an equivalent amount of RNA-PPh2, showing an approximately 12-fold increase in fluorescence, indicating that chemiluminescence is restored after the reaction, but the intensity is low. The red line represents the chemiluminescence spectrum after adding 10 equivalents of RNA-PPh2, with an intensity of 60 au, demonstrating that the reaction is slow when RNA is not ligated and there is no proximity-driven effect; strong chemiluminescence intensity only occurs when another reactant is in significant excess.

[0167] Response test of probe NPP to different concentrations of microRNA substrate

[0168] The two components of the NPP probe, RNA-N3CL and RNA-PPh2, were kept at a concentration of 100 nM and mixed with the substrate microRNA-21 in a buffer solution (100 mM Tris·HCl pH = 7.4, 150 mM NaCl) in a test system (total volume 100 μL). The substrate concentrations were 100 fM, 10 pM, 100 pM, 10 nM, 50 nM, 100 nM, and 150 nM. Both the experimental and control groups were reacted at 37°C.

[0169] After the reaction was completed, the chemiluminescence intensity of the system was measured, and the results are as follows: Figure 6As shown. Figure 6 A shows that the chemiluminescence intensity significantly increases with increasing microRNA-21 concentration. Based on Figure 6 The chemiluminescence intensity data corresponding to each concentration point measured in A were linearly fitted with chemiluminescence intensity as the ordinate and the logarithm of microRNA-21 concentration (Log C) as the abscissa, resulting in the following: Figure 6 The linear fitting curve shown in B. (As shown in the image) Figure 6 As shown in Figure B, within the concentration range of 100 fM to 150 nM, there is a good linear relationship between the chemiluminescence intensity and the logarithm of the concentration, with a linear correlation coefficient R² = 0.994.

[0170] Figure 6 The trend of response intensity change shown in A is Figure 6 B. Data sources and foundations for linear fitting. Figure 6 B is the answer. Figure 6 A. Quantitative characterization of data patterns; the combination of these two methods verifies that the probe system possesses high sensitivity and quantitative detection capability within the stated concentration range.

[0171] NPP probe anti-interference capability test

[0172] To evaluate the anti-interference ability of the NPP probe in complex biological environments, the two components of the NPP probe, RNA-N3CL and RNA-PPh2, were both mixed at a concentration of 400 nM in a buffer solution (100 mM Tris·HCl, pH=7.4, 150 mM NaCl) in a test system (total volume 100 μL). The control probe TCT was not included here, as its performance is clearly inferior to the NPP probe; therefore, subsequent quantitative analysis focused only on the probe of this invention. The control group was treated with the substrate microRNA-21. The three experimental groups were treated with common cellular oxidizing and reducing small molecules (5 mM each), including reducing cysteine ​​and tris(2-carboxyethyl)-phosphine (TCEP) and oxidizing H2O2. Both the experimental and control groups were reacted at 37°C, and the chemiluminescence kinetics curves were measured using a multi-mode microplate reader. Each experiment was repeated three times, and significance analysis was performed.

[0173] The results are as follows Figure 7 As shown, in the standard control group, the NPP probe exhibited a significant chemiluminescence signal after binding to miR-21. However, in the experimental group, the addition of the three small molecules mentioned above did not significantly increase the chemiluminescence signal intensity, indicating that the NPP probe system possesses good anti-interference performance. Specifically:

[0174] In a reducing environment containing 5 mM Cys or TCEP, the NPP probe did not show significant chemiluminescence enhancement, indicating that it did not respond to reducing substances.

[0175] Similarly, under the oxidizing conditions of 5 mM H2O2, the probe signal did not increase significantly.

[0176] This result demonstrates that the chemiluminescence process triggered by the Staudinger reaction of the NPP probe exhibits high resistance to interference, maintaining excellent detection performance even in complex physiological environments. Notably, this resistance to interference makes the NPP probe suitable not only for in vitro experimental conditions but also provides a solid foundation for the detection of miRNAs in real biological samples. Furthermore, compared to the conventional probe TCT described above, the NPP probe exhibits higher sensitivity and stronger environmental adaptability, further validating the technical advantages of this invention.

[0177] Selectivity testing of probe NPP

[0178] Both components of the NPP probe, RNA-N3CL and RNA-PPh2, were kept at a concentration of 100 nM and mixed in a buffer solution (100 μL) as a blank control group. Ten additional experiments were set up. One group contained only 100 nM RNA-N3CL, and eight groups were prepared by mixing the probe with small molecules commonly found in different cell types that may interfere with the reaction, including L-cysteine ​​(5 mM), bovine serum albumin (5 mM), homocysteine ​​(5 mM), glutathione (5 mM), glucose (5 mM), hydantoin (5 mM), phenylboronic acid (5 mM), and ovalbumin (5 mM). MicroRNA-21 (50 nM) was added to the tenth group as a standard control group.

[0179] The results are as follows Figure 8 The results showed that no significant chemiluminescence recovery was observed in the experimental group without the target template sequence. Only in the microRNA-21 experimental group was the chemiluminescence intensity significantly enhanced, indicating that the probe NPP has good selectivity for the target sequence and can be used in physiological environments without being subject to competitive interference from abundant biomolecules.

[0180] Specificity test of probe NPP

[0181] The two components of the NPP probe, RNA-N3CL and RNA-PPh2, were both tested at a concentration of 100 nM and mixed in a buffer solution (100 μL) as a blank control group. Three other experimental groups were set up. In two groups, the NPP probe was mixed with different RNA templates, mir-A (100 nM) and mir-B (100 nM), respectively. MicroRNA-21 (50 nM) was added to the third group as a standard control group. The blank control group, experimental group, and standard control group were all reacted at 37 °C, and the chemiluminescence intensity at the maximum emission wavelength was measured using a microplate reader.

[0182] The results are as follows Figure 9 As shown, a significant enhancement of the chemiluminescent signal only occurs when the target sequence microRNA-21, which is perfectly complementary, is present. Other groups of competing sequences that are not perfectly complementary hinder probe recognition and binding on the template strand, reducing the likelihood of the Staudinger reaction and thus the occurrence of cyclic amplification. These results indicate that the probe NPP possesses good specificity and can specifically recognize and detect target sequences within the miRNA family under complex physiological conditions.

[0183] Cellular dark toxicity assay of probe NPP

[0184] Cell viability assessment tests can be performed when cells are in the exponential growth phase. Using 1×10⁻⁶ cells... 6 Cells were added to 96-well plates at a density of 100 μL / mL Opti-MEM medium and incubated for 24 h. NPP probe solutions containing 0, 2, 4, 8, 16, 32, 64, 132, and 200 μg of NPP probe were prepared in Opti-MEM medium, with 1 mL of each concentration, and coated with Lipofectamine 3000 transfection reagent. The control group contained only the same concentration of Lipofectamine 3000 transfection reagent as the experimental group, without NPP probe. Afterward, the old medium was removed from the plates, and the Opti-MEM medium containing different concentrations of NPP probe were added to the 96-well plates. A control group containing only Opti-MEM medium was set up. After 24 h of incubation, the medium in each well was removed, and 100 μL of freshly prepared medium containing MTT (5 mg / mL) was added to each well for another 4 h of incubation. After adding MTT, blue-purple crystals gradually deposited and adhered to the bottom of the 96-well plate. After removing the culture medium, 100 μL of DMSO was added to each well to dissolve the blue-purple crystals, and the plate was placed in the dark. Next, cell viability was calculated using the absorbance at 490 nm using a multi-sensor microplate reader.

[0185] The results are as follows Figure 10 The results showed that even at the maximum concentration of the NPP probe, the cell viability of both cell types remained above 85%. Furthermore, at the working concentration of NPP (2 μg), cell viability reached over 95%. In addition, in the control group, only Lipofectamine 3000 transfection reagent was added, and cell viability did not significantly decrease after incubation with live cells. These results indicate that the probe and transfection transfection method possess good biocompatibility and low cytotoxicity, and can be directly applied to the imaging detection of target miRNAs in live cell samples.

[0186] Chemiluminescence imaging assays of NPP probes on different cell lines: A certain number of A549 and 3T3 cells were seeded into black 96-well plates and cultured overnight to allow for full cell adhesion. Black plates were chosen to minimize inter-well light crosstalk and improve the signal-to-noise ratio. Since 3T3 cells had relatively low microRNA-21 expression levels, they served as a negative control group and underwent the same assay procedure. For chemiluminescence imaging, the cell culture medium was removed, each well was washed with 1×PBS buffer, and then different concentrations of NPP probes were added for chemiluminescence imaging of the cell analysis solution. The IVIS imaging system was used to detect the cells, and the responsiveness and specificity of the probe to endogenous microRNA-21 levels were verified by comparing the differences in chemiluminescence intensity between different cell lines. No excitation light source was applied during imaging; the imaging relied entirely on the probe's own chemiluminescence signal to avoid autofluorescence interference. The luminescence mode was set to chemiluminescence mode without filters. Finally, Indigo software was used to read and process the luminescence data.

[0187] The results are as follows Figure 11 As shown, A549 cells exhibited a significantly stronger luminescent signal than 3T3 cells, and the signal intensity increased with increasing probe concentration, confirming that the NPP probe can respond to endogenous microRNA-21 levels in situ and without labeling within living cells.

[0188] Assay for microRNA content in serum samples using probe NPP

[0189] In this study, 30 serum samples were collected from Liaoning Cancer Hospital. Informed consent was obtained from the hospital for each serum sample. In this experiment, the refrigerated centrifuge temperature was set to 4°C and the rotation speed to 16000 ×g. After centrifugation for 10 min, the supernatant was collected for subsequent testing. To simulate a real-world testing scenario, RNA extraction or purification was not performed on the serum; a one-step "extraction-free" detection procedure was directly adopted. 30 μL of each sample was taken, and two fractions of the miR-21-responsive NPP probe, RNA-N3CL and RNA-PPh2, were added to each sample, with a probe concentration of 1 μM for each group. The chemiluminescence intensity at the maximum emission wavelength was measured using a microplate reader. Each experiment was repeated three times, and significance analysis was performed. Simultaneously, total RNA was extracted from each sample and subjected to qRT-PCR. This method, currently the gold standard for microRNA detection, was used as a control.

[0190] The results are as follows Figures 12-13 As shown, the luminescence signal in serum samples from lung cancer patients was significantly higher than that in the healthy control group (p<0.001). Figure 13 ), and was positively correlated with the relative expression level of miR-21 measured by qRT-PCR ( Figure 12 This demonstrates that the probe can achieve direct, rapid, and highly specific detection of microRNA-21 in untreated complex clinical samples, and has good potential for clinical translation.

[0191] To enable those skilled in the art to more clearly understand the technical solution and beneficial effects of the present invention, the present invention has been further described above in conjunction with specific preparation methods and application experiments. It should be noted that the above embodiments are only used to exemplify the present invention and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, simple improvements, or conventional adaptive adjustments made based on the technical concept of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microRNA-responsive 1,2-dioxane chemiluminescent compound, characterized in that: The general structural formula is shown in I: I R1 is selected from one of 2-methyl-4-azidobenzyl, 3-methyl-4-azidobenzyl, and p-azidobenzyl; R2 is a carboxyl group.

2. The method for synthesizing 1,2-dioxane-based chemiluminescent compounds as described in claim 1, characterized in that, Includes the following steps: (1) At 0-30℃, the raw material is dissolved in an organic solvent, and an organic base, magnesium chloride and paraformaldehyde are added in sequence. The molar ratio of the raw material, organic base, paraformaldehyde and magnesium chloride is 1:3-4:2-3:4-5. The reaction mixture is added to the solvent. The reflux temperature is 12-24 h. After the reaction is completed, excess dichloromethane is added for dilution and the mixture is washed with dilute hydrochloric acid. The volume ratio of the reaction organic solvent to dichloromethane / dilute hydrochloric acid is 1:5-8. The organic phase is collected and the solvent is evaporated to obtain the intermediate product S-1. (2) In an organic solvent, S-1 and a triphenylphosphine compound containing an R2 group were added. After reacting for 0.5-3 h, the mixture was extracted, concentrated, and purified to obtain an intermediate product S-2 containing an R2 substitution, wherein the molar ratio of S-1 to the triphenylphosphine compound containing an R2 group was 1:1.2-3. (3) Dissolve S-2 in an organic solvent, slowly add an inorganic base solution, stir at room temperature for 2-5 h, add ethyl acetate and dilute hydrochloric acid to wash, combine the organic phases, and evaporate the solvent to obtain S-3; wherein, the molar ratio of compound S-2 to inorganic base solution is 1:1-10, and the volume ratio of reaction organic solvent to ethyl acetate / dilute hydrochloric acid is 1:5-8. (4) Under the protection of an inert gas, in an organic solvent at 20-30°C, S-3 and an organic base are dissolved in the organic solvent, and a bromide containing an R1 group is added. A condensation reaction occurs under the catalysis of the organic base. After recrystallization and purification, S-4 is obtained, wherein the molar ratio of S-3 to the bromide containing an R1 group is 1:2-5. (5) In an organic solvent, S-4 and methylene blue are dissolved in the organic solvent at 0°C. S-4 is oxidized under laser catalysis and purified by HPLC to obtain a 1,2-dioxane chemiluminescent compound of general formula I, wherein the molar ratio of S-4 to methylene blue is 1:0.2-0.

5.

3. The method according to claim 2, characterized in that: In step (1), the organic solvent is selected from one of dichloromethane, chloroform, acetone, tetrahydrofuran, and acetonitrile; In step (2), the organic solvent is selected from any one or a mixture of several of benzene, toluene, o-dichlorobenzene, DMF, and dichloromethane; In step (3), the inorganic base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium acetate, sodium ethoxide, and lithium hydroxide; In step (4), the organic solvent is selected from any one or a mixture of several of ethanol, acetic acid, acetic anhydride, and DMF; the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, and propanol; and the organic base is selected from any one of triethylamine, pyridine, and DIPEA.

4. A two-component probe system based on the microRNA-responsive 1,2-dioxane chemiluminescent compound of claim 1, characterized in that: It includes a first probe component and a second probe component: The first probe component has the following general formula II: II The second probe component has the following general formula III: III Wherein, the active group in general formula III is selected from diphenylphosphine or its salt; The first and second oligonucleotide sequences respectively hybridize complementaryly with two adjacent but different segments of the same target microRNA molecule; The first probe component and the second probe component are paired via Staudinger bioorthogonal reaction.

5. The two-component probe system according to claim 4, characterized in that: The target microRNA is microRNA-21.

6. The two-component probe system according to claim 4, characterized in that: The first oligonucleotide sequence is as shown in SEQ ID NO: 1; the second oligonucleotide sequence is as shown in SEQ ID NO:

2.

7. A chemiluminescent detection kit for detecting target microRNA in in vitro samples, characterized in that: Includes the two-component probe system of claim 4 and a buffer system suitable for nucleic acid hybridization and Staudinger reaction.

8. The use of the two-component probe system as described in claim 4 in the preparation of in vitro diagnostic reagents for detecting target microRNA-21.

9. The application according to claim 8, characterized in that: The application of the two-component probe system in the preparation of in vitro diagnostic reagents for early screening, auxiliary diagnosis, or prognostic assessment of non-small cell lung cancer.

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