A probe for detecting microRNA-21 based on biological orthogonal cyclic amplification reaction and a synthesis method and application thereof

Abstract

This invention discloses a probe for detecting microRNA-21 based on a bioorthogonal cyclic amplification reaction, its synthesis method, and its applications, belonging to the field of biomedical detection and molecular diagnostics technology. The probe IDCR comprises compounds RNA-Cy3Tz and RNA-ABN; this probe can achieve signal amplification through catalytic target cycling, with each miRNA molecule achieving a turnover rate of 2.2 × 10⁻⁶. 9 The detection limit is as low as 3.58×10 ‑18 This probe exhibits high specificity and a wide linear range of 100 aM to 100 nM. It can perform in-situ, real-time imaging in living cells and tissues without lysing cells, and can also be used for clinical serum sample detection, effectively distinguishing miRNA levels between lung cancer patients and healthy individuals. It holds significant promise for applications in non-invasive diagnostics and biomedical research.

Description

Technical Field

[0001] This invention relates to the fields of biomedical detection and molecular diagnostics, and in particular to a probe for detecting microRNA-21 based on a bioorthogonal cyclic amplification reaction, its synthesis method, and its application. 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. Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide, with most patients diagnosed at an advanced stage, missing the optimal treatment window and resulting in extremely low five-year survival rates. Studies have shown that various miRNAs (such as microRNA-21, miR-21) are abnormally highly expressed in lung cancer and other tumors, becoming potential disease biomarkers. Among them, miR-21 has been widely confirmed as an oncogene, exhibiting significant high expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) tissues and peripheral blood of patients. Its expression level is closely related to tumor proliferation, invasion, metastasis, and drug resistance, and it is considered a highly valuable molecular target for early diagnosis and prognostic assessment of lung cancer.

[0003] Currently, commonly used clinical methods for miRNA detection include quantitative real-time polymerase chain reaction (qRT-PCR), gene chips, and Northern blotting. However, these methods typically require lysis of tissue or blood samples and RNA extraction, which is cumbersome and cannot achieve in-situ real-time dynamic imaging within living cells. Furthermore, due to the extremely low levels of miRNA in blood (usually at femtomolar levels) and the presence of significant homologous sequence interference, conventional techniques have limited sensitivity and are prone to false positives, failing to meet the ultra-sensitivity and specificity requirements for early lung cancer diagnosis. Although low-dose computed tomography (LDCT) is currently the primary method for lung cancer screening, it suffers from a high false positive rate and the risk of overdiagnosis, and it is difficult to provide functional information about lesions at the molecular level.

[0004] Bioorthogonal reactions, particularly the inverse electron demand Diels-Alder reaction (IEDDA), have been widely used in in vivo labeling and bioimaging due to their high reaction rate, excellent biocompatibility, and high specificity. However, current technologies have not yet achieved an integrated system for ultrasensitive and highly specific in situ detection and imaging of miRNAs in complex biological environments, and in particular, there is a lack of practical probes that can be used simultaneously for body fluid detection and in vivo imaging, and support the early molecular diagnosis of lung cancer. Summary of the Invention

[0005] This invention provides a probe for detecting microRNA-21 based on a bioorthogonal cyclic amplification reaction, its synthesis method, and its application, in order to overcome the above-mentioned problems and improve the detection sensitivity and accuracy of microRNA-21.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a probe for detecting microRNA-21 based on a bioorthogonal cyclic amplification reaction, wherein the probe IDCR comprises the compounds RNA-Cy3Tz and RNA-ABN;

[0008] The structural formula of the compound RNA-Cy3Tz is shown in Formula I:

[0009]

[0010] Formula I;

[0011] The structural formula of the compound RNA-ABN is shown in Formula II:

[0012]

[0013] Formula II.

[0014] In another aspect, this invention provides a method for synthesizing the probe for detecting microRNA-21 based on a bioorthogonal cyclic amplification reaction, comprising the steps of synthesizing the compound RNA-Cy3Tz and the compound RNA-ABN, respectively; wherein, the method for synthesizing the compound RNA-Cy3Tz is as follows:

[0015]

[0016] S1: Preparation of compound 4

[0017] 45.7 mmol of compound 1, p-iodoaniline, was dissolved in 50 mL of 20% hydrochloric acid and cooled to 0°C to obtain a first solution. Then, 47.0 mmol of sodium nitrite was dissolved in 75 mL of water to obtain a sodium nitrite solution. The sodium nitrite solution was added dropwise to the first solution over 20 minutes, and the mixture was stirred at 0°C for 1.5 hours to obtain a second solution. 111 mmol of SnCl2·2H2O was dissolved in 35 mL of concentrated hydrochloric acid and added dropwise to the second solution over 5 minutes. The mixture was stirred at 0°C for 1 hour to obtain a suspension. The suspension was filtered, and the solid precipitate was collected. The precipitate was dissolved in 1 M sodium hydroxide solution to obtain an alkaline aqueous solution. The alkaline aqueous solution was extracted multiple times with dichloromethane, and all organic phases of the extracts were combined, dried with anhydrous magnesium sulfate, and then concentrated under vacuum to obtain compound 2.

[0018] 30.0 mmol of compound 2 and 36.0 mmol of 3-methyl-2-butane were heated and refluxed in 200 mL of acetic acid for 3 h. After reflux, acetic acid was removed under reduced pressure to obtain crude product. The crude product was purified by column chromatography to obtain compound 3.

[0019] 7 mmol of compound 3 was dissolved in 5 mL of acetonitrile, and 35 mmol of 3-bromopropionic acid was added. The mixture was heated at 120 °C for 12 h in a pressure-resistant tube. After the reaction was completed, the resulting suspension was filtered, and the filtrate was washed with acetone until the acetone was colorless. Then it was dried under vacuum to obtain compound 4.

[0020] S2: Preparation of compound 7

[0021] 25 mmol of 4-hydrazinosulfonic acid and 25 mmol of 3-methyl-2-butanone were dissolved in 50 mL of acetic acid and heated under reflux at 120 °C overnight. After the reaction was completed, the acetic acid was removed by rotary evaporation under reduced pressure to obtain an oily substance. The oily substance was dissolved in methanol, and 10 mL of saturated potassium hydroxide isopropanol solution was slowly added. A solid precipitated, and the solid was filtered. The obtained solid was washed with isopropanol and dried under vacuum at 40 °C for 12 h to obtain intermediate compound 6.

[0022] 7.0 mmol of intermediate compound 6 was dissolved in 5 mL of iodoethane and heated at 120 °C for 12 h to obtain a suspension. The suspension was filtered, and the resulting solid was washed with acetone until the acetone became colorless. Then it was dried in vacuum at 40 °C for 12 h to obtain compound 7.

[0023] S3: Preparation of compound Cy3-I

[0024] 1.63 mmol of compound 4 and 1.63 mmol of compound 7 were dissolved in 2 mL of pyridine, followed by the addition of 1.63 mmol of triethylformyl phosphate. The mixture was stirred at 100 °C for 3 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the product was purified by reversed-phase high-performance liquid chromatography. The fraction containing the target product was collected, and after lyophilization to remove the solvent from the fraction, compound Cy3-I was obtained, with a retention time of 21-22 minutes.

[0025] S4: Preparation of compound 10

[0026] 4 mmol of 3-hydroxypropionitrile, 1.2 mmol of zinc trifluoromethanesulfonate (Zn(OTf)2), 20 mmol of acetonitrile, and 60 mmol of anhydrous hydrazine were mixed and reacted under nitrogen atmosphere at 70°C for 40 h with stirring to obtain a reaction solution. The reaction solution was cooled with ice water, and then 40 mmol of sodium nitrite dissolved in 20 mL of ice water was slowly added to obtain a sodium nitrite aqueous solution for further reaction, resulting in a mixture. During the addition of the sodium nitrite aqueous solution, 1 M hydrochloric acid was slowly added simultaneously until the gas escaped and the pH of the system was 3.

[0027] The mixture was removed by rotary evaporation to obtain a residue. Ethyl acetate was added to the residue, and the mixture was filtered. The filtrate was evaporated, and the crude product was purified by column chromatography to obtain solid compound 9.

[0028] 1.0 mmol of compound 9 was dissolved in dichloromethane, and 1.2 mmol of triethylamine and 1.2 mmol of methanesulfonyl chloride MsCl were added sequentially. The mixture was stirred at room temperature for 10 min. After the reaction was completed, TLC analysis showed that the polar position of the starting compound 9 was essentially colorless. The reaction was complete, and the organic layer was separated. The organic layer was washed with water, and then dried with anhydrous sodium sulfate using a dryer. The desiccant was removed by filtration, and the filtrate was evaporated. The residue was purified by column chromatography to obtain solid compound 10.

[0029] S5: Preparation of compound Cy3Tz

[0030] In a 30 mL microwave reaction tube, 0.005 mmol Pd2(dba)3, 0.020 mmol ligand 1,2,3,4,5-pentaphenyl-1'-(di-tert-butylphosphino)ferricyanide, 0.303 mmol compound 10, 0.104 mmol compound Cy3-I, and 0.5 mmol N,N-dicyclohexylmethylamine were dissolved in 3.5 mL anhydrous N,N-dimethylformamide. The reaction was carried out in a microwave reactor under inert nitrogen protection and heated by microwave radiation. After the reaction solution was cooled to room temperature, the product was purified by reversed-phase high-performance liquid chromatography. The fraction containing the target product was collected, and after lyophilization to remove the solvent from the fraction, compound Cy3-Tz was obtained.

[0031] S6: Preparation of compound RNA-Cy3Tz

[0032] The first terminal amino-modified oligonucleotide 5'-NH3-UGAUAAGCUA-3' was dissolved in 0.1 mM sodium bicarbonate buffer at pH 8.45 to obtain the third solution; then, the compound Cy3Tz obtained in S5 was dissolved in the organic solvent dimethyl sulfoxide (DMSO) to obtain the fourth solution; the third and fourth solutions were mixed and reacted at room temperature for 3 hours to obtain a reaction mixture; the reaction process was monitored by high performance liquid chromatography (HPLC) to ensure that the major product was generated and there were no obvious side reactions; the reaction mixture was purified by reversed-phase HPLC, and the fraction containing the target product was collected and immediately lyophilized to remove the solvent from the fraction, yielding the compound that is the 5'-terminal chemiluminescent probe, named RNA-Cy3Tz, where "Tz" represents the tetrazine functional group and "Cy3" represents the fluorescent molecular unit.

[0033] Furthermore, the synthesis method of compound RNA-ABN is as follows:

[0034] The second-terminal amino-modified oligonucleotide 5'-UCAACAUCAGU-NH3-3' was dissolved in 0.1 mM sodium bicarbonate buffer at pH 8.45 to obtain the fifth solution; the 7-azabenzonorbornene derivative was dissolved in the organic solvent DMSO to obtain the sixth solution; the fifth and sixth solutions were mixed and reacted at room temperature for 3 hours to obtain the reaction solution; the reaction process was monitored by high performance liquid chromatography to ensure that the major product was generated and there were no obvious side reactions; the reaction solution was purified by reversed-phase high performance liquid chromatography, the fraction containing the target product was collected, and the solvent in the fraction was removed by freeze drying to obtain the purified compound RNA-ABN.

[0035] Furthermore, in step S6 of the method for synthesizing compound RNA-Cy3Tz, the molar ratio of compound Cy3Tz to the first terminal amino-modified oligonucleotide is 30:1.

[0036] Furthermore, in the method for synthesizing the compound RNA-ABN, the molar ratio of the 7-azabenzonorbornene derivative to the second terminal amino-modified oligonucleotide is 30:1.

[0037] Furthermore, in step S5 of the method for synthesizing compound RNA-Cy3Tz, the microwave radiation heating conditions are: heating reaction at 50°C for 60 minutes.

[0038] Furthermore, in steps S3 and S5 of the method for synthesizing compound RNA-Cy3Tz, the column chromatography is silica gel column chromatography, and the eluent used is a mixture of hexane and ethyl acetate in a volume ratio of 1:1.

[0039] Furthermore, in steps S3 and S5 of the synthesis methods for compounds RNA-ABN and RNA-Cy3Tz, the specific conditions for the reversed-phase high-performance liquid chromatography purification are as follows: the chromatographic column used is a Phenomenex Clarity Oligo-MS column (2.1 mm × 150 mm, 2.6 μm); mobile phase A is an aqueous solution containing triethylamine and hexafluoroisopropanol; mobile phase B is methanol; the volume ratio of triethylamine, hexafluoroisopropanol, and water in mobile phase A is 0.4:30:1000.

[0040] The gradient elution program is as follows: first, elute with a mixture of 95% mobile phase A and 5% mobile phase B for 1 minute, then increase the proportion of mobile phase B to 50% within 15 minutes; the flow rate is 0.8-1.2 mL / min, and the column temperature is 20-60℃.

[0041] In another aspect, the present invention provides the application of the probe for detecting microRNA-21 based on bioorthogonal cyclic amplification reaction in the early diagnosis of lung cancer. When detecting tumors, the concentration of the probe is 1 μM, that is, the concentrations of both compound RNA-Cy3Tz and compound RNA-ABN are 1 μM.

[0042] Furthermore, it also includes buffer solutions;

[0043] The detection buffer solution comprises 100 mM Tris·HCl and 150 mM NaCl, wherein the pH of Tris·HCl is 7.4.

[0044] The beneficial effects of this invention are:

[0045] The probe for detecting microRNA-21 based on a bioorthogonal cyclic amplification reaction disclosed in this invention can achieve catalytic target cycling, with each miRNA molecule achieving a turnover rate of up to 2.2 × 10⁻⁶. 9 It reached 3.58×10 -18 With its ultra-low detection limit, excellent specificity, and wide linear range of 100 aM to 100 nM, this probe enables rapid and ultrasensitive detection of microRNA-21 in complex biological environments. It allows for in situ, real-time detection and imaging without cell lysis, significantly simplifying procedures and enhancing applicability. Furthermore, this probe supports in situ imaging in live cells and tissues, and has successfully detected miRNAs in clinical serum samples from lung cancer patients. It effectively distinguishes miRNA levels between lung cancer patients and healthy individuals, demonstrating significant potential for non-invasive diagnostics and biomedical research. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 The absorption spectra of compound Cy3-I in different solvents;

[0048] Figure 2 The absorption spectra of compound Cy3Tz in different solvents;

[0049] Figure 3 Normalized absorbances for compounds Cy3-I and Cy3Tz;

[0050] Figure 4 The fluorescence spectra of compound Cy3-I in different solvents;

[0051] Figure 5 The fluorescence spectra of compound Cy3Tz in different solvents;

[0052] Figure 6 The fluorescence spectra of compounds Cy3-I and Cy3Tz under the same conditions are shown.

[0053] Figure 7 The results show the fluorescence intensity at the maximum emission wavelength under different reaction conditions of compound Cy3Tz.

[0054] Figure 8 This invention provides an analysis of the fluorescence response and catalytic performance of the IDCR probe against the target microRNA-21, wherein... Figure 8 A represents the fluorescence intensity at the maximum emission wavelength after the probe has been incubated with different concentrations of microRNA-21 for 2 hours. Figure 8 B is the normalized image of catalytic turnover rate;

[0055] Figure 9 The anti-interference capability test results of the probe provided by this invention;

[0056] Figure 10 The selectivity test results of the probe provided by this invention;

[0057] Figure 11 The results of the target test of the probe provided by this invention with microRNA-21 and mismatched bases;

[0058] Figure 12 The cytotoxicity test results of the probe provided in this invention;

[0059] Figure 13 The imaging test results of the probe provided by this invention in PANC-1 cells;

[0060] Figure 14 The imaging test results of the probe provided by this invention in A549 cells;

[0061] Figure 15 Imaging test results of the probe provided by this invention in MCF-7 cells;

[0062] Figure 16 The probe provided by this invention was used to image cross-sections at different heights in mouse lung tissue sections and tumor sections from mice after A549 (human lung cancer cells) xenografting. Figure 16 A shows fluorescence imaging images of tumor sections at different heights. Figure 16 B shows fluorescence imaging images of mouse lung tissue at different heights; Figure 16 C is a 3D imaging image of a tumor slice; Figure 16 D is a 3D image of a mouse lung tissue section;

[0063] Figure 17 The results of detecting microRNA-21 in normal human serum and lung cancer patient serum using the probe and qPCR technology provided by this invention are shown below. Figure 17 A is a schematic diagram of human serum sample testing. Figure 17 B represents the fluorescence intensity detection results of the probe in the serum of 8 normal individuals and 22 lung cancer patients in the presence of microRNA-21. Figure 17 C represents the fold change in microRNA-21 expression in the serum of 8 healthy individuals and 22 lung cancer patients detected by qPCR. Detailed Implementation

[0064] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0065] Example 1:

[0066] Preparation of compound Cy3Tz

[0067] The structural formula of compound Cy3Tz:

[0068]

[0069] The preparation method of compound Cy3Tz is as follows:

[0070]

[0071] Compound 2:

[0072]

[0073] 10 g of p-iodoaniline (compound 1, 45.7 mmol) was dissolved in 50 mL of 20% hydrochloric acid and cooled to 0°C to obtain a first solution. Then, 3.24 g of sodium nitrite (47.0 mmol) was dissolved in 75 mL of water to obtain a sodium nitrite solution. The sodium nitrite solution was added dropwise to the first solution over 20 minutes, and the mixture was stirred at 0°C for 1.5 hours to obtain a second solution. 25 g of SnCl₂·2H₂O (111 mmol) was dissolved in 35 mL of concentrated hydrochloric acid and added dropwise to the second solution over 5 minutes. The mixture was stirred at 0°C for 1 hour to obtain a suspension. The suspension was filtered, and the solid precipitate was collected. The precipitate was dissolved in 1 M sodium hydroxide solution to obtain an alkaline aqueous solution. The alkaline aqueous solution was extracted multiple times with CH₂Cl₂. All organic phases were combined, dried with anhydrous magnesium sulfate, and then dried and concentrated under vacuum (40°C, 12 h) to obtain 8.3 g (78%) of compound 2 as a brown powder. 1 HNMR (500MHz, CDCl3) δ7.48 (d, J=8.7Hz, 2H), 6.61 (d, J=8.7Hz, 2H), 5.18 (bs, 1H), 3.55 (bs, 2H). 13 CNMR (126 MHz, CDCl3) δ 150.9, 137.9, 114.4, 80.4. ESI mass spectrometry (ESI) calculated m / z value: [C6H8N2I] + [M+H]+: 235.0; Measured value: 235.0.

[0074] Compound 3:

[0075]

[0076] 7 g of compound 2 (30.0 mmol) and 3 g of 3-methyl-2-butane (3.7 mL, 36.0 mmol) were refluxed in 200 mL of acetic acid for 3 h. After 3 h, the acetic acid was removed by vacuum distillation to obtain a crude product, which was then purified by silica gel column chromatography (eluent: hexane and ethyl acetate in a volume ratio of 1:1) to give 5.4 g (65%) of compound 3, which was a brown oil. 1HNMR (500MHz, CDCl3) δ7.60(dd,J=8.1,1.6Hz,1H),7.57(d,J=1.2Hz,1H),7.26(d,J=8.0Hz,1H),2.23(s,3H),1.26(s,6H). 13 CNMR(126MHz, CDCl3)δ188.3,153.5,148.3,136.7,130.7,121.9,90.0,54.1,23.0,15.4.MS(ESI)m / z[C 11 H 13 NI] + [M+H] + Calculated value: 286.0; Measured value: 286.0.

[0077] Compound 4:

[0078]

[0079] 2 g (7 mmol) of compound 3 was dissolved in 5 mL of acetonitrile, and 5.3 g (35 mmol) of 3-bromopropionic acid was added to a pressure-resistant tube and heated at 120 °C for 12 h. The resulting suspension was filtered and washed with acetone until the washing acetone was colorless. The crude product was dried under vacuum to give 1.9 g (Y = 61%) of a pale yellow solid, which is compound 4. 1 HNMR (500MHz, Chloroform- d )δ7.88,7.88,7.87,7.86,7.77,7.76,7.50,7.48,4.48,4.46,4.45,2.91,2.77,2.75,2.74,1.49.MS(ESI)m / z[C 14 H 17 NIO2] + [M+H] + Calculated value: 358.0; Measured value: 358.0.

[0080] Compound 6:

[0081]

[0082] 5 g (25 mmol) of 4-hydrazinosulfonic acid and 2.18 g (25 mmol, 2.7 mL) of 3-methyl-2-butanone were dissolved in 50 mL of acetic acid and reacted under reflux at 120 °C overnight for 12–16 hours. After the reaction, the acetic acid was removed by rotary evaporation under reduced pressure at 40–60 °C and a vacuum of ≤10 mmHg. The resulting oily substance was dissolved in methanol, and 10 mL of a saturated potassium hydroxide isopropanol solution was slowly added. After the solid precipitated, the suspension was filtered, and the filtered solid was washed with isopropanol and dried under vacuum at 40 °C for 12 h to obtain 3.85 g (56% yield) of compound 6, which was a yellow powder. Intermediate compound 6 can be used directly without further purification. MS(ESI) m / z [C 11 H 14 NO3S] + [M+2H] + Calculated value: 240.1; Measured value: 240.1.

[0083] Compound 7:

[0084]

[0085] 2 g (7.0 mmol) of intermediate compound 6 was dissolved in 5 mL of iodoethane and heated at 120 °C for 12 h in a pressure-resistant tube. The resulting suspension was filtered, and the filtered solid was washed with acetone until the acetone became colorless. After drying the solid under vacuum, 1.35 g (70% yield) of pink solid compound 7 was obtained. 1 HNMR(500MHz,DMSO-d6)δ8.00(s,1H),7.88(d,J=8.3Hz,1H),7.81(d,J=8.1Hz ,1H),4.46(q,J=6.8Hz,2H),2.80(s,3H),1.52(s,6H),1.42(t,J=7.0Hz,3H). 13 CNMR(126MHz,DMSO-d6)δ196.9,149.4,141.6,140.52,126.3,120.7,114.74,54.2,43.2,21.8,14.0,12.6.MS(ESI)m / z[C · H 17 NO3S] + [M+H] + Calculated value: 268.1; Measured value: 268.1.

[0086] Compound 9:

[0087]

[0088] Zn(OTf)₂ (427 mg, 1.2 mmol), 3-hydroxypropionitrile (285 mg, 4 mmol), acetonitrile (1.0 mL, 20 mmol), and anhydrous hydrazine (7.7 mL, 60 mmol) were added to a 50 mL reaction flask equipped with a magnetic stir bar. The reaction flask was stirred in an oil bath at 60°C for 40 h under nitrogen atmosphere. The reaction solution was cooled with ice water, and then 2.8 g (40 mmol) of sodium nitrite dissolved in 20 mL of ice water was slowly added. Subsequently, 1 M hydrochloric acid was slowly added during this process, at which point the solution turned a bright red color and gas was released. The addition of 1 M hydrochloric acid continued until gas was released and the pH reached 3, at which point the addition was stopped, yielding a mixture. The mixture was rotary evaporated to remove water and solvent. Ethyl acetate was added to the residue, and the solid was removed by filtration. The filtrate was evaporated, and the crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to give 180 mg of a deep pink solid compound 9 (yield 32%). 1 HNMR (500MHz, DMSO-) d 6)δ3.46(t, J =7.5Hz,4H),4.14(t, J =10Hz, 4H). 13 CNMR (125MHz, DMSO-) d 6) 38.4, 60.0, 168.8; HRMS[M+H] + m / z[C6H 11 N4O2] + Calculated value: 171.0877, measured value: 171.1.

[0089] Compound 10:

[0090]

[0091] In a 50 mL flask, compound 9 (180 mg, 1.0 mmol) was dissolved in dichloromethane, and triethylamine (1.2 mmol) was added, followed by methanesulfonyl chloride (MsCl) (137 mg, 1.2 mmol). The reaction solution was stirred at room temperature for 10 min. The reaction was checked by TLC to confirm the formation of the target product. After the reaction was complete and the target product was formed, the organic layer was separated, washed with water, dried over anhydrous sodium sulfate and evaporated. The residue was purified by silica gel column chromatography to give 238 mg of red solid compound 10 (85% yield). 1 HNMR(500MHz,CDCl3)δ2.99(s,3H),3.04(s,3H),3.73(t, J =7.5Hz,2H),4.83(dt, J =10,5Hz,2H).13 CNMR(125MHz, CDCl3)δ21.4,34.7,37.7,66.4,166.3,168.4;MS[M+H] + m / zcalcd.for[C6H 11 N4O3S] + Calculated value: 219.05, measured value: 219.05.

[0092] Compound Cy3-I:

[0093]

[0094] Cy3-I

[0095] Compound 4 (584.85 mg, 1.63 mmol) and compound 7 (500 mg, 1.63 mmol) were dissolved in pyridine (2 mL), followed by the addition of triethylformyl phosphate (242 mg, 1.63 mmol). The mixture was stirred at 100 °C for 3 hours, and the reaction progress was monitored by thin-layer chromatography. When the reaction was complete, the product was purified by reversed-phase high-performance liquid chromatography. The fraction containing the target product was collected, and after lyophilization to remove the solvent from the fraction, pure compound Cy3-I (298 mg, 27%) was obtained. This is an asymmetric pink solid with a retention time of 21-22 minutes. 1 HNMR(400MHz,MeOD)δ8.55(t,J=13.4Hz,1H),7.96(d,J=1.4Hz,1H),7.93(dd,J=8. 3,1.6Hz,1H),7.90(d,J=1.6Hz,1H),7.77(dd,J=8.4,1.6Hz,1H),7.43(d,J=8.4Hz ,1H),7.22(d,J=8.4Hz,1H),6.54(t,J=12.9Hz,2H),4.42(t,J=7.1Hz,2H),4.24(q ,J=7.2Hz,2H),2.84(t,J=7.1Hz,2H),1.78(s,6H),1.76(s,6H).MS:m / zcalcd.forC 28 H 30 IN2O5S - =633.09, found633.06.

[0096] In a 30 mL microwave reaction tube, Pd2(dba)3 (4.62 mg, 0.005 mmol) and ligands 1,2,3,4,5-pentaphenyl-1'-(di-tert-butylphosphino)ferricyanide (14.34 mg, 0.020 mmol), compound 10 (66 mg, 0.303 mmol), compound Cy3-I (66 mg, 0.104 mmol), and N,N-dicyclohexylmethylamine (98.39 mg, 0.5 mmol) were dissolved in anhydrous N,N-dimethylformamide (3.5 mL). The reaction was carried out under nitrogen protection and heated by microwave radiation in a microwave reactor at 50°C for 60 minutes. After the reaction was completed, the reaction solution was cooled to room temperature, and the product was purified by reversed-phase high-performance liquid chromatography. The fraction containing the target product was collected, and after lyophilization to remove the solvent from the fraction, pure compound Cy3-Tz (39 mg, yield 60%) was obtained as a purple solid. 1 HNMR(600MHz,MeOD)δ8.60(t,J=13.4Hz,1H),8.35(d,J=16.2Hz,1H),8.05(d,J=1.4Hz ,1H),7.99–7.91(m,2H),7.82(dd,J=8.3,1.4Hz,1H),7.61(d,J=16.2Hz,1H),7.46(dd ,J=19.0,8.3Hz,2H),6.56(dd,J=13.4,10.7Hz,2H),4.47(t,J=7.1Hz,2H),4.26(q,J= 7.2Hz,2H),3.00(s,3H),2.88(s,2H),1.84(s,6H),1.81(s,6H),1.44(t,J=7.3Hz,3H). 13 CNMR(126MHz,Chloroform-d)δ180.06,178.34,177.07,171.18,169.55,151.62,148.50,146.54,146.43,145.76,137.18,131.56,12 6.73,125.39,125.24,125.15,116.92,116.19,109.30,108.39,54.52,53.72,36.56,32.79,32.41,26.04,17.60.MS:m / zcalcd.forC 33 H 35 N6O5S - =627.24, found627.25.

[0097] Characterization and performance testing of compounds Cy3-I and Cy3Tz:

[0098] (1) The absorption spectra of compounds Cy3-I and Cy3Tz were measured respectively.

[0099] Cy3-I and Cy3Tz were prepared into 100 nM working solutions, and their absorption spectra were measured in methanol, dichloroisocyanurate, acetonitrile, water, and DMSO, respectively. The results are as follows: Figures 1-2 As shown, Cy3-I and Cy3Tz absorb at 500 nm–600 nm in the near-infrared region, which can be used for cell imaging.

[0100] Figure 3 Normalized absorption spectra of Cy3-I and Cy3Tz. Figure 3 The results show that the absorption of Cy3Tz has a slight red shift compared to Cy3-I.

[0101] (2) Determination of fluorescence spectra of compounds Cy3-I and Cy3Tz

[0102] Cy3-I and Cy3Tz were prepared into 100 nM working solutions, and their fluorescence spectra were tested in methanol, dichloroisocyanuric acid, acetonitrile, water and DMSO, respectively.

[0103] from Figures 4-5 The fluorescence spectra of Cy3-I and Cy3Tz can be seen in the 550 nm – 700 nm range, in the near-infrared region, which can be used for cell imaging.

[0104] Figure 6 Normalized absorption spectra of Cy3-I and Cy3Tz. Figure 6 The results showed that the fluorescence of Cy3Tz was basically quenched compared to Cy3-I, which verified that tetrazine effectively quenched the fluorescence of the Cy3 parent compound.

[0105] (3) Solution experiments were conducted on Cy3Tz.

[0106] Since tetrazines can undergo bio-orthogonal reactions (such as 7-azabenzonorbornene ABN) with olefins with high ring strain, to verify the difficulty of restoring fluorescence in solution systems using Cy3Tz, 100 μL of Cy3Tz working solution was placed in ABN solutions of equal concentration and incubated at different temperatures (37℃, 60℃) and for different times (2h, 24h), and the fluorescence intensity of the system was detected. The experimental results are as follows: Figure 7As shown, after incubation at 37°C for 2 hours, only negligible fluorescence enhancement was observed (not shown in the figure). To further evaluate the inherent stability of the system, the reaction was monitored for up to 24 hours at the same temperature (37°C 24h group in the figure), and only a slight fluorescence recovery was observed. A temperature-dependent reaction rate was observed; increasing the temperature to 60°C accelerated the reaction. After incubation at 60°C for 2 hours, the fluorescence intensity increased to 80.431 au, and further increased to 135.968 au after 24 hours, indicating that increasing the temperature accelerates the reaction. Importantly, at a physiological temperature of 37°C, the background fluorescence enhancement remained at a low level (69.44 au), indicating that the reaction system composed of Cy3Tz and ABN has high stability and is suitable for practical biological applications.

[0107] Example 2:

[0108] (1) Preparation of compound RNA-Cy3Tz

[0109] The first terminal amino-modified oligonucleotide 5'-NH3-UGAUAAGCUA-3' was dissolved in 0.1 mM sodium bicarbonate buffer at pH 8.45 to obtain a third solution; then, the compound Cy3Tz obtained in step S5 of Example 1 was dissolved in the organic solvent dimethyl sulfoxide (DMSO) to obtain a fourth solution; the third and fourth solutions were mixed and reacted at room temperature for 3 hours to obtain a reaction mixture; the molar ratio of the compound Cy3Tz to the first terminal amino-modified oligonucleotide was 30:1; the reaction mixture was purified by reversed-phase high-performance liquid chromatography, and the fraction containing the target product was collected and immediately lyophilized to remove the solvent from the fraction to obtain the compound RNA-Cy3Tz, which is the 5'-terminal chemiluminescent probe RNA-Cy3Tz (where "Tz" represents the tetrazine functional group and "Cy3" represents the fluorescent molecular unit).

[0110] (2) Preparation of compound RNA-ABN

[0111] The second-terminal amino-modified oligonucleotide 5'-UCAACAUCAGU-NH3-3' was dissolved in 0.1 mM sodium bicarbonate buffer at pH 8.45 to obtain the fifth solution; the 7-azabenzonorbornene derivative was dissolved in the organic solvent DMSO to obtain the sixth solution, with a molar ratio of 7-azabenzonorbornene derivative to the second-terminal amino-modified oligonucleotide of 30:1; the fifth and sixth solutions were mixed and reacted at room temperature for 3 hours to obtain the reaction solution; the reaction solution was purified by reversed-phase high-performance liquid chromatography, and the fraction containing the target product was collected, lyophilized to remove the solvent from the fraction, and the purified compound RNA-ABN was obtained.

[0112] In practical applications, miR-21 was chosen as the model target because it is a recognized oncogenic microRNA that is significantly upregulated in lung cancer. Two short amino-modified RNA sequences (UGAUAAGCUA and UCACAUCAGU) complementary to the target microRNA were designed, with Cy3Tz and a 7-azabenzonorbornene derivative ABN attached to their ends, respectively, forming RNA-Cy3Tz and RNA-ABN. These were purified and packaged by HPLC (High-performance liquid chromatography) and termed the probe IDCR.

[0113] Performance testing of the IDCR probe

[0114] (1) The fluorescence intensity at the maximum emission wavelength and its catalytic turnover performance of the probe after incubation with different concentrations of microRNA-21 for 2 hours were measured:

[0115] To confirm the template-dependent fluorescence activation, the concentrations of the two components of the probe IDCR, RNA-Cy3Tz and RNA-ABN, were both kept at 1 μM. These were mixed with the template microRNA-21 in a 100 μL buffer solution (100 mM Tris·HCl, pH = 7.4, and 150 mM NaCl). After incubation at 37°C for 2 h, fluorescence spectra were measured using a multi-mode microplate reader and a micro-fluorescence spectrometer, and the fluorescence intensity at the maximum emission wavelength was recorded. Each experiment was repeated three times, and statistical significance analysis was performed.

[0116] Test results: such as Figure 8 As shown, the probe IDCR showed a good response to the target. With increasing template concentration, the fluorescence of the cyanine dye parent material, which had been quenched by the tetrazine group, gradually recovered, and a significant signal enhancement was observed even at a low concentration (100 aM). Figure 8 A) indicates that the probe IDCR has good detection capability for low concentrations of target miRNA; subsequently, the limit of detection (LOD) of the probe IDCR was quantitatively calculated using the 3σ method, and the LOD was found to be 3.58 × 10⁻⁶. -18 M.

[0117] The superior sensitivity of the probe IDCR stems from its efficient catalytic turnover capability and signal amplification mechanism. Figure 8 B). Quantitative analysis results show a significant negative correlation between target concentration and catalytic efficiency: Calculations show that the turnover number of a single target molecule is approximately 4.7 at a target concentration of 100 nM, increases to 265.8 when the target concentration decreases to 1 nM, and reaches as high as 2.3 × 10⁻⁶ when the target concentration is as low as 10 fM.8 In the next cycle, when the target concentration is as low as 100 aM, the turnover rate reaches as high as 2.2 × 10⁻⁶. 9 This cycle. Correspondingly, under the condition of minimum target concentration, the apparent catalytic turnover rate of the system ( k cat (app) Approximately 1.8 × 10 7 min -1 That is, a single target molecule can catalyze nearly 1 × 10⁻⁶ per minute. 7 Secondary reaction.

[0118] This exceptional amplification efficiency aligns perfectly with the catalytic mechanism proposed in this invention. Under low target concentration conditions, a higher proportion of the probe can participate in multiple cyclic reactions, sequentially completing hybridization, the reverse electron-demanding Diels-Alder reaction (IEDDA reaction), and achieving probe release and recycling through the reverse Diels-Alder dissociation process. It is this combination of powerful signal amplification and optimized reaction kinetics that endows this probe with ultra-high sensitivity at the single-digit angstrom level, exhibiting significant performance advantages compared to traditional one-to-one detection methods.

[0119] The above conclusions indicate that the probe IDCR assay system has high detection sensitivity due to the signal amplification process caused by template sequence cycling, laying a solid foundation for its accurate detection of low-abundance target miRNAs in a variety of complex samples.

[0120] (2) Test the anti-interference capability of the probe:

[0121] Test method: Prepare the probe IDCR working solution so that the final concentration of both components (RNA-Cy3Tz and RNA-ABN) in the buffer solution test system (100 μL, containing 100 mM Tris-HCl, pH 7.4, 150 mM NaCl) is 1 μM. This serves as a template-free probe blank control group. The following four parallel experiments were set up: (1) Positive experimental group: 50 nM target microRNA-21 (Mir-21) was added to the above working solution; (2) Reducing substance interference group: 5 mM L-cysteine ​​(Cysteine) was added to the positive experimental group system; (3) Oxidizing substance interference group: 5 mM H2O2 was added to the positive experimental group system; (4) Single component probe control group: containing only 1 μM RNA-Cy3Tz, used to evaluate the fluorescence background of the probe component itself. After incubation at 37°C for 8 h, the fluorescence intensity at the maximum emission wavelength was measured using an ultra-micro fluorescence spectrometer. Each experiment was repeated three times.

[0122] Test results: such as Figure 9As shown, no significant fluorescence recovery was observed in the experimental group without the target template sequence. Only in the experimental group did the fluorescence intensity gradually increase over time, indicating that the probe IDCR has good selectivity for the target sequence and can be applied in physiological environments without competitive interference from abundant biomolecules.

[0123] (3) Test the selectivity of the probe:

[0124] Test Method: The IDCR working solution was prepared such that the final concentration of both components, RNA-Cy3Tz and RNA-ABN, in a buffer solution test system (100 μL, containing 100 mM Tris-HCl, pH 7.4, 150 mM NaCl) was 1 μM. This served as a template-free probe blank control group. Ten experimental groups were also set up: one group had 50 nM of the target microRNA-21 added to the working solution, while the other nine groups had different types of potential interfering agents added. The interfering agents were: glutathione, DL homocysteine, L-cysteine, hydantoin, phenylboronic acid, glucose, bovine serum albumin (BSA), and human serum albumin (Ovalbumin), with a concentration of 5 mM for each. All groups were incubated at 37°C for 2 h, and the fluorescence intensity at the maximum emission wavelength was measured using a micro-fluorescence spectrometer.

[0125] Test results: such as Figure 10 As shown, under the same conditions, the fluorescence intensity of the system was significantly enhanced only when the target microRNA-21 was present, while the presence of other potential interfering substances (such as glutathione, cysteine, proteins, and carbohydrates) did not cause significant changes in the fluorescence signal. These results indicate that the IEDDA (Inverse Electron-Demand Diels–Alder Reaction)-based probe IDCR exhibits good bioorthogonality and selectivity, can stably recognize target miRNAs in complex physiological environments, and is not easily interfered with by common biomolecules, providing an important guarantee for its reliable application in various biological samples. Furthermore, the relatively stable detection results help the probe IDCR to perform its recognition function in practical applications across various biological samples.

[0126] (4) Testing the specificity of the probe:

[0127] Test method: Prepare the probe IDCR working solution so that the final concentration of both components (RNA-Cy3Tz and RNA-ABN) in the buffer solution test system (100 μL, containing 100 mM Tris-HCl, pH 7.4, 150 mM NaCl) is 1 μM, which serves as the blank control group. Two other experimental groups were set up: (1) 100 nM of target microRNA-21 was added to the above working solution; (2) 100 nM of the incompletely complementary sequence Mis-a (a sequence that is highly homologous to microRNA-21 but has a base mismatch) was added to the same working solution and mixed. After all systems were reacted at 37℃ for 2 h, the fluorescence intensity at the maximum emission wavelength was measured using an ultra-micro fluorescence spectrometer.

[0128] Test results: such as Figure 11 As shown, significant fluorescence enhancement only occurs when the target sequence microRNA-21, which is perfectly complementary, is present (group "Right" in the figure). Non-perfectly complementary competing sequences hinder probe recognition and binding on the template strand (group "Wrong" in the figure), failing to induce significant fluorescence enhancement, reducing the likelihood of the reaction, and consequently reducing the occurrence of cyclic amplification. These results demonstrate that this probe possesses high specificity for target sequences and can effectively distinguish between perfectly and non-perfectly complementary RNA sequences, providing a foundation for the specific detection of target miRNAs under complex physiological conditions.

[0129] (5) Probe cytotoxicity test results

[0130] Assay Methods: Before imaging analysis of endogenous miRNAs in live cells, the cytotoxicity of the probe IDCR was first evaluated. Different concentrations of RNA-Cy3Tz and RNA-ABN (concentration gradients: 0 µM, 0.6 µM, 1.2 µM, 2.5 µM, 5 µM, 7.5 µM, 10 µM, 15 µM, 20 µM, 30 µM) were co-transfected into cells at a density of 5 × 10⁶ cells / year. 3 In human breast cancer cells (MCF-7), after culturing for another 24 h, cell viability was detected by the MTT assay, and cell survival rate at each concentration was calculated.

[0131] Test results: such as Figure 12 As shown, within the tested concentration range (0–30 µM), cell viability showed a slight decreasing trend with increasing probe concentration, but when the probe IDCR was added at the maximum concentration (30 μM), cell viability remained above 85%. These results indicate that the probe IDCR has no significant toxic effect on cell growth and is suitable for subsequent long-term imaging analysis of endogenous miRNAs in live cells.

[0132] (6) Imaging tests of the probe in different cells:

[0133] Test Methods: To verify the imaging ability of the probe IDCR on endogenous microRNA-21 in different cell lines, single-photon confocal imaging was performed on microRNA-21 in three cell lines: PANC-1 (human pancreatic ductal adenocarcinoma cells), A549 (human lung cancer cells), and MCF-7 (human breast adenocarcinoma cells). In the experimental group, two portions of the probe IDCR, RNA-Cy3Tz and RNA-ABN (both at a final concentration of 1 μM), were transfected into the three cell lines using Lipofectamine 3000 in Opti-MEM medium and incubated at 37°C for 6 hours. The control group was transfected only with RNA. Cy3Tz (Cy3Tz) 21). Single-photon confocal imaging was performed using 561 nm excitation, and fluorescence emission and reception wavelengths were selected from 580-680 nm. The images were then overlaid with nuclear dye (Hoechst) and bright-field images for analysis.

[0134] Test results: such as Figures 13-15 As shown, in tumor cells with different high expression of microRNA-21 (PANC-1, A549, MCF-7), the fluorescence intensity of the experimental group with the simultaneous addition of RNA-Cy3Tz and RNA-ABN was significantly higher than that of the control group with only RNA-Cy3Tz, demonstrating that the fluorescence recovery of the probe IDCR depends on the intracellular conversion of its two components by endogenous microRNA. Simultaneous recognition and activation of miRNA-21; while single-component RNA-Cy3Tz cannot independently complete fluorescence activation, producing only a small amount of background fluorescence, further confirming that this probe can specifically reflect the upregulated expression level of miRNA-21 in tumor cells through the synergistic response of RNA-Cy3Tz and RNA-ABN. At the same time, this probe can specifically achieve fluorescence imaging of various endogenous microRNA-21 in tumor cells, exhibiting good response specificity and imaging reliability.

[0135] (7) Imaging test of probes on mouse lung tissue sections and tumor sections after A549 (human lung cancer cells) xenograft mice.

[0136] Test method: A549 cells were inoculated into the axillary region of the forelimb of mice in the tumor group to construct a tumor model, and the tumors were cultured until the tumor volume reached 200 mm. 3Afterwards, the mice in the tumor group and the control group were dissected to obtain tumor tissue from the tumor model mice (tumor group) and normal lung tissue from the control mice. Both tissue samples were covered with embedding medium, and the cryostat was set to -20°C. After the embedding medium changed from transparent to white and solidified, 200 μm thick tissue sections were cut for subsequent imaging. The prepared tissue sections were placed on slides in cell culture dishes for laser confocal imaging, and two portions of the IDCR probe, RNA-Cy3Tz and RNA-ABN, were added. The incubation time for each probe was 2 h. Single-photon confocal microscopy was used for excitation at 561 nm, and the fluorescence emission and reception bands were selected at 580-680 nm. Olympus FV10-ASW 3.0 Viewer software was used to process the depth imaging of the tissues to obtain three-dimensional images.

[0137] Test results: such as Figure 16 As shown, the IDCR probe can clearly distinguish A549 xenograft tumor slices ( Figure 16 A) Compared with normal mouse lung tissue sections ( Figure 16 B), and in the 3D reconstructed image ( Figure 16 C- Figure 16 D) showed significant spatial distribution differences. Notably, this probe achieved deep tissue penetration imaging at 120 μm, effectively detecting the expression levels of target miRNAs within the tissue. These results demonstrate that the IDCR probe not only possesses precise detection capabilities for in vitro tissue samples but also serves as a potential auxiliary tool for in vivo diagnosis, providing a novel technical approach for tumor microenvironment analysis.

[0138] (8) Identification of target microRNAs in serum samples using probes and qRT-PCR

[0139] In this study, a total of 30 serum samples were collected from Liaoning Cancer Hospital. Informed consent was obtained from the hospital for each serum sample. In this experiment, the temperature of the refrigerated centrifuge was first set to 4°C and the rotation speed to 16000 g. After centrifuging the serum samples for 10 min, the supernatant was collected for subsequent tests. Figure 17 A is a schematic diagram of human serum sample testing.

[0140] For qRT-PCR experiments, RNA was first extracted from serum samples. The process included: extracting total RNA from serum samples according to the miRNeasySerum / Plasma kit instructions. Next, reverse transcription of the target miRNA and internal control gene was performed: the target microRNA was reverse transcribed into cDNA using a reverse transcriptase instrument according to the TaqMan® MicroRNA Reverse Transcription Kit instructions. Finally, quantitative real-time PCR analysis was performed: after mixing the premixed sample using the TaqMan Universal Master Mix II real-time quantitative PCR kit, quantification was performed on a real-time quantitative PCR instrument, and the Ct value was measured. Subsequently, 2... △△Ct Bar graphs showing the corresponding microRNA content in different serum samples were obtained using analytical methods. miR-16 was used as an internal control gene in serum, and each experiment was repeated three times. Significance analysis was then performed.

[0141] For the fluorescence detection method using this probe, the steps are as follows: Take 30 μL of the supernatant from each sample and add two portions of the miR-21 responsive probe IDCR, RNA-Cy3Tz and RNA-ABN, to it. The concentration of each probe is 1 μM, and incubate at 37°C for 2 h. Measure the chemiluminescence intensity at the maximum emission wavelength using a microplate reader. Repeat each experiment three times and calculate the significance.

[0142] from Figure 17 As can be seen, the IDCR probe can distinguish between serum samples from lung cancer patients (L1-L22) and normal human serum samples (N1-N8). Furthermore, to determine the reliability of the IDCR fluorescence detection results, qRT-PCR was used for auxiliary verification. Figure 17 B- Figure 17 As shown in C, the trends of the detection results for lung cancer patients and normal populations are basically consistent with those obtained by the fluorescence detection method (qPCR). These results indicate that this probe has the potential to be applied to the detection of target miRNA sequences in actual serum samples, and can clearly screen samples with abnormal levels. Furthermore, because the probe's pre-detection preparation is relatively simple, eliminating the complex pre-extraction and in vitro amplification procedures of qRT-PCR, it reduces the time cost of detection, thus demonstrating good prospects for both biological and practical applications.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probe IDCR for detecting microRNA-21 based on bio-orthogonal cyclic amplification reaction, characterized in that, The probe IDCR consists of the compounds RNA-Cy3Tz and RNA-ABN; The structural formula of the compound RNA-Cy3Tz is shown in Formula I: Formula I; The structural formula of the compound RNA-ABN is shown in Formula II: Formula II.

2. The method for synthesizing the probe IDCR for detecting microRNA-21 based on the bio-orthogonal amplification reaction according to claim 1, characterized in that, The synthesis method of compound RNA-Cy3Tz is as follows: S1: Preparation of compound 4 45.7 mmol of p-iodoaniline was dissolved in 50 mL of 20% hydrochloric acid and cooled to 0°C to obtain a first solution. Then, 47.0 mmol of sodium nitrite was dissolved in 75 mL of water to obtain a sodium nitrite solution. The sodium nitrite solution was added dropwise to the first solution over 20 minutes, and the mixture was stirred at 0°C for 1.5 hours to obtain a second solution. 111 mmol of SnCl2·2H2O was dissolved in 35 mL of concentrated hydrochloric acid and added dropwise to the second solution over 5 minutes. The mixture was stirred at 0°C for 1 hour to obtain a suspension. The suspension was filtered, and the solid precipitate was collected. The precipitate was dissolved in 1M sodium hydroxide solution to obtain an alkaline aqueous solution. The alkaline aqueous solution was extracted multiple times with dichloromethane. All organic phases were combined, dried, and concentrated to obtain compound 2. 30.0 mmol of compound 2 and 36.0 mmol of 3-methyl-2-butane were heated and refluxed in 200 mL of acetic acid for 3 h. After reflux, the acetic acid was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain compound 3. 7 mmol of compound 3 was dissolved in 5 mL of acetonitrile, 35 mmol of 3-bromopropionic acid was added, and the mixture was heated at 120 °C for 12 h. After the reaction was completed, the resulting suspension was filtered, and the filtrate was washed with acetone until the acetone was colorless. Then it was dried to obtain compound 4. S2: Preparation of compound 7 25 mmol of 4-hydrazinosulfonic acid and 25 mmol of 3-methyl-2-butanone were dissolved in 50 mL of acetic acid and heated under reflux at 120 °C overnight. After the reaction was completed, the acetic acid was removed under reduced pressure by rotary evaporation to obtain an oily substance. The oily substance was dissolved in methanol, and then 10 mL of saturated potassium hydroxide isopropanol solution was added to react. After the solid precipitated, it was filtered, and the obtained solid was washed and dried to obtain intermediate compound 6. 7.0 mmol of intermediate compound 6 was dissolved in 5 mL of iodoethane and heated at 120 °C for 12 h to obtain a suspension. The suspension was filtered, and the resulting solid was washed with acetone until the acetone became colorless. The solid was dried to obtain compound 7. S3: Preparation of compound Cy3-I 1.63 mmol of compound 4 and 1.63 mmol of compound 7 were dissolved in 2 mL of pyridine, followed by the addition of 1.63 mmol of triethylformyl phosphate. The mixture was stirred at 100 °C for 3 hours. After the reaction was completed, the product was purified by reversed-phase high-performance liquid chromatography. The fraction containing the target product was collected, and after lyophilization to remove the solvent from the fraction, compound Cy3-I was obtained. S4: Preparation of compound 10 4 mmol of 3-hydroxypropionitrile, 1.2 mmol of zinc trifluoromethanesulfonate (Zn(OTf)2), 20 mmol of acetonitrile, and 60 mmol of anhydrous hydrazine were mixed and reacted under nitrogen atmosphere at 70°C for 40 h with stirring to obtain a reaction solution. After cooling, 40 mmol of sodium nitrite dissolved in 20 mL of ice water was added to obtain an aqueous sodium nitrite solution for further reaction, resulting in a mixture. During the addition of the sodium nitrite solution, 1 M hydrochloric acid was added simultaneously until gas escaped and the pH of the system reached 3, at which point the addition of hydrochloric acid was stopped. The mixture was removed by rotary evaporation to obtain a residue. Ethyl acetate was added to the residue, and the mixture was filtered. The filtrate was evaporated, and the crude product was purified by column chromatography to obtain solid compound 9. 1.0 mmol of compound 9 was dissolved in dichloromethane, and 1.2 mmol of triethylamine and 1.2 mmol of methanesulfonyl chloride MsCl were added sequentially. The mixture was stirred at room temperature for 10 min. After the reaction was completed, the organic layer was separated, washed with water, dried with anhydrous sodium sulfate and evaporated. The residue was purified by column chromatography to obtain solid compound 10. S5: Preparation of compound Cy3Tz 0.005 mmol Pd2(dba)3, 0.020 mmol ligand 1,2,3,4,5-pentaphenyl-1'-(di-tert-butylphosphino)ferricyanide, 0.303 mmol compound 10, 0.104 mmol compound Cy3-I, and 0.5 mmol N,N-dicyclohexylmethylamine were dissolved in 3.5 mL of anhydrous N,N-dimethylformamide. The reaction was carried out under inert gas protection in a microwave reactor at 50°C for 60 minutes. After the reaction was completed, the reaction solution was cooled to room temperature, and the product was purified by reversed-phase high-performance liquid chromatography. The fraction containing the target product was collected, and after lyophilization to remove the solvent from the fraction, compound Cy3-Tz was obtained. S6: Preparation of compound RNA-Cy3Tz The first terminal amino-modified oligonucleotide 5'-NH3-UGAUAAGCUA-3' was dissolved in 0.1 mM sodium bicarbonate buffer at pH 8.45 to obtain a third solution; then, the compound Cy3Tz obtained in S5 was dissolved in the organic solvent dimethyl sulfoxide (DMSO) to obtain a fourth solution; the third and fourth solutions were mixed and reacted at room temperature for 3 hours to obtain a reaction mixture; the reaction mixture was purified by reversed-phase high-performance liquid chromatography, and the fraction containing the target product was collected and immediately lyophilized to remove the solvent from the fraction to obtain the compound RNA-Cy3Tz; the molar ratio of the compound Cy3Tz to the first terminal amino-modified oligonucleotide was 30:

1.

3. The method for synthesizing the probe IDCR for detecting microRNA-21 based on bioorthogonal cyclic amplification reaction according to claim 2, characterized in that, The synthesis method of compound RNA-ABN is as follows: The fifth solution was obtained by dissolving the second-terminal amino-modified oligonucleotide 5'-UCAACAUCAGU-NH3-3' in 0.1 mM sodium bicarbonate buffer at pH 8.45; the sixth solution was obtained by dissolving the 7-azabenzonorbornene derivative in the organic solvent DMSO. After mixing the fifth and sixth solutions, the mixture was reacted at room temperature for 3 hours to obtain a reaction solution. The reaction solution was purified by reversed-phase high-performance liquid chromatography, and the fraction containing the target product was collected. The solvent in the fraction was removed by freeze drying to obtain the purified compound RNA-ABN. The molar ratio of 7-azabenzonorbornene derivative to the second terminal amino-modified oligonucleotide was 30:

1.

4. The method for synthesizing the probe IDCR for detecting microRNA-21 based on bioorthogonal cyclic amplification reaction according to claim 2, characterized in that, In steps S3 and S5 of the method for synthesizing compound RNA-Cy3Tz, the column chromatography is silica gel column chromatography, and the eluent used is a mixture of hexane and ethyl acetate in a volume ratio of 1:

1.

5. The method for synthesizing the probe IDCR for detecting microRNA-21 based on a bioorthogonal cyclic amplification reaction according to claim 3, characterized in that, In steps S3 and S5 of the synthesis methods for compounds RNA-ABN and RNA-Cy3Tz, the specific conditions for the reversed-phase high-performance liquid chromatography purification are as follows: the chromatographic column used is a Phenomenex Clarity Oligo-MS column; mobile phase A is an aqueous solution containing triethylamine and hexafluoroisopropanol; mobile phase B is methanol; the volume ratio of triethylamine, hexafluoroisopropanol, and water in mobile phase A is 0.4:30:1000. The gradient elution program was as follows: first, elute with a mixture of 95% mobile phase A and 5% mobile phase B for 1 minute, then increase the proportion of mobile phase B to 50% over 15 minutes, with a flow rate of 10 mL / min and a column temperature of 26 °C.

6. The application of the probe IDCR for detecting microRNA-21 based on bioorthogonal cyclic amplification reaction according to claim 1 in the preparation of early diagnostic reagents for lung cancer, characterized in that, When detecting lung cancer tumors, the concentration of the probe IDCR in the reagent is 1 μM.

7. The application of the probe IDCR for detecting microRNA-21 based on bioorthogonal cyclic amplification reaction as described in claim 6 in the preparation of early diagnostic reagents for lung cancer, characterized in that, The reagents also include a detection buffer solution; The detection buffer solution comprises 100 mM Tris·HCl and 150 mM NaCl, wherein the pH of Tris·HCl is 7.4.

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