Silver nano-cluster and preparation method and application thereof

Abstract

The invention belongs to the technical field of detection, and particularly relates to a silver nanocluster and a preparation method and application thereof. The preparation method of the silver nano-cluster comprises the following steps: (1) mixing and incubating a denatured DNA solution, a silver ion solution and a reducing agent to obtain the silver nano-cluster; wherein the DNA is a three-chain DNA, the three-chain DNA is formed by embedding a double-chain DNA into a third DNA chain through a Hoogsteen hydrogen bond or an anti-Hoogsteen hydrogen bond, the nucleotide sequence of the double-chain DNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the third DNA chain is as shown in any one of SEQ ID NO.2 to SEQ ID NO.7. The invention also discloses a preparation method of the DNA. The silver nanocluster is excellent in stability and high in quantum yield; and when the silver nanocluster is used for detecting miRNA, the sensitivity and the specificity are high, and the correlation between the detection result of a clinical sample and the detection result of gold standard RT-qPCR is high.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a silver nanocluster, its preparation method, and its application. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide, characterized by high heterogeneity and aggressiveness. Its local invasion, distant metastasis, and recurrence and drug resistance after treatment seriously threaten patients' lives and health. Traditional diagnostic methods, such as imaging examinations (ultrasound, mammography) and tissue biopsy, while core clinical approaches, have significant limitations in early detection of small lesions, monitoring of residual lesions after treatment, and accurate prognostic assessment. In recent years, molecular biology research has revealed that microRNAs (miRNAs) are key regulators of breast cancer development—by targeting and regulating the expression of target genes, miRNAs can influence malignant behaviors such as tumor cell proliferation, apoptosis, invasion, and metastasis. Furthermore, the miRNA expression profiles in the serum, plasma, and other bodily fluids of breast cancer patients show characteristic changes, closely related to tumor subtype, clinical stage, treatment response, and prognosis. Compared to traditional biomarkers, miRNAs offer advantages such as high stability in bodily fluids, ease of detection, and dynamic monitoring, providing new potential tools for early non-invasive diagnosis of breast cancer, refined molecular subtyping, and the development of personalized treatment strategies. However, their clinical translation relies on efficient and accurate detection technologies.

[0003] Advances in miRNA detection technology are the core driving force behind its clinical application. Currently, miRNA detection technology has formed a complete chain of "discovery-validation-clinical translation": Next-generation sequencing (NGS), with its unbiased and high-throughput characteristics, has become a powerful tool for discovering novel miRNA biomarkers and mapping global expression profiles; microarray technology has been widely used in medium- and high-throughput screening and differential expression analysis; real-time quantitative PCR (RT-qPCR), due to its excellent sensitivity, specificity, and quantitative accuracy, is considered the "gold standard" for the detection of known miRNAs. However, existing technologies have significant limitations: NGS is costly and involves complex data analysis; microarrays have relatively limited sensitivity and specificity; and RT-qPCR relies on pre-designed primers, making it difficult to meet diverse detection needs. To overcome these bottlenecks, sample processing, data analysis standardization, and technological integration have become key directions. Emerging rapid detection technologies based on nanomaterials and biosensors are booming, aiming to achieve rapid, low-cost point-of-care testing, bringing new hope for early breast cancer screening and dynamic monitoring.

[0004] Silver nanoclusters (AgNCs), composed of several to hundreds of silver atoms, possess properties such as high stability, good biocompatibility, ultra-small size, high quantum yield, excellent photostability, and low toxicity, making them a focus of attention in the field of biosensing. Among them, DNA-templated silver nanoclusters (DNA-AgNCs) exhibit highly tunable fluorescence properties due to the diversity of DNA sequences and structures—by altering the DNA template base sequence and length, the fluorescence emission wavelength can be adjusted from blue light to near-infrared. Modification of the auxiliary strand can further optimize optical properties, and they have been applied to the detection of metal ions, ATP, nucleotide mutations, and cancer cells. However, traditional DNA-AgNCs are mostly single-template, single-cluster structures, exhibiting drawbacks such as low quantum yield, weak fluorescence signal intensity, and poor signal stability, severely limiting their practical application and sensitivity improvement as biosensor probes. Therefore, overcoming these technical bottlenecks is crucial for DNA-AgNCs to leverage their advantages in miRNA detection. Summary of the Invention

[0005] Based on this, the present invention provides a method for constructing ultra-bright photoluminescent silver nanoclusters TAT-AgNCs using a rigid programmable triple-stranded DNA (TAT) scaffold. These silver nanoclusters have an ultra-high photoluminescence quantum yield (PLQY) of up to 98.89%, which can be used to detect miRNAs with high sensitivity.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: The present invention provides a method for preparing silver nanoclusters, the method comprising: (1) mixing denatured DNA solution with silver ion solution to obtain a mixture; (2) mixing the mixture with a reducing agent and incubating to obtain silver nanoclusters; wherein the DNA is triple-stranded DNA, the triple-stranded DNA is composed of double-stranded DNA embedded in a third DNA strand through Hoogsteen hydrogen bonds or anti-Hoogsteen hydrogen bonds, the nucleotide sequence of the double-stranded DNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the third DNA strand is as shown in any one of SEQ ID NO.2 to SEQ ID NO.7.

[0007] Preferably, in the above preparation method, the silver ion solution is AgNO3 solution; and / or the reducing agent is NaBH4.

[0008] Preferably, in the above preparation method, the molar ratio of denatured DNA, silver ions and reducing agent is 1:(10-24):(2-3.5).

[0009] More preferably, in the above preparation method, the molar ratio of denatured DNA, silver ions and reducing agent is 1:17:3.5.

[0010] Preferably, in the above preparation method, the incubation temperature is 25℃~37℃; and / or the incubation time is 30h~40h.

[0011] Preferably, in the above preparation method, the method for preparing denatured DNA includes: mixing DNA solution and magnesium ions, and then denaturing at 95℃~98℃ for 8min~12min.

[0012] In another aspect, the present invention provides a silver nanocluster prepared according to the above-described preparation method.

[0013] In another aspect, the present invention provides a reagent or kit for detecting miRNA, comprising the silver nanoclusters described above.

[0014] Preferably, the reagents or kits described above further include ESDA amplification reaction reagents and / or miRNA extraction reagents.

[0015] More preferably, in the above-described reagents or kits, the ESDA amplification reaction reagent includes a hairpin probe, the sequence of which is one or more of the sequences shown in SEQ ID NO.8 to SEQ ID NO.10.

[0016] In another aspect, the present invention provides the application of the above-mentioned silver nanoclusters or the above-mentioned reagents in the preparation of products for diagnosing breast cancer, wherein the serum of the subject expresses one or more of miR-122, miR-222 or miR-221.

[0017] The beneficial effects of this invention include at least the following: the silver nanoclusters provided by this invention exhibit excellent stability and high quantum yield; and when using these silver nanoclusters to detect miRNAs, the sensitivity and specificity are high, such as showing good linearity for different concentrations of miR-122, miR-222, and miR-221, with limits of detection of 0.32 pM, 0.66 pM, and 4.68 pM, respectively; in addition, the results of testing clinical samples show high correlation with the gold standard RT-qPCR results, with correlation r values ​​of 0.7716, 0.8731, and 0.9487, respectively. Attached Figure Description

[0018] Figure 1Fluorescence spectra, quantum yields, fluorescence lifetimes, and transmission electron microscopy (TEM) images of silver nanoclusters prepared with A-C34 single-chain, AT-C34 double-chain, or different TAT triple-chains; where A represents the fluorescence spectra of silver nanoclusters prepared with A-C34 single-chain, AT-C34 double-chain, or TAT-34-34 triple-chain; B represents the fluorescence intensity of silver nanoclusters prepared with different TAT triple-chains; C represents the quantum yield and fluorescence lifetime of silver nanoclusters prepared with TAT-34-34 triple-chain; and D represents the TEM image of silver nanoclusters prepared with TAT-34-34 triple-chain. Figure 2 UV-Vis absorption spectrum of silver nanoclusters prepared for TAT-34-34 triple chain; Figure 3 The effect of different preparation conditions on the fluorescence intensity of silver nanoclusters prepared for TAT-34-34 triple chains; where A represents the fluorescence intensity of silver nanoclusters prepared with different concentrations of NaBH4 aqueous solution, B represents the fluorescence intensity of silver nanoclusters prepared with different AgNO3 aqueous solutions, and C represents the fluorescence intensity of Mg... 2+ The effect of incubation temperature on the fluorescence intensity of the prepared silver nanoclusters; D represents the fluorescence intensity curves for different incubation temperatures and times. Figure 4 This invention demonstrates the detection of miRNA using silver nanoclusters (TAT-AgNCs) combined with ESDA amplification. A represents a schematic diagram illustrating the feasibility of using TAT-AgNCs as the detection probe; B is a gel electrophoresis image of the amplification products after ESDA amplification; C shows the detection of miR-122 without ESDA amplification; and D shows the detection of miR-122 after ESDA amplification. Figure 5 This is a performance test of the silver nanoclusters (TAT-AgNCs) combined with ESDA amplification for detecting miRNAs in this invention; wherein, A, B and C are the sensitivity of detecting miR-122, miR-222 and miR-221, respectively; D, E and F are the specificity of detecting miR-122, miR-222 and miR-221, respectively. Figure 6 This study presents a correlation analysis between the detection of serum miRNAs using silver nanoclusters (TAT-AgNCs) combined with ESDA amplification and the gold standard RT-qPCR. A, B, and C represent the detection of miR-122, miR-222, and miR-221 in serum, respectively. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0021] In a first aspect, embodiments of the present invention provide a method for preparing silver nanoclusters, the method comprising: (1) mixing denatured DNA solution with silver ion solution to obtain a mixture; (2) mixing the mixture with a reducing agent and incubating to obtain silver nanoclusters; The DNA is triple-stranded DNA, which is composed of a double-stranded DNA strand intercalated into a third DNA strand via Hoogsteen hydrogen bonds or anti-Hoogsteen hydrogen bonds. The nucleotide sequence of the double-stranded DNA is shown in SEQ ID NO.1, and the nucleotide sequence of the third DNA strand is shown in any one of SEQ ID NO.2 to SEQ ID NO.7.

[0022] It should be noted that this invention provides a method for constructing ultra-bright photoluminescent silver nanoclusters TAT-AgNCs using a rigid programmable triple-stranded DNA (TAT) scaffold. These silver nanoclusters possess an ultra-high photoluminescence quantum yield (PLQY) of up to 98.89%, enabling the detection of miRNAs with high sensitivity. Specifically, the double-stranded DNA must contain a sequence of homopurein-pyrimidine base pairs (i.e., one strand consists entirely of purine A / G, and the complementary strand consists entirely of pyrimidine T / C); the third strand (usually an oligonucleotide) is embedded in the major groove of the DNA double helix via Hoogsteen hydrogen bonds or anti-Hoogsteen hydrogen bonds, specifically binding to the bases on the purine strand.

[0023] In some specific examples, the silver ion solution in the above preparation method is AgNO3 solution; and / or the reducing agent is NaBH4.

[0024] It should be noted that the silver ion solution and reducing agent used in the preparation method of the present invention are known in the art, including but not limited to the silver ion solution and reducing agent listed above.

[0025] In some specific examples, the molar ratio of denatured DNA, silver ions and reducing agent in the above preparation method is 1:(10-24):(2-3.5).

[0026] It should be noted that the molar ratio of denatured DNA, silver ions, and reducing agent in this invention affects the performance of the prepared silver nanoclusters. The molar ratio in this invention can be 1:(10-24):(2-3.5), such as 1:13:3, 1:17:3, or 1:20:3.5. In particular, the silver nanoclusters prepared with a molar ratio of 1:17:3.5 exhibit superior performance.

[0027] In some specific examples, the incubation temperature in the above preparation method is 25℃-37℃; and / or the incubation time is 30h-40h.

[0028] It should be noted that the incubation temperature in this invention is preferably 25℃~37℃, such as 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃ or 36℃, etc., and the incubation time based on this temperature can be 30h-40h, such as 33h, 35h or 37h.

[0029] In some specific examples, the denatured DNA preparation method described above includes: mixing the DNA solution with magnesium ions and then denaturing it at 95℃~98℃ for 8min~12min.

[0030] It should be noted that the addition of magnesium ions during the preparation of denatured DNA in this invention can enhance the fluorescence intensity of the prepared silver nanoclusters. The magnesium ions used are those known in the art, such as magnesium acetate.

[0031] Secondly, embodiments of the present invention provide a silver nanocluster prepared according to the above-described preparation method.

[0032] It should be noted that the silver nanoclusters in this invention exhibit high stability, and when used to detect miRNAs, they demonstrate high sensitivity and specificity. For example, they show good linearity for different concentrations of miR-122, miR-222, and miR-221, with limits of detection of 0.32 pM, 0.66 pM, and 4.68 pM, respectively. Furthermore, the results of testing clinical samples show high correlation with the gold standard RT-qPCR results, with correlation r values ​​of 0.7716, 0.8731, and 0.9487, respectively.

[0033] Thirdly, embodiments of the present invention provide a reagent or kit for detecting miRNA, which includes the silver nanoclusters described above.

[0034] It should be noted that the uniqueness of the silver nanoclusters in this invention lies not only in their excellent fluorescence performance but also in the inherent metastability of their triple-strand structure. Based on this, this invention develops an enzyme-mediated strand displacement-off (EDTO) sensing strategy: targeting miRNA to trigger a strand displacement amplification reaction to generate a large amount of product. This strategy achieves a detection limit in the femtomolar to picomolar (fM-pM) range and exhibits excellent single-base discrimination ability for various cancers. The successful validation of this technology in clinical samples, coupled with convenient "one-pot" operation, strongly emphasizes its potential for laboratory-to-bedside applications in fields such as precision medicine. Specifically, based on the fluorescence-enhancing properties of the triple-cluster silver probe, the triple-cluster probe is applied by using strand displacement. By adding a target, the "side-by-side" triple-cluster structure is opened, thereby achieving the enhanced fluorescence turn-off effect. Furthermore, compared with traditional miRNA detection methods (e.g., RT-qPCR-based detection methods), the EDTO biosensor constructed in this invention greatly simplifies the workflow, enabling single-step detection of miRNA. More importantly, it eliminates the reliance on specialized equipment such as qPCR thermal cyclers; only a standard microplate reader is needed for signal acquisition.

[0035] It should be noted that the silver nanoclusters in this invention can be prepared into reagents or kits for detecting miRNA, and the forms of the reagents or kits are known in the art.

[0036] In some specific examples, the reagents or kits described above may also include ESDA amplification reaction reagents and / or miRNA extraction reagents.

[0037] It should be noted that the silver nanoclusters in this invention can be combined with ESDA amplification reaction reagents to improve detection accuracy. Specifically, miRNA is first amplified using an ESDA amplification reaction to obtain amplification products, and the reaction between the silver nanoclusters and the amplification products can improve reaction sensitivity and accuracy. Furthermore, the ESDA amplification reaction reagents may also include one or more of HP, Klenow fragments, Nb.BbvCI, dNTPs, 10X rCutSmart™ Buffer, or enzyme-free water. In addition, when the target substance is serum, miRNA can be extracted first using a miRNA extraction reagent before detection.

[0038] In some specific examples, the ESDA amplification reaction reagents in the above-described reagents or kits include hairpin probes, the sequences of which are one or more of the sequences shown in SEQ ID NO. 8 to SEQ ID NO. 10.

[0039] Fourthly, embodiments of the present invention provide the application of the above-mentioned silver nanoclusters or the above-mentioned reagents in the preparation of products for diagnosing breast cancer, wherein the serum of the subject expresses one or more of miR-122, miR-222 or miR-221.

[0040] It should be noted that the silver nanoclusters in this invention are particularly suitable for the detection of miR-122, miR-222, or miR-221 in serum. Good linearity is observed for different concentrations of miR-122, miR-222, and miR-221, with limits of detection of 0.32 pM, 0.66 pM, and 4.68 pM, respectively. Furthermore, the results of the clinical sample tests show high correlation with the gold standard RT-qPCR results, with correlation r values ​​of 0.7716, 0.8731, and 0.9487, respectively.

[0041] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0042] The sequences used in the following examples are shown in Table 1 below.

[0043] Table 1. Sequence information used in the examples

[0044] Preparation Examples and Comparative Examples Example 1 (1) Add Milli-Q water (pure water) to the DNA template (TAT-34-34 triple strand, abbreviated as 34-34, which is formed by the combination of TT-C34 and A-C34 in Table 1 above), and then add 4 mM magnesium acetate; then denature at 95 °C for 10 min, and then slowly cool to room temperature to obtain a denatured DNA solution (concentration of 100 μM). (2) Add AgNO3 aqueous solution (1400 μM) to the above denatured DNA solution (volume ratio 1:1), then vortex in the dark for 1 min and in an ice bath for 30 min to obtain a mixed solution; (3) Add NaBH4 aqueous solution (sodium borohydride aqueous solution) (freshly prepared, concentration of 350 μM) to the above mixed solution (volume ratio of AgNO3 aqueous solution is 1:1), vortex for 1 min, and incubate at 25°C for 36 hours to obtain silver nanoclusters TAT-AgNCs, and store at 4°C.

[0045] Examples 2 to 6 Examples 2 to 6 are largely the same as Example 1, except that the DNA templates are different. Otherwise, they are the same as Example 1, and different silver nanoclusters are prepared. The DNA templates used in Examples 2 to 6 are shown below: Example 2: TAT-34-22 triple chain, abbreviated as 34-22, is formed by combining TT-C34 and A-C22 from Table 1 above; Example 3: TAT-34-33 triple chain, abbreviated as 34-33, is formed by the combination of TT-C34 and A-C33 in Table 1 above; Example 4: TAT-34-43 triple chain, abbreviated as 34-43, is formed by the combination of TT-C34 and A-C43 in Table 1 above; Example 5: TAT-34-44 triple chain, abbreviated as 34-44, is formed by the combination of TT-C34 and A-C44 in Table 1 above; Example 6: TAT-34-55 triple chain, abbreviated as 34-55, is formed by combining TT-C34 and A-C55 from Table 1 above.

[0046] Three Examples 7 to 10 Examples 7 to 10 are largely the same as Example 1, except that the concentration of the NaBH4 aqueous solution is different. Otherwise, they are the same as Example 1, and different silver nanoclusters are prepared. The concentrations of the NaBH4 aqueous solution in Examples 7 to 10 are 200 μM, 850 μM, 1700 μM and 3400 μM, respectively.

[0047] Examples 11 to 14 Examples 11 to 14 are largely the same as Example 1, except that the concentration of the AgNO3 aqueous solution is different. Otherwise, they are the same as Example 1, and different silver nanoclusters are prepared. The concentrations of the AgNO3 aqueous solution in Examples 11 to 14 are 1000 μM, 1400 μM, 2000 μM and 2400 μM, respectively.

[0048] Example 15 Example 15 is largely the same as Example 1, except that magnesium acetate was not added during the denaturation process in Example 15. Otherwise, the silver nanoclusters were prepared.

[0049] Examples 16 to 17 Examples 16 and 17 are largely the same as Example 1, except that the incubation temperature and incubation in step (3) are different, while the rest are the same as in Example 1, and different silver nanoclusters are prepared; wherein, the incubation temperatures of Example 16 and Example 17 are 4℃ and 37℃, respectively, and the incubation times are 0h to 80h, respectively.

[0050] Comparative Example 1 Comparative Example 1 is largely the same as Example 1, except that the DNA template is different, while everything else is the same as Example 1. In Comparative Example 1, the DNA template is the T-C34 single strand in Table 1 above, and silver nanoclusters T-AgNCs are prepared.

[0051] Comparative Example 2 Comparative Example 1 is largely the same as Example 1, except that the DNA template is different, while everything else is the same as Example 1. In Comparative Example 1, the DNA template is AT-C34 double strand, and silver nanoclusters AT-AgNCs are prepared. The AT-C34 double strand is formed by the combination of A-C34 and T-C34 in Table 1 above.

[0052] Characterization test The silver nanoclusters prepared in Example 1, Comparative Examples 1 and 2 were subjected to fluorescence spectroscopy tests, and the results are as follows: Figure 1 As shown in Figure A (the small image in the upper right corner is a photograph of the actual fluorescent sample).

[0053] The silver nanoclusters prepared in Examples 1 to 6 were subjected to fluorescence spectroscopy testing, and the results are as follows: Figure 1 As shown in B, the results show that the silver nanoclusters (34-34) prepared in Example 1 have the strongest fluorescence.

[0054] The silver nanoclusters prepared in Example 1 were subjected to fluorescence lifetime detection, and the results are as follows: Figure 1 As shown in Figure C, the results show that the fluorescence lifetime (τ) of the silver nanocluster is 4.40 ns and the fluorescence quantum yield (QY) is as high as 98.89%, indicating its excellent fluorescence performance.

[0055] The silver nanoclusters prepared in Example 1 were tested using transmission electron microscopy, and the results are as follows: Figure 1 As shown in Figure D (the small image in the upper right corner is a particle size distribution histogram), the results show that the prepared silver nanoclusters have good dispersibility, with the diameter of the silver nanoclusters mainly concentrated around 2 nm, and the size uniformity is high.

[0056] The silver nanoclusters prepared in Example 1, Comparative Examples 1 and 2 were subjected to UV-Vis absorption spectroscopy tests, and the results are as follows: Figure 2 As shown, the results indicate that the formation of more than 500 peaks signifies the successful preparation of the silver clusters, while the peak at 570 corresponds to its excitation wavelength.

[0057] The silver nanoclusters prepared in Examples 1 and 7 to 10 were subjected to fluorescence spectroscopy tests, and the results are as follows: Figure 3 As shown in A, the silver nanoclusters prepared in Example 1 and Examples 11 to 14 were also subjected to fluorescence spectroscopy tests, and the results are as follows. Figure 3As shown in Figure B, it can be seen from the above that the fluorescence is strongest when the molar ratio of DNA, AgNO3, and NaBH4 in the solution is 1:17:3.5.

[0058] The silver nanoclusters prepared in Examples 1 and 15 were subjected to fluorescence spectroscopy testing, and the results are as follows: Figure 3 As shown in C, the results show that Mg 2+ It can enhance fluorescence intensity.

[0059] The silver nanoclusters prepared in Examples 1 and 16 to 17 were subjected to fluorescence spectroscopy tests, and the results are as follows: Figure 3 As shown in Figure D, the results show that the fluorescence intensity at 25℃ and 37℃ is higher than that at 4℃ at different incubation times. Before 20 hours, the fluorescence intensity at 37℃ is higher than that at 25℃, and after 20 hours, the fluorescence intensity at 25℃ is higher than that at 37℃.

[0060] Performance testing (I) Stability test of silver nanoclusters (1) The sequence at the loop of the silver nanocluster prepared in Example 1 was designed to destroy the structure of the three cluster probes and obtain the comparative silver nanocluster; (2) The fluorescence intensity was detected after the silver nanoclusters prepared in Example 1 and the comparative silver nanoclusters were combined with miR-122, miR-222 and miR-221 respectively.

[0061] The results showed that the fluorescence intensity of the three miRNAs bound to the control silver nanoclusters was slightly lower than that of the silver nanoclusters prepared in Example 1, but the change was not significant. This indicates that the fluorescence intensity of the silver nanoclusters bound to miRNAs in this invention does not change drastically due to changes in the loop sequence, demonstrating its excellent fluorescence stability.

[0062] (ii) miRNA amplification test As can be seen from the above, the fluorescence intensity of the silver nanoclusters prepared in Example 1 binding with miRNA is relatively weak. Therefore, this test introduces enzyme chain displacement amplification (ESDA) technology to pre-amplify the target before binding it with the silver nanoclusters for detection. The schematic diagram of the detection principle is shown below. Figure 4 As shown in Figure A, the specific details are as follows: After the target miRNA activates the amplification system, it generates a large number of specific nucleic acid fragments, which combine complementaryly with the silver nanoclusters to destroy their three-cluster structure, triggering fluorescence quenching (turn-off); while samples without targets cannot trigger amplification, and the fluorescence signal of the silver nanoclusters will remain stable, thereby achieving the purpose of detecting miRNA.

[0063] The miRNA was amplified using ESDA to obtain amplification products. The ESDA amplification conditions were as follows: the corresponding hairpin probe was heated at 95°C for 10 minutes and cooled to room temperature to obtain MHP. Then, the amplification system was prepared according to the following formula: 2 μL miRNA, 10 μL 1 μMHP, 1 μL 5 U / μL Klenow fragment, 3 μL 20 U / μL Nb.BbvCI, 12.5 μL 5 mM dNTPs, 5 μL 10X rCutSmart@Buffer, and 16.5 μL enzyme-free water. After incubation at 37°C for 2 hours, the enzyme was inactivated by heating to 80°C for 20 minutes. Thus, amplified miRNA targets of different concentrations were obtained. The hairpin probe sequences corresponding to different miRNAs are shown in Table 2 below.

[0064] Table 2 Hairpin probe sequences corresponding to different miRNAs

[0065] The amplified products were then tested using polyacrylamide gel electrophoresis (PAGE), and the results are as follows: Figure 4 As shown in Figure B, the results indicate that ESDA can successfully amplify miRNA.

[0066] (III) Construction of miRNA detection method Fluorescence spectroscopy was performed after miR-122 was combined with the silver nanoclusters prepared in Example 1, and a blank sample (without miR-122) was used as a control. The test results are as follows: Figure 4 As shown in Figure C, the results indicate that when ESDA is not used and only miR-122-mediated turn-off is present, the fluorescence signal containing miR-122 is reduced by only 3.58% compared to the blank control; miR-122 was amplified using the amplification method described in (II) above to obtain the miR-122 amplification product. Then, the miR-122 amplification product was combined with the silver nanoclusters prepared in Example 1, and fluorescence spectroscopy was performed. A blank sample (without miR-122) was used as a control. The test results are as follows: Figure 4 As shown in Figure D, the results indicate that when ESDA was used and only miR-122-mediated turn-off was present, the fluorescence signal of the miR-122-containing amplified product was reduced by 58.60% compared to the blank control. These results demonstrate that the ESDA-mediated TAT-AgNCs probe turn-off sensor has the potential to achieve highly sensitive detection of miRNAs.

[0067] (iv) Specificity and sensitivity analysis of miRNA detection methods (1) Sensitivity Different concentrations of miR-122 were amplified using the amplification method described in (II) above to obtain different miR-122 amplification products. These products were then combined with the silver nanoclusters prepared in Example 1, and fluorescence intensity was measured using fluorescence spectroscopy. Curves showing the relationship between different miR-122 concentrations and fluorescence intensity were then plotted. The results are as follows: Figure 5 As shown in Figure A, the results indicate that the fluorescence intensity and the logarithm of the miR-122 concentration maintain a good linear relationship between 1 pM and 100 nM. The linear equation is ΔI = 232.18lgcmiR-122 + 2957.14, the linear correlation coefficient R = 0.9905, and the limit of detection (LOD) is 0.32 pM.

[0068] The detection limit of miR-222 was tested according to the above-described test method for miR-122 detection limit, and the results are as follows. Figure 5 As shown in Figure B, the results indicate that the fluorescence intensity and the logarithm of the miR-222 concentration maintain a good linear relationship between 1 pM and 100 nM, with the linear equation being ΔI = 264.75lgcmiR-222 + 3376.97 (R = 0.9926) and the limit of detection (LOD) being 0.66 pM.

[0069] The detection limit of miR-221 was tested using the same method as described above for miR-122, and the results are as follows: Figure 5 As shown in Figure C, the results show that the fluorescence intensity and the logarithm of the miR-221 concentration maintain a good linear relationship between 1 pM and 100 nM, with the linear equation being ΔI = 336.05lgcmiR-221 + 3891.14 (R = 0.9904) and the limit of detection (LOD) being 4.68 pM.

[0070] (2) Specificity In the following tests, miR-221, miR-222, and miR-122 and their mismatch sequences are shown in Table 3.

[0071] Table 3 miR-221, miR-222, and miR-122 and their mismatch sequences

[0072] miR-122 was mismatched to form miR-122-SM, miR-122-DM, and miR-122-TM, with a blank control, miR-221 control, and miR-222 control set up. Then, miR-122, mismatched miR-122, the blank control, and the miR-221 and miR-222 controls were amplified according to the amplification method described in (II) above to obtain different amplified products. These different amplified products were then combined with the silver nanoclusters prepared in Example 1, and fluorescence spectroscopy was performed to obtain fluorescence intensity. The difference ΔI between the amplified products and the blank control was calculated. The results are as follows: Figure 5 As shown in Figure D, the results demonstrate that the silver nanoclusters exhibit excellent specificity for miR-122.

[0073] miR-222 was mismatched to form miR-222-SM, miR-222-DM, and miR-222-TM, with a blank control, miR-122 control, and miR-221 control set up. Then, miR-222, mismatched miR-222, the blank control, and the miR-122 and miR-221 controls were amplified according to the amplification method described in (II) above to obtain different amplified products. These different amplified products were then combined with the silver nanoclusters prepared in Example 1, and fluorescence spectroscopy was performed to obtain fluorescence intensity. The difference ΔI between the fluorescence intensity and the blank control was calculated. The results are as follows: Figure 5 As shown in Figure E, the results demonstrate that the silver nanoclusters exhibit excellent specificity for miR-222.

[0074] miR-221 was mismatched to form miR-221-SM, miR-221-DM, and miR-221-TM, with a blank control, miR-222 control, and miR-122 control also set up. Then, miR-221, mismatched miR-221, the blank control, and the miR-222 and miR-122 controls were amplified according to the amplification method described in (II) above to obtain different amplification products. These different amplification products were then combined with the silver nanoclusters prepared in Example 1, and fluorescence spectroscopy was performed to obtain fluorescence intensity. The difference ΔI between the fluorescence intensity and the blank control was calculated. The results are as follows: Figure 5 As shown in Figure F, the results demonstrate that the silver nanoclusters exhibit excellent specificity for miR-221.

[0075] The above results demonstrate that the biosensing strategy of this invention can achieve high sensitivity and high specificity detection of miR-122, miR-221, and miR-222.

[0076] (v) Clinical sample testing To evaluate the clinical application potential of the EDTO biosensor, plasma samples were collected from 10 healthy volunteers and 50 breast cancer patients. Total miRNAs in plasma were extracted using a commercially available kit (miRcute Serum Plasma miRNA Extraction and Isolation Kit (DP503)). The expression levels of miRNAs were analyzed in parallel using the miRNA detection methods described above and RT-qPCR kits (Takar's qPCR_RR820A and reverse transcription_RR047A, with the detection steps as described above).

[0077] The results showed that the two detection methods yielded relatively consistent results, with a strong correlation observed between the two methods used to detect miR-222 and miR-221, with Pearson's r values ​​of 0.8731 and 0.9487*, respectively (see [link to Pearson's method]). Figure 6 A and Figure 6 B); the correlation obtained for miR-122 was modest (Pearson's r = 0.7716), which can be attributed to the relatively low levels of miR-221 expression in most samples, leading to large variability in the analysis (see B). Figure 6 C). The above results further demonstrate the high sensitivity, specificity, and stability of the miRNA detection method of this invention for potential applications in liquid biopsy-based cancer diagnosis.

[0078] 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 it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing silver nanoclusters, characterized by, The preparation method comprises: (1) mixing a denatured DNA solution with a silver ion solution to obtain a mixed solution; (2) mixing the mixed solution with a reducing agent and incubating to obtain silver nanoclusters; wherein the DNA is triple-stranded DNA, the triple-stranded DNA is formed by inserting a third DNA strand into a double-stranded DNA through Hoogsteen hydrogen bonding or reverse Hoogsteen hydrogen bonding, the nucleotide sequence of the double-stranded DNA is shown in SEQ ID NO. 1, and the nucleotide sequence of the third DNA strand is shown in any one of SEQ ID NO. 2 to SEQ ID NO.

7.

2. The preparation method of claim 1, wherein the silver ion solution is an AgNO3 solution; and / or the reducing agent is NaBH4; and / or the molar ratio of the denatured DNA, the silver ion and the reducing agent is 1: (10-24): (2-3.5). The molar ratio of the denatured DNA, the silver ion and the reducing agent is 1:17:3.

5.

4. The preparation method of any one of claims 1 to 3, wherein the incubation temperature is 25-37℃; and / or the incubation time is 30-40h. The preparation method of the denatured DNA comprises mixing a DNA solution with magnesium ions and denaturing at 95-98℃ for 8-12min.

3. The production method according to claim 2, characterized by, The silver nanoclusters are prepared by the preparation method of any one of claims 1 to 5. The silver nanoclusters of claim 6. The reagent or kit further comprises ESDA amplification reaction reagents and / or miRNA extraction reagents. The ESDA amplification reaction reagents comprise hairpin probes, and the sequence of the hairpin probes is one or more of the sequences shown in SEQ ID NO. 9 to SEQ ID NO.

11.

5. The production method according to any one of claims 1 to 3, characterized by, 10. Use of the silver nanoclusters of claim 6 or the reagent of any one of claims 7 to 9 in the preparation of a product for diagnosing breast cancer, wherein the serum of the subject expresses one or more of miR-122, miR-222 or miR-221.

6. Silver nanoclusters characterized in that, ​ 7. A reagent or a kit for detecting miRNA, characterized by, ​ 8. The reagent or kit according to claim 7, wherein ​ 9. The reagent or kit according to claim 8, wherein ​ ​

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