Single nucleotide mutation detection probe and method integrating dynamic signal and accumulated signal

Abstract

The invention relates to the technical field of biological detection, in particular to a single nucleotide mutation detection probe and method integrating a dynamic signal and an accumulated signal. The single nucleotide mutation detection probe comprises a DNA hairpin H1, an RNA base embedded hairpin Sub, a DNA single strand E1, a DNA single strand E2 and a DNA hairpin H2, wherein the DNA hairpin H1, the RNA base embedded hairpin Sub, the DNA single strand E1 and the DNA single strand E2 form a DNA enzyme structure; the 5'terminal of the DNA hairpin H2 is modified with carboxyl molecules, and the 3 'terminal of the DNA hairpin H2 is modified with ferrocene molecules. The hairpin probe recognition technology is combined with DNA enzyme, the purpose of cyclic detection is achieved through the thermodynamic chain competition effect, the sensitivity of single base mutation detection is improved through an electrogenerated chemiluminescence detection method for indirectly detecting a mononucleotide mutant by recognizing an intermediate product chain in a cyclic reaction, and the detection sensitivity of the mononucleotide mutant is improved. And the accuracy of single base mutation recognition is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a single nucleotide mutation detection probe and method integrating dynamic signals and cumulative signals. Background Art

[0002] Single nucleotide mutations, as important biomarkers of human disease and drug resistance, are crucial for disease diagnosis and monitoring, as well as personalized precision medicine. By exploring the mechanisms of single nucleotide mutations, building mutation models, and studying genome evolution and population genetics, we can gain a deeper understanding of gene function and regulatory mechanisms, uncovering the mechanisms of mutation. Therefore, single nucleotide mutation detection is crucial in the analysis of disease mechanisms, precision medicine, and biotechnology development.

[0003] Traditional single-nucleotide mutation detection methods, such as enrichment culture, PCR (RT-qPCR), and next-generation sequencing (NGS), rely on amplification to qualitatively and quantitatively analyze and detect trace amounts of nucleic acids. These methods are complex to operate and require extremely high sample quality. Their capabilities for detecting mutant genes are limited and incompatible with routine clinical use. Furthermore, response strategies for nucleic acid single-base mutation detection mostly focus on a single detection mode based on a steady-state system. These strategies have their own limitations in distinguishing single-base mutations, resulting in high false-positive rates and insufficient accuracy in accurately identifying drug-resistance mutations, making them incapable of dynamically responding to and tracking targets.

[0004] The synergistic action of multiple units within biological dynamic networks, coupled with a high degree of multifunctional integration, are beneficial to currently developed artificial biosensor devices in terms of recognition capability, sensitivity, and specificity. A significant amount of research has been devoted to developing artificial dynamic networks that replicate the functions of natural processes. In actual molecular diagnostics, the use of enzyme-containing or enzyme-free assembly of biological dynamic networks can evolve a variety of dynamic molecular networks with different biological functions, and establish analytical methods for multi-signal coordinated responses. However, biological dynamic networks typically operate under non-equilibrium and dissipative conditions with greater complexity and functionality, making it difficult to capture dynamic details using static or averaged measurement methods. Furthermore, model construction often ignores environmental influences and metabolites, making it difficult to maintain high recognition capabilities and detection efficiency in complex background sample systems. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a single nucleotide mutation detection probe and method that integrates dynamic signals and cumulative signals.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the present invention provides a single nucleotide mutation detection probe that integrates dynamic signals and cumulative signals, comprising a DNA hairpin H1, an RNA base-intercalating hairpin Sub, a DNA single strand E1, a DNA single strand E2, and a DNA hairpin H2; Among them, DNA hairpin H1, RNA base embedded hairpin Sub, DNA single strand E1, and DNA single strand E2 constitute the DNA enzyme structure; The 5' end of the DNA hairpin H2 is modified with a carboxyl molecule, and the 3' end is modified with a ferrocene molecule.

[0007] The second aspect of the present invention provides a single nucleotide mutation detection platform that integrates dynamic signals and cumulative signals, which comprises the single nucleotide mutation detection probe described in the first aspect.

[0008] The third aspect of the present invention provides a method for preparing the above-mentioned single nucleotide mutation detection platform, comprising the following steps: annealing the DNA hairpin H1 and the RNA base embedded hairpin Sub separately, then adding equimolar amounts of DNA single strand E1 and DNA single strand E2 for mixed incubation to obtain a DNA enzyme-based circular recognition probe solution.

[0009] A fourth aspect of the present invention provides a method for detecting single nucleotide mutants that integrates dynamic signals and cumulative signals, which comprises using the above-mentioned single nucleotide mutation detection platform for detection.

[0010] The fifth aspect of the present invention provides the use of the above-mentioned single nucleotide mutation detection platform in the preparation of products for detecting single nucleotide mutations.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a single-nucleotide mutation detection probe and platform that integrates dynamic and cumulative signals. This approach combines hairpin probe recognition technology with DNA enzymes, achieving cyclic detection through thermodynamic chain competition. Furthermore, an electrochemiluminescence detection method indirectly detects single-nucleotide mutations by identifying intermediate chains in the cyclic reaction, improving the sensitivity of single-base mutation detection and enhancing the accuracy of single-base mutation identification. The specific mechanism is as follows: upon addition of a single-nucleotide mismatch target, T, pairs with the DNA hairpin H1 in the cyclic recognition probe, releasing it and subsequently pairing with DNA single strands E1 and E2. DNA single strands E1 and E2 simultaneously pair with an RNA base intercalating hairpin, Sub, linking hairpin H1 to form a DNA enzyme structure. DNA enzyme cleavage then occurs, cleaving the RNA base intercalating hairpin, Sub, into two fragments (Sub-fraction 1 and Sub-fraction 2). Sub-fraction 1 undergoes a chain displacement reaction with DNA hairpin H1, subsequently displacing the single-nucleotide mismatch target, T, and restoring hairpin H1. This results in a dynamic readout of the fluorescence signal, enabling a dynamic response of the detection signal. At the same time, Sub-fragmentation part 2 pairs with the DNA hairpin H2, thereby releasing the ferrocene molecule, resulting in the readout of the electrochemical signal. Since the cyclic reaction product Sub-fragmentation part 2 continues to accumulate with the reaction, the result of each dynamic response is accumulated in the electrochemical signal. Therefore, the reaction platform can not only realize each dynamic signal response, but also record the total amount of the response through the electrochemical signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which constitute a part of the present disclosure, are used to provide a further understanding of the present disclosure. The exemplary embodiments of the present disclosure and their descriptions are used to explain the present disclosure and do not constitute an improper limitation to the present disclosure.

[0013] Figure 1 Schematic diagram of the detection principle of an embodiment of the present invention; Figure 2 This is a gel electrophoresis characterization diagram of the DNA enzyme recognition and displacement probe system constructed in Example 1 of the present invention; Figure 3 This is the construction of the cyclic detection system in Example 2 of the present invention; wherein A is a gel electrophoresis characterization diagram of multiple cycles, B is a dynamic fluorescence spectrum diagram of whether a single cycle contains RNA base embedded in the hairpin over time, and C is a fluorescence spectrum diagram of the cyclic response of the cyclic detection system to the target T; Figure 4This is the performance verification of the circulating probe in Example 3 of the present invention; wherein A is the fluorescence intensity response of the circulating probe to different concentrations of T; B is the standard curve of the fluorescence intensity response of the circulating probe to different concentrations of T; C is the repeatability evaluation of the circulating probe; D is the selectivity evaluation of the circulating probe for the target; E is the dynamic fluorescence response graph of the circulating probe for single-base mismatch recognition; F is the specificity evaluation of the circulating probe for single-base mismatch recognition ability; Figure 5 Schematic diagram of an electrochemiluminescence detection platform with a planar electrode covering a hole to separate the sensing electrode and the luminescent electrode in Example 4 of the present invention; Figure 6 The electrochemiluminescence detection method in Example 4 of the present invention is used to detect the cyclic reaction products and indirectly detect single nucleotide mutants; wherein A is the electrochemiluminescence signal response standard curve of the ferrocene-based recognition probe to different S2 concentrations; B is the electrochemiluminescence signal response of the ferrocene-based recognition probe to different S2 concentrations. DETAILED DESCRIPTION

[0014] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0015] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0016] In the absence of conflict, the embodiments of the present invention and the features thereof may be combined with each other.

[0017] As previously mentioned, existing technologies for detecting single nucleotide mutations mostly focus on a single detection model based on steady-state systems. These methods have their own limitations in distinguishing single-base mutations, resulting in high false-positive rates and insufficient accuracy in accurately identifying drug-resistant mutations, making it impossible to dynamically respond to and track targets. Biological dynamic networks typically operate under non-equilibrium and dissipative conditions characterized by increased complexity and functionality. Static or averaged measurement methods struggle to capture dynamic details, and model construction often overlooks environmental influences and metabolites, making it difficult to maintain high recognition and detection efficiency in complex background sample systems.

[0018] In view of this, the present invention provides a single nucleotide mutation detection platform that integrates dynamic signals and cumulative signals, combines hairpin probe recognition technology with DNA enzyme, and achieves the purpose of cyclic detection through thermodynamic chain competition effect; at the same time, an electrochemiluminescence detection method for indirectly detecting single nucleotide mutants by identifying the intermediate product chain in the cyclic reaction is used to improve the sensitivity of single-base mutation detection and enhance the accuracy of single-base mutation identification.

[0019] A first typical embodiment of the present invention provides a single nucleotide mutation detection probe integrating dynamic signals and cumulative signals, comprising a DNA hairpin H1, an RNA base-intercalating hairpin Sub, a DNA single strand E1, a DNA single strand E2, and a DNA hairpin H2; The 5′ end of the DNA hairpin H1 is modified with a fluorescent group, the 3′ end is modified with a fluorescent quenching group, and the RNA base embedded in the middle of the hairpin Sub is embedded with riboadenosine (rA); The DNA hairpin H1 and the RNA base embedded hairpin Sub are linked by DNA single strand E1 and DNA single strand E2 to form a DNA enzyme structure, forming a DNA enzyme-based recognition and displacement probe.

[0020] Among them, after the hairpin probe H1 responds to the target T, it activates the DNA enzyme and cuts the hairpin Sub. The resulting broken chain competes with the target T, causing it to detach from the hairpin probe H1. H1 restores the closed loop, realizing the "detection-reset" cycle and breaking through the single detection limitation of the static probe; the dynamic recovery characteristics of fluorescence provide a new way to monitor mutations in real time.

[0021] The 5' end of the DNA hairpin H2 is modified with a carboxyl molecule, and the 3' end is modified with a ferrocene molecule.

[0022] In one or more embodiments, the fluorescent group modified at the 5' end of the DNA hairpin H1 includes but is not limited to ROX, HEX, FAM, TET, TAMRA, JOE, Cy3, Cy5, etc., and is not specifically limited here. In the present invention, the fluorescent group modified at the 5' end of the DNA hairpin H1 is FAM.

[0023] A second typical embodiment of the present invention provides a single nucleotide mutation detection platform that integrates dynamic signals and cumulative signals, which includes the above-mentioned single nucleotide mutation detection probe.

[0024] A third typical embodiment of the present invention provides a method for preparing the above-mentioned single nucleotide mutation detection platform, comprising the following steps: annealing the DNA hairpin H1 and the RNA base embedded hairpin Sub separately, then adding DNA single strand E1 and DNA single strand E2 for mixed incubation to obtain a DNA enzyme-based circular recognition probe solution.

[0025] In one or more embodiments, the annealing condition is heating at 95° C. for 5 minutes and naturally cooling to room temperature.

[0026] In one or more embodiments, the incubation temperature is 25° C. to 35° C., and the incubation time is 1 h to 3 h.

[0027] In the present invention, the incubation temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, etc., and the incubation time can be 1h, 1.5h, 2h, 2.5h or 3h, etc., but is not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0028] In one or more embodiments, the final concentration of DNA hairpin H1 is 400nM~600nM, the final concentration of RNA base intercalating hairpin Sub is 1μM~5μM, the final concentration of single-stranded DNA E1 is 50nM~200nM, and the final concentration of single-stranded DNA E2 is 50nM~200nM.

[0029] In one or more embodiments, the concentrations of the single-stranded DNA E1 and the single-stranded DNA E2 are the same or different.

[0030] In one or more embodiments, the concentrations of the single-stranded DNA E1 and the single-stranded DNA E2 are the same.

[0031] In the present invention, the final concentration of the DNA hairpin H1 can be 400nM, 450nM, 500nM, 550nM or 600nM, etc., the final concentration of the RNA base embedded in the hairpin Sub can be 1μM, 2μM, 3μM, 4μM or 5μ, etc., the final concentration of the MDNA single chain E1 can be 50nM, 100nM, 150nM or 200nM, etc., and the final concentration of the DNA single chain E2 can be 50nM, 100nM, 150nM or 200nM, etc., but are not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0032] In one or more embodiments, the preparation method further includes the step of preparing a sensing substrate assembled with recognition probes, specifically: pouring PDMS on ITO glass and curing it to form a PDMS film, cutting the PDMS film into a desired shape and placing it on ITO glass to make an electrolytic cell, and then incubating the annealed DNA hairpin H2 on the electrode surface to obtain a sensing substrate assembled with recognition probes.

[0033] In one or more embodiments, the steps of preparing a sensor substrate assembled with a recognition probe are specifically as follows: PDMS is poured on ITO glass and solidified to form a PDMS film, the PDMS film is cut into a desired shape and placed on ITO glass to make an electrolytic cell, chitosan with amino-terminal activation treatment is incubated on the electrode surface and the DNA hairpin H2 that has been annealed and carboxyl-terminal activated is added dropwise to the electrode surface to cause carboxylamide reaction coupling and fixation to obtain a sensor substrate assembled with a recognition probe.

[0034] In one or more embodiments, the chitosan after amino-terminal activation is incubated on the electrode surface by dissolving 1% chitosan in a solution of 1-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), stirring at room temperature to activate the amino end of chitosan, obtaining amino-terminal activated chitosan, and dropping it onto the ITO electrode sensing pole for incubation.

[0035] In one or more embodiments, the annealing and carboxyl end activation treatment of the DNA hairpin H2 is specifically as follows: the annealing conditions are heating at 95°C for 5 min and naturally cooling to room temperature to obtain the annealed DNA hairpin H2; EDC and NHS are weighed in a HEPES buffer solution, the annealed DNA hairpin H2 is added and stirred at room temperature to activate the carboxyl end of the DNA hairpin H2.

[0036] In one or more embodiments, the DNA hairpin H2 after annealing and carboxyl end activation treatment is added dropwise to the surface of the ITO electrode and incubated at 4°C for 4 hours to couple the DNA hairpin H2 with chitosan through a carboxylamine reaction and fix it to the ITO electrode sensing pole, thereby obtaining a sensing substrate assembled with a recognition probe.

[0037] In one or more embodiments, the PDMS curing condition is heating at 60 °C for 3 h.

[0038] In one or more embodiments, the molar ratio of EDC to NHS solution is 1:1 to 1.5.

[0039] In one or more embodiments, the molar ratio of EDC to NHS solution is 1:1.2.

[0040] In one or more embodiments, stirring is performed at room temperature for 1 hour to activate the amino termini of chitosan, thereby obtaining amino termini-activated chitosan.

[0041] In one or more embodiments, in the HEPES buffer, the concentration of HEPES is 0.02 M, the concentration of NaCl is 0.1 M, and the concentration of MgCl2 is 0.1 M.

[0042] In one or more embodiments, the incubation concentration of the DNA hairpin H2 is 1 μM to 5 μM, and the amount of DNA hairpin H2 added is 5 to 15 μL.

[0043] In the present invention, the incubation concentration of the DNA hairpin H2 can be 1 μM, 2 μM, 3 μM, 4 μM or 5 μ, and the added amount of the DNA hairpin H2 can be 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL or 15 μL, but is not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0044] A fourth typical embodiment of the present invention provides a method for detecting single nucleotide mutants that integrates dynamic signals and cumulative signals, including using the above-mentioned single nucleotide mutation detection platform for detection.

[0045] In one or more embodiments, the detection method comprises the following steps: S1. Annealing a DNA hairpin H1 and an RNA base embedded in a hairpin Sub in the presence of a buffer solution, adding a DNA single strand E1 and a DNA single strand E2 for incubation to construct a cyclic detection system, obtaining an initial fluorescence signal F0 of the cyclic detection system, adding the test DNA and incubating together to trigger a DNA enzyme digestion reaction, and obtaining a fluorescence signal F1 that changes over time; determining whether a single nucleotide mutation occurs in the test DNA by the difference between the initial fluorescence signal value F0 and the post-reaction fluorescence signal F1; S2. After the fluorescence signal stabilizes, the DNA to be tested is added again to obtain a fluorescence signal F2 that changes with time; the above steps are repeated to obtain fluorescence signals F3, F4, F5, etc. that change with time; and the change curves of the fluorescence signal values ​​F1, F2, F3, F4, F5, etc. are used to determine whether the DNA to be tested is cycled detected; S3, obtaining DNA enzyme digestion cycle reaction products through single and multiple cycle reactions; S4. Obtaining the ferrocene-based molecular recognition probe system in Ru(bpy)3 2+ The initial electrochemiluminescence signal ECL0 of the ferrocene molecule in the co-reaction electrochemiluminescence system with tri-n-propylamine is obtained; then the cyclic reaction product obtained in S3 is added and incubated together to obtain the electrochemiluminescence signal ECL1; S5. Determine whether the cyclic reaction product Sub fragment 2 is present by comparing the difference between the initial electrochemiluminescence signal ECL0 and the electrochemiluminescence signal ECL1 after co-incubation, and indirectly determine whether a single nucleotide mutation occurs.

[0046] In one or more embodiments, in step S1, the buffer is HEPES buffer.

[0047] In one or more embodiments, in the HEPES buffer, the concentration of HEPES is 0.02 M, the concentration of NaCl is 0.1 M, and the concentration of MgCl2 is 0.1 M.

[0048] In one or more embodiments, in step S1, the fluorescence spectrum is scanned under the conditions of excitation at 495 nm and scanning emission range of 510-600 nm to obtain an initial fluorescence signal F0.

[0049] In one or more embodiments, the concentration of the test DNA (or target T) is 40-100 nM.

[0050] In the present invention, the concentration of the DNA to be tested (or target T) can be 40 nM, 50 nM, 60 nM, 80 nM or 100 nM, etc., but is not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0051] In one or more embodiments, steps S1 to S3 combine hairpin probe recognition technology with DNA enzymes, and achieve the purpose of probe cycling detection through thermodynamic chain competition effects. The difference between the initial fluorescence signal F0 and the post-reaction fluorescence signal F1 is used to determine whether a single nucleotide mutation has occurred in the DNA to be tested. The specific reaction mechanism is as follows: the DNA sequence to be tested pairs with the DNA hairpin H1 in the cycling recognition probe and releases it, which then pairs with DNA single strands E1 and E2. DNA single strands E1 and E2 simultaneously pair with the RNA base embedded hairpin Sub, linking hairpin H1 to form a DNA enzyme structure. Then, a DNA enzyme cleavage reaction occurs, breaking the ribonucleotide embedded in the hairpin Sub into two parts. The broken part 1 of Sub undergoes a chain displacement reaction with the DNA hairpin H1, which then displaces the single nucleotide mismatch target T, restoring hairpin H1, resulting in a dynamic readout of the fluorescence signal. The change in the dynamic signal can be used to determine whether the nucleotide site is mutated.

[0052] It should be further explained that in determining whether a single nucleotide mutation has occurred in the DNA to be tested by the difference between the initial fluorescent signal F0 and the fluorescent signal F1 after the reaction, the determination of the difference between the initial fluorescent signal F0 and the fluorescent signal F1 after the reaction means that the fluorescent signal F1 after the reaction is higher than the initial fluorescent signal F0, and the specific size of the difference does not affect the determination of the result (whether a mutation has occurred).

[0053] In one or more embodiments, in step S4, the Ru(bpy)3 2+ Ru(bpy)3 in the co-reaction electrochemiluminescence system with tri-n-propylamine 2+ The final concentration is 100 μM, and the final concentration of tri-n-propylamine is 10 mM.

[0054] In one or more embodiments, in steps S4 to S5, whether a single nucleotide mutation occurs can be indirectly determined by identifying the intermediate product chain (Sub breakage portion 2) in the cyclic reaction, thereby further improving the sensitivity of single-base mutation detection and enhancing the accuracy of single-base mutation identification.

[0055] The above detection method detects single nucleotide mutations based on the dual modes of dynamic signals and cumulative signals, realizing the detection of single nucleotide mutations based on biological signals, and can also dynamically respond to and track the target. Compared with the traditional amplification-based method for qualitative and quantitative detection of single nucleotide mutations, the method provided by the present invention is simple to operate, has higher sensitivity and specificity, and is particularly suitable for complex background sample systems.

[0056] It should be further explained that the sequences of probes, single-stranded molecules, etc. in the single nucleotide mutation detection platform can be designed and implemented by those skilled in the art based on the mutation sites in the DNA to be detected. In principle, any means that can implement the detection method disclosed in the present invention should fall within the scope of protection of the present invention.

[0057] A fifth typical embodiment of the present invention provides the use of a single nucleotide mutation detection platform in the preparation of products for detecting single nucleotide mutations.

[0058] In one or more embodiments, the product can be a kit.

[0059] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0060] As an example, the nucleic acid sequences used in the examples of this application are shown in Table 1 below.

[0061] Table 1 Nucleotide sequences

[0062] Figure 1 The gene expression detection mechanism of the present invention is as follows: After the addition of a single nucleotide target T, T pairs with the DNA hairpin H1 in the circulating recognition probe and releases it, which then pairs with the DNA single strands E1 and E2. DNA single strands E1 and E2 simultaneously pair with the RNA base embedded in the hairpin Sub, linking the hairpin H1 to form a DNA enzyme structure. A DNA enzyme cleavage reaction then occurs, breaking the ribonucleotide embedded in the hairpin Sub into two parts. Sub-fragmented portion 1 undergoes a chain displacement reaction with the DNA hairpin H1, subsequently displacing the target T and restoring the hairpin H1, resulting in a dynamic readout of the fluorescence signal. Sub-fragmented portion 2 pairs with the DNA hairpin H2, releasing the ferrocene molecule and leading to an electrochemical signal readout.

[0063] Example 1 This example demonstrates the construction of a DNA enzyme-based recognition and displacement probe. H1, H2, and Sub were heated at 10 μM in HEPES buffer (0.02 M HEPES, 0.1 M NaCl, 0.1 M MgCl2) at 95°C for 5 minutes, followed by natural cooling to enhance the stability of the hairpin structure. The DNA hairpins were then mixed and incubated with E1 and E2 at 30°C to construct a DNA enzyme-based recognition and displacement probe.

[0064] Gel electrophoresis was used to characterize the construction of the DNAzyme-based recognition and displacement probe, including: DNAzyme structure formation, RNA base embedding into the hairpin Sub cleavage, target T displacement and cleavage product capture. First, the target T is captured by the hairpin H1 and combined with the auxiliary chain and substrate chain to form a DNAzyme structure with enzyme cleavage. Figure 2 As shown in A, lanes 1 to 4 show the results of the hairpin H1 capturing the target nucleic acid and the auxiliary chain to form a trimer structure. With the addition of the substrate hairpin Sub, it was observed that the sixth lane 2+ The buffer system produced a relatively high fifth channel (without Mg 2+ ) A new band with a faster migration rate indicates that the formation of DNA enzyme structure is related to the Mg-containing 2+ Under the conditions of , the substrate hairpin Sub is cut.

[0065] The product chains after enzyme cleavage were further characterized. Figure 2B, Lanes 1 to 4 show single bands of simulated product chains and the binding bands of product chains and corresponding binding chains. In lane 5, in the presence of probe H1, target T, and product chain S1, the color of the binding band of S1 to T is significantly darker than that of the other bands, indicating that there are more bound product chains, that is, product chain S1 has stronger binding to target T than hairpin H1. In lane 6, DNAzyme structure formation causes substrate chain cleavage. Addition of capture hairpin H2, which contains product S2, reveals the appearance of binding bands of S2 and H2. Significant band overlap is observed at the positions of the S1 and T binding bands, and the S2 band disappears, while the S1 band becomes lighter, indicating that the substrate chain is cleaved and the cleaved product chains S1 and S2 bind to T and H2, respectively.

[0066] Example 2 This embodiment further constructs a circulation detection system based on embodiment 1.

[0067] H1, H2, and Sub were heated at 95°C for 5 minutes at a concentration of 10 μM in HEPES buffer (0.02 M HEPES, 0.1 M NaCl, 0.1 M MgCl2). The mixture was then cooled to enhance the stability of the hairpin structure. The DNA hairpins were then mixed and incubated with E1 and E2 at 30°C to construct the DNA enzyme-based recognition and displacement probe. The target T1 was added and incubated at 30°C for 2 hours as one cycle. The same amount of target T was added and incubated at 30°C for 2 hours as a second cycle. The above loading and incubation steps were repeated to establish a cyclic assay.

[0068] Gel electrophoresis was performed on the initial state of the first cycle, the final state of the first cycle, the final state of the second cycle, and the final state of the third cycle to verify the cycle detection of the system. Figure 3 As shown in Figure A, lanes 5 to 8 represent the initial and final states of the first cycle, and the final states of the second and third cycles, respectively. The color of the binding band for product S1 and T was observed to gradually deepen with increasing reaction cycles, indicating the accumulation of this product binding chain and confirming the construction of the cyclic assay.

[0069] Based on the RNA base embedding hairpin Sub, the non-embedded hairpin Sub2 was designed and the dynamic fluorescence curves were compared. The annealed Sub and Sub2 were mixed and incubated with the annealed H1, H2, E1, and E2 in HEPES buffer (0.02 M HEPES, 0.1 M NaCl, 0.1 M MgCl2). The final concentration of hairpin H1 was 500 nM, the final concentration of DNA single-strand E1 and E2 was 100 nM, the final concentration of hairpin Sub and Sub2 was 2 μM, and the final concentration of hairpin H2 was 2 μM. Target T was added, and the dynamic fluorescence spectrum that changed with time was obtained by continuous scanning under the conditions of excitation at 495 nm and scanning emission range of 510~600 nm. The results are shown in Figure 2. Figure 3 As shown in B, the fluorescence spectrum curve of the hairpin Sub with RNA base embedded shows a trend of rapidly rising in a short period of time and then gradually falling back to the initial value. The fluorescence spectrum curve of the hairpin Sub2 with non-RNA base embedded only shows an increase in fluorescence value, further confirming that the breakage and displacement reaction of the hairpin Sub restores the hairpin probe.

[0070] The annealed H1, H2, E1, E2 and Sub were mixed and incubated in HEPES buffer (0.02 M HEPES, 0.1 M NaCl, 0.1 M MgCl2). The final concentration of hairpin H1 was 500 nM, the final concentration of DNA single-strand E1 and E2 was 100 nM, the final concentration of hairpin Sub and Sub2 was 2 μM, and the final concentration of hairpin H2 was 2 μM. After adding target T, the fluorescence spectrum was scanned under the conditions of excitation 495 nm and scanning emission range of 510~600 nm, which was used as the initial fluorescence signal. Subsequently, the fluorescence signal value was measured at intervals of 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 90 minutes. After the fluorescence signal recovered to the initial value, target T was added and the fluorescence signal value was measured again at the above time intervals. Figure 3 As shown in Figure B, the fluorescence signal value shows a trend of rapid increase in a short period of time, followed by a gradual decrease and then returning to the initial value. The reintroduction of the target chain repeats this signal change, confirming the establishment of a cyclic detection system.

[0071] Example 3 3.1 Performance evaluation of recyclable probes: In order to ensure the accuracy and reliability of the experimental results, a standard curve for the detection was first established. H1, H2, and Sub were heated at 95°C for 5 minutes at a concentration of 10 μM in HEPES buffer (0.02 M HEPES, 0.1 M NaCl, 0.1 M MgCl2), and naturally cooled to improve the stability of the hairpin structure. Subsequently, the above DNA hairpins were mixed and E1 and E2 were added and incubated at 30°C to construct a DNA enzyme-based recognition and displacement probe. The final concentration of hairpin H1 was 500 nM, the final concentration of DNA single-strand E1 and E2 was 100 nM, the final concentration of hairpin Sub was 2 μM, and the final concentration of hairpin H2 was 2 μM. Different concentrations of target T (final concentrations of 0, 40, 50, 60, 80, and 100 nM) were added to the system to form the recognition and displacement probe. The fluorescence intensity was measured after incubation at 30°C for 5 minutes. The results are shown in Figure 2. Figure 4 As shown in AB, the fluorescence intensity showed a significant dose-dependence with the increase of target T concentration.

[0072] By keeping the concentration of T constant (100 nM), repeated detection analysis was performed. Figure 4 As shown in Figure C, the stability and accuracy of this method are further verified.

[0073] 3.2 Specificity of recyclable probes A single base mismatch was designed at the position where T and hairpin H1 were paired as a mismatch target (SNV1). In addition, a random DNA chain was used as a negative target (Random, E. coli 、 S. aureus, S. flexneri Specifically, 100 nM T and equal concentrations of other negative targets and recognition and displacement probes were mixed in HEPES buffer for reaction, followed by DNA enzyme digestion and fluorescence intensity measurement.

[0074] First, prepare the recognition and displacement probe solution, where the final concentration of H1 is 500 nM, the final concentration of DNA single strands E1 and E2 is 100 nM, the final concentration of hairpin Sub is 2 μM, and the final concentration of hairpin H2 is 2 μM. 100 nM T and 100 nM of other negative targets (Random, E. coli 、 S. aureus, S. flexneri ), with excitation at 495 nm and scanning emission range of 510-600 nm, scan the fluorescence spectrum and record the fluorescence values ​​that change over time. The results are as follows Figure 4 As shown in D, by comparing the peak values ​​of the fluorescence time curve, the system has a good selective response to the target T.

[0075] Mixed solutions of target T and the single-base mismatch target SNV1 were prepared at different ratios, totaling 100 nM. The target T:SNV1 ratios were 1:100, 1:1000, 1:5000, and 1:10000, respectively. The mixed targets were added to the cyclic detection system solution, and fluorescence intensity was measured at intervals of 3, 5, 10, 15, and 20 minutes, using excitation at 495 nm and a scanning emission range of 510–600 nm. Figure 4 The results shown in EF indicate that the peak value of the temporal fluorescence spectrum decreases with the decrease of the target T content and shows a trend of rapidly rising in a short period of time and then falling; the probe can detect the above-mentioned mixed target solutions containing target T, and the detection efficiency can reach 0.01%, highlighting the application potential of this method in single-base mutation detection.

[0076] The specific cleavage of DNA enzymes coupled with chain displacement competition increases the single-base discrimination ability by 10 times (vs. traditional hairpin probes); the cyclic amplification effect reduces the detection limit to 0.01%, meeting the clinical needs of extremely low-abundance mutations.

[0077] Example 4 Verification of the Signal Generated by the Cyclic Reaction Product Simulated Chain S2 and the Electrode Modified with H2 First, PDMS (dimethylsiloxane) was cast onto an ITO glass and cured at 60°C for 3 hours to form a PDMS membrane. The PDMS membrane was then cut into the desired shape and placed on the ITO glass. The prepared PDMS membrane was then placed on the ITO glass to create an electrolytic cell.

[0078] 1% chitosan was dissolved in a solution of 1-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (molar ratio EDC:NHS = 1:1.2) at room temperature and stirred for 1 hour to activate the amino termini of the chitosan. The solution was then dripped onto the sensing electrode of an indium tungsten (ITO) electrode and dried at 37°C for 15 minutes before use. Three mg of EDC and 1.5 mg of NHS were weighed and dissolved in 0.5 mL of HEPES buffer (0.02 M HEPES, 0.1 M NaCl, 0.1 M MgCl2). Annealed DNA hairpin H2 was added and stirred at room temperature for 6 hours to activate the carboxyl termini of the DNA hairpin H2. 10 μL of this solution was dripped onto the surface of an ITO electrode and incubated at 4°C for 4 hours to allow the DNA hairpin H2 to couple with chitosan via a carboxyamide reaction and immobilize on the sensing electrode of the ITO electrode. Prepare an electrolyte containing 1M KCl, 10 mM tri-n-propylamine and 100 μM tris(2,2'-bipyridyl)ruthenium(II) chloride and add it to the luminescent electrode (such as Figure 5The electrochemiluminescence detection platform was constructed as shown in the figure. The initial electrochemiluminescence signal (ECL0) of the electrode was measured with a driving voltage range of 2 V to 5 V, a scan rate of 100 mV / s, and a photomultiplier tube voltage of 480 V. 10 μL of the cyclic reaction product, the simulated chain S2, was added dropwise to the H2-modified electrode surface for a 2-h reaction, and the post-reaction electrochemiluminescence signal (ECL1) was measured. Figure 6 The results shown in AB indicate that the electrochemiluminescence signal difference increases with the increase of S2 concentration, verifying the feasibility of indirect detection of targets by electrochemiluminescence.

[0079] Example 5 Preparation of a single nucleotide mutation detection platform integrating dynamic and cumulative signals This example prepared a single nucleotide mutation detection platform that integrates dynamic and cumulative signals. The platform includes preparing a DNA enzyme-based circulating recognition probe solution and constructing a sensor substrate assembled with the recognition probe. The construction process is as follows: S1. Construct a DNA enzyme-based cycling recognition probe. Heat the DNA hairpins H1 and Sub at 10 μM concentrations in HEPES buffer (0.02 M HEPES, 0.1 M NaCl, 0.1 M MgCl2) at 95°C for 5 minutes each. Allow to cool naturally to enhance the stability of the hairpin structure. Subsequently, mix the DNA hairpins and add E1 and E2. Incubate at 30°C to construct a DNA enzyme-based cycling recognition probe, yielding a DNA enzyme-based cycling recognition probe solution. The final concentration of hairpin H1 is 500 nM, the final concentrations of E1 and E2 are 100 nM, and the final concentration of hairpin Sub is 2 μM.

[0080] S2. Assemble the sensing substrate with the recognition probe. PDMS (dimethylsiloxane) was cast onto an indium tin oxide (ITO) glass and cured at 60°C for 3 h to form a PDMS membrane. The PDMS membrane was cut into the desired shape and placed on the ITO glass. The prepared PDMS membrane was then placed on the ITO glass to create an electrolytic cell. Chitosan that had been activated at the amino end was incubated on the electrode surface. A DNA hairpin (H2) that had been annealed and activated at the carboxyl end was then added dropwise to the electrode surface to couple and immobilize the chitosan via a carboxyamide reaction. Simultaneously, an electrolyte solution containing 1 M KCl, 10 mM tri-n-propylamine, and 100 μM tris(2,2'-bipyridyl)ruthenium(II) chloride was prepared. The initial electrochemiluminescence (ECL0) signal from the electrode was measured using a drive voltage range of 2 V to 5 V, a scan rate of 100 mV / s, and a photomultiplier tube voltage of 480 V.

[0081] S3. The single nucleotide mutation detection platform is used as follows: the sample DNA is heated at 95°C for 15 minutes, then added to a DNA enzyme-based cyclic recognition probe solution. The fluorescence spectrum is scanned with an excitation wavelength of 495 nm and an emission range of 510–600 nm, using this as the initial fluorescence signal F0. The sample is then incubated at 30°C for 2 hours. Fluorescence intensities F1, F2, and so on are measured at intervals of 5, 10, 15, 30, 45, 60, and 90 minutes, respectively. Fluorescence intensities F1, F2, and so on are measured after 5 minutes and compared with the initial fluorescence signal F0 to determine whether the sample has mutated. After the fluorescence signal returns to its initial value, the sample to be tested can be added and the fluorescence signal measurements repeated at these intervals to verify the cyclic system. The reaction solution, after 2 hours of incubation, is then added dropwise to the H2-modified electrode surface prepared in S2 and allowed to react for 2 hours. The post-reaction electrochemiluminescence signal ECL1 is measured, and the difference between ECL1 and ECL0 indirectly determines whether the sample has mutated.

[0082] Although the present invention is disclosed as above, the scope of protection disclosed by the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A single nucleotide mutation detection probe integrating dynamic signals and cumulative signals, characterized in that: Includes DNA hairpin H1, RNA base embedded hairpin Sub, DNA single strand E1, DNA single strand E2 and DNA hairpin H2; Among them, DNA hairpin H1, RNA base embedded hairpin Sub, DNA single strand E1, and DNA single strand E2 constitute the DNA enzyme structure; The 5′ end of the DNA hairpin H2 is modified with a carboxyl molecule, and the 3′ end is modified with a ferrocene molecule; The 5' end of the DNA hairpin H1 is modified with a fluorescent group, and the 3' end is modified with a fluorescence quenching group.

2. A single nucleotide mutation detection platform integrating dynamic signals and cumulative signals, characterized in that: It comprises the single nucleotide mutation detection probe according to claim 1.

3. The method for preparing the single nucleotide mutation detection platform according to claim 2, characterized in that: The following steps are involved: The DNA hairpin H1 and the RNA base embedded hairpin Sub are annealed separately, and then the DNA single strand E1 and the DNA single strand E2 are added and mixed and incubated to obtain a DNA enzyme-based circular recognition probe solution.

4. The preparation method according to claim 3, wherein The method also includes the steps of preparing a sensing substrate assembled with recognition probes, specifically: pouring PDMS on ITO glass and curing it to form a PDMS film, cutting the PDMS film into the required shape and placing it on the ITO glass to make an electrolytic cell, and then incubating the annealed DNA hairpin H2 on the electrode surface to obtain a sensing substrate assembled with recognition probes.

5. A method for detecting single nucleotide mutants that integrates dynamic signals and cumulative signals, characterized in that: Detection is performed using the single nucleotide mutation detection platform according to any one of claims 1-2.

6. The detection method according to claim 5, wherein The following steps are involved: S1: Annealing a DNA hairpin H1 and an RNA base embedded in a hairpin Sub in the presence of a buffer solution, adding E1 and E2 for incubation to construct a cyclic detection system, obtaining an initial fluorescence signal F0 of the cyclic detection system, adding the test DNA and incubating together to trigger a DNA enzyme digestion reaction, and obtaining a fluorescence signal F1 that changes over time; determining whether a single nucleotide mutation occurs in the test DNA by the difference between the initial fluorescence signal value F0 and the fluorescence signal F1 after the reaction; S2. After the fluorescence signal stabilizes, the DNA to be tested is added again to obtain a fluorescence signal F2 that changes with time; the above steps are repeated to obtain fluorescence signals F3, F4, F5, etc. that change with time; and the change curves of the fluorescence signal values ​​F1, F2, F3, F4, F5, etc. are used to determine whether the DNA to be tested is cycled detected; S3, after a single or multiple cycle reaction, obtaining the DNA enzyme digestion cycle reaction product and the Sub-fragmented portion 2; S4. Obtaining the ferrocene-based molecular recognition probe system in Ru(bpy)3 2+ The initial electrochemiluminescence signal ECL0 of the ferrocene molecule in the co-reaction electrochemiluminescence system with tri-n-propylamine is obtained; then the cyclic reaction product Sub fragmentation part 2 obtained in S3 is added and incubated together to obtain the electrochemiluminescence signal ECL1; S5. Determine whether the cyclic reaction product Sub fragment 2 is present by the difference between the initial electrochemiluminescence signal ECL0 and the electrochemiluminescence signal ECL1 after co-incubation, and indirectly determine whether a single nucleotide mutation occurs.

7. The detection method according to claim 6, wherein The following steps are involved: The DNA hairpin H1 and the RNA base embedded in the hairpin Sub are annealed in the presence of a buffer solution, and E1 and E2 are added for incubation to construct a circular detection system.

8. The detection method according to claim 6, wherein In step S4, the Ru(bpy)3 2+ Ru(bpy)3 in the co-reaction electrochemiluminescence system with tri-n-propylamine 2+ The final concentration is 100 μM, and the final concentration of tri-n-propylamine is 10 mM.

9. Use of the single nucleotide mutation detection platform according to claim 2 in the preparation of products for detecting single nucleotide mutations.

10. The use according to claim 9, characterized in that The product is a test kit.

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