MiRNA electrochemical detection kit and detection method based on one-step entropy-driven reaction loop

Abstract

A miRNA electrochemical detection kit based on a one-step method entropy-driven reaction loop comprises a plurality of functional chains and TDN probe skeleton chains used for synthesizing an entropy-driven tetrahedral DNA reaction loop system, the functional chains comprise auxiliary chains T-A1 and A2 and a signal chain Biotin-A3, and four chains of a TDN probe comprise TetraA, TetraB, TetraC and TetraD. The miRNA electrochemical detection kit detection method based on the one-step entropy-driven reaction loop is used for the miRNA electrochemical detection method, a tetrahedral DNA nanostructure is used as a support and a substrate of the entropy-driven reaction loop, and the spatial arrangement of probes on the surface of an electrode is optimized, so that the hybridization efficiency and the entropy-driven reaction efficiency are improved, and the detection sensitivity is improved. And necessary structural interaction is promoted. By utilizing the thermodynamic principle of entropy, a signal amplification strategy without nuclease dependence is realized, and the reaction efficiency equivalent to that of a homogeneous liquid phase is achieved. A signal amplification mode of directly performing entropy driving reaction on the surface of the electrode is adopted, so that the detection time and the operation steps are greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of biosensor detection, and in particular to a miRNA electrochemical detection kit and a detection method based on a one-step entropy-driven reaction circuit. Background Art

[0002] In modern medical research, microRNA (miRNA) has attracted extensive attention as an important gene regulatory factor. MiRNA regulates gene expression by binding to the mRNA of the target gene, thereby affecting cell growth, differentiation and apoptosis. Studies have shown that abnormal expression of miRNA is closely related to the occurrence and development of many diseases, especially cancer. Therefore, it is of great clinical significance to develop highly sensitive and specific detection methods to identify these biomarkers at an early stage.

[0003] Traditional miRNA detection methods usually rely on PCR technology. Although it has high sensitivity, it has high requirements for equipment and operation, which limits its application in primary care. In recent years, electrochemical biosensors have gradually become an important tool for miRNA detection due to their high sensitivity, low cost and portability. In this regard, sensors based on tetrahedral DNA nanostructure probes have attracted widespread attention due to their programmability and adjustability. However, existing electrochemical methods usually require additional enzymatic reactions, which increases the complexity and cost of the system. In order to overcome the above limitations, researchers began to explore the potential of entropy-driven reactions. Entropy-driven reactions use thermodynamic principles to increase the configurational entropy of the reaction system to achieve signal amplification without additional enzymes. This novel reaction mechanism not only simplifies the detection process, but also improves sensitivity. Therefore, electrochemical sensors that combine entropy-driven reaction loops with tetrahedral DNA nanostructure probes are expected to significantly improve the detection performance of miRNA and provide new solutions for early cancer diagnosis. Summary of the invention

[0004] The present invention provides a miRNA electrochemical detection kit and a detection method based on a one-step entropy-driven reaction circuit.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] A miRNA electrochemical detection kit based on a one-step entropy-driven reaction loop includes several functional chains and various TDN probe backbone chains for synthesizing an entropy-driven tetrahedral DNA reaction loop system. The functional chains include auxiliary chains T-A1 and A2 and a signal chain Biotin-A3. The four chains of the TDN probe include TetraA, TetraB, TetraC and TetraD.

[0007] Preferably, the A2 chain is 18 bases long, the average chain length of TetraB, TetraC and TetraD is 55 bases, and the 5' end is labeled with a thiol group. The sequences of TetraB, TetraC, TetraD and A2 chains are fixed and remain unchanged when detecting different target miRNAs, while the sequences of T-A1, Biotin-A3 and TetraA are changed accordingly according to different target miRNAs, wherein the 5' end of the auxiliary chain T-A1 generally requires the addition of 15 T bases; the Tetra A chain is 99 bases long, and the 5' end has a probe with a sequence designed according to the target miRNA and a fixed sequence, which is a special sequence forming an EDTC system; the 3' end of the signal chain Biotin-A3 chain is labeled with biotin.

[0008] Preferably, the sequences of T-A1, Biotin-A3 and TetraA are designed with a fixed template.

[0009] A detection method of a miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit comprises the following steps:

[0010] S1. Design and synthesize each probe chain in the entropy-driven tetrahedral DNA reaction circuit (EDTC) system according to the sequence of the target miRNA;

[0011] S2, modifying the tetrahedral DNA nanostructure (TDN) probe on the surface of the electrochemical gold electrode and combining with auxiliary chains T-A1 and A2 to form the initial state of the EDTC system;

[0012] S3, the miRNA to be detected is mixed with Biotin-A3 and then added to the electrode reaction system. miR-96 will recognize and trigger the entropy-driven reaction of the system, replacing Biotin-A3 into the EDTC system, so that it is fixed on the electrode surface through the TDN probe to achieve the amplification of the detection signal;

[0013] S4. Avidin-HRP conjugated with horseradish peroxidase is captured through biotin-avidin binding, and the electrochemical signal is generated by the enzyme catalysis process to further achieve cascade signal amplification.

[0014] Preferably, in step S1, four tetrahedral probe backbone chains TetraA, TetraB, TetraC and TetraD, auxiliary chains T-A1, A2 and signal chain A3 are designed and synthesized according to the miRNA sequence, and equal concentrations of the four tetrahedral probe backbone chains are dissolved in TM hybridization buffer with a final concentration of 1 μM, and TCEP with a final concentration of 30 mM is added; the solution is heated to 95°C for 5 min to fully unwind the nucleic acid chains, and then quickly transferred to 4°C and maintained for more than 30 s, and the four nucleic acid chains will hybridize in pairs to form a tetrahedral structure; the three vertices at the bottom of TetraB, TetraC and TetraD contain thiol groups, which are used to interact with the gold electrode surface to fix the tetrahedral probe in an orderly manner on the electrode surface; a single-stranded probe extends from the top of TetraA, which is used to hybridize with the auxiliary chains T-A1 and A2 to form an EDTC initial state, and there is a gap of 6 thymine (T) bases between the probe and the tetrahedral backbone.

[0015] Preferably, the hybridization buffer contains 1 mM Na2HPO4, 1 mM KH2PO4, 200 mM NaCl, 2 mM MgCl2.

[0016] Preferably, in step S2, the auxiliary chains T-A1 and A2 can be hybridized with the TDN probe after it has been modified on the electrode surface, or can be modified on the electrode surface after annealing and hybridization with the four chains of the TDN probe.

[0017] Preferably, in step S3, the target miRNA chain is mixed with the signal chain Biotin-A3 and then added to the reaction system. The target miRNA chain will hybridize with the six vacant bases on the substrate of the EDTC system to form an unstable four-stranded intermediate. Due to the entropy-driven release of the auxiliary chain T-A1 that is weakly bound to the substrate, the target miRNA is completely integrated into the EDTC system structure and exposes the three vacant bases in the middle of the Tetra-A chain; this newly exposed area will bind to Biotin-A3, further promoting the displacement of the auxiliary chain A2 and the target miRNA from the intermediate; the newly released miRNA will then start a new cycle to drive the cyclic amplification of the signal.

[0018] Preferably, the optimized reaction time is 30 minutes, and the optimized concentration of signal chain Biotin-A3 added is 5 μM.

[0019] Preferably, in step S4, the added Avidin-HRP enzyme protein is diluted 1:1000 in 1×PBS containing 0.1% BSA, and the reaction time is 20 min.

[0020] Compared with the prior art, the present invention is based on a one-step entropy-driven reaction loop of the miRNA electrochemical detection kit detection method for the miRNA electrochemical detection method, using tetrahedral DNA nanostructures as the support and substrate of the entropy-driven reaction loop, optimizing the spatial arrangement of the probe on the electrode surface, thereby improving the hybridization efficiency and the entropy-driven reaction efficiency, and promoting the necessary structural interactions. By utilizing the thermodynamic principle of entropy, a signal amplification strategy without nuclease dependence is realized, and the reaction efficiency equivalent to that of a homogeneous liquid phase is achieved. The signal amplification method of directly performing entropy-driven reactions on the electrode surface is adopted, which greatly reduces the detection time and operation steps; the entropy-driven reaction loop is adopted, which depends on the triggering of specific sequences, has ultra-high selectivity, and can specifically amplify and detect target molecules; the cascade signal amplification is adopted, and the entropy-driven reaction loop and the catalytic amplification system of peroxidase are integrated, which has high sensitivity and can improve the sensitivity to extremely trace miRNA; the present invention is simple in design, and the gold electrode modified with the functionalized nucleic acid tetrahedral structure probe has universality. For the detection of different miRNA sequences, it only needs to change and synthesize the corresponding nucleic acid chain according to the template to complete the detection. The present invention can have a detection limit as low as 74aM and a dynamic detection range of 100aM to 100pM. The expression level of miR-96 from B[a]PDE exposed cells was verified, and it was found that the miR-96 level was significantly increased. The detection results were consistent with the qRT-PCR method, which shows the potential of this method in early cancer diagnosis and biomarker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a schematic flow chart of the detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit.

[0022] Figure 2 It is the probe sequence of the miRNA electrochemical detection kit based on the one-step entropy-driven reaction circuit of the present invention.

[0023] Figure 3 The invention provides a probe design template for the detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit.

[0024] Figure 4 This is the first experimental diagram of the detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction loop of the present invention - a PAGE characterization diagram for EDTC feasibility evaluation.

[0025] Figure 5This is the second experimental diagram of the detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction loop of the present invention - an electrochemical method diagram for characterizing the gradual modification process of the electrode surface; wherein, (A) cyclic voltammogram; (B) square wave voltammogram; (C) electrochemical impedance spectroscopy, the inset is an equivalent circuit diagram for calculating impedance; (D) timed charge diagram.

[0026] Figure 6 This is the third experimental diagram of the detection method of the miRNA electrochemical detection kit based on the one-step entropy-driven reaction circuit of the present invention - the optimization diagram, wherein (A) assembly mode optimization; (B) reaction time optimization; (C) A3 probe concentration optimization.

[0027] Figure 7 This is the fourth experimental diagram of the detection method of the miRNA electrochemical detection kit based on the one-step entropy-driven reaction circuit of the present invention - a comparison diagram of the reaction efficiency of the entropy-driven reaction circuit on the TDN-based electrode surface and in the homogeneous liquid phase.

[0028] Figure 8 The fifth experimental figure of the detection method of the miRNA electrochemical detection kit based on the one-step entropy-driven reaction loop of the present invention - (A) The specificity evaluation of the constructed biosensor for different miRNAs was carried out with and without the addition of target molecules, and all the corresponding miRNA concentrations were 1pM. (B) The repeatability of the constructed biosensor. (C) The stability of the constructed biosensor. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to the accompanying drawings.

[0030] A miRNA electrochemical detection kit based on a one-step entropy-driven reaction loop comprises several functional chains and various TDN (tetrahedral DNA nanostructure) probe backbone chains for synthesizing an entropy-driven tetrahedral DNA reaction loop (EDTC) system, wherein the functional chains comprise auxiliary chains T-A1, A2 and a signal chain Biotin-A3, wherein the A2 chain is 18 bases long, and the four TDN probe chains comprise TetraA, TetraB, TetraC and TetraD; TetraB, TetraC and TetraD have an average chain length of 55 bases, and are labeled with thiol groups at the 5' end; the sequences of TetraB, TetraC, TetraD and A2 chains remain fixed and unchanged when detecting different target miRNAs; and the sequences of T-A1, Biotin-A3 and TetraA are changed accordingly according to different target miRNAs, wherein the 5' end of the auxiliary chain T-A1 generally requires the addition of 15 T bases; Tetra The A chain is 99 bases long, and the 5' end has a probe with a sequence designed according to the target miRNA and a fixed sequence, which is a special sequence that forms an EDTC system; the 3' end of the signal chain Biotin-A3 chain is labeled with biotin.

[0031] Taking miR-96 as an example, the sequences of the functional chain and the four chains of the TDN probe are as follows Figure 2 The sequence designs of T-A1, Biotin-A3 and TetraA have fixed templates and only need to be changed according to the sequences of the corresponding target miRNAs. The sequence design method is as follows Figure 3 shown.

[0032] like Figure 1 As shown, a detection method of a miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit comprises the following steps:

[0033] S1. Design and synthesize each probe chain in the entropy-driven tetrahedral DNA reaction circuit (EDTC) system according to the sequence of the target miRNA;

[0034] In step S1, taking miR-96 as an example, four tetrahedral probe backbone chains TetraA, TetraB, TetraC and TetraD, auxiliary chains T-A1, A2 and signal chain A3 are designed and synthesized according to the miR-96 sequence. The four tetrahedral probe backbone chains of equal concentration are dissolved in TM hybridization buffer with a final concentration of 1 μM, and TCEP with a final concentration of 30 mM is added; the solution is heated to 95°C for 5 minutes to fully unwind the nucleic acid chains, and then quickly transferred to 4°C for more than 30 seconds, and the four nucleic acid chains will hybridize in pairs to form a tetrahedral structure. The three vertices at the bottom of TetraB, TetraC and TetraD contain thiol groups, which are used to interact with the gold electrode surface to fix the tetrahedral probe on the electrode surface in an orderly manner. The single-stranded probe extending from the top of TetraA is used to hybridize with the auxiliary chains T-A1 and A2 to form the EDTC initial state, and there is a gap of 6 thymine (T) bases between the probe and the tetrahedral backbone.

[0035] The hybridization buffer contained 1 mM Na2HPO4, 1 mM KH2PO4, 200 mM NaCl, and 2 mM MgCl2.

[0036] Among them, the tetrahedral DNA nanostructure serves as a scaffold and substrate for the entropy-driven reaction circuit, optimizing the spatial arrangement of the probes on the electrode surface, thereby improving the hybridization efficiency and entropy-driven reaction efficiency and promoting the necessary structural interactions.

[0037] S2, modifying the tetrahedral DNA nanostructure (TDN) probe on the surface of the electrochemical gold electrode and combining with auxiliary chains T-A1 and A2 to form the initial state of the EDTC system;

[0038] In step S2, the auxiliary chains T-A1 and A2 can be hybridized with the TDN probe after it has been modified on the electrode surface, or can be annealed and hybridized with the four chains of the TDN probe and then modified on the electrode surface.

[0039] Among them, Figures 4 to 8 As shown, the experiment showed that the final signal detection effect was slightly better after the six chains were annealed and hybridized together and then modified on the electrode surface.

[0040] S3, the miRNA to be detected is mixed with Biotin-A3 and then added to the electrode reaction system. miR-96 will recognize and trigger the entropy-driven reaction of the system, replacing Biotin-A3 into the EDTC system, so that it is fixed on the electrode surface through the TDN probe to achieve the amplification of the detection signal;

[0041] In step S3, the target miRNA chain is mixed with the signal chain Biotin-A3 and added to the reaction system. The target miRNA chain will hybridize with the six vacant bases on the EDTC system substrate to form an unstable four-stranded intermediate. Due to the entropy-driven release of the auxiliary chain T-A1 that binds weakly to the substrate, the target miRNA is completely integrated into the EDTC system structure and exposes the three vacant bases in the middle of the Tetra-A chain; this newly exposed area will bind to Biotin-A3, further promoting the displacement of the auxiliary chain A2 and the target miRNA from the intermediate; the newly released miRNA will then start a new cycle, driving the cyclic amplification of the signal.

[0042] Among them, the 15 T bases at the 5' end of the T-A1 chain are more easily replaced by the target miRNA when combined with the Tetra-A chain in the EDTC system.

[0043] Among them, after the entropy-driven reaction loop is completed, Biotin-A3 is fixed on the electrode surface, and biotin is exposed to the top of the TDN probe, which is easier to be bound by the subsequent Avidin-HRP enzyme and trigger an electrochemical reaction.

[0044] Among them, Figures 4 to 8 As shown, after experiments, the optimized reaction time is 30 minutes, and the optimized signal chain Biotin-A3 addition concentration is 5 μM.

[0045] Among them, due to the rigid support structure of the TDN probe, the entire entropy-driven reaction system will be carried out in a solution far away from the electrode surface, which greatly reduces the inhibitory effect of the electrode surface on the reaction efficiency and improves the signal amplification capability.

[0046] S4. Avidin-HRP conjugated with horseradish peroxidase is captured through biotin-avidin binding, and the electrochemical signal is generated by the enzyme catalysis process to further achieve cascade signal amplification.

[0047] In step S4, the added Avidin-HRP enzyme protein was diluted 1:1000 in 1× PBS containing 0.1% BSA, and the reaction time was 20 min.

[0048] The present invention is based on a one-step entropy-driven reaction loop of the miRNA electrochemical detection kit detection method for the miRNA electrochemical detection method, using a tetrahedral DNA nanostructure as a support and substrate for the entropy-driven reaction loop, optimizing the spatial arrangement of the probe on the electrode surface, thereby improving the hybridization efficiency and the entropy-driven reaction efficiency, and promoting the necessary structural interactions. By utilizing the thermodynamic principle of entropy, a signal amplification strategy without nuclease dependence is realized, and a reaction efficiency equivalent to that of a homogeneous liquid phase is achieved. The signal amplification method of directly performing an entropy-driven reaction on the electrode surface is adopted, which greatly reduces the detection time and operation steps; the entropy-driven reaction loop is adopted, which depends on the triggering of a specific sequence, has ultra-high selectivity, and can specifically amplify and detect target molecules; the cascade signal amplification is adopted, and the entropy-driven reaction loop and the catalytic amplification system of peroxidase are integrated, which has high sensitivity and can improve the sensitivity to extremely trace miRNA; the present invention is simple in design, and the gold electrode modified with a functional nucleic acid tetrahedral structure probe has universality. For the detection of different miRNA sequences, it only needs to change and synthesize the corresponding nucleic acid chain according to the template to complete the detection. The present invention can have a detection limit as low as 74aM and a dynamic detection range of 100aM to 100pM. The expression level of miR-96 from B[a]PDE exposed cells was verified, and it was found that the miR-96 level was significantly increased. The detection results were consistent with the qRT-PCR method, which shows the potential of this method in early cancer diagnosis and biomarker detection.

[0049] Finally, it should be noted that the above embodiments only illustrate the technical solutions of the present invention, rather than limiting it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit, characterized in that: It includes several functional chains and various TDN probe backbone chains for synthesizing the entropy-driven tetrahedral DNA reaction loop system. The functional chains include auxiliary chains T-A1, A2 and signal chain Biotin-A3. The four chains of TDN probes include TetraA, TetraB, TetraC and TetraD.

2. The miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 1, characterized in that: The A2 chain is 18 bases long, and the average chain length of TetraB, TetraC, and TetraD is 55 bases. The 5' end is labeled with a thiol group. The sequences of TetraB, TetraC, TetraD, and A2 chains are fixed and unchanged when detecting different target miRNAs, while the sequences of T-A1, Biotin-A3, and TetraA are changed accordingly according to different target miRNAs. The 5' end of the auxiliary chain T-A1 generally needs to add 15 T bases; The Tetra A chain is 99 bases long, with a probe with a sequence designed according to the target miRNA and a fixed sequence at the 5' end, which is a special sequence that forms an EDTC system; the 3' end of the signal chain Biotin-A3 chain is labeled with biotin.

3. The miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 2, characterized in that: The sequences of T-A1, Biotin-A3 and TetraA were designed with a fixed template.

4. A detection method of a miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit, characterized in that: The following steps are involved: S1. Design and synthesize each probe chain in the entropy-driven tetrahedral DNA reaction circuit (EDTC) system according to the sequence of the target miRNA; S2, modifying the tetrahedral DNA nanostructure (TDN) probe on the surface of the electrochemical gold electrode and combining with auxiliary chains T-A1 and A2 to form the initial state of the EDTC system; S3, the miRNA to be detected is mixed with Biotin-A3 and then added to the electrode reaction system. miR-96 will recognize and trigger the entropy-driven reaction of the system, replacing Biotin-A3 into the EDTC system, so that it is fixed on the electrode surface through the TDN probe to achieve the amplification of the detection signal; S4. Avidin-HRP conjugated with horseradish peroxidase is captured through biotin-avidin binding, and the electrochemical signal is generated by the enzyme catalysis process to further achieve cascade signal amplification.

5. The detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 4, characterized in that: In step S1, four tetrahedral probe backbone chains TetraA, TetraB, TetraC and TetraD, auxiliary chains T-A1, A2 and signal chain A3 are designed and synthesized according to the miRNA sequence, and the four tetrahedral probe backbone chains of equal concentration are dissolved in TM hybridization buffer with a final concentration of 1 μM, and TCEP with a final concentration of 30 mM is added; the solution is heated to 95°C for 5 min to fully unwind the nucleic acid chains, and then quickly transferred to 4°C for more than 30 seconds, and the four nucleic acid chains will hybridize in pairs to form a tetrahedral structure; The three vertices at the bottom of TetraB, TetraC and TetraD contain thiol groups, which are used to interact with the gold electrode surface to fix the tetrahedral probes on the electrode surface in an orderly manner; the single-stranded probe extending from the top of TetraA is used to hybridize with the auxiliary chains T-A1 and A2 to form the initial state of EDTC, and there are 6 thymine (T) bases between the probe and the tetrahedral skeleton.

6. The detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 4, characterized in that: The hybridization buffer contained 1 mM Na2HPO4, 1 mM KH2PO4, 200 mM NaCl, 2 mM MgCl2.

7. The detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 4, characterized in that: In step S2, the auxiliary chains T-A1 and A2 can be hybridized with the TDN probe after it has been modified on the electrode surface, or can be annealed and hybridized with the four chains of the TDN probe and then modified on the electrode surface.

8. The detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 4, characterized in that: In step S3, the target miRNA chain is mixed with the signal chain Biotin-A3 and added to the reaction system. The target miRNA chain will hybridize with the six vacant bases on the EDTC system substrate to form an unstable four-stranded intermediate. Due to the entropy-driven release of the auxiliary chain T-A1 that binds weakly to the substrate, the target miRNA is completely integrated into the EDTC system structure and exposes the three vacant bases in the middle of the Tetra-A chain; this newly exposed area will bind to Biotin-A3, further promoting the displacement of the auxiliary chain A2 and the target miRNA from the intermediate; the newly released miRNA will then start a new cycle, driving the cyclic amplification of the signal.

9. The detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 4, characterized in that: The optimized reaction time was 30 minutes, and the optimized concentration of signal chain Biotin-A3 was 5 μM.

10. The detection method of the miRNA electrochemical detection kit based on a one-step entropy-driven reaction circuit according to claim 4, characterized in that: In step S4, the added Avidin-HRP enzyme protein was diluted 1:1000 in 1× PBS containing 0.1% BSA, and the reaction time was 20 min.

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