Entropy driving module biosensor based on miRNA response as well as preparation method and application of entropy driving module biosensor

Abstract

The invention discloses an entropy driving module biosensor based on miRNA response and a preparation method and application thereof, and belongs to the technical field of biological detection. The entropy driving module biosensor comprises DNA frame nucleic acid, and the DNA frame nucleic acid is composed of a substrate chain C123, a substrate chain S123, a fuel chain F1, a fuel chain F2 and a DNA frame TP. Characteristic miRNA of macrophages M1 and M2 is used for performing specific response on an entropy driving module on DNA framework nucleic acid, so that after chain displacement and fuel chain combination, a chain with a fluorophore is released, and miRNA is released to realize circulation of a target object, so that amplification of a fluorescence signal is realized; the sensor performs typing detection on macrophages M1 and M2 by using a fluorescence method, and compared with other immunology, morphology and other methods, the method is simple to operate, weak in false positive signal, high in reaction speed, rapid in typing when the M1 and M2 cells are detected, accurate in quantification and high in sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to an entropy-driven module biosensor based on miRNA response, a preparation method and an application thereof. Background Art

[0002] Macrophages are key cells in tissue homeostasis and inflammation, performing essential tissue-specific functions and protecting organisms from infection. Functionally, macrophages can be divided into classically activated pro-inflammatory M1 macrophages and selectively activated anti-inflammatory M2 macrophages. A well-controlled M1 / M2 balance is important to avoid pathological or acute responses. This control can be effectively achieved through macrophage phenotypic plasticity. The M1 / M2 balance is dynamic and is controlled by the activity of intracellular signaling mediators activated by exogenous stimuli. Imbalance between M1 and M2 macrophages can cause damage to the body, so accurate classification of M1 and M2 becomes crucial.

[0003] At present, various methods for macrophage typing have been established, including traditional flow cytometry to identify surface antibodies, immunohistochemistry, and real-time fluorescence quantitative PCR to detect cytokines. However, these methods still face some problems due to the complex operation steps and one-sided analysis results. It is urgent to establish a rapid, accurate, sensitive and highly specific typing method to type macrophages M1 and M2. Summary of the invention

[0004] In view of the problems of complex operation steps and one-sided analysis results of macrophage typing methods in the prior art, the present invention provides an entropy-driven module biosensor based on miRNA response and its preparation method and application, which realizes more sensitive and specific typing of M1 and M2 macrophages.

[0005] The present invention is achieved through the following technical solutions: An entropy-driven module biosensor based on miRNA response includes a DNA framework nucleic acid, wherein the DNA framework nucleic acid is composed of a substrate chain C123, a substrate chain S123, a fuel chain F1, a fuel chain F2, and a DNA framework TP; The DNA framework TP is composed of three chains, TP1, TP2 and TP3, the nucleotide sequence of TP1 is shown in SEQ ID No.1, the nucleotide sequence of TP2 is shown in SEQ ID No.2, and the nucleotide sequence of TP3 is shown in SEQ ID No.3; The nucleotide sequence of the fuel chain F1 is shown in SEQ ID No. 4; The substrate chain C123 is composed of three chains, C1, C2 and C3, the nucleotide sequence of C1 is shown in SEQ ID No.5, the nucleotide sequence of C2 is shown in SEQ ID No.6, and the nucleotide sequence of C3 is shown in SEQ ID No.7; The nucleotide sequence of the fuel chain F2 is shown in SEQ ID No.8; The substrate chain S123 is composed of three chains, S1, S2 and S3. The nucleotide sequence of S1 is shown in SEQ ID No.9, the nucleotide sequence of S2 is shown in SEQ ID No.10, and the nucleotide sequence of S3 is shown in SEQ ID No.11.

[0006] Furthermore, in the substrate chain C123 in the entropy-driven module, the 5' end of C2 is modified with Cy5, and the 3' end of C1 is modified with BHQ3; in the substrate chain S123 in the entropy-driven module, the 5' end of S2 is modified with FAM, and the 3' end of S1 is modified with BHQ1.

[0007] The present invention also discloses a method for preparing the entropy-driven module biosensor based on miRNA response, comprising the following steps: (1) Preparation of DNA framework TP: TP1, TP2, and TP3 are mixed and reacted to obtain DNA framework TP; (2) Preparation of substrate chain C123: C1, C2, and C3 are mixed and reacted to obtain substrate chain C123; (3) Preparation of substrate chain S123: S1, S2, and S3 are mixed and reacted to obtain substrate chain S123; (4) Preparation of DNA framework nucleic acid: DNA framework TP is mixed with substrate chain C123, substrate chain S123, fuel chain F1 and fuel chain F2 to obtain DNA framework nucleic acid.

[0008] Furthermore, in step (1), the molar ratio of TP1, TP2 and TP3 is 1:1:1; in step (2), the molar ratio of C1, C2 and C3 is 1:1:1; in step (3), the molar ratio of S1, S2 and S3 is 1:1:1; Furthermore, in step (4), the molar ratio of the DNA framework TP to the substrate chain C123, the substrate chain S123, the fuel chain F1, and the fuel chain F2 is 1:1:1:1:1.

[0009] Furthermore, in step (1), the mixed reaction conditions of TP1, TP2, and TP3 are heated at 95° C. for 5 min and then slowly cooled to room temperature; in step (2), the mixed reaction conditions of C1, C2, and C3 are heated at 95° C. for 5 min and then slowly cooled to room temperature; in step (3), the mixed reaction conditions of S1, S2, and S3 are heated at 95° C. for 5 min and then slowly cooled to room temperature.

[0010] Furthermore, in step (4), the DNA framework TP reacts with the substrate chain C123, the substrate chain S123, the fuel chain F1, and the fuel chain F2 at room temperature for 3 h.

[0011] In the present invention, the entropy-driven module biosensor based on miRNA response is used in macrophage typing.

[0012] Furthermore, the macrophages are macrophages M1 and macrophages M2, and the components to be detected are RAW264.7 cell M1 characteristic miRNA-miR-145a-5p and RAW264.7 cell M2 characteristic miRNA-Let-7c-1 as the objects to be detected.

[0013] Furthermore, the entropy-driven module biosensor based on miRNA response was reacted with the mixed reaction of miRNA- miR-145a-5p and / or M2 signature miRNA- Let-7c-1 at 37 °C for 5 h.

[0014] In the present invention, the M1 characteristic miRNA-miR-145a-5p specifically binds to the substrate chain C123 of the DNA framework TP, triggering an entropy-driven reaction, and then binds to F1 on the other end of the DNA framework TP, and the C2 chain falls off, generating a fluorescent signal; the M2 characteristic miRNA-Let-7c-1 specifically binds to the substrate chain S123 on the DNA framework TP, triggering an entropy-driven reaction, and then binds to F2 on the other end of the DNA framework TP, and the S2 chain falls off, generating a fluorescent signal.

[0015] M1 characteristic miRNA-miR-145a-5p specifically binds to C123, while M2 characteristic miRNA-Let-7c-1 specifically binds to S123. After entropy-driven reaction, the substrate chains bind to F1 and F2 respectively, thereby releasing C2 with cy5 and S2 with FAM labeling respectively, and miRNA can be released to achieve the circulation of the target and amplification of the fluorescence signal.

[0016] Beneficial Effects The entropy-driven module based on miRNA response of the present invention is used for the biosensor of macrophage typing, which utilizes the characteristic miRNA of macrophages M1 and M2 to specifically respond to the entropy-driven module on the DNA framework nucleic acid, so that after chain displacement and binding with the fuel chain, the chain with the fluorophore is released, and the miRNA is released to realize the circulation of the target, thereby realizing the amplification of the fluorescence signal. The sensor uses the fluorescence method to perform typing detection of macrophages M1 and M2. Compared with other immunological, morphological and other methods, this method is simple to operate, has weak false positive signals, and fast reaction speed. In addition, when detecting M1 and M2 cells, the typing is rapid, the quantification is accurate, and the sensitivity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the entropy-driven module biosensor based on miRNA response; a is a schematic diagram of macrophage M1 and M2 typing, and b is the DNA nucleic acid framework responding to the M1 characteristic miRNA-miR-145a-5p and the M2 characteristic miRNA-Let-7c-1 respectively, and then amplifying the signal through the entropy-driven module.

[0018] Figure 2 This is a polypropylene gel electrophoresis diagram of the DNA framework nucleic acid and entropy-driven response in Example 2 of the present invention; Figure 3 This is a fluorescence image of the DNA framework nucleic acid after entropy driving in Example 2 of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] Example 1 The entropy-driven module biosensor of miRNA response in the present invention comprises a DNA framework nucleic acid, wherein the DNA framework nucleic acid is composed of a substrate chain C123, a substrate chain S123, a fuel chain F1, a fuel chain F2, and a DNA framework TP; The DNA framework TP is composed of three chains, TP1, TP2 and TP3, the nucleotide sequence of TP1 is shown in SEQ ID No.1, the nucleotide sequence of TP2 is shown in SEQ ID No.2, and the nucleotide sequence of TP3 is shown in SEQ ID No.3; The nucleotide sequence of the fuel chain F1 is shown in SEQ ID No. 4 (the fuel chain F1 consists of the fuel chain corresponding to M1 and a sequence connected to the DNA framework TP); The substrate chain C123 is composed of three chains, C1, C2 and C3. The nucleotide sequence of C1 is shown in SEQ ID No.5, the nucleotide sequence of C2 is shown in SEQ ID No.6, the nucleotide sequence of C3 is shown in SEQ ID No.7, the 5' end of C2 is modified with a Cy5 fluorescent group, and the 3' end of C1 is modified with a BHQ3 quenching group; The nucleotide sequence of the fuel chain F2 is shown in SEQ ID No. 8 (F2 consists of the fuel chain corresponding to M2 and a sequence connected to the DNA framework TP); The substrate chain S123 is composed of three chains, S1, S2 and S3. The nucleotide sequence of S1 is shown in SEQ ID No.9, the nucleotide sequence of S2 is shown in SEQ ID No.10, and the nucleotide sequence of S3 is shown in SEQ ID No.11. The 5' end of S2 is modified with a FAM fluorescent group, and the 3' end of S1 is modified with a BHQ1 quenching group.

[0021] The nucleotide sequences of SEQ ID No. 1 to 11 are shown in Table 1 below: Table 1 RAW264.7 cell M1 characteristic miRNA-miR-145a-5p, RAW264.7 cell M2 characteristic miRNA-Let-7c-1 are to be detected; the schematic diagram of the entropy-driven module biosensor based on miRNA response for macrophage M1 and M2 typing is shown in the figure Figure 1 As shown, Figure 1 a is a schematic diagram of the classification of macrophages M1 and M2. Figure 1 b is that the DNA nucleic acid framework responds to the M1 characteristic miRNA-miR-145a-5p and the M2 characteristic miRNA-Let-7c-1 respectively, and then amplifies the signal through the entropy driving module, so that M1M2 typing can be performed.

[0022] Example 2 (1) Dilute 100 μM TP1, TP2, and TP3 to 3 μM with TE buffer, add 200 μL TP1, 200 μL TP2, and 200 μL TP3, mix well, and react in a water bath at 95 °C for 5 min. Slowly cool to room temperature and place in -20 °C to obtain DNA framework TP, which is stored in a refrigerator. (2) Take 50 μL C1, 50 μL C2, and 50 μL C3 stored in TE buffer at equal concentration (3 μM), mix them, heat at 95 °C for 5 min, and then slowly cool to room temperature to obtain C123; (3) Take 50 μL S1, 50 μL S2, and 50 μL S3 stored in TE buffer of equal concentration (3 μM), mix them, heat at 95 °C for 5 min, and then slowly cool to room temperature to obtain S123; (4) Pipette equal moles of DNA framework TP, C123, S123, F1, and F2 (50 μL), mix well, and react at room temperature for 3 h to obtain DNA framework nucleic acid; (5) Add 1 μM M1 characteristic miRNA-miR-145a-5p and M2 characteristic miRNA-Let-7c-1 to the DNA framework nucleic acid in step (4) and mix them. Incubate the mixture at 37°C for 5 h. During this period, the miRNA and the substrate chain undergo an entropy-driven reaction to release the fluorophore-modified chain, and achieve target circulation and signal amplification.

[0023] (6) In order to prove the successful construction of DNA framework nucleic acid and the entropy-driven response, a polyacrylamide gel electrophoresis experiment was carried out. 12% PAGE gel was used for electrophoresis at 130 V for 1.5 h. Channel 1 was DNA framework TP (TP), channel 2 was DNA framework TP and nucleic acid bound to substrate chain C123 and fuel chain F1 (TP-CF), channel 3 was DNA framework TP and nucleic acid bound to substrate chain S123 and fuel chain F2 (TP-SF), channel 4 was DNA framework nucleic acid (TP-CF-SF), channel 5 was DNA framework nucleic acid and M1 characteristic miRNA-miR-145a-5p, M2 characteristic miRNA-Let-7c-1 entropy-driven response (TP-CF-SF+M+L, M refers to miR-145a-5p, L refers to Let-7c-1); the polyacrylamide gel electrophoresis results are shown in the figure. Figure 2 As shown in the figure, it can be seen that the DNA framework nucleic acid is successfully constructed, and the entropy-driven reaction can be initiated in the presence of M1 characteristic miRNA-miR-145a-5p and M2 characteristic miRNA-Let-7c-1.

[0024] (7) The fluorescence change is detected by a fluorescence instrument. The fluorescence image after DNA framework nucleic acid entropy driving is as follows: Figure 3 As shown by Figure 3 It can be seen from the figure that the fluorescence signal can be detected when the M1 characteristic miRNA-miR-145a-5p and the M2 characteristic miRNA-Let-7c-1 are present, while the signal intensity is almost 0 when the target miRNA is absent.

Claims

1. An entropy-driven module biosensor based on miRNA response, characterized in that: It includes a DNA framework nucleic acid, which is composed of a substrate chain C123, a substrate chain S123, a fuel chain F1, a fuel chain F2, and a DNA framework TP; The DNA framework TP is composed of three chains, TP1, TP2 and TP3, the nucleotide sequence of TP1 is shown in SEQ ID No.1, the nucleotide sequence of TP2 is shown in SEQ ID No.2, and the nucleotide sequence of TP3 is shown in SEQ ID No.3; The nucleotide sequence of the fuel chain F1 is shown in SEQ ID No. 4; The substrate chain C123 is composed of three chains, C1, C2, and C3, the nucleotide sequence of C1 is shown in SEQ ID No.5, the nucleotide sequence of C2 is shown in SEQ ID No.6, and the nucleotide sequence of C3 is shown in SEQ ID No.7; The nucleotide sequence of the fuel chain F2 is shown in SEQ ID No.8; The substrate chain S123 is composed of three chains, S1, S2 and S3. The nucleotide sequence of S1 is shown in SEQ ID No.9, the nucleotide sequence of S2 is shown in SEQ ID No.10, and the nucleotide sequence of S3 is shown in SEQ ID No.

11.

2. The entropy-driven module biosensor based on miRNA response according to claim 1, characterized in that: In the substrate chain C123, the 5' end of C2 is modified with Cy5, and the 3' end of C1 is modified with BHQ3; in the substrate chain S123, the 5' end of S2 is modified with FAM, and the 3' end of S1 is modified with BHQ1.

3. A method for preparing an entropy-driven module biosensor based on miRNA response according to claim 1 or 2, characterized in that: The following steps are involved: (1) Preparation of DNA framework TP: TP1, TP2, and TP3 are mixed and reacted to obtain DNA framework TP; (2) Preparation of substrate chain C123: C1, C2, and C3 are mixed and reacted to obtain substrate chain C123; (3) Preparation of substrate chain S123: S1, S2, and S3 are mixed and reacted to obtain substrate chain S123; (4) Preparation of DNA framework nucleic acid: DNA framework TP is mixed with substrate chain C123, substrate chain S123, fuel chain F1 and fuel chain F2 to obtain DNA framework nucleic acid.

4. The method for preparing the entropy-driven module biosensor based on miRNA response according to claim 3, characterized in that: In step (1), the molar ratio of TP1, TP2 and TP3 is 1:1:1; in step (2), the molar ratio of C1, C2 and C3 is 1:1:1; in step (3), the molar ratio of S1, S2 and S3 is 1:1:

1.

5. The method for preparing the entropy-driven module biosensor based on miRNA response according to claim 3, characterized in that: In step (4), the molar ratio of DNA framework TP to substrate chain C123, substrate chain S123, fuel chain F1, and fuel chain F2 is 1:1:1:1:

1.

6. The method for preparing the entropy-driven module biosensor based on miRNA response according to claim 3, characterized in that: In step (1), the mixed reaction conditions of TP1, TP2, and TP3 are heated at 95°C for 5 min and then slowly cooled to room temperature; in step (2), the mixed reaction conditions of C1, C2, and C3 are heated at 95°C for 5 min and then slowly cooled to room temperature; in step (3), the mixed reaction conditions of S1, S2, and S3 are heated at 95°C for 5 min and then slowly cooled to room temperature.

7. The method for preparing the entropy-driven module biosensor based on miRNA response according to claim 3, characterized in that: Step (4) DNA framework TP is reacted with substrate chain C123, substrate chain S123, fuel chain F1 and fuel chain F2 at room temperature for 3 hours.

8. Use of the entropy-driven module biosensor based on miRNA response according to claim 1 or 2 in macrophage typing.

9. The use of the entropy-driven module biosensor based on miRNA response in macrophage typing according to claim 8, characterized in that: The macrophages are macrophages M1 and macrophages M2, and the components to be detected are RAW264.7 cell M1 characteristic miRNA-miR-145a-5p and RAW264.7 cell M2 characteristic miRNA-Let-7c-1 as the objects to be detected.

10. The use of the entropy-driven module biosensor based on miRNA response in macrophage typing according to claim 8, characterized in that: The entropy-driven module biosensor based on miRNA response was reacted with miRNA-miR-145a-5p and / or M2 signature miRNA-Let-7c-1 at 37°C for 5 h.

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