Electrochemical sensor for detecting nucleic acid miR-205 and application thereof

Abstract

The invention discloses an electrochemical sensor for detecting nucleic acid miR-205 and application of the electrochemical sensor. The sensor comprises an oligonucleotide probe Probe1, an oligonucleotide probe Probe2, a SplintR ligase, a T7RNA polymerase, an ssDNA-MB connected with methylene blue and a gold electrode. The sensor is used for detecting and analyzing the expression quantity of nucleic acid miR-205, and the detection method of the sensor has good linearity and sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a chemical sensor for detecting nucleic acid miR-205 and an application thereof. Background Art

[0002] The integration of cell-free systems and inducible synthetic genetic circuits has promoted the advancement of diagnostic and therapeutic methods. Biosensors developed based on cell-free systems contain specifically designed gene circuits that initiate the transcription and translation of reporter molecules in the presence of targets. This system has been used to detect specific nucleic acid sequences, small molecules, metal ions, and biological molecules including antibodies. However, traditional cell-free biosensors often use RNA fluorescent aptamers or fluorescent proteins for back-end signal output, which has high requirements for the conformation of the output RNA and requires a long detection time. Therefore, exploring a back-end signal output method that can produce high signal output and is simple and convenient is of great significance to the construction of cell-free biosensors. Electrochemistry has attracted the attention of more and more researchers due to its ultra-high sensitivity and portability. The present invention combines the advantages of cell-free expression technology and electrochemical detection for the first time to construct a bioelectrochemical detection method and apply it to the analysis and detection of the miR-205 gene.

[0003] MicroRNA-205 (miR-205) is located in the second intron of the L642587 locus on human chromosome 1 (1q32.2). It is 110 bases long and was first detected in human cells in 2007. Numerous studies have demonstrated that miR-205 is closely associated with the development and progression of various human malignancies. The biological effects of miR-205 vary depending on the cancer tissue and type, and can even exhibit bidirectional effects. The development and progression of various human malignancies, such as breast cancer, lung cancer, thyroid cancer, and renal cancer, are closely linked to miR-205. miR-205 regulates tumor cell proliferation, differentiation, apoptosis, invasion, and infiltration, with its mechanisms of action varying depending on the tumor type. Therefore, detecting miR-205 gene expression is of great clinical significance. Therefore, developing a method for analyzing and detecting miR-205 is crucial. Summary of the Invention

[0004] The purpose of the present invention is to provide a chemical sensor for detecting nucleic acid miR-205 and its application.

[0005] The following embodiments are provided to achieve the objects of the present invention.

[0006] In one embodiment, the present invention provides an electrochemical sensor for detecting nucleic acid miR-205, comprising oligonucleotide probe Probe 1, oligonucleotide probe Probe 2, SplintR ligase, T7 RNA polymerase, ssDNA-MB linked to methylene blue, and a gold electrode. Probe 1 comprises a T7 RNA polymerase promoter sequence and a recognition sequence that hybridizes with the 5' half of the miR-205 region, and Probe 2 comprises a template sequence and a recognition sequence that hybridizes with the 3' half of the miR-205 region. The gene sequence of miR-205 is (SEQ ID NO: 1):

[0007] 5'-UAGCUUAUCAGACUGAUGUUGA-3'.

[0008] Note: In order to comply with the WIPOST.26 standard, the subsequent sequence table uses "t" instead of "u" to represent uracil in RNA sequences.

[0009] Preferably, in the electrochemical sensor of the present invention, the probe Probe1 has a nucleic acid sequence as shown in SEQ ID NO:2.

[0010] The gene sequence of probe Probe1 is (SEQ ID NO: 2):

[0011] 5'-TGATAAGCTACCCTATAGTGAGTCGTATTAATTTCGCGACAACACGCGAAATTAATACGACTCACTATAGGG-3'.

[0012] Preferably, the electrochemical sensor of the present invention, probe Probe2, has a nucleic acid sequence as shown in SEQ ID NO:3.

[0013] The gene sequence of probe Probe2 is (SEQ ID NO: 3):

[0014] 5'-CTGTTGAGTAGAGTGTGAGCTCCTCAACATCAGTC-3'.

[0015] Furthermore, in the electrochemical sensor of the present invention, ssDNA-MB is modified on the electrode surface, and its nucleic acid sequence is shown in SEQ ID NO:4.

[0016] The sequence of the ssDNA is (SEQ ID NO: 4):

[0017] 5'-SH-CTGTTGAGTAGAGTGTGAGCTCCTCAA-MB-3'.

[0018] Another object of the present invention is to provide an electrochemical sensor of the present invention for detecting nucleic acid miR-205 (miR-205).

[0019] To this end, the present invention provides a method for detecting nucleic acid miR-205 using the electrochemical sensor of the present invention, comprising the following steps:

[0020] 1) Probe 1, Probe 2, and miR-205 are mixed in a buffer solution, and Probe 1 and Probe 2 hybridize with a portion of miR-205 respectively;

[0021] 2) Adding SplintR ligase, T7 RNA polymerase, and NTPs to the hybridized miR-205 solution, using SplintR ligase to connect to a DNA template with RNA transcription activity, and transcribing the RNA chain (Output RNA) by T7 RNA polymerase;

[0022] 3) Output RNA is dropped onto the electrode surface, where it complements the ssDNA-MB modified on the gold electrode surface to form an RNA-DNA complex, which changes the state of the electrode surface and causes a change in the current, thus completing the detection.

[0023] Preferably, in the above detection method of the present invention, the concentration of ssDNA modified on the electrode surface is 10 μM.

[0024] Furthermore, the above-mentioned detection method of the present invention further includes electrode activation, comprising placing a gold electrode on an electrode polishing cloth, polishing it with 0.3μm Al2O3 and 0.05μm Al2O3 powder, placing it in a 0.5M H2SO4 solution for activation, modifying the surface of the gold electrode with a complementary DNA modified with a thiol group at one end and methylene blue (MB) at the other end, and then blocking nonspecific binding sites with mercaptoethanol.

[0025] In one embodiment, the present invention provides a method for detecting miR-205 using the electrochemical sensor of the present invention, comprising the following steps:

[0026] 1) Add 0.22 μL (10 μM) Probe 1, 0.22 μL (10 μM) Probe 2, and 0.22 μL miR-205 to 1 μL of a buffer solution containing 100 mM Tris-HCl and 0.5 M KCl. Then add 6.86 μL of RNAse-free water and mix. Heat to 95°C for 3 minutes and slowly cool to room temperature to obtain complementary hybridization solutions of Probe 1 and Probe 2 with miR-205, respectively.

[0027] 2) Add 1 μL of 10× SplintR buffer and 0.5 μL of SplintR ligase (25 U / μL) to the complementary hybridization solution in the previous step, mix, and incubate the mixed solution at 37°C for 30 min. Take 5 μL of the incubated mixed solution, add 5 μL of 10× T7 RNA polymerase reaction buffer, 2.5 μL of dithiothreitol (100 mM), 2 μL of NTPs (25 mM each NTP), 1.25 μL of recombinant RNA inhibitor (20 U / μL), 5 μL of T7 RNA polymerase (50 U / μL) and 29.25 μL of RNAse-free water, and incubate the mixed solution at 37°C for 16 h to obtain the transcribed RNA chain (Output RNA);

[0028] 3) Take 10 μl of the transcribed output RNA solution from step 2 and drop it on the electrode surface. After a certain reaction time, rinse with distilled water to remove nonspecifically bound substances. Detect the signal using electrochemical SWV (square wave voltammetry) in PBS and measure the change in signal before and after.

[0029] Terminology: NTPs represent various free nucleotides that are used to provide the raw materials for RNA synthesis. The corresponding nucleotides are provided according to the RNA sequence to be synthesized.

[0030] The beneficial effects of the present invention are as follows:

[0031] (1) A novel miR-205 gene detection method was designed using a cell-free RNA expression system.

[0032] (2) No need for complex PCR amplification and complex sequencing equipment.

[0033] (3) It overcomes the shortcomings of traditional cell-free biosensors that often use RNA fluorescent aptamers or fluorescent proteins for back-end signal output, have high requirements on the conformation of the output RNA, and require a long detection time.

[0034] (4) The electrochemical detection method of the present invention detects MiR-205 with good linearity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the principle of the electrochemical sensor of the present invention for detecting miR-205;

[0036] Figure 2 This is a graph showing the feasibility of the impedance test for detecting the miR-205 gene in Example 5;

[0037] Figure 3 This is an electrochemical detection diagram of the experimental feasibility of detecting the miR-205 gene in Example 5;

[0038] Figure 4 This is the working curve for detecting the miR-205 gene in Example 5. DETAILED DESCRIPTION

[0039] The following examples are provided to describe the present invention in more detail. However, the following examples are provided only to help further understand the present invention and are not intended to limit the present invention. Those skilled in the art will appreciate that any equivalent substitutions made to the present invention, or corresponding improvements, still fall within the scope of protection of the present invention.

[0040] Example 1

[0041] The principle of the electrochemical electrode sensor for detecting mir-205 gene is as follows Figure 1 The sensor's main components involve two probes, Probe 1 and Probe 2, expressed in a cell-free system, and a modified ssDNA-MB attached to a gold electrode. The design of the two probes is as follows: Probe 1 contains the T7 RNA polymerase promoter sequence and a recognition sequence that hybridizes with the 5' half of the miR-205 region, as shown in SEQ ID NO: 2:

[0042] 5'-TGATAAGCTACCCTATAGTGAGTCGTATTAATTTCGCGACAACACGCGAAATTAATACGACTCACTATAGGG-3'.

[0043] Probe 2 consists of a template sequence and a recognition sequence that hybridizes to the 3' half of the miR-205 region, and its sequence is shown in SEQ ID NO: 3:

[0044] 5'-CTGTTGAGTAGAGTGTGAGCTCCTCAACATCAGTC-3'.

[0045] The sequence of miR-205 is shown in SEQ ID NO: 1:

[0046] 5'-UAGCUUAUCAGACUGAUGUUGA-3'.

[0047] The sequence of ssDNA-MB is shown in SEQ ID NO: 4:

[0048] 5'-SH-CTGTTGAGTAGAGTGTGAGCTCCTCAA-MB-3'.

[0049] Example 2 Preparation of electrochemical sensor electrodes for detecting miR-205

[0050] The steps for preparing the sensor electrode based on the cell-free RNA expression system are as follows:

[0051] 1) Electrode Activation: Place a gold electrode (0.5 mm diameter) on an electrode polishing cloth and polish with 0.3 μm Al2O3 and 0.05 μm Al2O3 powders. Cyclic scan in 0.5 M H2SO4 solution until stable, followed by sonication in ethanol and then ultrapure water. Subsequently, immerse the gold electrode in 0.50 M sulfuric acid (H2SO4) and scan the cyclic voltammogram over the potential range of 0-1.2 V until a stable cyclic voltammogram with three distinct oxidation peaks and one reduction peak is obtained. Remove the gold electrode and rinse it with ultrapure water for later use.

[0052] 2) 10mM thiol (-SH) and methylene blue (MB) modified ssDNA-MB was dissolved in a buffer solution containing 1.0mM DTT to open the disulfide bonds. 10uL of the solution was then added dropwise to the surface of a gold electrode and incubated at 37°C for 2 hours to allow the ssDNA-MB to covalently bind to the gold electrode surface via the Au-S bond. To prevent nonspecific adsorption on the electrode surface, 1.0mM mercaptoethanol was added to the electrode surface and reacted at 37°C for 30 minutes. Next, the amplification solution was placed on the gold electrode surface and incubated at 37°C for 0.5 hours. After each treatment step, the gold electrode was rinsed with ultrapure water to remove excess unreacted and nonspecifically adsorbed substances, resulting in a ssDNA-MB modified gold electrode.

[0053] The sequence of the ssDNA-MB is 5'-SH-CTGTTGAGTAGAGTGTGAGCTCCTCAA-MB-3'.

[0054] Example 3 Preparation of RNA Chain Orthogonal to ssDNA-MB (Output RNA)

[0055] The process of cell-free RNA expression based on the cell-free RNA expression system sensor is as follows:

[0056] Under a clean bench environment, 0.22 μL (10 μM probe 1), 0.22 μL (10 μM probe 2), and 0.22 μL miR-205 were added to 1 μL (100 mM Tris-HCl, pH 7.4 and 0.5 M KCl) in 6.86 μL RNAse-free water, heated at 95 ° C for 3 minutes, and then slowly cooled to room temperature. 1 μL 10× SplintR buffer and 0.5 μL SplintR ligase (25 U / μl) were added, and the mixture was incubated at 37 ° C for 30 minutes. 5 μL of the above solution was taken and added to 5 μL 10× T7 RNA polymerase reaction buffer, 2.5 μL dithiothreitol (100 mM), 2 μL NTP, and each NTP 25 mM, 1.25 μL recombinant RNA inhibitor (20 U / μL), 5 μL T7 RNA polymerase 50 U / μL, and 29.25 μL RNAse-free water, the mixture was incubated at 37°C for 16 h to prepare the transcription output RNA.

[0057] Example 4 miR-205 detection

[0058] Take 10 μL of the Output RNA solution prepared in Example 3 and drop it onto the surface of the ssDNA-MB modified gold electrode prepared in Example 2. Incubate at 37°C for 30 minutes, rinse with distilled water to remove nonspecific adsorbed substances, and immerse in 2.0 mL of PBS (10 mM, pH 7.4) as an electrolyte for electrochemical detection. All electrochemical measurements were performed on a three-electrode system, with Ag / AgCl (3.0 M KCl) as the reference electrode, a platinum wire electrode as the counter electrode, and a gold electrode as the working electrode. Electrochemical measurements were performed using the Square Wave Voltammetry (SWV) method in the potential range of -0.5 V to -0.1 V, under the conditions of Incr E = 0.004, Amplitude = 0.025, Frequency = 60 Hz, and Quiet Time = 2 sec.

[0059] Example 5 Detection Method of Nucleic Acid MiR-205

[0060] 1. Electrochemical impedance spectroscopy is used to characterize the state of the electrode under different modification conditions.

[0061] All electrode impedance measurements were performed in [Fe(CN)6] 3- / 4-The electrochemical parameters were as follows: Init E (V) = 0.209, High Freq (Hz) = 1e+6, Low Freq (Hz) = 0.1, Imp FT, Amplitude (V) = 0.005, Quiet Time (s) = 2 Cycles (.1-1 Hz) = 1. The black curve represents the impedance of a bare gold electrode, the red curve represents the impedance of a gold electrode modified with 10 μM ssDNA-MB, and the green curve represents the impedance measured after a cell-free expression reaction in the presence of 0.0005 μM miR-205 and incubation with an ssDNA-MB-modified electrode for 30 minutes.

[0062] See the results Figure 2 , Figure 2 The red curve shows that the capture probe has been successfully modified onto the electrode surface (the impedance of the red curve is greater than that of the black curve). Furthermore, when the output RNA is present, the electrode impedance (green curve) is much greater than when the target is absent (red curve), demonstrating that the addition of the target can trigger the cell-free RNA expression reaction. The output RNA of the cell-free expression product is captured on the electrode surface by hybridization with the template DNA, significantly increasing the surface resistance of the electrode.

[0063] 2. Validation of electrochemical analysis method

[0064] Under electrochemical square wave voltammetry conditions, the electrochemical spectrum of a gold electrode modified with 10 μM ssDNA-MB was first measured in 1× PBS buffer at pH 7.4 (red). This electrode was then incubated with the output RNA from a cell-free expression reaction in the presence of 0.0005 μM miR-205 and the ssDNA-MB-modified electrode for 30 minutes, and the electrochemical spectrum was measured under the same conditions (black).

[0065] See the results Figure 3 , we can see that the peak current drops significantly, indicating that square wave voltammetry can be used for detection.

[0066] 3. Working curve of detecting miR-205 gene

[0067] Under the conditions that the miR-205 contents were 0.001μM, 0.0005μM, 0.0001μM, 0.00005μM, 0.000005μM, 0.000001μM, 0.0000005μM, and 0.00000005μM, respectively, the square wave voltammetry detection frequency was 60Hz, the concentration of the gold electrode modified ssDNA-MB was 10μM, the modification time was 3 hours, and the incubation time with the output RNA produced after the miR-205 cell-free expression reaction was 30 minutes. The standard curve was constructed with the current drop value as the vertical axis. The results are shown in FIG. Figure 4 The results showed that the detection method had good linearity.

[0068] It should be understood that the application of the present invention is not limited to the above examples. Those skilled in the art can make improvements or changes based on the above description. All these improvements and changes should fall within the scope of protection of the claims of the present invention.

Claims

1. An electrochemical sensor for detecting nucleic acid miR-205, characterized in that include: Oligonucleotide probe Probe1, oligonucleotide probe Probe2, SplintR ligase, T7 RNA polymerase, ssDNA-MB connected with methylene blue and a gold electrode, wherein the probe Probe1 contains a promoter sequence of T7 RNA polymerase and a recognition sequence that can hybridize with the 5' half of the miR-205 region, and the probe Probe2 consists of a template sequence and a recognition sequence that can hybridize with the 3' half of the miR-205 region. 2 . The electrochemical sensor according to claim 1 , wherein the probe Probe 1 has a nucleic acid sequence as shown in SEQ ID NO:

2. 3 . The electrochemical sensor according to claim 1 , wherein the probe Probe 2 has a nucleic acid sequence as shown in SEQ ID NO:

3. 4 . The electrochemical sensor according to claim 1 , wherein the ssDNA-MB is located on the electrode surface and has a nucleic acid sequence as shown in SEQ ID NO:

4.

5. The method for detecting miR-205 nucleic acid using the electrochemical sensor according to any one of claims 1 to 4, comprising the following steps: 1) Probe 1, Probe 2, and miR-205 are mixed in a buffer solution, and Probe 1 and Probe 2 hybridize with a portion of miR-205 respectively; 2) Add SplintR ligase, T7 RNA polymerase and NTPs to the hybridized miR-205 solution, SplintR ligase is used to connect to a DNA template with RNA transcription activity, and the RNA chain (Output RNA) is transcribed by T7 RNA polymerase; 3) Output RNA is dropped onto the electrode surface, where it complements the ssDNA-MB modified on the electrode surface to form an RNA-DNA complex, thereby changing the state of the electrode surface and causing a change in the current, thereby completing the detection. The detection method according to claim 5 , wherein the concentration of ssDNA connected to the electrode surface is 10 μM.

7. The detection method according to claim 5, further comprising electrode activation, comprising placing a gold electrode on an electrode polishing cloth, polishing it with 0.3 μm Al2O3 and 0.05 μm Al2O3 powder, activating the electrode in a 0.5M H2SO4 solution, modifying the surface of the gold electrode with complementary DNA modified with a thiol group at one end and methylene blue at the other end, and blocking nonspecific binding sites with mercaptoethanol.

8. The detection method according to claim 5, wherein step 1) specifically comprises adding 0.22 μL (10 μM) probe Probe1, 0.22 μL (10 μM) probe Probe2, and 0.22 μL miR-205 to 1 μL of a buffer solution containing 100 mM Tris-HCl and 0.5 M KCl, then adding 6.86 μL of RNAse-free water, mixing, heating to 95°C, reacting for 3 minutes, and slowly cooling to room temperature to obtain complementary hybridization solutions of probe Probe1 and probe Probe2 with miR-205, respectively.

9. The detection method according to claim 8, wherein the step 2) specifically comprises adding 1 μL of 10×SplintR buffer and 0.5 μL of SplintR ligase (25 U / μl) to the complementary hybridization solution, mixing the mixture, incubating the mixed solution at 37°C for 30 min, taking 5 μL of the incubated mixed solution, adding 5 μL of 10×T7 RNA polymerase reaction buffer, 2.5 μL of dithiothreitol (100 mM), 2 μL of NTPs (25 mM each NTP), 1.25 μL of recombinant RNA inhibitor (20 U / μL), 5 μL of T7 RNA polymerase (50 U / μL) and 29.25 μL of RNAse-free water, and incubating the mixed solution at 37°C for 16 h to obtain a transcribed RNA chain (Output RNA).

10. The detection method according to claim 8, wherein step 3) comprises taking 10 μl of the output RNA solution transcribed in step 2), dropping it on the electrode surface, reacting for a certain period of time, washing away non-specifically bound substances with distilled water, and detecting the result by electrochemical SWV in PBS, and measuring the change in signal before and after.

Images