Preparation method of miRNA-141 photoelectrochemical biosensor for regulating GaN hole transport based on G-quadruplex and detection method of miRNA-141

Abstract

The invention relates to a preparation method of a photoelectrochemical biosensor of miRNA-14 based on G-quadruplex regulation and control of GaN hole transport and a detection method of miRNA-141. An Au / GaN electrode with excellent photoelectric conversion efficiency is adopted, a hairpin DNA HP1 serving as a miRNA-141 detection probe is fixed on the surface of the electrode through an Au-S bond, then a hairpin DNAHP2 is introduced, and a hybrid chain amplification reaction (HCR) is started; and HP1 and HP2 with a large amount of G form a G-quadruplex under the induction of proper metal ions, so that the electron-hole pair separation efficiency of the electrode can be efficiently improved, and a photoelectric signal is remarkably enhanced, thereby realizing the quantitative detection of the miRNA-141. The method has the advantages of being high in sensitivity (the detection limit is as low as 0.13 fmol / L), rapid in detection, small in sample dosage and the like, a high-sensitivity technical means is provided for early diagnosis of related diseases, the method can be expanded to be applied to detection of other short-sequence and low-abundance miRNA, including but not limited to detection of other short-sequence and low-abundance miRNA such as miRNA-122 and miRNA-21 through replacement of aptamer sequences, and the application prospect is wide. The applicability is wide.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of analytical detection, in particular to a preparation method of a photoelectrochemical biosensor for miRNA-141 based on G-quadruplex regulation of GaN hole transport and a detection method of miRNA-141. BACKGROUND

[0002] Micro ribonucleic acid (miRNA) is a kind of endogenous small RNA molecule with a length of about 20-24 nucleotides, which plays a key role in cell gene expression regulation. Among them, miRNA-141 as a member of the miRNA-200 family has been confirmed to not only play a regulatory role in the occurrence and development of various cancers, but also participate in the pathological process of immune abnormal diseases and organ fibrosis, and its abnormal expression is closely related to the pathological mechanism of various diseases. Therefore, establishing a high-sensitivity miRNA-141 detection method has important clinical value for early diagnosis of diseases.

[0003] At present, miRNA detection technologies mainly include reverse transcription polymerase chain reaction (RT-PCR), Northern blotting and chip technology. However, these technologies have limitations: RT-PCR is complex and easy to be contaminated, Northern blotting has low sensitivity and complicated steps, and chip technology has large sample demand and high cost. These defects limit the application of miRNA-141 detection in early diagnosis, and therefore a new technology with high sensitivity, rapid detection and small sample amount needs to be developed. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a preparation method of a photoelectrochemical biosensor for miRNA-141 based on G-quadruplex regulation of GaN hole transport and a detection method of miRNA-141, which has the characteristics of simple structure, low cost, high sensitivity and excellent selectivity.

[0005] To achieve the above purpose, the present application adopts the following technical solution: a preparation method of a photoelectrochemical biosensor for miRNA-141 based on G-quadruplex regulation of GaN hole transport, the photoelectrochemical biosensor comprises a detection electrode, a counter electrode, a reference electrode and a detection solution, and comprises the following steps: Step A1: preparing the miRNA-141 detection electrode: gold with a set thickness is sputtered on the surface of a GaN wafer by a sputtering method; a hairpin DNA HP1 aptamer with a partial G-quadruplex sequence is fixed on the electrode surface by incubation to form an Au-S bond, thereby forming a miRNA-141 detection probe; the miRNA-141 detection probe has a hairpin structure of G-quadruplex and is connected to the electrode surface through the Au-S bond; Step A2: the hairpin structure can base-pair with miRNA-141; Step A3: the detection solution contains a hairpin DNA HP2 used to initiate the hybridization chain reaction, and the hairpin DNA HP2 combines with the hairpin DNA HP1 to form a DNA nanowire, thereby enhancing the photoelectric signal.

[0006] In a preferred embodiment, the electrode substrate is gold-plated GaN.

[0007] In a preferred embodiment, the hairpin DNA HP1 with a partial G-quadruplex is connected to the electrode surface through an Au-S bond to form a miRNA-141 detection probe.

[0008] In a preferred embodiment, the photoelectrochemical biosensor can detect the target miRNA-141 by the following steps: the hairpin DNA HP1 aptamer base-pairs with the miRNA-141 to open the hairpin structure, thereby triggering the signal amplification process.

[0009] In a preferred embodiment, the hybridization chain reaction further includes that after the hairpin DNA HP1 structure is opened, the hairpin DNA HP1 and the hairpin DNA HP2 cross-hybridize through specific base-pairing to form a stable G-rich DNA nanowire structure.

[0010] In a preferred embodiment, the G-quadruplex is formed under the action of metal ions to enhance the photoelectric signal.

[0011] The application also provides a detection method for miRNA-141, which uses the above-mentioned preparation method of a photoelectrochemical biosensor for miRNA-141 based on G-quadruplex-regulated GaN hole transport to prepare a photoelectrochemical biosensor for miRNA-141 based on G-quadruplex-regulated GaN hole transport: in the detection process, when miRNA-141 exists in the sample to be detected, the miRNA-141 base-pairs with the hairpin DNA HP1 to open the hairpin structure of the HP1, thereby triggering the HCR of the HP2 and the HP1 in the detection solution to form a stable DNA nanowire structure; under the action of metal ions such as magnesium, the G-rich sequence in the DNA nanowire is stacked to form a G-quadruplex structure; by measuring the photocurrent response of the electrode under light, the presence and concentration of the miRNA-141 are quantitatively judged and analyzed according to the change value of the photocurrent.

[0012] Compared with the prior art, the application has the following beneficial effects: 1. The introduction of the Au layer solves the problems of high chemical stability and difficulty in modification of GaN, and Au / GaN forms a metal semiconductor heterojunction, which inhibits the recombination of photo-generated carriers through a Schottky barrier, thereby realizing the optimization of carrier separation and the strengthening of targeted recognition, and solving the problems of high carrier recombination rate of traditional semiconductor electrodes and unstable probe loading.

[0013] 2. The G-quadruplex promotes the rapid transport of photo-generated holes in GaN, enhances the photocurrent response, and thereby realizes the quantitative detection of miRNA-141. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The PEC sensor working principle schematic diagram of the preferred embodiment of the present application is shown in Figure 1 The PEC sensor working principle schematic diagram of the preferred embodiment of the present application is shown in

[0015] Figure 2 The PAGE analysis result schematic diagram of the preferred embodiment of the present application is shown in

[0016] Figure 3 The AFM image and the height distribution diagram thereof of the preferred embodiment of the present application are shown in

[0017] Figure 4 (A) of which is the photocurrent response curve of different modified electrodes in 5.00 mL, 0.0100 mol / L Tris-HCl solution (experimental conditions: miRNA-141 concentration 10.0 μmol / L, hairpin RNA1 and hairpin DNA HP2 concentrations are both 100 μmol / L, and the solution is 0.100 mol / L Tris-HCl buffer solution with pH 7.4); Figure 4 (B) of which is the electrochemical impedance spectroscopy (EIS) diagram of different modified electrodes in 5.00 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] mixed solution containing 0.100 mol / L KCl, and the experimental conditions are the same as above.

[0018] Figure 5 The schematic diagram showing the effects of incubation concentration and time of thiolated hairpin RNA1, incubation time of miRNA-141, and subsequent incubation time of hairpin RNA1 / hairpin DNA HP2 mixture on photocurrent signal is shown in. In the experiment, the concentrations of hairpin RNA1 and hairpin DNA HP2 are both 500 nmol / L, the concentration of miRNA-141 is 10.0 fmol / L, and the solution is 0.100 mol / L Tris-HCl buffer solution (pH 7.4).

[0019] Figure 6The relationship between the photocurrent response and the concentration of miRNA-141: (A) is the photocurrent curve of adding different concentrations of miRNA-141 (a-g corresponds to the concentration of 0, 0.500 fmol / L, 1.00 fmol / L, 10.0 fmol / L, 100 fmol / L, 1.00 pmol / L and 10.0 pmol / L); (B) is the corresponding relationship diagram of the photocurrent change value and the concentration of miRNA-141, and the interpolation diagram is the linear fitting curve of the concentration range of 0.500 fmol / L-10.0 pmol / L (n=3). The solution is 0.100 mol / L Tris-HCl buffer (pH 7.4).

[0020] Figure 7 The specific detection results of the PEC sensor for miRNA-141. In the control experiment, the concentrations of miRNA-122, miRNA-210, miRNA-26a, miRNA-126 and miRNA-21 are all 1.00 pmol / L, and the concentration of miRNA-141 is 10.0 fmol / L. The solution is 0.100 mol / L Tris-HCl buffer (pH 7.4). DETAILED DESCRIPTION

[0021] The application will be further described below in conjunction with the drawings and examples.

[0022] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0023] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application; as used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form, and furthermore, it should be understood that when the terms "comprise" and / or "include" are used in the specification, they mean the presence of a feature, step, operation, device, component and / or combination thereof.

[0024] A preparation method of a photoelectrochemical biosensor for miRNA-141 based on G-quadruplex regulation of GaN hole transport and a photoelectrochemical detection method for miRNA-141, referring to Figures 1-7 The sensor has the characteristics of simple structure, low cost, high sensitivity and excellent selectivity.

[0025] To achieve the above object, the application adopts the following technical scheme: taking Au / GaN electrode with excellent photoelectric conversion efficiency as a substrate, fixing hairpin DNA HP1 containing part G-quadruplex sequence on the electrode surface through Au-S bond to construct miRNA-141 detection probe; miRNA-141 in the system to be detected is paired with hairpin DNA HP1 and makes the latter open, further introducing hairpin DNA HP1 and hairpin DNA HP2 to start hybrid chain amplification reaction, forming DNA long chain nanowire; under the action of metal ions, the G-rich sequence at the tail end of the hairpin in the nanowire is folded and stacked to form a large number of G-quadruplexes, which improves the separation efficiency of the electrode electron-hole pairs and enhances the photoelectric signal, so that the quantitative detection of miRNA-141 is realized.

[0026] The technical scheme of the application mainly includes the following aspects: 1) Sensing system: the sensing system includes a three-electrode system (Au / GaN working electrode modified with HP1, platinum counter electrode, mercury reference electrode), detection solution containing hairpin DNA HP1, hairpin DNA HP2 and metal ions.

[0027] 2) The preparation of the Au / GaN working electrode is as follows: a certain thickness of gold is sputtered on the surface of the GaN wafer by sputtering method; the solution containing thiol-modified hairpin DNA HP1 aptamer (HP1-SH) is co-incubated with the Au / GaN electrode, and the hairpin DNA HP1 of miRNA-141 is fixed on the electrode surface through Au-S bond to construct the miRNA-141 detection probe.

[0028] 3) The DNA sequence includes: HP1 (hairpin 1), HP2 (hairpin 2), and HP1 modified with thiol (HP1-SH). They have the following characteristics and functions respectively: HP1 contains a sequence complementary to miRNA-141 and part G-quadruplex sequence; after HP1 recognizes miRNA-141, the exposed single-stranded region (such as “AAAGATGTGGTCTT”) of the hairpin can be paired with the complementary region (“TGGGTGTTAAGACCA”) of HP2 to start HCR and form G-rich DNA nanowire; part G-quadruplex sequence “AGGGCGGGTGGG” in HP1 can be folded into G-quadruplex under the induction of metal ions (such as Mg²⁺); the thiol group (-SH) of HP1-SH is the key group for realizing “Au-S bond fixation”.

[0029] All G-containing sequences (such as “AGGGCGGGTGGG” in HP1) can be folded into G-quadruplex under the induction of metal ions (such as Mg²⁺), which improves the separation efficiency of the electron-hole pairs.

[0030] This invention utilizes metal ions to induce the stacking of G-rich sequences at the hairpin ends of DNA long-chain nanowires to form a large number of G-quadruplexes, which can efficiently improve the electron-hole pair separation efficiency of Au / GaN electrodes, enhance photoelectric signals, and achieve high-sensitivity detection of miRNA-141.

[0031] The miRNA-141 photoelectrochemical sensor provided by this invention is not only applicable to the detection of miRNA-141, but can also be extended to the detection of other short-sequence, low-abundance miRNAs by adjusting the aptamer sequence, thus having wide applicability.

[0032] The miRNA-141 detection method provided by this invention has a wide detection range (0.500 fmol / L~10.0 pmol / L) and a low detection limit (0.13 fmol / L), exhibiting higher sensitivity and selectivity compared to existing technologies. This invention uses the detection of miRNA-141 as an example for detailed explanation. As a key microRNA involved in the pathological processes of various diseases, the accurate detection of miRNA-141 is of great significance for early disease diagnosis. This invention effectively improves the sensitivity and specificity of detection by constructing an HCR system and utilizing the property of G-quadruplexes to enhance GaN hole transport capacity.

[0033] Specifically, this invention relates to the construction of a photoelectrochemical analysis method for microRNA-141 based on the GaN hole transport properties regulated by the G-quadruplex. This invention utilizes a specifically designed hairpin RNA structure to achieve precise identification and signal amplification of miRNA-141, providing a new approach for microRNA detection.

[0034] Preparation of Au / GaN electrodes: An n-type GaN wafer (sapphire substrate) was selected and ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and ultrapure water for 5 min each, then dried with nitrogen. Using a JGP-450 (or similar) magnetron sputtering system, the GaN wafer was placed in the sputtering chamber and evacuated to a vacuum level ≤5×10⁻⁻. 4 Pa; Argon gas (flow rate 10~20 sccm) is introduced as sputtering gas, sputtering power is set to 50~100 W, sputtering time is 10~60 s; after sputtering, the gold-plated GaN electrode is annealed at 400℃ for 3 min to obtain a stable Au / GaN heterojunction electrode.

[0035] Examples of the DNA sequences of HP1, HP2, and HP1-SH are shown in Table 1, forming a hairpin DNA HP1 structure containing a partial G-quadruplex sequence. This structure is designed to specifically bind to the target miRNA-141, ensuring the specificity and accuracy of the detection.

[0036] Table 1: Nucleic acid sequences (5'-3') required for detecting miRNA-141: Preparation of PEC sensor: HP1-SH is immobilized on the Au / GaN electrode surface by the following steps: Au / GaN is used as the electrode substrate, heated at 95°C for 3-10 min, then cooled to room temperature (25°C) for at least 2-4 h. Then, 10-50.0 μL of 2.00-8.00 μmol / L (preferably 5.00 μmol / L) HP1-SH aptamer solution is deposited on the Au / GaN electrode surface and incubated at 37°C for 1-3 h (preferably 2 h) to form a stable covalent connection between HP1 and the electrode through Au-S bond. This step constructs the miRNA-141 detection probe, which is the core of high-sensitivity detection, because the formation of G-quadruplex can significantly enhance the photoelectric signal. Then, the modified electrode is thoroughly washed with ultrapure water to remove weakly bound or unbound aptamer solution.

[0037] Detection of miRNA-141: The solution containing miRNA-141 (concentration range 0.500 fmol / L-10.0 pmol / L, which is the detection linear range, corresponding to the possible concentration of miRNA-141 in the actual sample to be detected) is added to the surface of the Au / GaN electrode modified with HP1 and incubated at 37°C for 20-60 min to open the conformation of HP1. This step is the key to miRNA-141 detection, and sufficient incubation can ensure specific binding between miRNA-141 and the hairpin DNA HP1.

[0038] Hybridization chain amplification reaction: After opening the hairpin DNA HP1, the detection solution (containing 300-800 nmol / L HP1 and 300-800 nmol / L HP2, 10-50 mmol / L metal ions (such as Mg² + or K + , ), 0.100 mol / L Tris-HCl (pH 7.4)) is added to the system; incubate for 1.0-2.5 hours (preferably 2.5 hours) to start the hybridization chain amplification reaction to form G-rich sequence DNA nanowires, and promote the stacking of G-rich sequences to form G-quadruplex, improve the electron-hole pair separation ability of the electrode, and further improve the detection sensitivity.

[0039] Performance test of PEC sensor: Atomic force microscope (AFM) is used to characterize the "hairpin DNA HP1 + hairpin DNA HP2 + miRNA-141" complex, and the photocurrent-time curve and electrochemical impedance spectrum (EIS) are tested to verify the feasibility of the sensor.

[0040] Optimization of experimental conditions: including the incubation concentration and time of thiol-modified hairpin DNA HP1, the incubation time of miRNA-141, and the subsequent incubation time of hairpin DNA HP1 and hairpin DNA HP2 mixture to ensure that the sensor obtains the best sensitivity and selectivity. Thiol-modified hairpin DNA HP1 (HP1-SH): incubation concentration 2.00-8.00 μmol / L, incubation time 1-3 h (preferably 5.00 μmol / L, 2 h); miRNA-141: incubation time 20-60 minutes (adjust according to the concentration to be tested, low concentration samples are recommended for 60 minutes); hairpin DNA HP1 and HP2 mixture: incubation concentration is 300-800 nmol / L (preferably 500 nmol / L each), incubation time 1.0-2.5 h (preferably 2.5 h); Performance evaluation: Under the optimal conditions, test the influence of different concentrations of miRNA-141 on the sensing response signal, and evaluate the performance indicators (including detection limit, linear range and selectivity) of the sensor. The above evaluation indicators are crucial for verifying the practicality and reliability of the sensor.

Claims

1. A preparation method of a photoelectrochemical biosensor of miRNA-141 based on G-quadruplex regulating GaN hole transport, the photoelectrochemical biosensor comprising a detection electrode, a counter electrode, a reference electrode, a detection solution, characterized in that, The method comprises the following steps: Step A1: preparing the miRNA-141 detection electrode: gold of a set thickness is sputtered on the surface of a GaN wafer by a sputtering method; a hairpin DNA HP1 aptamer with a partial G-quadruplex sequence is fixed on the electrode surface by incubation to form an Au-S bond, thereby forming a miRNA-141 detection probe; the miRNA-141 detection probe has a G-quadruplex hairpin structure and is connected to the electrode surface through an Au-S bond; Step A2: the hairpin structure can base-pair with miRNA-141; Step A3: the detection solution contains a hairpin DNA HP2 used to initiate a hybridization chain reaction, which combines with the hairpin DNA HP1 to form a DNA nanowire and enhance the photoelectric signal.

2. The preparation method of the photoelectrochemical biosensor based on G-quadruplex-regulated miRNA-141 for hole transport in GaN according to claim 1, characterized in that, The electrode substrate is gold-plated GaN.

3. The preparation method of the photoelectrochemical biosensor based on G-quadruplex-regulated miRNA-141 for hole transport in GaN according to claim 1, characterized in that, The hairpin DNA HP1 with a partial G-quadruplex is connected to the electrode surface through an Au-S bond to form a miRNA-141 detection probe.

4. The preparation method of the photoelectrochemical biosensor based on G-quadruplex-regulated miRNA-141 for hole transport in GaN according to claim 1, characterized in that, The photoelectric chemical biosensor can detect the target miRNA-141 through the following steps: the hairpin DNA HP1 aptamer base-pairs with the miRNA-141 to open the hairpin structure, thereby triggering a signal amplification process.

5. The method of claim 1, wherein the method is characterized by: The hybridization chain reaction further comprises that, after the hairpin DNA HP1 structure is opened, the hairpin DNA HP1 and the hairpin DNA HP2 cross-hybridize through specific base-pairing to form a stable G-rich DNA nanowire structure.

6. The method of claim 1, wherein the method is characterized by: G-quadruplexes are formed under the action of metal ions to enhance the photoelectric signal.

7. A method for detecting miRNA-141, characterized by The photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 is prepared by the preparation method of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the photoelectric chemical biosensor based on G-quadruplex-regulated GaN hole transport for miRNA-141 according to any one of the preparation methods of the

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