A miRNA photoelectrochemical biosensor based on magnetic COF composite material and its preparation method and application

Abstract

The present invention discloses a miRNA photoelectrochemical biosensor based on a magnetic COF composite material, and a preparation method and application thereof. The sensor utilizes Fe3O4@D-A COF-HP1 to efficiently enrich the target miRNA from a complex sample, and triggers a catalytic hairpin self-assembly (CHA) reaction through ZnSe QDs-HP2, so that a large number of ZnSe QDs are connected to the Fe3O4@D-A COF surface, and then the magnetic complex is captured to the surface of a magnetic working electrode by magnetic adsorption, and the photocurrent signal change is recorded. ZnSe QDs can induce the photocurrent polarity reversal of the COF composite material. Within a certain concentration range, the greater the concentration of the target miRNA, the more obvious the photocurrent polarity reversal response. Based on the excellent photoelectric properties of the donor-acceptor type COF material, the photocurrent polarity reversal strategy, the CHA signal amplification strategy and the magnetic separation technology are organically combined to achieve highly sensitive detection of miRNA in complex samples, with the advantages of strong specificity and simple and rapid detection methods, and universality for the detection of different miRNAs.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional biomaterials and biosensors, and in particular relates to a miRNA photoelectrochemical biosensor based on a magnetic COF composite material, and a preparation method and application thereof. Background Art

[0002] MicroRNA (miRNA) is an endogenous non-coding RNA composed of 19-24 nucleotides. MiRNA is abnormally expressed in the occurrence and development stages of various diseases and can be used as a marker for non-invasive diagnosis. Therefore, the development of analytical methods for sensitive detection of miRNA is of great significance for clinical diagnosis. However, due to the short sequence length, low concentration, high sequence homology and easy degradation of miRNA, accurate detection of miRNA is extremely challenging. Currently, the most widely used miRNA detection methods include blotting, reverse transcription polymerase chain reaction and microarray. Among them, the blotting method is the standard method for detecting miRNA, but this method is time-consuming, low in sensitivity and requires a large amount of samples. Although the reverse transcription polymerase chain reaction method has the advantages of high sensitivity and good specificity, the required primers are short, resulting in low detection accuracy of this method. In addition, the reverse transcription step in this method increases the complexity of the experiment and increases the cost of the experiment. Although the microarray analysis method can achieve multiple detection of miRNA, its low reproducibility, cross-hybridization and nonspecific adsorption reduce the accuracy of the detection. Therefore, it is necessary to develop a highly specific, sensitive and simple miRNA detection method.

[0003] Photoelectrochemical (PEC) biosensors rely on the transfer of electrons and holes between photosensitive materials and electrodes to convert biochemical indicators into readable photoelectric signals. They have high sensitivity and low background signals, which are more advantageous than traditional detection methods. In addition, this sensing technology has been widely used in various biological detections due to its low cost, simple operation and easy miniaturization. However, the traditional PEC sensor analyzes the photoelectric signal based on the enhancement or weakening of the background signal during the detection process. Its disadvantage is that coexisting interferents can easily cause false positive or false negative signals, resulting in reduced detection specificity and sensitivity. In view of the above problems, the photocurrent polarity reversal strategy is an ideal choice. The construction of the photocurrent polarity reversal type photoelectrochemical biosensor can achieve the change of the polarity of the photocurrent in the presence of the target, thus having the advantages of high sensitivity and high selectivity.

[0004] In PEC biosensors, the selection of photoelectric active materials is crucial. Some traditional photoelectric materials, such as sulfides, carbon nitrides, oxides, etc., are widely used in the field of PEC sensing due to their good photoelectric properties. However, for most PEC sensors constructed with traditional active materials, electron donors, such as ascorbic acid and hydrogen peroxide, need to be added during detection to compensate for electron consumption and obtain stable PEC signals. At the same time, the transfer of photoelectrons between photoelectric active materials and electron donors is an intermolecular transfer process, which greatly reduces the efficiency and stability of electron transfer, thereby limiting the improvement of the intensity of the photocurrent response signal. Electron donor-acceptor type (DA) covalent organic framework material (COF) is a new type of semiconductor composed of electron-donating groups and electron-withdrawing groups. PEC analysis based on DA COF can obtain good photoelectric signals without adding electron donors to the detection solution. In addition, DA COF can realize intramolecular electron transfer and has a self-enhancement effect, which is expected to improve the performance of PEC sensing and has potential application prospects in the field of PEC sensing. Therefore, it is of great significance to develop a new photocurrent polarity reversal system based on the excellent photoelectric properties of donor-acceptor COF materials and construct a PEC biosensor that can be used for sensitive detection of miRNA. Summary of the invention

[0005] The purpose of the present invention is to combine the catalytic hairpin assembly (CHA) signal amplification strategy with magnetic separation technology to develop a photocurrent polarity reversal type photoelectrochemical biosensor based on magnetic COF composite materials for highly sensitive and specific detection of miRNA in complex samples. Figure 1As shown in the figure, first, the hairpin DNA (HP1) modified Fe3O4@DA COF (Fe3O4@DA COF-HP1) efficiently enriches the target miRNA from complex samples through base complementary pairing and opens the hairpin structure of HP1. Subsequently, the introduction of hairpin DNA HP2 (ZnSe QDs-HP2) modified with ZnSe quantum dots (QDs) initiates the CHA reaction and replaces the miRNA. The released miRNA then hybridizes with the next hairpin structure HP1, triggering the next CHA reaction. This cycle allows a large number of ZnSe QDs to be captured on the surface of Fe3O4@DA COF, thereby achieving signal amplification. Finally, the magnetic complex is introduced to the surface of the magnetic working electrode by magnetic adsorption, and the PEC signal change is detected under light, thereby achieving highly sensitive and specific detection of the target. Among them, ZnSe QDs, as a photosensitive material, matches DA COF in energy level, which is not only conducive to the transfer of photogenerated carriers, but also can induce the change of photocurrent polarity of the magnetic COF composite material (Fe3O4@DA COF). The proposed novel photocurrent polarity reversal system based on ZnSe QDs-induced magnetic COF composites is beneficial to improve the specificity of PEC biosensors. In addition, by replacing the corresponding HP1 and HP2, different types of miRNA can be detected.

[0006] Based on the above purpose, the technical solutions involved in the present invention are as follows:

[0007] (1) Preparation of Fe3O4@DA COF-HP1

[0008] a. Preparation of Fe3O4

[0009] First, FeCl3·6H2O (0.24-1.88 g) was dissolved in 10-80 mL ethylene glycol. Subsequently, 0.40-1.86 g sodium acetate and 0.10-1.00 g sodium citrate were added in sequence and stirred for 10-90 min. The mixed solution was transferred to a stainless steel reactor with a polytetrafluoroethylene liner and reacted at 150-220 ° C for 6-20 h. After cooling to room temperature, the product was collected with a magnet, washed several times with deionized water and ethanol, and then vacuum dried.

[0010] b. Preparation of Fe3O4@DA COF

[0011] Weigh 10-50 mg of Fe3O4, disperse it in 5-50 mL of acetonitrile solution, and ultrasonically treat for 10-60 min. Then add 1,3,5-tri(4-aminophenyl)benzene (0.1-0.3 mmol) and shake slowly on a shaker at room temperature for 2-8 h. Subsequently, add 0.1-0.4 mmol of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde and 1-8 mL of glacial acetic acid (6-12 mol / L), continue shaking at room temperature for 12-36 h, separate the product with an external magnetic field, wash it with methanol three times, and finally dry it in vacuum.

[0012] c. Preparation of carboxylated Fe3O4@DA COF (Fe3O4@DA COF-PAA)

[0013] Mix 0.5-2 mL of polyacrylic acid (PAA) solution (5 mg / mL) with 0.5-2 mL of an aqueous solution containing 0.1 M EDC / NHS (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide), and shake in the dark for 10-40 min. Then, add 0.5-2 mL of Fe3O4@DA COF solution (4 mg / mL). After shaking the mixed solution for 12-36 h, wash it three times with ultrapure water and perform magnetic separation. Finally, disperse the product in 2 mL of ultrapure water and store it at 4°C for later use.

[0014] d. Preparation of Fe3O4@DA COF-HP1

[0015] 10-200 μL of carboxylated Fe3O4@DA COF dispersion was mixed with 10-200 μL of 0.1M EDC / NHS solution, shaken for 10-40 min, magnetically separated and washed with phosphate buffered saline (PBS) solution (0.1M, pH=7.4), then 10-200 μL of PBS solution containing HP1 (100 μM) was added, and then reacted at 25-40°C for 40-90 min to obtain Fe3O4@DA COF-HP1. After magnetic separation and washing with PBS solution, the remaining active sites were blocked with 1% bovine serum albumin (BSA), and then redispersed in 100 μL of PBS solution for use.

[0016] (2) Preparation of ZnSe QDs-HP2

[0017] a. Preparation of ZnSe QDs

[0018] Under nitrogen atmosphere, 28-240 mg of NaBH4 was added to 1-8 mL of ultrapure water, and then 22-220 mg of Se powder was added to obtain NaHSe as a Se precursor. 100-400 mg of Zn(CH3COOH)2 was dissolved in 5-40 mL of ultrapure water, stirred with N2 bubbling at 90-110 ° C, and then 200-500 μL of mercaptopropionic acid (MPA) was added. Under N2 protection, the pH value of the solution was adjusted to 9-11 with 0.1-1 M NaOH, and 1-5 mL of NaHSe solution was quickly injected into the above solution. After reacting for 2-6 hours, an MPA-coated ZnSe QDs solution was obtained. After extraction with ethanol three times, the unreacted reagents were removed by centrifugation at 8000 rpm to obtain purified ZnSe QDs. Ultrapure water was added for ultrasonic dispersion to obtain a 1-4 mg / mL ZnSe QDs dispersion, which was stored at 4 ° C in the dark for later use.

[0019] b. Preparation of ZnSe QDs-HP2

[0020] 10-200 μL of ZnSe QDs dispersion was added to 200 μL PBS (0.1M, pH=7.4) solution containing 0.1M EDC / NHS, and reacted for 20-60 min. After centrifugation and washing with PBS, 50-300 μL PBS containing 150 μM HP2 was added, reacted at 37°C for 1-3 h, and centrifuged and washed 3 times with PBS to obtain ZnSe QDs-HP2. Finally, it was blocked with 1% BSA and redispersed in 200 μL PBS solution and stored at 4°C for later use.

[0021] (3) Preparation of photoelectrochemical biosensors

[0022] First, 10-100 μL of Fe3O4@DA COF-HP1 solution and 10-100 μL of ZnSe QDs-HP2 solution were added to 10-100 μL of miRNA samples with different concentrations and reacted for 10-60 min. Since there are more binding sites between HP2 and HP1, the addition of HP2 can trigger CHA, and miRNA is displaced and enters the next cycle. The obtained Fe3O4@DA COF-HP1 / ZnSe QDs-HP2 complex was dispersed in 100 μL of PBS solution after magnetic separation and PBS washing. Finally, the dispersion (10-30 μL) was dropped on the surface of the magnetic indium tin oxide (ITO) electrode, and the magnetic complex was quickly captured by the electrode. PEC test was carried out in PBS solution under xenon lamp irradiation. Among them, the saturated calomel electrode and the platinum electrode were used as the reference electrode and the counter electrode, respectively, and together with the working electrode, they formed a three-electrode system.

[0023] The miRNAs are: miRNA-1, miRNA-16, miRNA-21, miRNA-34a, miRNA-107, miRNA-133a, miRNA-138, miRNA-141, miRNA-146a, miRNA-155, miRNA-373, etc.

[0024] Principle of the invention: The Fe3O4@DA COF in the present invention has excellent photoelectric properties and strong magnetism, and is modified with a large number of probes HP1. When the target miRNA is present, the HP1-modified Fe3O4@DA COF can efficiently enrich the target from a complex sample. Therefore, the use of Fe3O4@DA COF can simplify the experimental operation steps and aggregate the analytes, thereby improving the sensitivity of the biosensor. The presence of the target miRNA opens the HP1 hairpin structure of Fe3O4@DA COF-HP1, but because there are more binding sites between HP2 and HP1, the addition of ZnSe QDs-HP2 can trigger the CHA reaction and release miRNA to enter the next cycle, so that more ZnSe QDs can be captured on the Fe3O4@D-ACOF surface, which is beneficial to the detection of low-concentration targets. Finally, the magnetic complex is introduced to the surface of the magnetic working electrode by magnetic adsorption, and the PEC signal changes are detected under light to achieve highly sensitive and specific detection of the target. Its advantages are:

[0025] (1) High sensitivity. The DA COF and ZnSe QDs used in the present invention are both optoelectronic materials with good optoelectronic properties. The introduction of extremely low concentrations of the target can cause obvious photocurrent signal output; on the other hand, the catalytic hairpin self-assembly strategy is adopted to achieve signal amplification by cyclically releasing the target miRNA.

[0026] (2) Strong specificity. Common other miRNAs and electroactive / electroinert interferents have no interference with the detection of target miRNA. The reason is that: on the one hand, the present invention is based on the design of two hairpin structure DNA sequences (HP1 and HP2) based on the target miRNA sequence. The target miRNA binds to HP1 through base complementary pairing to open the HP1 hairpin structure, so that HP2 can bind to HP1 through base complementary pairing, triggering the CHA reaction. Other miRNA sequences are different and cannot trigger the CHA reaction; on the other hand, only the target miRNA can induce the binding of ZnSe QDs-HP2 and Fe3O4@D-ACOF-HP1, triggering the reversal of the photocurrent polarity, but the presence of other miRNAs and electroactive / electroinert interferents cannot induce the binding of ZnSe QDs-HP2 and Fe3O4@DA COF-HP1, and cannot change the photocurrent polarity. Therefore, they have no interference with this detection system.

[0027] (3) The detection method is simple and rapid. Fe3O4@D-ACOF-HP1, ZnSe QDs-HP2, and target miRNA are placed in the same container to achieve rapid identification and form a photocurrent polarity reversal system ZnSe QDs / / Fe3O4@DA COF. Through magnetic-assisted rapid separation and adsorption onto the magnetic ITO electrode, an immediate photoelectrochemical polarity reversal signal can be detected, achieving highly sensitive and selective detection of the target.

[0028] (4) This method is universal. It only requires designing the corresponding hairpin structure DNA (HP1 and HP2) according to the target miRNA sequence to detect different miRNAs.

[0029] In summary, the present invention is based on a magnetic COF composite material, organically combines the photocurrent polarity reversal strategy, the catalytic hairpin self-assembly signal amplification strategy and the magnetic separation technology to construct a miRNA photoelectrochemical biosensor, which has the advantages of high sensitivity, high specificity, universality, simplicity, speed and easy operation. It can realize the detection of low-concentration miRNA in complex samples and has good application prospects for instant and clinical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a flow chart of the preparation of the miRNA photoelectrochemical biosensor based on the magnetic COF composite material;

[0031] Figure 2 is a photoelectric response diagram of the photoelectrochemical biosensor for detecting miRNA-138;

[0032] Figure 3 It is a standard curve diagram of the photoelectrochemical biosensor detecting miRNA-138;

[0033] Figure 4 This is a graph showing the selectivity experimental data of the photoelectrochemical biosensor. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0035] In the following examples, all oligonucleotides were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0036] Example 1 Preparation of Fe3O4@DA COF-HP1 and ZnSe QDs-HP2

[0037] (1) Preparation of Fe3O4@DA COF-HP1

[0038] a. Preparation of Fe3O4

[0039] First, FeCl3·6H2O (1.36 g) was dissolved in 40 mL of ethylene glycol. Subsequently, 1.44 g of sodium acetate and 0.40 g of sodium citrate were added in sequence and stirred for 50 min. The mixed solution was transferred to a stainless steel reactor with a polytetrafluoroethylene liner and reacted at 200 °C for 14 h. After cooling to room temperature, the product was collected with a magnet, washed several times with deionized water and ethanol, and then vacuum dried.

[0040] b. Preparation of Fe3O4@DA COF

[0041] Weigh 30 mg of Fe3O4, disperse it in 30 mL of acetonitrile solution, and ultrasonicate for 30 min. Then add 1,3,5-tri(4-aminophenyl)benzene (0.24 mmol) and shake slowly for 6 h at room temperature on a shaker. Subsequently, add 0.36 mmol of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde and 6 mL of glacial acetic acid (12 mol / L), continue shaking at room temperature for 24 h, separate the product with an external magnetic field, wash it 3 times with methanol, and finally dry it in vacuum.

[0042] c. Preparation of carboxylated Fe3O4@DA COF (Fe3O4@DA COF-PAA)

[0043] 1 mL of PAA solution (5 mg / mL) was mixed with 1 mL of aqueous solution containing 0.1 M EDC / NHS and shaken in the dark for 30 min. Then, 1 mL of Fe3O4@DA COF solution (4 mg / mL) was added. After shaking the mixed solution for 24 h, it was washed with ultrapure water three times and subjected to magnetic separation. Finally, the product was dispersed in 2 mL of ultrapure water and stored at 4 °C for later use.

[0044] d. Preparation of Fe3O4@DA COF-HP1

[0045] 100 μL of carboxylated Fe3O4@DA COF dispersion was mixed with 100 μL of 0.1M EDC / NHS solution, shaken for 30 min, washed with PBS solution, and then added with 100 μL of PBS solution containing HP1 (100 μM), and then reacted at 37°C for 60 min to obtain Fe3O4@D-ACOF-HP1, wherein the sequence of HP1 (hairpin DNA) is 5'-NH2-(CH2)6-CGGCCTGATTCACAACACCAGCTCCATGTGTAGAAGCTGGTGTTGTGAATC-3'. After washing with PBS and blocking with 1% BSA, it was redispersed in 100 μL of PBS solution for use.

[0046] (2) Preparation of ZnSe QDs-HP2

[0047] a. Preparation of ZnSe QDs

[0048] Under nitrogen atmosphere, 132 mg of NaBH4 was added to 4 mL of ultrapure water, and then 110 mg of Se powder was added to obtain NaHSe as a Se precursor. 229 mg of Zn(CH3COOH)2 was dissolved in 25 mL of ultrapure water, stirred with N2 bubbling at 100 °C, and then 375 μL of MPA was added. Under N2 protection, the pH value of the solution was adjusted to 10 with 1 M NaOH, and 2.85 mL of NaHSe solution was quickly injected into the above solution. After reacting for 5 hours, an MPA-coated ZnSe QDs solution was obtained. After three extractions with ethanol, the unreacted reagents were removed by centrifugation at 8000 rpm to obtain purified ZnSe QDs. Ultrapure water was added for ultrasonic dispersion to obtain a 2 mg / mL ZnSe QDs dispersion, which was stored in the dark at 4 °C for later use.

[0049] b. Preparation of ZnSe QDs-HP2

[0050] 100 μL of ZnSe QDs was added to 200 μL PBS (0.1M, pH=7.4) solution containing 0.1M EDC / NHS and reacted for 30 min. After centrifugation and washing with PBS, 200 μL PBS containing 150 μM HP2 was added, reacted at 37°C for 2 h, and centrifuged and washed 3 times with PBS to obtain ZnSe QDs-HP2, wherein the sequence of the HP2 (hairpin structure DNA) is 5'-NH2-(CH2)6-CACCAGCTTCTACACATGGAGCTGGTGTTGTGAATCCATGTGTAGA-3'. Finally, it was blocked with 1% BSA, and then redispersed in 200 μL PBS solution and stored at 4°C for future use.

[0051] Example 2 Detection of miRNA in standard samples and human serum samples

[0052] Based on the Fe3O4@DA COF-HP1 and ZnSe QDs-HP2 prepared in Example 1, 10 μL of Fe3O4@D-ACOF-HP1 solution and 100 μL of ZnSe QDs-HP2 solution were added to 100 μL of standard solution / human serum solution containing different concentrations of miRNA and reacted for 30 min. The obtained Fe3O4@DA COF-HP1 / ZnSe QDs-HP2 complex was washed with PBS and magnetically separated and dispersed in 100 μL of PBS solution. Finally, the dispersion (20 μL) was dropped on the surface of the magnetic ITO electrode, and the magnetic complex was quickly captured by the electrode. PEC test was performed in PBS solution under xenon lamp irradiation.

[0053] Example 3 Detection of miRNA-138

[0054] Based on the process of Example 1 and Example 2, miRNA-138 was used as the target model for detection. The miRNA-138 sequence was 5'-AGCUGGUGUUGUGAAUCAGGCCG-3', and the sequences of HP1 and HP2 in the prepared Fe3O4@DA COF-HP1 and ZnSeQDs-HP2 were designed according to the sequence of the target miRNA-138. The photocurrent response curves were recorded when different miRNA-138 standard concentrations were added. The concentrations of the miRNA-138 standard solutions from a to j were 0fM, 1fM, 5fM, 10fM, 50fM, 100fM, 1pM, 10pM, 30pM, and 50pM, respectively. The results are shown in the attached figure. Figure 2 As the concentration of miRNA-138 increases, the intensity of the polarity-reversed photocurrent signal increases. Figure 3 As shown, it can be observed that the PEC photocurrent signal size and the logarithm of the miRNA-138 concentration show a good linear relationship between 1fM–10pM. The linear correlation equation is ΔI(nA)=672.26logC miRNA -138(fM)+1380.66(R 2 =0.9924). ΔI = I–I0, where I and I0 are the photocurrent values ​​in the presence and absence of miRNA-138, respectively. The calculated detection limit is 0.42 fM, indicating that the sensor has excellent analytical performance. The above experimental results show that the analytical method we proposed has application value in sensitive detection of miRNA.

[0055] Example 4 Selectivity and Anti-interference Experiment

[0056] In the selectivity and anti-interference experiments, the concentration of the target miRNA-138 was 1pM, and the names and concentrations of the other interfering substances used were as follows: miRNA-155 (10pM), miRNA-141 (10pM), miRNA-21 (10pM) and miRNA-107 (10pM). According to the steps of the above-mentioned Examples 1, 2 and 3, other miRNAs were used to replace miRNA-138 for selective detection, and other miRNAs were mixed with miRNA-138 for anti-interference detection. The results are shown in the attached Figure 4 As shown, compared with miRNA-138, the introduction of other miRNAs did not cause the polarity reversal of the photocurrent signal, which was basically close to the blank signal, indicating that the method of the present invention has excellent selectivity for the detection of the target miRNA-138. In addition, after the other miRNAs were mixed with miRNA-138, the polarity reversal of the photocurrent and the signal intensity after the reversal were not affected, indicating that the sensor proposed by the present invention has excellent anti-interference performance for the detection of the target miRNA.

[0057] The above description is a detailed description of a feasible embodiment of the present invention, but the embodiment is not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A miRNA photoelectrochemical biosensor based on a magnetic COF composite material, characterized in that: The electron donor-acceptor covalent organic framework material DA COF is used as the photoelectric active material of the photoelectrochemical biosensor; The photoelectrochemical biosensor comprises: Fe3O4@DA COF-HP1, ZnSe QDs-HP2; Its characteristics are also that: the target miRNA can bind to HP1 through base complementary pairing to open the HP1 hairpin structure of Fe3O4@DA COF-HP1, so that HP2 of ZnSe QDs-HP2 can bind to HP1 through base complementary pairing to trigger the CHA reaction; the target miRNA can induce the binding of ZnSe QDs-HP2 and Fe3O4@DA COF-HP1, triggering the reversal of the photocurrent polarity.

2. The method for preparing a miRNA photoelectrochemical biosensor based on a magnetic COF composite material according to claim 1, characterized in that: The method comprises the following steps: a. Preparation of Fe3O4@DA COF composite material Fe3O4@DA COF-HP1 modified with hairpin structure DNA HP1; b. Preparation of hairpin structure DNA HP2 modified ZnSe quantum dots ZnSe QDs-HP2; c. Preparation of photoelectrochemical biosensor: Fe3O4@DA COF-HP1 solution and ZnSe QDs-HP2 solution were added to a solution containing different concentrations of miRNA. After the reaction, the obtained Fe3O4@DA COF-HP1 / ZnSe QDs-HP2 complex was washed with phosphate buffer solution and magnetically separated, and redispersed in PBS solution. Finally, the dispersed solution was dropped on the surface of the magnetic indium tin oxide electrode, and the magnetic complex was quickly captured by the electrode to prepare the miRNA photoelectrochemical biosensor.

3. The method for preparing a miRNA photoelectrochemical biosensor based on a magnetic COF composite material according to claim 2, characterized in that: In step a, the Fe3O4@DA COF composite material is prepared by using Fe3O4 as a precursor through an in-situ polymerization reaction of 1,3,5-tri(4-aminophenyl)benzene and 2,5-dimethoxyphenylene-1,4-dicarboxaldehyde.

4. Use of a miRNA photoelectrochemical biosensor based on a magnetic COF composite material as claimed in claim 1 or a miRNA photoelectrochemical biosensor based on a magnetic COF composite material prepared by the preparation method according to any one of claims 2 to 3 in the detection of miRNA for non-diagnostic purposes.

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