Photoelectrochemical-colorimetric dual-mode biosensor and application thereof in miRNA detection

Abstract

The invention belongs to the technical field of biological analysis and detection, and particularly relates to a photoelectrochemical-colorimetric dual-mode biosensor and application thereof in miRNA detection. According to the biosensor prepared by the invention, the bifunctional CuO-Cu2O nanoflower and the three-dimensional network g-C3N4 / AuNPs electrode are integrated, and an enzyme-assisted strand displacement signal amplification mechanism is combined, so that dual-mode collaborative detection of a target miRNA-21 is realized: in a photoelectrochemical mode, the detection limit reaches 0.35 fM (the linear range is 1 fM to 10 fM), and response is performed based on photocurrent polarity reversal; the colorimetric mode is as follows: the detection limit is 3.9 fM (the linear range is 10 fM to 10 nM), and TMB (Tetramethylbenzidine) color development visualization analysis is carried out. Single-mode interference is effectively eliminated through double-signal independent output and cross validation, and the method is suitable for high-reliability detection of trace miRNA (such as a breast cancer marker miRNA-21) in complex samples such as serum. According to the invention, the defects of insufficient sensitivity (pM level), poor anti-interference performance and high platform complexity in the prior art are overcome, and an efficient tool is provided for early diagnosis of cancers.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection technology, specifically relating to a photoelectrochemical-colorimetric dual-mode biosensor and its application in miRNA detection. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] MicroRNAs (miRNAs) are endogenous small RNAs composed of 20-24 nucleotides that play important regulatory roles in various biological processes, primarily by targeting and modifying or inhibiting the translation of messenger RNA (mRNA). Currently, as key non-coding RNAs that can regulate gene expression, cancer cell proliferation and metastasis, and promote chemotherapy resistance, microRNAs have the potential to become important biomarkers and prognostic indicators for cancer. For example, miRNA-21 has been identified as a biomarker for breast cancer, lung cancer, and other cancers. Therefore, developing simple and reliable biosensors for accurate and sensitive detection of miRNAs is crucial for improving the early diagnosis, treatment, and prevention of diseases.

[0004] However, due to issues such as low miRNA expression and homology sequence interference, existing detection technologies have the following limitations: (1) Insufficient sensitivity: The detection limit of colorimetric methods and other methods that rely on nanozyme catalysis is usually in the pM range, which is difficult to meet the needs of early diagnosis; (2) Weak anti-interference ability: Single signal sensors (such as electrochemical and fluorescence methods) are easily affected by sample matrix, instrument, operator and non-standard analysis environment, thus lacking the reliability and accuracy of quantitative determination; (3) High platform complexity: Photoactive materials (such as TiO2) and nanozymes (such as Fe3O4) need to be constructed separately, resulting in poor interface compatibility.

[0005] Therefore, those skilled in the art urgently need to develop a dual-mode collaborative sensing platform to overcome the above limitations. Summary of the Invention

[0006] To address the needs of existing technologies, the present invention aims to provide a photoelectrochemical-colorimetric dual-mode biosensor and its application in miRNA detection. This invention develops a nanoprobe possessing both optical and enzymatic properties for the construction of a dual-mode biosensor. The designed dual-mode sensing platform exhibits unique accuracy in complex real-world sample analysis, based on the independent signal response and switching mechanism of miRNA-21 detected by photocurrent polarity switching and visual colorimetric methods. This offers significant potential for advancements in food safety analysis, environmental monitoring, and clinical diagnostics.

[0007] Specifically, the present invention provides the following technical solution: In a first aspect, the present invention provides a photoelectrochemical-colorimetric dual-mode biosensor, comprising: a photoactive electrode, an immobilized trapping probe, a bifunctional nanoprobe, and a component for recognizing amplification. The photoactive electrode is composed of an indium tin oxide (ITO) conductive substrate, a three-dimensional network carbon nitride (3D g-C3N4) layer supported on the surface of the conductive substrate, and gold nanoparticles (AuNPs) modified on the surface of the three-dimensional network carbon nitride layer. The immobilized capture probe is a hairpin DNA H3 immobilized on the surface of AuNPs via Au-N coordination bonds, and its sequence is SEQ ID NO:3; The bifunctional nanoprobe is formed by coupling CuO-Cu2O nanoflowers with exported DNA via amino bonds; The identification amplification component comprises hairpin DNA H1 and hairpin DNA H2, whose sequences are SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0008] Preferably, the CuO-Cu2O nanoflowers have a porous flower-like structure with a size of 450~550 nm, and are a composite crystalline phase of Cu2O (JCPDS05-0667) and CuO (JCPDS 45-0937); The three-dimensional network carbon nitride has a BET specific surface area of ​​120~125 m² / g and an average pore size of 20~25 nm.

[0009] A second aspect of the present invention provides an application of the photoelectrochemical-colorimetric dual-mode biosensor described in the first aspect in miRNA detection.

[0010] A third aspect of the present invention provides a method for detecting miRNA using the photoelectrochemical-colorimetric dual-mode biosensor described in the first aspect, comprising the following steps: S1. Hybridize the target miRNA-21 with hairpin DNA H1 to form a miRNA-21 / H1 complex; add CuO-Cu2O modified hairpin DNA H2, and form H1-H2 double strands through strand displacement reaction and release miRNA-21; then cut the H1-H2 double strands under the action of exonuclease III to release the exported DNA-CuO-Cu2O complex. S2. The output DNA-CuO-Cu2O complex is incubated with a photoactive electrode containing hairpin DNA H3, allowing the output DNA strand in the complex to hybridize with H3 through base complementary pairing. Then, the products modified on the photoactive electrode are subjected to photoelectrochemical signal detection. S3. Mix the output DNA-CuO-Cu2O complex with H2O2 and TMB solution, and then detect the colorimetric signal.

[0011] Preferably, in step S1, the concentration of hairpin DNA H1 is 2.2~2.6 μM, the final concentration of CuO-Cu2O modified hairpin DNA H2 is 1.8~2 μM, and the molar ratio of CuO-Cu2O modified hairpin DNA H2 to hairpin DNA H1 is 1:1.2~1.6; the temperature of the strand displacement reaction is 36~37℃, and the incubation time is 0.5~2 h; the CuO-Cu2O modified hairpin DNA H2 is obtained by EDC / NHS activating the carboxyl groups on the surface of CuO-Cu2O, reacting it with aminated hairpin DNA H2 in PBS buffer solution (pH 7.4) for 12 h, and then purifying it by centrifugation.

[0012] Preferably, in step S1, the digestion conditions of the exonuclease III are: reaction at 35~38℃ in buffer for 25~35 min, enzyme concentration of 1.5~2.5 U / μL, and the buffer is a mixture of NEBuffer and 0.1 M PBS (pH 7.4).

[0013] Preferably, in step S2, the output DNA-CuO-Cu2O complex is purified by magnetic separation and washed ≥3 times with PBS buffer (pH 7.4).

[0014] Preferably, in step S2, the method for preparing the photoactive electrode includes the following steps: (1) Mix lauric acid, copper nitrate and 1,3,5-benzenetricarboxylic acid in n-butanol, disperse by ultrasonication and then carry out a solvothermal reaction. The resulting precipitate is heated and reacted to obtain CuO-Cu2O nanoflowers. (2) Mix cyanuric acid and melamine in deionized water. After stirring and centrifuging at room temperature, the mixture is vacuum dried. After heating, the product is obtained as a three-dimensional network carbon nitride. (3) The three-dimensional network of carbon nitride is sequentially coated on ITO, AuNPs are electrodeposited, hairpin DNA H3 is fixed, and the sealing operation is performed to obtain the product.

[0015] More preferably, in step (1), the ratio of lauric acid, copper nitrate, 1,3,5-benzenetricarboxylic acid, and n-butanol is (50~55) mg:(80~82) mg:(45~50) mg:(25~35) mL; the temperature of the solvothermal reaction is 120~150℃, and the time is 3~6 h; the heating reaction is carried out by heating to 330~360℃ at a heating rate of 3~5℃ / min and holding at that temperature for 1.5~3 h. In step (2), the molar ratio of cyanuric acid to melamine is 1:1; the heating reaction is carried out by heating at a rate of 3~5℃ / min to 500~600℃ and holding at that temperature for 3~5 h. In step (3), the electrodeposition conditions are: chloroauric acid solution concentration 0.5~1 wt%, deposition potential -0.1~-0.2 V, deposition time 15~25 s; the hairpin DNA H3 concentration is 2~2.5 mM; the blocking operation refers to blocking non-specific sites with 6-mercaptohexanol, the concentration of 6-mercaptohexanol is 5~10 mM.

[0016] Preferably, in step S2, the detection conditions for the photoelectrochemical signal are: bias voltage of 0.05~0.15 V, excitation light source wavelength of 430~460 nm, buffer system of PBS buffer (pH 7.4), and photocurrent reading time of 8~12 s after illumination.

[0017] Preferably, in step S3, the colorimetric reaction system for detecting the colorimetric signal comprises TMB, H2O2, and PBS buffer, wherein the volume ratio of TMB, H2O2, and PBS buffer (pH 7.4) is 1:1:5~7; the concentration of TMB is 1.5~2.5 mM, and the concentration of H2O2 is 8~12 mM; the reaction temperature is room temperature, the reaction time is 8~12 min, and the detection wavelength is 650~654 nm.

[0018] Preferably, the concentration of miRNA-21 is quantified by photocurrent reversal value or absorbance value.

[0019] A fourth aspect of the present invention provides a kit for detecting miRNA-21, comprising the photoelectrochemical-colorimetric dual-mode biosensor described in the first aspect, and further comprising: (a) Exonuclease III (Exo III); (b) 3,3',5,5'-Tetramethylbenzidine (TMB) colorimetric solution; (c) Hydrogen peroxide solution; (d) Phosphate-buffered saline (PBS), pH 7.4.

[0020] The beneficial effects achieved by one or more of the above technical solutions of the present invention are as follows: (1) This invention integrates dual-functional nanomaterials to solve the problem of multi-material interface compatibility and simplify the sensor assembly process, specifically including: CuO-Cu2O nanoflowers: possess both photoactivity (triggering photocurrent reversal) and peroxidase activity (catalyzing H2O2-TMB color development), replacing traditional discrete photoactive materials and nanoenzymes; 3D g-C3N4 electrode substrate: High specific surface area (124 m² / g) and uniform mesoporous structure (25 nm pore size) enhance electron transfer efficiency and improve signal response.

[0021] (2) The biosensor prepared by this invention has ultra-high sensitivity and a wide linear range, breaking through the sensitivity limitations of traditional colorimetric methods (pM level) and single-signal sensors, and meeting the early diagnostic needs of trace miRNAs (such as the cancer biomarker miRNA-21), including: Photoelectrochemical mode (PEC): Detection limit up to 0.35 fM, linear range covering 1 fM to 10 fM; Colorimetric mode (CL): Detection limit is 3.9 fM, and the linear range extends to 10 fM~10 nM.

[0022] (3) This invention achieves dual-signal independent verification: the target analyte is detected simultaneously through two independent mechanisms: photoelectrochemical (photocurrent polarity reversal) and colorimetry (TMB color development); it is resistant to environmental interference: dual-signal cross-verification can effectively eliminate single-signal errors caused by sample matrix, instrument fluctuations or operational variations (such as when the photocurrent is affected by spatial steric hindrance, the colorimetric signal can still respond accurately), and is suitable for the precise analysis of complex biological samples (such as serum and tissue fluid). Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the photoelectrochemical-colorimetric dual-mode biosensor prepared in Example 1 of the present invention; Figure 2 This is a transmission electron microscope image of CuO-Cu2O prepared in Example 1 of the present invention; Figure 3 This is a scanning electron microscope image of CuO-Cu2O prepared in Example 1 of the present invention; Figure 4 This is a transmission electron microscope image of 3D g-C3N4 prepared in Example 1 of the present invention; Figure 5This is a scanning electron microscope image of 3D g-C3N4 prepared in Example 1 of the present invention; Figure 6 The PXRD pattern of CuO-Cu2O prepared in Example 1 of this invention; Figure 7 The attached diagram shows the adsorption-desorption process of 3D g-C3N4 prepared in Example 1 of this invention; Figure 8 Polyacrylamide gel electrophoresis image of the nucleic acid signal amplification strategy for the photoelectrochemical-colorimetric dual-mode biosensor structure in this embodiment of the invention; Figure 9 The photocurrent response characterization diagram shows the construction process of the photoelectrochemical-colorimetric dual-mode biosensor prepared in Example 1 of this invention. Figure 10 This is a signal response characterization diagram of the photoelectrochemical-colorimetric dual-mode biosensor prepared in Example 1 of the present invention; Figure 11 The image shows the analytical performance of the photoelectrochemical-colorimetric dual-mode biosensor prepared in Example 1 of this invention. Figure 12 The image shows the analytical performance of the photoelectrochemical-colorimetric dual-mode biosensor prepared in Example 1 of this invention. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0025] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0026] Example 1 This embodiment provides a method for fabricating a photoelectrochemical-colorimetric dual-mode biosensor. (1) Preparation and synthesis of multifunctional nanoprobe CuO-Cu2O: First, 5.1 g of lauric acid, 82 mg of copper nitrate, 48 mg of 1,3,5-benzenetricarboxylic acid, and 30 mL of n-butanol were added to a 100 mL beaker. After sonication for 10 minutes, the mixture was stirred at room temperature for half an hour. Then, the obtained blue precursor was transferred to a reaction vessel and heated at 140 °C for 5 h. After cooling to room temperature, the suspension was purified by centrifugation with ethanol, and the blue precipitate was dried at 60 °C. Finally, the obtained solid was transferred to a tube furnace and heated to 350 °C at a heating rate of 5 °C / min and held for 2 h. After natural cooling, CuO-Cu2O was obtained.

[0027] (2) Synthesis of 3D g-C3N4: 1 mol of cyanuric acid and 1 mol of melamine were dissolved in 30 mL of ultrapure water and stirred at room temperature for 12 h. Then, the mixture was centrifuged and freeze-dried under vacuum. Finally, the dried white powder was heated to 550 °C at a rate of 5 °C / min in a muffle furnace and held for 4 h to obtain pale yellow 3D g-C3N4.

[0028] (3) Preparation of Au NPs: Take 1 mL of 1% HAuCl4 solution and dilute it into 100 mL of ultrapure water. Heat to boiling and quickly add 2.5 mL of 1% trisodium citrate. Keep boiling for 15 min. After stopping heating, keep it still and let it cool naturally to obtain gold nanoparticles (Au NPs).

[0029] (3) Construction of the dual-mode PEC-CL biosensor: 10 μL of the target compound (miRNA-21) at different concentrations (1 fm, 10 fm, 100 fm, 1 pm, 10 pm, 100 pm, 1 nm, 10 nm), 90 μL of MB-Au-H1-MCH (2.5 μM), and 80 μL of CuO-Cu2O modified H2 (2 μM) were mixed and reacted in an oven at 37 °C for 2 h. 20 μL of Exo III (2 U / μL) and buffer (including 15 μL NEBuffer and 12 μL PBS) were added to the above system, and the reaction was carried out at 37 °C for 0.5 h to obtain the output DNA. Finally, the DNA was inactivated at 70 °C for 20 min.

[0030] According to the Watson-Crick principle, miRNA-21 and H1 form a double strand through base complementarity. In the presence of CuO-Cu2O modified H2, this double strand undergoes a strand displacement reaction to form the H1-H2 complex, simultaneously releasing miRNA-21 for reuse in cycle 1 (e.g., ...). Figure 1 (As shown in Figure 1A). Then, under the action of Exo III, the double strand formed by H1-H2 is cleaved to produce output DNA-Cu2O-CuO, while the remaining H1 continues to hybridize with CuO-Cu2O modified H2 to form the H1-H2 complex.

[0031] The preparation process of MB-Au-H1-MCH is as follows: 5 mL of prepared gold nanoparticles were mixed with 1 mL of Fe3O4-NH2 magnetic microspheres (MB) and incubated overnight to obtain the Fe3O4@Au conjugate (MB-Au). 250 μL of MB-Au was mixed with 250 μL of 2.5 μM hairpin DNA 1 (H1) and stirred at 4 °C for 16 h to prepare MB-Au--H1. 40 μL of 10 mM 6-mercaptohexanol (MCH) was added to the above solution, and the mixture was incubated at room temperature for 40 min to specifically cap the ends, thus obtaining the final product.

[0032] Experiment Example 2 This experimental example characterizes the structure and properties of the multifunctional nanoprobes CuO-Cu2O and 3D g-C3N4 prepared in Example 1.

[0033] like Figure 2 As shown, transmission electron microscopy reveals that the CuO-Cu2O nanoflowers are approximately 500 nanometers in size, with a rough surface and multiple pores; Figure 3 As shown, CuO-Cu2O nanoflowers exhibit a polyhedral structure, with uniform distribution, rough surface, and multiple pores.

[0034] like Figure 4 As shown, transmission electron microscopy reveals that the basic unit of 3D g-C3N4 is an ultrathin nanosheet with a lateral dimension of 1–10 micrometers; Figure 5 As shown, scanning electron microscopy reveals that 3D g-C3N4 has an interconnected network structure with an average pore size of approximately 25 nm, and is composed of ultrathin nanosheet units.

[0035] like Figure 6 As shown, the X-ray powder diffraction (XRD) pattern shows that the diffraction peaks of CuO-Cu2O are completely matched with the standard phase cards of CuO (JCPDS No. 45−0937) and Cu2O (JCPDS No. 05−0667), proving the successful preparation of the composite material.

[0036] like Figure 7 As shown, the porosity and surface area of ​​bulk C3N4 and 3D g-C3N4 were determined by nitrogen adsorption isotherm at 77 K. The porosity of 3D g-C3N4 was much higher than that of bulk C3N4, with a BET specific surface area of ​​124 m² / g and a Langmuir specific surface area of ​​139 m² / g.

[0037] Experiment Example 2 This experimental example strongly demonstrates the feasibility of the enzyme-assisted and strand displacement amplification strategy through polyacrylamide gel electrophoresis (PAGE).

[0038] Lanes 1 and 2 correspond to single-stranded target miRNAs 21 and H1, respectively.

[0039] A taller band was observed in lane 3, indicating that H1 can be opened by the target and form a target / H1 hybridization complex.

[0040] A bright band was obtained in lane 4, which corresponds to H2.

[0041] When H2 was added to a mixture of H1 and miRNA-21, a low-migration band was observed (lane 5 compared to lane 3), indicating that H2 was able to recognize and bind to H1.

[0042] When the above mixture was co-incubated with Exo III, a new band with faster migration speed was obtained (lane 6 compared to lane 5), which corresponds to the cleavage product (output DNA) when H2 coexists with target miRNA-21, H2 and H3.

[0043] The migration rate in lane 8 was slower than that in lane 7 (H3), which confirms that the formation of the H3 / output DNA complex was successful.

[0044] In the 9th sample band, two bands corresponding to H2 and H3 can still be clearly seen, which indicates that there is no interaction between H2 and H3 in the absence of exported DNA (i.e., target miRNA-21).

[0045] Experimental Example 1 This experimental example utilizes the photoelectrochemical-colorimetric dual-mode biosensor prepared in Example 1 to detect photoelectrochemical signals.

[0046] The testing process is as follows Figure 1 As shown in Figure 1C, the specific test process is as follows: (1) Electrode preparation: The ITO electrode was polished and then cleaned with acetone, ethanol and ethanol / sodium hydroxide aqueous solution for 15 minutes in sequence. Then, it was ultrasonically cleaned with ultrapure water under ultrasonic treatment. After drying with nitrogen, 10 μL of 3D g-C3N4 (2 mg / mL) was dropped onto the surface of the ITO electrode and then dried at room temperature to form a thin film.

[0047] In a 1% chloroauric acid solution, a layer of gold particles was electrodeposited on the ITO surface (gold nanoparticle deposition) at a potential of -0.2 V for 20 s. 10 μL of 2.5 μM H3 was dropped onto the electrode, and the particles were assembled at 4 °C through coordination interactions between nitrogen atoms in H3 and gold atoms. Then, 10 mM 6-mercaptohexanol (MCH) was incubated on the modified electrode surface at room temperature for 40 min to specifically seal the ends. Finally, 10 μL of the magnetically separated output DNA-Cu2O-CuO was incubated on the modified electrode at 37 °C for 2 hours to obtain the final product.

[0048] (2) PEC measurements were performed in PBS buffer (5 mL, 0.1 mol / L, pH 7.0) at room temperature. An LED lamp (wavelength λ = 460 nm; radiant flux Φ = 976 mW) was used as the excitation source in the experiment and was switched on and off in a 10 s-20 s-10 s mode at a potential of 0.0 V.

[0049] Test phenomena such as Figure 9 As shown, curve a represents the photocurrent response of the ITO electrode. Modifying the ITO electrode with 3D g-C3N4 yields an anodic photocurrent response (curve b). Coating the modified electrode surface with gold nanoparticles (Au NPs) results in an enhanced photocurrent response (curve c), thanks to the good conductivity of Au NPs. Continuous modification of the electrode surface with hairpin DNA2 (curve d) and 6-mercaptohexanol (curve e) leads to a progressively weakening photocurrent response, primarily due to charge repulsion and steric hindrance. When DNA-CuO-Cu2O is assembled (base complementary pairing) on ​​the electrode surface, the photocurrent reverses, resulting in a cathodic photocurrent (curve f). Each modification step of the sensor exhibits a corresponding change in the PEC signal, confirming the successful step-by-step construction of the sensor and enabling the detection of miRNA-21 in its PEC mode.

[0050] Test results are as follows Figure 11 As shown, the designed PEC biosensor exhibits excellent analytical performance for the target analyte. First, in the absence of miRNA-21, an anodic photocurrent with an intensity of 0.1 μA is observed (curve a). When 1 fM of miRNA-21 is present, the anodic photocurrent switches to a cathodic photocurrent (curve i). With increasing target analyte concentration, the cathodic photocurrent continuously increases (curves b to i). From 1 fM to 10 fM, the change in photocurrent shows a strong positive correlation with the logarithm of the miRNA-21 concentration, with a linear regression equation of I = -0.1174 log c - 1.0663 (R = 0.9976), and a detection limit (LOD) of 0.35 fM (e.g., ...). Figure 11(As shown in B).

[0051] Experimental Example 2 This experimental example utilizes the photoelectrochemical-colorimetric dual-mode biosensor prepared in Example 1 to detect colorimetric signals.

[0052] Test procedure: With the assistance of an external magnetic force, 200 μL of the supernatant was mixed with 200 μL of H2O2, 200 μL of TMB and 1400 μL of PBS. After 10 min, the absorbance value at 652 nm was recorded using a UV absorbance spectrophotometer.

[0053] Test results: The DNA-Cu2O-CuO complex can effectively catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide, resulting in significant changes in the color and absorption signal of the TMB solution, thus enabling the visualization and colorimetric detection of miRNA-21.

[0054] Specifically, such as Figure 10 The image shows the UV absorption spectra with and without the target compound. No significant absorbance or color change was observed in the absence of miRNA-21 (curve a). In contrast, a distinct blue color was observed in the presence of 10 pM miRNA-21, with a maximum absorption peak at 652 nm (curve b), indicating that CuO-Cu2O exhibits excellent peroxidase-like properties.

[0055] like Figure 12 As shown, the absorbance of the characteristic tetramethylbenzidine oxide (oxTMB) peak of the developed colorimetric sensor increases linearly with increasing miRNA-21 concentration (10 fM ~ 10 nM, curves a ~ h). Furthermore, the linear relationship between ΔI and the logarithm (lg c) of the miRNA-21 concentration is described by the equation ΔI = 0.08299 lg c + 0.3836, with a correlation coefficient (R) of 0.9907 and a detection limit (LOD) of 3.9 fM. Figure 12 As shown in B.

[0056] The nucleotide sequences are shown in Table 1: Table 1

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A photoelectrochemical-colorimetric dual-mode biosensor, characterized in that, include: Photoactive electrodes, immobilized capture probes, bifunctional nanoprobes, and recognition amplification components; The photoactive electrode is composed of an indium tin oxide conductive substrate, a three-dimensional network carbon nitride layer loaded on the surface of the conductive substrate, and gold nanoparticles modified on the surface of the three-dimensional network carbon nitride layer. The immobilized capture probe is a hairpin DNA H3 immobilized on the surface of AuNPs via Au-N coordination bonds, and its sequence is SEQ ID NO:3; The bifunctional nanoprobe is formed by coupling CuO-Cu2O nanoflowers with exported DNA via amino bonds; The identification amplification component comprises hairpin DNA H1 and hairpin DNA H2, whose sequences are SEQ ID NO:1 and SEQ ID NO:2, respectively.

2. The photoelectrochemical-colorimetric dual-mode biosensor as described in claim 1, characterized in that, The CuO-Cu2O nanoflowers have a porous flower-like structure with a size of 450~550 nm, and are a composite crystalline phase of Cu2O and CuO. The three-dimensional network carbon nitride has a BET specific surface area of ​​120~125 m² / g and an average pore size of 20~25 nm.

3. The application of the photoelectrochemical-colorimetric dual-mode biosensor according to any one of claims 1 to 2 in miRNA detection.

4. A method for miRNA detection using a photoelectrochemical-colorimetric dual-mode biosensor according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1. Hybridize the target miRNA-21 with hairpin DNA H1 to form a miRNA-21 / H1 complex; add CuO-Cu2O modified hairpin DNA H2, and form H1-H2 double strands through strand displacement reaction and release miRNA-21; then cut the H1-H2 double strands under the action of exonuclease III to release the exported DNA-CuO-Cu2O complex. S2. The output DNA-CuO-Cu2O complex is incubated with a photoactive electrode containing hairpin DNA H3, allowing the output DNA strand in the complex to hybridize with H3 through base complementary pairing. Then, the products modified on the photoactive electrode are subjected to photoelectrochemical signal detection. S3. Mix the output DNA-CuO-Cu2O complex with H2O2 and TMB solution, and detect the colorimetric signal.

5. The miRNA detection method of the photoelectrochemical-colorimetric dual-mode biosensor as described in claim 3, characterized in that, In step S1, the concentration of hairpin DNA H1 is 2.2~2.6 μM, the final concentration of CuO-Cu2O modified hairpin DNA H2 is 1.8~2 μM, and the molar ratio of CuO-Cu2O modified hairpin DNA H2 to hairpin DNA H1 is 1:1.2~1.

6. Preferably, the chain displacement reaction is carried out at a temperature of 36-37°C and an incubation time of 0.5-2 h. Preferably, the CuO-Cu2O modified hairpin DNA H2 is obtained by reacting the carboxyl groups on the surface of EDC / NHS-activated CuO-Cu2O with aminoated hairpin DNA H2 in PBS buffer solution for 12 h, followed by centrifugation purification. Preferably, the digestion conditions of the exonuclease III are: reaction at 35-38°C in buffer for 25-35 min, enzyme concentration of 1.5-2.5 U / μL, and the buffer is a mixture of NE Buffer and 0.1 M PBS buffer.

6. The miRNA detection method of the photoelectrochemical-colorimetric dual-mode biosensor as described in claim 3, characterized in that, In step S2, the output DNA-CuO-Cu2O complex is purified by magnetic separation and washed with PBS buffer ≥3 times.

7. The miRNA detection method of the photoelectrochemical-colorimetric dual-mode biosensor as described in claim 3, characterized in that, In step S2, the method for preparing the photoactive electrode includes the following steps: (1) Mix lauric acid, copper nitrate and 1,3,5-benzenetricarboxylic acid in n-butanol, disperse by ultrasonication and then carry out a solvothermal reaction. The resulting precipitate is heated and reacted to obtain CuO-Cu2O nanoflowers. (2) Mix cyanuric acid and melamine in deionized water. After stirring and centrifuging at room temperature, the mixture is vacuum dried. After heating, the product is obtained as a three-dimensional network carbon nitride. (3) The three-dimensional network of carbon nitride is sequentially coated onto ITO, AuNPs are electrodeposited, hairpin DNA H3 is fixed, and the sealing operation is performed to obtain the product. Preferably, in step (1), the ratio of lauric acid, copper nitrate, 1,3,5-benzenetricarboxylic acid, and n-butanol is (50~55) mg:(80~82) mg:(45~50) mg:(25~35) mL; the temperature of the solvothermal reaction is 120~150℃, and the time is 3~6h; the heating reaction is carried out by raising the temperature to 330~360℃ at a rate of 3~5℃ / min and holding it at that temperature for 1.5~3h. In step (2), the molar ratio of cyanuric acid to melamine is 1:1; the heating reaction is carried out by heating at a rate of 3~5℃ / min to 500~600℃ and holding at that temperature for 3~5 h. In step (3), the electrodeposition conditions are: chloroauric acid solution concentration 0.5~1 wt%, deposition potential -0.1~-0.2 V, deposition time 15~25 s; the hairpin DNA H3 concentration is 2~2.5 mM; the blocking operation refers to blocking non-specific sites with 6-mercaptohexanol, the concentration of 6-mercaptohexanol is 5~10 mM.

8. The miRNA detection method of the photoelectrochemical-colorimetric dual-mode biosensor as described in claim 7, characterized in that, In step S2, the detection conditions for the photoelectrochemical signal are: bias voltage of 0.05~0.15 V, excitation light source wavelength of 430~460 nm, buffer system of PBS buffer, and photocurrent reading time of 8~12 s after illumination.

9. The miRNA detection method of the photoelectrochemical-colorimetric dual-mode biosensor as described in claim 7, characterized in that, In step S3, the colorimetric reaction system for detecting the colorimetric signal includes TMB, H2O2, and PBS buffer, with a volume ratio of TMB, H2O2, and PBS buffer of 1:1:5~7; the concentration of TMB is 1.5~2.5 mM, and the concentration of H2O2 is 8~12 mM; the reaction temperature is room temperature, the reaction time is 8~12 min, and the detection wavelength is 650~654 nm.

10. A kit for detecting miRNA-21, characterized in that, The photoelectrochemical-colorimetric dual-mode biosensor according to any one of claims 1 to 2 further comprises: (a) Exonuclease III; (b) 3,3',5,5'-Tetramethylbenzidine colorimetric solution; (c) Hydrogen peroxide solution; (d) Phosphate buffer.

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