Fabrication of a BiOI homojunction pre-enhancement platform based on electron-conductive DNA orbital modification for DNA walker-guided photoelectrochemical polarity reversal sensors

Abstract

The application belongs to the field of immunoassay and biosensing technology, and provides a preparation method of a BiOI homojunction photoelectrochemical sensor with DNA track pre-enhancement and signal inversion. First, a morphologically engineered BiOI homojunction is constructed, which produces a strong stable base cathode photocurrent by promoting efficient charge separation and interface transmission. More importantly, a double helix DNA track is rationally designed, which actively promotes electron transfer instead of inhibition, thereby significantly enhancing the initial PEC signal and effectively eliminating the inherent signal quenching effect of the traditional DNA interface. When miRNA-125b is recognized, the DNA walker activated by catalytic hairpin assembly (CHA) amplification migrates along the DNA track, guides the in-situ accumulation of dopamine (PDA) as a signal inversion medium, and induces a controllable photocurrent to reverse from the cathode to the anode. According to the different photoelectrochemical signal intensities of different concentrations of the to-be-detected substance, the detection of miRNA-125b is realized.

Description

Technical Field

[0001] This invention relates to a novel construction of an "on-off-on" sensing strategy that modulates the "self-sensitization" effect using a Ferris wheel-shaped DNA nanostructure, and a method for preparing a novel CoS / CoCdS in-situ heterojunction optoelectronic substrate material. First, a CoS / CoCdS heterojunction with a "self-sensitization" effect is generated in situ to provide an initial "on" signal as the optoelectronic substrate. Subsequently, triggered by the target compound MC-LR, a Ferris wheel-shaped DNA structure with steric hindrance is formed through a DNA cascade reaction. This structure acts as a steric hindrance layer covering the electrode surface, hindering electron transfer and causing the signal to briefly switch to an "off" state. By specifically modifying AgInS2 QDs at the ends of the DNA Ferris wheel-shaped structure and constructing a ternary heterojunction using the band-matching principle, a clear signal switching from "off" to "on" is achieved, thereby enabling highly sensitive detection of MC-LR. This invention belongs to the field of novel functional materials and biosensing detection technology. Background Technology

[0002] Microcystin-LR (MC-LR), a highly toxic cyclic heptapeptide released by cyanobacteria, poses a serious threat to human health. It primarily targets the liver, causing acute liver injury and even liver failure by inhibiting protein phosphatase. Long-term low-dose exposure can lead to liver fibrosis and liver cancer. In addition, it also has nephrotoxicity, neurotoxicity, and immunosuppressive effects, and has been listed as a potential human carcinogen. Therefore, the development of highly sensitive detection methods is crucial for public health protection.

[0003] While combining nucleic acid aptamers with photoelectrochemical (PEC) sensing platforms can significantly improve detection performance, their application in the analysis of trace toxins (such as MC-LR) in the environment and food remains limited by low signal amplification efficiency and weak electrical signal response. This study constructed a DNA cascade reaction-driven photoelectrochemical sensing platform based on MC-LR triggering of the target analyte. During signal amplification, a DNA nanoassembly with a "Ferris wheel" shaped topology was successfully constructed using a CHA-HCR cascade reaction triggered by MC-LR. This nanostructure acts as a steric hindrance layer on the surface of the photoactive material at the sensing interface, hindering the transfer of electrons and holes, thereby significantly suppressing the photocurrent signal. This process transitions the sensing system from an initial signal "on" state to a signal "off" mode. Subsequently, the generated "Ferris wheel" shaped DNA nanostructure exposes specific base recognition sites at its ends, enabling efficient and specific anchoring of DNA single strands modified with AgInS2 QDs. AgInS2 QDs, acting as photosensitizers, can achieve band matching with the substrate material CoS / CoCdS to construct a stable ternary heterojunction structure. This heterojunction effectively promotes the separation and migration of photogenerated carriers, significantly recovering the previously quenched photocurrent signal and allowing the sensing system to re-enter the signal "on" state, thereby achieving highly sensitive and specific detection of MC-LR.

[0004] This approach significantly enhances detection selectivity through the highly specific design of nucleic acid sequences and effectively overcomes the technical bottleneck of inaccurate detection of low-concentration target substances due to weak signals by utilizing a cascade amplification mechanism. The generated "Ferris wheel-shaped" DNA nanostructure exposes specific base recognition sites at its ends, enabling efficient and specific anchoring of DNA single strands modified with AgInS2 QDs. AgInS2 QDs, acting as a photosensitizer, achieve band matching with the substrate material CoS / CoCdS, constructing a stable ternary heterojunction structure. This heterojunction effectively promotes the separation and migration of photogenerated carriers, significantly restoring the previously quenched photocurrent signal, allowing the sensing system to re-enter the signal "on" state, ultimately achieving highly sensitive and selective dual-signal response detection of the target substance MC-LR. This dual detection strategy effectively reduces background interference through an "on-off-on" signal conversion mechanism, significantly improving the signal-to-noise ratio and detection reliability. Simultaneously, this strategy achieves two signal amplifications and verifications in a single detection, providing a reliable analytical platform for the accurate detection of trace target substances in complex matrices. Summary of the Invention

[0005] One of the objectives of this invention is to achieve "self-sensitization" of the CoS / CoCdS heterojunction by generating the hydroxyl layer in situ, thereby constructing a stable and efficient photoelectric conversion substrate, providing an initial "on" signal, and laying a good foundation for subsequent signal switching.

[0006] The second objective of this invention is to form a "Ferris wheel"-shaped DNA structure with steric hindrance effect through a DNA cascade reaction in the presence of the target MC-LR. This structure can partially hinder electron transfer, causing the signal to switch to a temporary "off" state.

[0007] The third objective of this invention is to use this DNA structure as a signal amplification platform, modify its periphery with AgInS2 QDs, and use its photosensitization effect to efficiently recover the signal, realize the switch from "off" to "on", and thus complete the detection of MC-LR with high specificity and high sensitivity.

[0008] The technical solution of the present invention is as follows: 1. A method for fabricating an "on-off-on" type PEC sensor based on a Ferris wheel-shaped DNA structure, characterized by comprising the following steps: (1) Cut the ITO glass into 2 × 0.8 cm pieces. 2 The strips were ultrasonically cleaned in sequence with detergent, acetone, ethanol and deionized water for 30 min and then dried at 60 ℃ for 24 h. (2) Take 8.0 µL of CoS / CoCdS in-situ heterojunction material dispersion with a concentration of 2.0 ~ 10.0 mg / mL and drop it onto the electrode surface; (3) Add 6.0 µL of citrate-stabilized gold nanoparticle solution to the electrode surface to introduce high-density thiol reactive binding sites, air dry at room temperature, rinse the electrode surface with deionized water, and air dry at room temperature. (5) Take 10.0 µL of the pre-prepared DNA Ferris wheel structure solution with a concentration of 1.0 ~ 5.0 nmol / L and add it to the electrode surface. Incubate at 4°C for 14 hours to fix the DNA to the electrode surface through Au-S bonds. Rinse the electrode surface with deionized water and air dry at room temperature. (6) Continue to add 5.0 µL of 2.0 ~ 10.0 mmol / L 6-mercapto-1-hexanol (HT) aqueous solution to the modified electrode to block the non-specific active sites on the electrode surface. Rinse the electrode surface with deionized water and air dry at room temperature. (7) Finally, 5.0 µL of DNA strands with AgInS2 QDs linked to them and 0.2 ~ 1.0 μmol / L were added to the surface of the above HT blocking electrode as photosensitizers and incubated at 37°C for 1 h to allow them to specifically bind to the electrode interface through DNA hybridization reaction. A PEC sensor that can be used to detect MC-LR was successfully constructed and stored in a 4°C refrigerator for later use.

[0009] 2. The method for preparing an "on-off-on" type PEC sensor based on a Ferris wheel-shaped DNA structure as described in claim 1, wherein the preparation of the CoS / CoCdS heterojunction material is characterized by the following steps: (1) Preparation of CoS First, 7.8 mmol of dimethylimidazole was dissolved in 80 mL of anhydrous methanol to prepare solution A. Then, 1.2 mmol of cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in 80 mL of anhydrous methanol to prepare solution B. Solution A was slowly poured into solution B, and the mixture was stirred continuously until homogeneous. The mixture was then allowed to stand overnight for aging. The resulting product was washed several times with anhydrous methanol and dried to obtain the ZIF-67 precursor. 40 mg of the ZIF-67 precursor was dispersed in 20 mL of ethanol to form a homogeneous dispersion (solution C). Separately, 180 mg of thioacetamide was dissolved in 40 mL of ethanol to prepare solution D. Under continuous vigorous stirring, solution D was slowly added to solution C, and the reaction was allowed to proceed for 45 min. The mixture was then transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 180 °C for 1–5 h. After the reaction was completed, the reactor was allowed to cool naturally to room temperature. The resulting solid product was washed with methanol multiple times and dried at 60°C and atmospheric pressure for 26 hours to finally obtain a black solid CoS material. (2) Preparation of CoS / CoCdS heterojunction materials 24 mg of CoS and 20 mg of CdCl2 were weighed and dissolved sequentially in 40 ml of anhydrous ethanol. After vigorous stirring at room temperature for 1.5 h, the mixture was transferred to a polytetrafluoroethylene reactor and reacted at 150 °C for 2–8 h. Subsequently, the mixture was washed several times with anhydrous methanol and dried non-vacuum for 12 h to obtain a black CoS / CoCdS in-situ heterojunction material.

[0010] 3. The method for preparing an "on-off-on" type PEC sensor based on a Ferris wheel-shaped DNA structure as described in claim 1, wherein the preparation of the AgInS2QDs material is characterized by the following steps: First, 0.1 mmol AgNO3 and 0.4 mmol In(NO3)3 were weighed and dissolved in 50 mL of deionized water, respectively. After vigorous stirring, the solutions were transferred to a flask. Then, 8.3 μL MPA and 3 mL 0.6 mmol Na2S·9H2O were added to the solution under vigorous stirring. The reaction mixture was then heated under reflux at 100 °C for 1–5 h. After the reaction was complete, the system was cooled to room temperature, and the resulting precipitate was washed repeatedly with anhydrous ethanol to remove impurities. The resulting orange precipitate was then redispersed in 50 mL of deionized water to form a homogeneous dispersion. Finally, the dispersion was stored at 4 °C in the dark for subsequent experiments.

[0011] 4. The method for preparing an "on-off-on" type PEC sensor based on a Ferris wheel-shaped DNA structure as described in claim 1, for the detection of MC-LR, characterized in that the steps are as follows: (1) The photoelectrochemical workstation was used to test the three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared BiOI homojunction photoelectrochemical sensor based on DNA orbital pre-enhancement and signal inversion was used as the working electrode. A 100 W LED lamp was used as the irradiation source, and the lamp was switched on and off every 20 seconds. The bias voltage was set to 0 V. (2) In 10 mL of PBS buffer solution containing 0 to 0.2 mol / L ascorbic acid at pH = 6.81 to 8.04, the photoelectric signals generated in the antigens of different concentrations were detected, and the working curve was plotted; (3) Replace the MC-LR solution with the sample solution to be tested for detection.

[0012] Beneficial results of the present invention (1) This invention successfully prepared an in-situ heterojunction CoS / CoCdS composite material using ZIF-67 as a precursor through a controllable derivatization strategy, and further optimized the construction of a hydroxyl layer on its surface. Due to its wide specific surface area, the hydroxyl layer effectively promoted the oxidation process of surface hydroxyl groups by holes, realizing the efficient separation of electron-hole pairs, thereby constructing a high-performance signal "on" type photoelectric sensing substrate.

[0013] (2) The present invention triggers a cascade reaction by the target MC-LR to drive the formation of a “Ferris wheel” shaped DNA nanostructure. This structure causes the photoelectric signal to enter the “off” state through the steric hindrance effect.

[0014] (3) This invention utilizes the DNA nanostructure end to bind AgInS2 QDs to form a ternary heterojunction, and reconstructs and enhances the electron transfer pathway through energy level matching to realize the signal re-"turning on".

[0015] (4) The photoelectrochemical sensor prepared by the present invention realizes reliable detection of MC-LR. The “on-off-on” signal switching strategy significantly improves the detection sensitivity and specificity, providing a high-precision analytical method for the detection of trace toxins in complex matrices. Detailed Implementation

[0016] (The present invention will now be further described through specific embodiments, but is not limited thereto.) Example 1. A method for fabricating an "on-off-on" type PEC sensor based on a Ferris wheel-shaped DNA structure, characterized by comprising the following steps: (1) Cut the ITO glass into 2 × 0.8 cm pieces. 2 The strips were ultrasonically cleaned in sequence with detergent, acetone, ethanol and deionized water for 30 min and then dried at 60 ℃ for 24 h. (2) Take 8.0 µL of 2.0 mg / mL CoS / CoCdS in-situ heterojunction material dispersion and drop it onto the electrode surface; (3) Add 6.0 µL of citrate-stabilized gold nanoparticle solution to the electrode surface to introduce high-density thiol reactive binding sites, air dry at room temperature, rinse the electrode surface with deionized water, and air dry at room temperature. (5) Take 10.0 µL of the pre-prepared DNA Ferris wheel structure solution with 1.0 nmol / L and add it to the electrode surface. Incubate at 4℃ for 14 hours to fix the DNA to the electrode surface through Au-S bonds. Rinse the electrode surface with deionized water and air dry at room temperature. (6) Continue to add 5.0 µL of 2.0 mmol / L 6-mercapto-1-hexanol (HT) aqueous solution to the modified electrode to seal the non-specific active sites on the electrode surface. Rinse the electrode surface with deionized water and air dry at room temperature. (7) Finally, 5.0 µL of DNA strands with AgInS2 QDs linked to AgInS2 were added to the surface of the above-mentioned HT blocking electrode as photosensitizers and incubated at 37°C for 1 h to allow it to specifically bind to the electrode interface through DNA hybridization reaction. A PEC sensor that can be used to detect MC-LR was successfully constructed and stored in a 4°C refrigerator for later use.

[0017] Example 2. A method for preparing an "on-off-on" type PEC sensor based on a Ferris wheel-shaped DNA structure, characterized by comprising the following steps: (1) Cut the ITO glass into 2 × 0.8 cm pieces. 2 The strips were ultrasonically cleaned in sequence with detergent, acetone, ethanol and deionized water for 30 min and then dried at 60 ℃ for 24 h. (2) Take 8.0 µL of CoS / CoCdS in-situ heterojunction material dispersion with a concentration of 4.0 mg / mL and drop it onto the electrode surface; (3) Add 6.0 µL of citrate-stabilized gold nanoparticle solution to the electrode surface to introduce high-density thiol reactive binding sites, air dry at room temperature, rinse the electrode surface with deionized water, and air dry at room temperature. (5) Take 10.0 µL of the pre-prepared DNA Ferris wheel structure solution with 2.0 nmol / L and add it to the electrode surface. Incubate at 4℃ for 14 hours to fix the DNA to the electrode surface through Au-S bonds. Rinse the electrode surface with deionized water and air dry at room temperature. (6) Continue to add 5.0 µL of 4.0 mmol / L 6-mercapto-1-hexanol (HT) aqueous solution to the modified electrode to block the non-specific active sites on the electrode surface. Rinse the electrode surface with deionized water and air dry at room temperature. (7) Finally, 5.0 µL of DNA strands with AgInS2 QDs linked to AgInS2 were dropped onto the surface of the above-mentioned HT blocking electrode as photosensitizers and incubated at 37°C for 1 h to allow it to specifically bind to the electrode interface through DNA hybridization reaction. A PEC sensor that can be used to detect MC-LR was successfully constructed and stored in a 4°C refrigerator for later use.

[0018] Example 3. A method for preparing an "on-off-on" type PEC sensor based on a Ferris wheel-shaped DNA structure, characterized by comprising the following steps: (1) Cut the ITO glass into 2 × 0.8 cm pieces. 2 The strips were ultrasonically cleaned in sequence with detergent, acetone, ethanol and deionized water for 30 min and then dried at 60 ℃ for 24 h. (2) Take 8.0 µL of CoS / CoCdS in-situ heterojunction material dispersion with a concentration of 6.0 mg / mL and drop it onto the electrode surface; (3) Add 6.0 µL of citrate-stabilized gold nanoparticle solution to the electrode surface to introduce high-density thiol reactive binding sites, air dry at room temperature, rinse the electrode surface with deionized water, and air dry at room temperature. (5) Take 10.0 µL of the pre-prepared DNA Ferris wheel structure solution with 3.0 nmol / L and add it to the electrode surface. Incubate at 4℃ for 14 hours to fix the DNA to the electrode surface through Au-S bonds. Rinse the electrode surface with deionized water and air dry at room temperature. (6) Continue to add 5.0 µL of 6-mercapto-1-hexanol (HT) aqueous solution to the modified electrode to seal the non-specific active sites on the electrode surface. Rinse the electrode surface with deionized water and air dry at room temperature. (7) Finally, 5.0 µL of DNA strands with AgInS2 QDs linked to AgInS2 were added to the surface of the above-mentioned HT blocking electrode as photosensitizers and incubated at 37°C for 1 h to allow it to specifically bind to the electrode interface through DNA hybridization reaction. A PEC sensor that can be used to detect MC-LR was successfully constructed and stored in a 4°C refrigerator for later use.

[0019] Example 4. The preparation of the CoS / CoCdS heterojunction material is characterized by comprising the following steps: (1) Preparation of CoS First, 7.8 mmol of dimethylimidazole was dissolved in 80 mL of anhydrous methanol to prepare solution A. Then, 1.2 mmol of cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in 80 mL of anhydrous methanol to prepare solution B. Solution A was slowly poured into solution B, and the mixture was stirred continuously until homogeneous. The mixture was then allowed to stand overnight. The resulting product was washed several times with anhydrous methanol and dried to obtain the ZIF-67 precursor. 40 mg of the ZIF-67 precursor was dispersed in 20 mL of ethanol to form a homogeneous dispersion (solution C). Separately, 180 mg of thioacetamide was dissolved in 40 mL of ethanol to prepare solution D. Under continuous vigorous stirring, solution D was slowly added to solution C, and the reaction was allowed to proceed for 45 min. The mixture was then transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 180 °C for 2 h. After the reaction was completed, the reactor was allowed to cool naturally to room temperature. The resulting solid product was washed with methanol multiple times and dried at 60°C and atmospheric pressure for 26 hours to finally obtain a black solid CoS material. (2) Preparation of CoS / CoCdS heterojunction materials 24 mg of CoS and 20 mg of CdCl2 were weighed and dissolved sequentially in 40 ml of anhydrous ethanol. After vigorous stirring at room temperature for 1.5 h, the mixture was transferred to a polytetrafluoroethylene reactor and reacted at 150 °C for 3 h. Subsequently, the mixture was washed several times with anhydrous methanol and dried in a non-vacuum environment for 12 h to obtain a black CoS / CoCdS in-situ heterojunction material.

[0020] Example 5. The preparation of the AgInS2QDs material, characterized by the following steps: First, 0.1 mmol AgNO3 and 0.4 mmol In(NO3)3 were weighed and dissolved in 50 mL of deionized water, respectively. After vigorous stirring, the solutions were transferred to a flask. Then, 8.3 μL MPA and 3 mL 0.6 mmol Na2S·9H2O were added to the solution under vigorous stirring. The reaction mixture was then heated under reflux at 100 °C for 2 h. After the reaction was complete, the system was cooled to room temperature, and the resulting precipitate was washed repeatedly with anhydrous ethanol to remove impurities. The resulting orange precipitate was then redispersed in 50 mL of deionized water to form a homogeneous dispersion. Finally, the dispersion was stored at 4 °C in the dark for subsequent experiments.

[0021] Example 6. Detection for MC-LR, characterized by the following steps: (1) The photoelectrochemical workstation was used to test the three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared BiOI homojunction photoelectrochemical sensor based on DNA orbital pre-enhancement and signal inversion was used as the working electrode. A 100 W LED lamp was used as the irradiation source, and the lamp was switched on and off every 20 seconds. The bias voltage was set to 0 V. (2) In 10 mL of PBS buffer solution containing 0.1 mol / L ascorbic acid at pH = 7.38, the photoelectric signals generated in the analyte antigen at different concentrations were detected, and the working curve was plotted; (3) Replace the MC-LR solution with the sample solution to be tested for detection.

Claims

1. A method for fabricating a photoelectrochemical polarity reversal sensor guided by a DNA walker, based on a BiOI homojunction pre-enhancement platform with electron-conductive DNA orbital modification, characterized in that... Includes the following steps: (1) Cut the ITO glass into 2 × 0.8 cm pieces. 2 The strips were ultrasonically cleaned in sequence with detergent, acetone, ethanol and deionized water for 30 min and then dried at 60 ℃ for 24 h. (2) Take 10.0 µL of BiOI micron-sized sheet dispersion with a concentration of 2.0 ~ 10.0 mg / mL and add it dropwise to the electrode surface; (3) Take 10.0 µL of BiOI nanoflower dispersion with a concentration of 2.0 ~ 10.0 mg / mL and add it vertically to the surface of BiOI microsheet dispersion droplets. After evaporation at 37℃ for 2 hours, a uniform BiOI homojunction film is formed on the ITO surface. (4) Add 10.0 µL of citrate-stabilized gold nanoparticle solution to the electrode surface to introduce high-density thiol reactive binding sites, air dry at room temperature, rinse the electrode surface with deionized water, and air dry at room temperature. (5) Take 10.0 µL of reduced DNA orbital hairpin (HS-HP5) solution with a concentration of 1.0 ~ 5.0 nmol / L and add it to the electrode surface. Incubate at 4°C for 14 hours to allow HS-HP5 to be covalently fixed on the gold nanoparticle modification layer through Au-S bonds. Rinse the electrode surface with deionized water and air dry at room temperature. (6) Continue to add 5.0 µL of 2.0 ~ 10.0 mmol / L 6-mercapto-1-hexanol (HT) aqueous solution to the modified electrode to block the non-specific active sites on the electrode surface. Rinse the electrode surface with deionized water and air dry at room temperature. (7) Add 5.0 µL of DNA walker solution of 0.2 ~ 1.0 μmol / L to the surface of the above HT blocking electrode; (8) Finally, 5.0 µL of 0.2 ~ 1.0 μmol / L dopamine (PDA)-HP6 covalent coupling solution was added to the electrode surface containing the DNA walker solution. The electrode surface was incubated at room temperature for 1.5 hours and rinsed with deionized water to obtain the DNA orbital modified BiOI homojunction pre-enhancement platform for photoelectrochemical signal inversion detection of miRNA-125b sensor. The platform was stored in a 4 ℃ refrigerator for later use.

2. The method for a photoelectrochemical polarity reversal sensor guided by a DNA walker using a BiOI homojunction pre-enhancement platform based on electron-conductive DNA orbital modification as described in claim 1, wherein the preparation of the BiOI homojunction is characterized in that... The steps are as follows: (1) Preparation of BiOI microsheets First, solution A was prepared by dissolving 0.472 g of potassium iodide in 4 mL of deionized water. Simultaneously, solution B was prepared by dissolving 1.378 g of bismuth nitrate pentahydrate in 10 mL of glacial acetic acid. Both solutions were stirred separately at 500 rpm for 1 hour, then mixed and stirred for another 10 minutes to form a homogeneous precursor suspension. The suspension was transferred to a 50 mL PTFE-lined stainless steel autoclave and reacted at 180 °C for 12–24 hours. After natural cooling to room temperature, the reddish-brown precipitate was collected by centrifugation and purified by washing three times alternately with deionized water and anhydrous ethanol. Finally, the product was dried in a vacuum oven at 60 °C for 12 hours to obtain reddish-brown BiOI micron-sized flake powder. (2) Preparation of BiOI nanoflowers 0.332 g of potassium iodide was dissolved in 20 mL of deionized water (solution C), and 0.970 g of bismuth nitrate pentahydrate was dissolved in 20 mL of ethylene glycol (solution D). Both solutions were stirred for 30 minutes each. Then, solution C was slowly poured into solution D and stirred for 15 minutes. The resulting precursor was transferred to a 50 mL autoclave and reacted at 160°C for 12–24 hours. After naturally cooling to room temperature, the reddish-brown precipitate was collected by centrifugation and purified by washing three times alternately with deionized water and anhydrous ethanol. Finally, the product was dried in a vacuum oven at 60°C for 12 hours to obtain bright yellow BiOI nanoflower powder. (3) Preparation of BiOI homojunction First, BiOI microsheets and BiOI nanoflower powder were dispersed separately in deionized water and sonicated for 0.5 hours to prepare homogeneous suspensions with a concentration of 4 mg / mL. A pretreated ITO conductive glass slide was used as the substrate. The slide was pre-cut into 2.0 cm × 0.8 cm blocks and sonicated sequentially in deionized water, ethyl acetate, and deionized water for 30 minutes each. Finally, it was dried overnight in a 60°C oven. Using a micropipette, 10 μL of the BiOI microsheet suspension was vertically added to the center of the ITO surface to form an initial droplet. The key step involves immediately and vertically stacking a 10 μL BiOI nanoflower suspension at the center of the droplet to form a composite liquid column. The substrate supporting the liquid column is then placed horizontally in a 37°C forced-air drying oven and reacted in static air for 2 hours.

3. The method for fabricating a photoelectrochemical polarity reversal sensor guided by a DNA walker using a BiOI homojunction pre-enhancement platform based on electron-conductive DNA orbital modification as described in claim 1, wherein the fabrication of the reduced DNA orbital hairpin (HS-HP5) is characterized in that, The steps are as follows: (1) Preparation of the reduced-prototype track hairpin 100 μL of thiolized hairpin DNA (HSHP5, 100 μM) was mixed with freshly prepared TCEP solution (20 mM in TE buffer) and reduced at room temperature for 1 hour to break disulfide bonds. The solution was then heated at 95°C for 5 minutes and slowly cooled to room temperature for annealing to form a stable hairpin structure. (2) Fixing the replica track hairpin Take 10 μL of 2pM~2μM reduced and annealed HSHP5 solution and add it to the clean gold electrode surface. Incubate at 4℃ for 14 hours to form an ordered monolayer DNA probe through covalent Au-S bonds on the gold surface.

4. The method for a photoelectrochemical polarity reversal sensor guided by a BiOI homojunction pre-enhancement platform based on electron-conductive DNA orbital modification as described in claim 1, wherein the fabrication of the DNA walker is characterized in that... The steps are as follows: (1) Chain displacement reaction 10 μL of amino-modified trigger chain T1 (NH2-T1, 10 μM) was mixed with 40 μL of carboxyl-activated magnetic beads (MB-COOH) and incubated overnight at 4 °C to form the MB-T1 complex. After magnetic separation and washing with PBS (0.1 M, pH 7.38), the MB-T1 complex was resuspended in 100 μL of PBS. 20 μL of this MB-T1 suspension was hybridized with 20 μL of partially complementary pseudo-target chain T2 (10 μM) at 37 °C for 2 hours to form the MB-T1 / T2 complex, which was then washed and resuspended in 20 μL of PBS. To activate the cyclist, 10 μL of the target solution was mixed with the above complex solution and incubated at 37 °C for 2 hours. The target specifically recognizes T1 and displaces T2; the released T2 chain is collected in the supernatant by magnetic separation. (2) Catalytic hairpin assembly reaction Take 10 μL of supernatant containing free T2 and mix it with an equal volume of solution containing four hairpin substrates (HP1, HP2, HP3, and HP4, 5 μM each), and incubate at 37°C for 4 hours. Through a T2-triggered strand displacement cascade reaction, a DNA walking organism with autonomous walking function is self-assembled.

5. The method for a photoelectrochemical polarity reversal sensor guided by a BiOI homojunction pre-enhancement platform based on electron-conductive DNA orbital modification as described in claim 1, wherein the preparation of the polydopamine (PDA)-HP6 covalent conjugate is characterized in that, The steps are as follows: (1) Preparation of polydopamine (PDA) First, 150 mg of dopamine hydrochloride was added to 26 mL of a water-ethanol mixture (water to ethanol volume ratio of 18:8), and stirred continuously at room temperature for 12 hours to form dopamine (PDA) nanoparticles via oxidative self-polymerization. After the reaction, the PDA dispersion was purified by centrifugation and redispersed in TrisHCl buffer (0.1 M, pH 8.0). (2) Pretreatment of hairpin DNA (HSHP6) Mix 100 μM of HSHP6 stock solution with a sufficient volume of freshly prepared tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) solution (20 mM in TE buffer) and reduce at room temperature for 1 hour to completely break any intermolecular or intramolecular disulfide bonds, ensuring that the terminal thiol groups are fully activated to their reduced form (-SH). Subsequently, heat the reduction solution at 95 °C for 5 minutes to denature the DNA, and then slowly cool it to room temperature ("slow annealing") to allow it to refold into the thermodynamically stable standard hairpin secondary structure. (3) Preparation of polydopamine (PDA)-HP6 covalent conjugate 100 μL of PDA dispersion was mixed with 100 μL of reduced and annealed HSHP6 solution and gently shaken at 4 °C for 12 hours. Under these alkaline conditions, the ortho-quinone groups on the PDA surface undergo a specific Michael addition reaction with the active thiol groups (-SH) at the ends of HSHP6 to form stable covalent thioether bonds (CS bonds), thereby generating the covalent complex PDAHP6.

6. A BiOI homojunction photoelectrochemical sensor based on DNA orbital pre-enhancement and signal inversion, prepared by the method described in claim 1, for the detection of miRNA-125b, characterized in that, The steps are as follows: (1) The photoelectrochemical workstation was used to test the three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared BiOI homojunction photoelectrochemical sensor based on DNA orbital pre-enhancement and signal inversion was used as the working electrode. A 100 W LED lamp was used as the irradiation source, and the lamp was switched on and off every 20 seconds. The bias voltage was set to 0 V. (2) In 10 mL of Tris-HCl buffer solution containing 0 to 0.2 mol / L ascorbic acid at pH = 6.81 to 8.04, the photoelectric signals generated in the antigens of different concentrations were detected, and the working curve was plotted; (3) Replace the miRNA-125b solution with the test sample solution for detection.