Preparation of heterojunction dual-photoelectrode type photoelectrochemical sensor based on multi-ball cascade DNA reaction amplification

Abstract

The invention belongs to the technical field of immunoassay and biosensing, and provides a novel construction mode of a heterojunction dual-photoelectrode photoelectric sensing strategy and a preparation method of an in-situ heterojunction MIO photocathode. The invention relates to a preparation method of a heterojunction dual-photoelectrode type photoelectrochemical sensor based on multi-ball cascade DNA reaction amplification. Specifically, the design is composed of an In2O3 / CdS photoanode and an in-situ formed MIL-68 / In2O3 (MIO) photocathode. The heterojunction dual-photoelectrode sensing strategy effectively prolongs an electron-hole transmission path, enhances photo-generated charge separation, and realizes' super-open 'PEC signal output, thereby providing a steady baseline for signal conversion. Meanwhile, a multi-sphere cascade DNA reaction is combined to output a three-dimensional DNA net structure to provide a large number of sites for embedding of a PEC quenching probe doxorubicin-ferrocene carboxylic acid (DOX-FcCOOH), obvious PEC signal quenching is realized by utilizing the efficient competitive electron transfer capability of the three-dimensional DNA net structure, and finally, a signal conversion system with'opening-super opening-closing 'multistage response characteristics is constructed. The detection of the nucleic acid marker Pax-5a gene of the B cell acute lymphatic leukemia is realized by detecting target objects with different concentrations.

Description

Technical Field

[0001] This invention relates to a novel construction of a heterojunction dual-photoelectrode photoelectric sensing strategy and a method for preparing an in-situ heterojunction MIO (MIO) photocathode material. Specifically, the method employs In2O3 / CdS as the photoanode material and in-situ formed MIL-68 / In2O3 (MIO) as the photocathode material. This method combines a multi-sphere cascade DNA reaction to output a DNA mesh structure, which is then "embedded" with a PEC quenching probe DOX-FcCOOH. This ultimately achieves multi-stage PEC signal switching, "on-super on-off," to fabricate a photoelectrochemical sensor for detecting the Pax-5a gene, a nucleic acid marker for B-cell acute lymphoblastic leukemia. This approach relates to the fields of novel functional materials and biosensor detection technologies. Background Art

[0002] B-cell acute lymphoblastic leukemia (ALL) is one of the most common acute lymphoblastic malignancies, with significantly increased morbidity and mortality, particularly among children. Despite recent advances in treatment, early and accurate diagnosis remains crucial for effective treatment. Current diagnostic methods, particularly those relying on protein biomarkers, often lack the sensitivity and specificity required for early detection. In contrast, nucleic acid-based technologies offer more precise and informative biomarkers, making them a promising alternative. Among them, the Pax-5a gene is a key regulator of cell differentiation and tissue development and is closely associated with the pathogenesis of B-cell ALL. Due to its critical role in B-cell ALL, Pax-5a could serve as a biomarker for early diagnosis. However, clinical research on Pax-5a nucleic acid detection remains limited, highlighting the urgent need for low-cost, efficient, and sensitive detection strategies.

[0003] Currently, there are many methods for detecting the Pax-5a gene, a nucleic acid marker for B-cell acute lymphoblastic leukemia. Common methods include electrochemical, fluorescence, and colorimetric methods. However, these methods all require cumbersome pretreatment steps, expensive equipment, and long processing times. Due to the separation of the excitation and detection sources, photoelectrochemical biosensors offer the advantages of low background signals and rapid response. Compared with traditional analytical methods, the photoelectrochemical biosensor employed in the present invention has the advantages of high sensitivity, good selectivity, ease of operation, and miniaturization. Therefore, the present invention fabricated a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode to achieve specific detection of Pax-5a.

[0004] This invention uses MIL-68 as a precursor, synthesizing In2O3 nanoclusters through calcination and oxidation to form a heterojunction with CdS as a photoanode. Simultaneously, the MIL-68 precursor is partially oxidized to form an in-situ heterojunction MIO as a photocathode, resulting in a heterojunction dual-photoelectrode sensor. This strategy effectively utilizes the unique advantages of the heterostructure. The synergistic effect of the band gradient and interfacial electric field between the two electrodes effectively prolongs the migration path of photogenerated electrons. Furthermore, the matching redox reaction at the electrode-electrolyte interface and the band structure form a synergistic promotion mechanism, significantly improving charge separation efficiency and ultimately achieving a significant increase in the photocurrent signal. This invention fabricated a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode. This sensor achieves quantitative detection of Pax-5a with high sensitivity, fast response, a wide detection range, a low detection limit, and excellent stability, selectivity, and reproducibility. The construction of this photoelectrochemical sensor provides a new approach for early screening of B-cell acute lymphoblastic leukemia. Summary of the Invention

[0005] One of the purposes of the present invention is to use MIL-68 to prepare In2O3 nanoclusters and CdS nanowires to form a heterojunction as a photoanode material, thereby improving the utilization rate of light by the photoactive material, while improving the photoelectric conversion efficiency, inhibiting the recombination of electron-hole pairs, and improving the stability of the photocurrent.

[0006] The second purpose of the present invention is to use MIL-68 partially oxidized to form an in-situ heterojunction MIO as a photocathode material, and form a heterojunction dual photoelectrode system with a CdS / In2O3 photoanode, thereby extending the photogenerated electron migration path. The external circuit provides a continuous internal driving force for charge transfer, rapidly improving the charge separation efficiency, and promoting a significant enhancement of the photoelectric signal.

[0007] The third purpose of the present invention is to utilize a multi-ball cascade DNA reaction to output a DNA network structure with an expansion effect, connect it to the PEC quenching probe DOX-FcCOOH through an "embedded" connection method, and utilize the competitive electron transfer ability of FcCOOH to achieve significant quenching of the photocurrent signal, thereby improving the sensitivity of detection.

[0008] The technical solutions of the present invention are as follows: A method for preparing a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode is characterized by comprising the following steps: (1) Cut the ITO glass into 2.0 × 0.8 cm 2 Long strips were ultrasonically cleaned with detergent powder, acetone, ethanol, and deionized water for 30 min and dried at 60 °C for 24 h; (2) Take 8.0 μL of 2.0 ~ 10.0 mg / mL In2O3 single material dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Take 8.0 μL of 2.0 ~ 10.0 mg / mL CdS single material dispersion and drop it onto the surface of the electrode with In2O3 attached, and let it dry at room temperature; (4) 8.0 μL of gold nanoparticle dispersion was added to the surface of the In2O3 / CdS electrode to connect DNA through Au-S bonds and dried at room temperature; (5) Continue to add 6.0 μL, 1 fM ~ 1 nM series of different concentrations of nucleic acid marker Pax-5a to the mixture of different nanoprobes NS-13 and NS-24, incubate and hybridize at 37 °C for 2.5 hours in PCR, trigger the multi-ball cascade reaction to output different DNA network structures, incubate at room temperature for 1 hour, and store in a refrigerator at 4 °C for use; (6) 8.0 μL of the synthesized DNA network solution was added dropwise to the electrode surface and incubated in a refrigerator for 14 to 15 hours. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (7) Continue to add 8.0 μL of 5.0 mM to 10 mM hexanethiol to the electrode surface to block the nonspecific active sites on the electrode surface. Rinse the electrode surface with phosphate buffer (pH = 7.38) and dry at room temperature. (8) 8.0 μL of doxorubicin-ferrocene carboxylic acid solution was added dropwise to the electrode surface. Incubated at room temperature for 2 h in the dark to connect the PEC quencher ferrocene via DNA. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. The resulting electrode was used as a photoanode. (9) 8.0 μL of 4.0–10.0 mg / mL MIO dispersion was added to the electrode surface and allowed to dry at room temperature to serve as the photocathode. (10) The modified electrode with In2O3 / CdS droplets was used as the working electrode, and the electrode with MIO droplets was used to connect to the platinum electrode, and then the reference electrode was connected to obtain a heterojunction dual-photoelectrode photoelectrochemical sensor based on In2O3 / CdS photoanode and in-situ heterojunction MIO photocathode.

[0009] 2. The photoanode material and the photocathode material are prepared as follows: (1) Preparation of In2O3 470 mg of indium nitrate tetrahydrate and 180 mg of terephthalic acid were dissolved in 54 mL of N,N-dimethylformamide and stirred until clear. Subsequently, the resulting mixture was transferred to an oil bath and maintained at 100°C for 1.5 to 2 hours. After cooling to room temperature, the mixture was centrifuged and washed three times with ethanol and deionized water, respectively, to collect the white precipitate. Dry it in a vacuum oven at 60°C for 12 hours to obtain MIL-68. Subsequently, the obtained white powder was calcined in a muffle furnace at 500°C and a heating rate of 5°C / min for 4 hours to obtain In2O3; (2) Preparation of CdS 308.48 mg of cadmium nitrate tetrahydrate and 228.36 mg of thiourea were dispersed in 20 mL of ethylenediamine at a molar ratio of 1:3. The solution was then transferred to a polytetrafluoroethylene reactor at 160°C for 20–30 hours. The mixture was then cooled to room temperature and centrifuged at 10,000 rpm (without washing) to remove the supernatant. The resulting product (precipitate) was then ultrasonically dispersed in a certain amount of deionized water. The mixture was refluxed in an oil bath (60°C) overnight, then washed several times by centrifugation with deionized water and ethanol, and dried at 60°C overnight to produce CdS. (3) Synthesis of MIO MIO microrods were prepared using an in situ oxidation method. First, 0.3766 g of terephthalic acid and 1.7328 g of indium nitrate tetrahydrate were dissolved dropwise in 20 mL of DMF with stirring. The solution was then transferred to a polytetrafluoroethylene reactor and heated at 125°C for 4–5 hours. After cooling to room temperature, the solution was washed several times with ethanol and water by centrifugation. Finally, the solution was dried at 80°C for 12 hours to obtain pure white MIO powder.

[0010] 3. Detection of the nucleic acid marker Pax-5a, comprising the following steps: (1) A photoelectrochemical workstation was used to test the three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the MIO-dropped electrode was used as the auxiliary electrode instead of the platinum electrode, and the prepared In2O3 / CdS-dropped electrode with multiple modifications was used as the working electrode for biosensing. A 100W LED lamp was used as the irradiation source. The light was turned on and off every 20 seconds, and the bias voltage was set to 0 V. (2) Detect the photoelectric signal generated by the antigen at different concentrations in 10 mL of phosphate buffer solution containing 0 to 0.2 mol / L ascorbic acid at pH = 6.81 to 8.04, and draw a working curve; (3) The sample solution to be tested is used instead of the B cell acute lymphoblastic leukemia Pax-5a solution for testing.

[0011] Beneficial results of the present invention (1) The present invention successfully synthesized a new cathode material MIO. The preparation method of this material is simple. Compared with traditional cathode photoactive materials, the electron transfer path inside the material is extended, which significantly improves the photoelectric conversion efficiency and solves the problem of low visible light utilization rate of traditional photoactive materials.

[0012] (2) In the present invention, In2O3 / CdS is used as the photoanode and MIO is used as the photocathode to construct a heterojunction dual-photoelectrode sensor. The synergistic effect of the energy band gradient and the interface electric field between the heterojunctions on the dual electrodes effectively extends the migration path of the photogenerated electrons. At the same time, the matching redox reaction and the energy band structure at the electrode-electrolyte interface form a synergistic promotion mechanism, which significantly improves the charge separation efficiency and enhances the detection sensitivity.

[0013] (3) In this invention, a multi-sphere cascade reaction is used to output a DNA mesh structure as a signal amplifier for the immobilized PEC quenching probe. By "inserting" the DOX-FcCOOH into the DOX-FcCOOH, the efficient competitive electron transfer ability of ferrocene is achieved, resulting in significant signal quenching and improved detection accuracy.

[0014] (4) The photoelectric chemical sensor prepared by the present invention is used for the quantitative detection of Pax-5a, a nucleic acid marker for B-cell acute lymphoblastic leukemia, and has a fast response speed, a wide linear range, a low detection limit, good stability, selectivity and reproducibility, and can achieve simple, fast, highly sensitive and specific detection. DETAILED DESCRIPTION

[0015] (The present invention will now be further described through specific embodiments, but is not limited thereto) Example 1. A method for preparing a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode comprises the following steps: (1) Cut the ITO glass into 2.0 × 0.8 cm 2 Long strips were ultrasonically cleaned with detergent powder, acetone, ethanol, and deionized water for 30 min and dried at 60 °C for 24 h; (2) Take 8.0 μL of 6.0 mg / mL In2O3 single material dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Take 8.0 μL of 4.0 mg / mL CdS single material dispersion and drop it onto the surface of the In2O3 electrode and let it dry at room temperature; (4) 8.0 μL of gold nanoparticle dispersion was added to the surface of the In2O3 / CdS electrode to connect DNA through Au-S bonds and dried at room temperature; (5) Continue to add 6.0 μL, 1 fM nucleic acid marker Pax-5a to the mixture of different nanoprobes NS-13 and NS-24, incubate and hybridize at 37°C for 2.5 hours in PCR, trigger the multi-ball cascade reaction to output different DNA network structures, incubate at room temperature for 1 hour, and store in a refrigerator at 4°C for use; (6) 8.0 μL of the synthesized DNA network solution was added dropwise to the electrode surface and incubated in a refrigerator for 14 h. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (7) 8.0 μL of 5.0 mM hexanethiol was added dropwise to the electrode surface to block the nonspecific active sites on the electrode surface. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (8) 8.0 μL of doxorubicin-ferrocene carboxylic acid solution was added dropwise to the electrode surface. Incubated at room temperature for 2 h in the dark to connect the ferrocene PEC quencher via DNA. The electrode surface was rinsed with phosphate buffer solution (pH = 7.38) and dried at room temperature. The resulting electrode was used as a photoanode. (9) Take 8.0 μL of 8.0 mg / mL MIO dispersion and drop it onto the electrode surface. Let it dry at room temperature to serve as the photocathode. (10) The modified electrode with In2O3 / CdS droplets was used as the working electrode, and the electrode with MIO droplets was used to connect to the platinum electrode, and then the reference electrode was connected to obtain a heterojunction dual-photoelectrode photoelectrochemical sensor based on In2O3 / CdS photoanode and in-situ heterojunction MIO photocathode.

[0016] Example 2. A method for preparing a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode comprises the following steps: (1) Cut the ITO glass into 2.0 × 0.8 cm 2 Long strips were ultrasonically cleaned with detergent powder, acetone, ethanol, and deionized water for 30 min and dried at 60 °C for 24 h; (2) Take 8.0 μL of 4.0 mg / mL In2O3 single material dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Take 8.0 μL of 6.0 mg / mL CdS single material dispersion and drop it onto the surface of the electrode with In2O3 and let it dry at room temperature; (4) 8.0 μL of gold nanoparticle dispersion was added to the surface of the In2O3 / CdS electrode to connect DNA through Au-S bonds and dried at room temperature; (5) Continue to add 6.0 μL, 10 fM nucleic acid marker Pax-5a to the mixture of different nanoprobes NS-13 and NS-24, incubate and hybridize at 37°C for 2.5 hours in PCR, trigger the multi-ball cascade reaction to output different DNA network structures, incubate at room temperature for 1 hour, and store in a refrigerator at 4°C for use; (6) 8.0 μL of the synthesized DNA network solution was added dropwise to the electrode surface and incubated in a refrigerator for 14 h. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (7) 8.0 μL of 5.0 mM hexanethiol was added dropwise to the electrode surface to block the nonspecific active sites on the electrode surface. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (8) 8.0 μL of doxorubicin-ferrocene carboxylic acid solution was added dropwise to the electrode surface. Incubated at room temperature for 2 h in the dark to connect the ferrocene PEC quencher via DNA. The electrode surface was rinsed with phosphate buffer solution (pH = 7.38) and dried at room temperature. The resulting electrode was used as a photoanode. (9) 8.0 μL of 6.0 mg / mL MIO dispersion was added to the electrode surface and allowed to dry at room temperature to serve as the photocathode. (10) The modified electrode with In2O3 / CdS droplets was used as the working electrode, and the electrode with MIO droplets was used to connect to the platinum electrode, and then the reference electrode was connected to obtain a heterojunction dual-photoelectrode photoelectrochemical sensor based on In2O3 / CdS photoanode and in-situ heterojunction MIO photocathode.

[0017] Example 3. A method for preparing a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode, comprising the following steps: (1) Cut the ITO glass into 2.0 × 0.8 cm 2 Long strips were ultrasonically cleaned with detergent powder, acetone, ethanol, and deionized water for 30 min and dried at 60 °C for 24 h; (2) Take 8.0 μL of 10 mg / mL In2O3 single material dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Take 8.0 μL of 8.0 mg / mL CdS single material dispersion and drop it onto the surface of the In2O3 electrode and let it dry at room temperature; (4) 8.0 μL of gold nanoparticle dispersion was added to the surface of the In2O3 / CdS electrode to connect DNA through Au-S bonds and dried at room temperature; (5) Continue to add 6.0 μL, 100 fM nucleic acid marker Pax-5a to the mixture of different nanoprobes NS-13 and NS-24, incubate and hybridize at 37°C for 2.5 hours in PCR, trigger the multi-ball cascade reaction to output different DNA network structures, incubate at room temperature for 1 hour, and store in a refrigerator at 4°C for use; (6) 8.0 μL of the synthesized DNA network solution was added dropwise to the electrode surface and incubated in a refrigerator for 14 h. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (7) 8.0 μL of 5.0 mM hexanethiol was added dropwise to the electrode surface to block the nonspecific active sites on the electrode surface. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (8) 8.0 μL of doxorubicin-ferrocene carboxylic acid solution was added dropwise to the electrode surface. Incubated at room temperature for 2 h in the dark to connect the ferrocene PEC quencher via DNA. The electrode surface was rinsed with phosphate buffer solution (pH = 7.38) and dried at room temperature. The resulting electrode was used as a photoanode. (9) Take 8.0 μL of 8 mg / mL MIO dispersion and drop it onto the electrode surface. Let it dry at room temperature to serve as the photocathode. (10) The modified electrode with In2O3 / CdS droplets was used as the working electrode, and the electrode with MIO droplets was used to connect to the platinum electrode, and then the reference electrode was connected to obtain a heterojunction dual-photoelectrode photoelectrochemical sensor based on In2O3 / CdS photoanode and in-situ heterojunction MIO photocathode.

[0018] Example 4. The anode and cathode heterojunction materials of the heterojunction dual photoelectrode system are prepared in the following steps: (1) Preparation of In2O3 470 mg of indium nitrate tetrahydrate and 180 mg of terephthalic acid were dissolved in 54 mL of N,N-dimethylformamide and stirred until clear. Subsequently, the resulting mixture was transferred to an oil bath and maintained at 100°C for 1.5 hours. After cooling to room temperature, the mixture was centrifuged and washed three times with ethanol and deionized water, respectively, to collect the white precipitate. Dry it in a vacuum oven at 60°C for 12 hours to obtain MIL-68. Subsequently, the obtained white powder was calcined in a muffle furnace at 500°C and a heating rate of 5°C / min for 4 hours to obtain In2O3; (2) Preparation of CdS 308.48 mg of cadmium nitrate tetrahydrate and 228.36 mg of thiourea were dispersed in 20 mL of ethylenediamine at a molar ratio of 1:3. The solution was then transferred to a polytetrafluoroethylene reactor at 160°C for 20 hours. The mixture was then cooled to room temperature and centrifuged at 10,000 rpm (without washing) to remove the supernatant. The resulting product (precipitate) was then ultrasonically dispersed in a certain amount of deionized water. The reaction was refluxed overnight in an oil bath (60°C), followed by washing several times with deionized water and ethanol by centrifugation, and dried at 60°C overnight to produce CdS. (3) Synthesis of MIO MIO microrods were prepared using an in situ oxidation method. First, 0.3766 g of terephthalic acid and 1.7328 g of indium nitrate tetrahydrate were dissolved dropwise in 20 mL of DMF with stirring. The solution was then transferred to a polytetrafluoroethylene reactor and incubated at 125°C for 4 hours. After cooling to room temperature, the solution was washed several times with ethanol and water by centrifugation. Finally, pure white MIO powder was obtained by drying at 80°C for 12 hours.

[0019] Example 5 The steps for preparing the cathode and anode heterojunction materials of the heterojunction dual photoelectrode system are as follows: (1) Preparation of In2O3 470 mg of indium nitrate tetrahydrate and 180 mg of terephthalic acid were dissolved in 54 mL of N,N-dimethylformamide and stirred until clear. Subsequently, the resulting mixture was transferred to an oil bath and maintained at 100°C for 1.7 hours. After cooling to room temperature, the mixture was centrifuged and washed three times with ethanol and deionized water, respectively, to collect the white precipitate. Dry it in a vacuum oven at 60°C for 12 hours to obtain MIL-68. Subsequently, the obtained white powder was calcined in a muffle furnace at 500°C and a heating rate of 5°C / min for 4 hours to obtain In2O3; (2) Preparation of CdS 308.48 mg of cadmium nitrate tetrahydrate and 228.36 mg of thiourea were dispersed in 20 mL of ethylenediamine at a molar ratio of 1:3. The solution was then transferred to a polytetrafluoroethylene reactor at 160°C for 25 hours. The mixture was then cooled to room temperature and centrifuged at 10,000 rpm (without washing) to remove the supernatant. The resulting product (precipitate) was then ultrasonically dispersed in a certain amount of deionized water. The reaction was refluxed in an oil bath (60°C) overnight, followed by washing several times with deionized water and ethanol by centrifugation, and dried at 60°C overnight to produce CdS. (3) Synthesis of MIO MIO microrods were prepared using an in situ oxidation method. First, 0.3766 g of terephthalic acid and 1.7328 g of indium nitrate tetrahydrate were dissolved dropwise in 20 mL of DMF with stirring. The solution was then transferred to a polytetrafluoroethylene reactor and incubated at 125°C for 4.5 hours. After cooling to room temperature, the solution was washed several times with ethanol and water by centrifugation. Finally, pure white MIO powder was obtained by drying at 80°C for 12 hours.

[0020] Example 6 The steps for preparing the cathode and anode heterojunction materials of the heterojunction dual photoelectrode system are as follows: (1) Preparation of In2O3 470 mg of indium nitrate tetrahydrate and 180 mg of terephthalic acid were dissolved in 54 mL of N,N-dimethylformamide and stirred until clear. Subsequently, the resulting mixture was transferred to an oil bath and maintained at 100°C for 2 hours. After cooling to room temperature, the mixture was centrifuged and washed three times with ethanol and deionized water, respectively, to collect the white precipitate. Dry it in a vacuum oven at 60°C for 12 hours to obtain MIL-68. Subsequently, the obtained white powder was calcined in a muffle furnace at 500°C and a heating rate of 5°C / min for 4 hours to obtain In2O3; (2) Preparation of CdS 308.48 mg of cadmium nitrate tetrahydrate and 228.36 mg of thiourea were dispersed in 20 mL of ethylenediamine at a molar ratio of 1:3. The solution was then transferred to a polytetrafluoroethylene reactor at 160°C for 30 hours. The mixture was then cooled to room temperature and centrifuged at 10,000 rpm (without washing) to remove the supernatant. The resulting product (precipitate) was then ultrasonically dispersed in a certain amount of deionized water. The mixture was refluxed in an oil bath (60°C) overnight, then washed several times by centrifugation with deionized water and ethanol, and dried at 60°C overnight to produce CdS. (3) Synthesis of MIO MIO microrods were prepared using an in situ oxidation method. First, 0.3766 g of terephthalic acid and 1.7328 g of indium nitrate tetrahydrate were dissolved dropwise in 20 mL of DMF with stirring. The solution was then transferred to a polytetrafluoroethylene reactor and incubated at 125°C for 5 hours. After cooling to room temperature, the solution was washed several times with ethanol and water by centrifugation. Finally, pure white MIO powder was obtained by drying at 80°C for 12 hours.

[0021] Example 7 The detection of Pax-5a, a nucleic acid marker for B-cell acute lymphoblastic leukemia, comprises the following steps: (1) The photoelectrochemical workstation was used for testing with a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the in-situ heterojunction MIO was used as the auxiliary electrode instead of the platinum electrode, and the prepared In2O3 / CdS composite photoelectrochemical sensor was used as the working electrode. A 100 W LED lamp was used as the irradiation source. The lamp was turned on and off every 20 seconds, and the bias voltage was set to 0 V. (2) Detect the photoelectric signal generated by the antigen at different concentrations in 10 mL of phosphate buffer solution containing 0.1 mol / L ascorbic acid at pH = 7.38, and draw a working curve; (3) The sample solution to be tested is used instead of the Pax-5a solution, a nucleic acid marker for B cell acute lymphoblastic leukemia, for testing.

Claims

1. A method for preparing a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode, characterized in that: The following steps are involved: (1) Cut the ITO glass into 2.0×0.8cm 2 Long strips were ultrasonically cleaned with detergent powder, acetone, ethanol, and deionized water for 30 min and dried at 60 °C for 24 h; (2) Take 8.0 μL of 2.0-10.0 mg / mL In2O3 single material dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Take 8.0 μL of 2.0-10.0 mg / mL CdS single material dispersion and drop it onto the surface of the In2O3 electrode and let it dry at room temperature; (4) 8.0 μL of gold nanoparticle dispersion was added dropwise to the surface of the In2O3 / CdS-coated electrode to connect DNA via Au-S bonds and allowed to dry at room temperature. (5) 6.0 μL of a series of different concentrations of nucleic acid marker Pax-5a (1 fM to 1 nM) was added dropwise to the mixture containing different nanoprobes NS-13 and NS-24. The mixture was incubated at 37°C for 2.5 hours in PCR to trigger the multi-sphere cascade reaction to output different DNA network structures. The mixture was incubated at room temperature for 1 hour and stored in a refrigerator at 4°C for later use. (6) 8.0 μL of the synthesized DNA network solution was added dropwise to the electrode surface and incubated in a refrigerator for 14–15 h. The electrode surface was rinsed with phosphate buffer (pH 7.38) and allowed to dry at room temperature. (7) 8.0 μL of 5.0 mM to 10 mM hexanethiol was added dropwise to the electrode surface to block the nonspecific active sites on the electrode surface. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. (8) 8.0 μL of doxorubicin-ferrocene carboxylic acid solution was added dropwise to the electrode surface and incubated at room temperature for 2 hours in the dark to connect the PEC quencher ferrocene via DNA. The electrode surface was rinsed with phosphate buffer (pH = 7.38) and dried at room temperature. The resulting electrode was used as a photoanode. (9) 8.0 μL of 4.0-10.0 mg / mL MIO dispersion was added dropwise to the electrode surface and allowed to dry at room temperature to serve as the photocathode. (10) The modified electrode with In2O3 / CdS droplets was used as the working electrode, and the electrode with MIO droplets was used to replace the platinum electrode for connection, and then the reference electrode was connected to obtain a heterojunction dual-photoelectrode photoelectrochemical sensor based on In2O3 / CdS photoanode and in-situ heterojunction MIO photocathode.

2. The method for preparing a heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode according to claim 1, wherein the preparation of the In2O3 / CdS photoanode and the in-situ heterojunction MIO photocathode substrate material is characterized in that: Here are the steps: (1) Preparation of In2O3 470 mg of indium nitrate tetrahydrate and 180 mg of terephthalic acid were dissolved in 54 mL of N,N-dimethylformamide and stirred until clear and transparent. Subsequently, the resulting mixture was transferred to an oil bath and maintained at 100°C for 1.5 hours. After cooling to room temperature, the mixture was centrifuged and washed three times with ethanol and deionized water, respectively, to collect the white precipitate. Dry it in a vacuum oven at 60°C for 12 hours to obtain MIL-68. Subsequently, the obtained white powder was calcined in a muffle furnace at 500°C and a heating rate of 5°C / min for 4 hours to obtain In2O3; (2) Preparation of CdS 308.48 mg of cadmium nitrate tetrahydrate and 228.36 mg of thiourea were dispersed in 20 mL of ethylenediamine at a molar ratio of 1:

3. The solution was then transferred to a polytetrafluoroethylene reactor at 160°C for 20 hours. The mixture was then cooled to room temperature and centrifuged at 10,000 rpm (without washing) to remove the supernatant. The resulting product (precipitate) was then ultrasonically dispersed in a certain amount of deionized water, refluxed in an oil bath (60°C) overnight, and then washed several times by centrifugation with deionized water and ethanol, and dried at 60°C overnight to produce CdS. (3) Synthesis of MIO MIO microrods were prepared using an in-situ oxidation method. First, 0.3766 g of terephthalic acid and 1.7328 g of indium nitrate tetrahydrate were dissolved dropwise in 20 mL of DMF with stirring. The solution was then transferred to a polytetrafluoroethylene reactor and incubated at 125°C for 4 hours. After cooling to room temperature, the solution was washed several times with ethanol and water by centrifugation. Finally, pure white MIO powder was obtained by drying at 80°C for 12 hours.

3. A heterojunction dual-photoelectrode photoelectrochemical sensor based on an In2O3 / CdS photoanode and an in-situ heterojunction MIO photocathode prepared by the preparation method according to claim 1, for detecting Pax-5a, a nucleic acid marker for B-cell acute lymphoblastic leukemia, characterized in that: Here are the steps: (1) A photoelectrochemical workstation was used for testing using a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the in-situ heterojunction MIO was used as the auxiliary electrode instead of the platinum electrode, and the prepared In2O3 / CdS composite photoelectrochemical sensor was used as the working electrode. A 100W LED light was used as the irradiation source. The light was turned on and off every 20 seconds, and the bias voltage was set to 0V. (2) Detecting the photoelectric signal generated by the antigen at different concentrations in 10 mL of phosphate buffer solution containing 0.1 mol / L ascorbic acid at pH 7.38, and plotting a working curve; (3) The sample solution to be tested is used instead of the Pax-5a solution, a nucleic acid marker for B cell acute lymphoblastic leukemia, for detection.