Photoelectrochemical sensor based on sequential amplification DNA circuit and preparation method thereof

Abstract

The application provides a photoelectrochemical sensor based on sequential amplification of DNA circuit and a preparation method thereof, relates to the field of biosensing, and comprises the following steps: S1, preparing a signal converter; S2, preparing a photoelectrode interface; S3, preparing a catalytic hairpin self-assembly module; S4, preparing a DNA walker walking module; S5, synthesizing the signal converter, the photoelectrode interface, the catalytic hairpin self-assembly module and the DNA walker walking module into a working electrode, respectively electrically connecting the working electrode, a reference electrode and a counter electrode through an electrochemical workstation, and installing a light source to obtain the photoelectrochemical sensor. The photoelectrochemical sensor provided by the application adopts sequential amplification of the catalytic hairpin self-assembly module in the upstream liquid phase and the DNA walker walking module in the downstream photoelectrode interface, and adopts DNA cascade signal amplification, so that the problems of signal crosstalk when multiple trace tumor markers are detected, and low detection sensitivity and low signal-to-noise ratio caused by low content of target proteins in blood are overcome.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology, specifically to a photoelectrochemical sensor based on sequentially amplified DNA circuits and its fabrication method. Background Technology

[0002] Breast cancer (BC) is a highly heterogeneous malignant tumor that seriously threatens the health of women worldwide. Survival and sensitivity to radiotherapy and chemotherapy vary significantly among different subtypes. Therefore, accurately distinguishing different molecular subtypes of breast cancer is crucial for early diagnosis, drug efficacy evaluation, and personalized treatment. Studies have shown that various specific membrane proteins, such as epithelial cell adhesion molecule (EpCAM), mucin 1 (MUC1), estrogen receptor protein (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor protein (PR), have become potential biomarkers for identifying breast cancer subtypes due to their close association with the occurrence, development, and metastasis of breast cancer.

[0003] Currently, detection technologies such as gene chips, immunohistochemistry, and genomics are widely used for the analysis of the aforementioned protein biomarkers. Gene chip detection can perform high-throughput parallel analysis of multiple biomarkers, completing the detection of multiple targets and more associated sites in a single experiment. Immunohistochemistry can directly visualize the in situ distribution of proteins in tissues, preserving the morphological background of tumor tissues. Genomics can directly detect the gene amplification status of biomarkers such as HER2, with higher accuracy than simple protein-level detection. However, the shortcomings of these detection methods in terms of sensitivity, convenience, and the ability to simultaneously detect multiple targets limit their application in clinical testing.

[0004] Cascaded catalytic DNA circuits (such as catalytic hairpin self-assembly and hybridization chain reactions) have been used in the detection of low-abundance tumor markers due to their excellent signal amplification capabilities. Photoelectrochemical detection technology, with its advantages of low background noise and high sensitivity due to the different forms of excitation light source and output signal energy, is also widely used in detection and analysis. By orderly arranging DNA substrate and the liquid interface on the photoelectrode surface, a sophisticated cascaded catalytic circuit nanostructure can be constructed, which significantly reduces crosstalk caused by random collisions and dissociation of components compared to all-liquid phase reactions. Therefore, developing a detection method combining sequentially amplified DNA circuits and photoelectrochemical sensing holds promise for achieving highly sensitive detection of cell surface proteins. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention aims to provide a photoelectrochemical sensor based on sequentially amplified DNA circuits and its preparation method, which solves the problems of low sensitivity and crosstalk in existing protein biomarker analysis methods. It can construct a fine cascaded catalytic circuit nanostructure by orderly arranging DNA substrates and liquid phase interfaces on the photoelectrode surface.

[0006] Specifically, on the one hand, the present invention provides a method for fabricating a photoelectrochemical sensor based on a sequentially amplified DNA circuit, which includes the following steps: S1: Preparation of signal converter: Ferric chloride hexahydrate, sodium acetate and polyethylene glycol are added to ethylene glycol and heated to 200℃ to synthesize ferric oxide magnetic material by hydrothermal method; A concentration of 100 µmol·L -1 Amino-functionalized capture strand DNA solution and concentration of 100 µmol·L -1 Equal volumes of the trigger strand DNA solution were mixed and heated to 95 °C, then cooled to room temperature to obtain signal-converting DNA double strands. This process was repeated three times to obtain three different signal-converting DNA double strands: DNA double strand one was obtained when the capture strand DNA solution was SEQ ID NO.1 and the trigger strand DNA solution was SEQ ID NO.4; DNA double strand two was obtained when the capture strand DNA solution was SEQ ID NO.2 and the trigger strand DNA solution was SEQ ID NO.5; and DNA double strand three was obtained when the capture strand DNA solution was SEQ ID NO.3 and the trigger strand DNA solution was SEQ ID NO.6. Two mL of oxalic anhydride solution was mixed with magnetic material and then mixed with three different signal-transforming DNA double strands to obtain three magnetic capture probes, which were used to specifically recognize EpCAM, MUC1 and HER2 proteins on the surface of tumor cells and to unify the recognition events of each protein into the release of the trigger strand. S2: Preparation of photoelectrode interface: FTO / BiOI was prepared by electrodeposition using a mixture of Bi(NO3)3·5H2O and KI as electrolyte, and FTO / BiVO4 was prepared by adding vanadium acetylacetonate and calcining. Using FTO / BiVO4 as working electrode, FTO / BiVO4 / NiFe-LDH heterojunction was prepared by electrodeposition using a mixture of Ni(NO3)2·6H2O and Fe(NO3)3·9H2O as electrolyte. FTO / BiVO4 / NiFe-LDH / DNA4 was prepared by covalently fixing hairpin DNA4 with 3-aminopropyltriethoxysilane and carboxyl groups. S3: Preparation of upstream liquid-phase catalytic hairpin self-assembly module: Heat DNA1, DNA2 and DNA3 chains of a certain concentration to 95 ℃, cool naturally and mix hairpin DNA1, DNA2 and DNA3 in equal concentration; S4: Preparation of the downstream electrode interface DNA walker module: Aminated DNA5 was heated to 95℃ and naturally cooled to obtain hairpin structured DNA5. 5 µmol·L⁻¹ was added... -1 CdTe QDs with activated carboxyl groups were covalently ligated at a set temperature to obtain hairpin DNA 5-CdTe QDs; S5: The working electrode is synthesized by combining the signal converter, photoelectrode interface, upstream liquid phase catalytic hairpin self-assembly module, and downstream electrode interface DNA walker walking module. The working electrode, reference electrode, and counter electrode are electrically connected through an electrochemical workstation, and a light source is installed to obtain a photoelectrochemical sensor.

[0007] Furthermore, in S1, the amounts of ferric chloride hexahydrate, sodium acetate, polyethylene glycol, and ethylene glycol are 1.35 g, 3.6 g, 1 g, and 30 mL, respectively.

[0008] Furthermore, in S2, the amounts of Bi(NO3)3·5H2O, KI, Ni(NO3)2·6H2O, and Fe(NO3)3·9H2O were 0.01 mmol, 0.01 mmol, and 0.125 mmol·L, respectively. -1 And 0.125 mmol.

[0009] Furthermore: In S3, the volume ratio of DNA1, DNA2, and DNA3 is 1:1:1, and the concentration of each is 5 µmol / L. -1 .

[0010] Furthermore, in S4, the covalent connection is set at a temperature of 80-95 ℃.

[0011] Furthermore, in S5, the reference electrode is a saturated Ag / AgCl electrode, the counter electrode is a platinum electrode, and the light source is an infrared laser pointer.

[0012] Furthermore, in S5, the sequential amplification method of the DNA circuit at different interfaces includes: Upstream liquid-phase catalytic hairpin self-assembly interface, the trigger DNA strand released by the target trigger signal conversion DNA double strand is added to the metastable hairpin DNA1, hairpin DNA2 and hairpin DNA3 solution, and incubated at 25 ℃ for 4 hours, triggering the amplification of the cyclic CHA signal to form a Y-shaped DNA walker; The DNA walker at the downstream photoelectrode interface is moved by adding the fully reacted Y-type DNA walker and hairpin DNA 5-CdTe quantum dots to FTO / BiVO4 / NiFe-LDH / DNA4 and incubating in a humid environment at 37 °C for 3 hours to realize the EDC DNA walker, thereby constructing the working electrode of FTO / BiVO4 / NiFe-LDH / DNA4 / 5-CdTe QDs.

[0013] The present invention also provides a photoelectrochemical sensor prepared by a method for preparing a photoelectrochemical sensor based on a sequentially amplified DNA circuit, comprising: a light source for providing illumination, an electrochemical workstation, and a working electrode, a reference electrode, and a counter electrode respectively electrically connected to the electrochemical workstation; The working electrode includes: Signal transducers: consist of a chain protection structure comprising a protein-specific aptamer nucleic acid chain and a signal triggering chain that hybridizes with it; Photoelectrode interface: including photoelectrode and BiVO4 / NiFe-LDH heterojunction and carboxylated hairpin DNA4 orbital chain sequentially modified on photoelectrode; Upstream liquid-phase catalytic hairpin self-assembly module: used to trigger DNA strands generated in response to target substances in solution and construct a three-legged Y-shaped DNA walker to amplify the signal; Downstream electrode interface DNA walker walking module: used for signal amplification at the photoelectrode interface.

[0014] Furthermore: the upstream liquid-phase catalytic hairpin self-assembly module includes three stable DNA hairpin structures. The target triggers the DNA strand to open one of the corresponding hairpins and trigger a cascade hybridization reaction, ultimately forming a three-legged Y-shaped DNA walker in the liquid phase. The downstream electrode interface DNA walker module includes a hairpin DNA4 orbital strand fixed on the photoelectrode interface. The three-legged Y-shaped DNA walker recognizes and hybridizes with the hairpin DNA4 orbital strand and walks along the orbital strand under the drive of the hairpin DNA5-CdTe quantum dots.

[0015] This invention also provides an application of a photoelectrochemical sensor in the highly sensitive detection of various protein biomarkers.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The photoelectrochemical sensor based on sequentially amplified DNA circuits provided by this invention employs sequential amplification via a catalytic hairpin self-assembly module in the upstream liquid phase and a DNA walker module at the downstream photoelectrode interface, overcoming the severe signal crosstalk in existing detection technologies for multiple trace tumor markers.

[0017] The photoelectrochemical sensor based on sequentially amplified DNA circuits provided by this invention overcomes the shortcomings of weak photocurrent signal changes in existing technologies by using BiVO4 / NiFe-LDH / CdTe QDs type heterojunction photoactive materials.

[0018] The photoelectrochemical sensor based on sequential amplification DNA circuit provided by this invention uses DNA cascade signal amplification, which overcomes the shortcomings of existing detection technologies, such as low detection sensitivity and low signal-to-noise ratio due to the low content of target proteins in blood.

[0019] The photoelectrochemical sensor application based on sequential amplification DNA circuit provided by this invention enables precise detection of cell surface proteins and analysis of breast cancer subtypes, providing technical support for precision treatment.

[0020] The signal converter provided by this invention can realize the conversion of various proteins into nucleic acid trigger chains, enabling universal protein detection. Attached Figure Description

[0021] Figure 1 is a flowchart of the photoelectrochemical sensor based on sequential amplification DNA circuit of the present invention; in, Figure 1 In the middle, A represents a general signal conversion; Figure 1 B represents the photogenerated electron transfer mechanism; Figure 1 C represents the upstream liquid-phase catalytic hairpin self-assembly module and the downstream electrode interface DNA walker walking module; Figure 1 D in the middle refers to the detection application; Figure 2 is a schematic diagram of the agarose gel electrophoresis characterization of the sequential amplification DNA circuit of the present invention; in, Figure 2 In the middle, A represents the targeted release of the signal conversion double strand; Figure 2 B represents the construction of the catalytic hairpin self-assembly module; Figure 2 C represents the walking mechanism of the DNA walker; Figure 3 is a schematic diagram of impedance, cyclic voltammetry curves and photocurrent characterization during the sensor modification and construction process of the present invention. in, Figure 3 In the diagram, A represents the impedance during the sensor modification and construction process; Figure 3 B in the figure represents the cyclic voltammetry curve; Figure 3 C represents the photocurrent. Figure 4 is a data characterization diagram of the photoelectrochemical sensor of the present invention for tumor surface protein detection and breast cancer cell typing. in, Figure 4 In section A, the photocurrent characterization of MCF-7 cells is shown. Figure 4 B represents the photocurrent characterization of SK-BR-3 cells; Figure 4C represents the photocurrent characterization of MDA-MB-231 cells; Figure 4 The bar chart in the middle (D) represents the analysis of different cell subtypes; Figure 4 E represents the relative protein expression levels of different cell subtypes. Detailed Implementation

[0022] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.

[0023] like Figures 1-4 As shown, this invention provides a method for fabricating a photoelectrochemical sensor based on a sequentially amplified DNA circuit. The invention will be further described in detail below with reference to embodiments: like Figure 1 As shown, a method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits includes the following steps: S1: Preparation of signal transducers: namely, three magnetic capture probes. FeCl3·6H2O 1.35g, CH3COONa 3.6g, and polyethylene glycol 30ml were added to ethylene glycol and heated to a set temperature to synthesize Fe3O4 magnetic material via a hydrothermal method. Subsequently, oxalic anhydride solution was mixed with the magnetic material and then mixed with the three signal transducer DNA double strands respectively to obtain three magnetic capture probes. These probes are used to specifically recognize EpCAM, MUC1, and HER2 proteins on the surface of tumor cells, and to uniformly convert the recognition events of each protein into the release of the trigger chain (TS).

[0024] Signal-converting DNA double strands consist of a capture strand and a trigger strand (TS), respectively. 100 µmol·L⁻¹ -1 Amino-functionalized chain-trapping solutions (such as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3) and 100 µmol·L -1 The corresponding trigger chain solutions (such as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6) were mixed in equal volumes, heated to 95 °C, incubated and assembled into three signal conversion DNAs.

[0025] The capture chain has aminolated aptamer sequences containing EpCAM, MUC1, and HER2 proteins targeting the surface of tumor cells, as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and corresponding trigger chains SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, i.e. SEQ ID NO.1: -CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGGGTT GGC CTG AAT ACT TTTTTT-C6-NH2- ; SEQ ID NO.2: -GCA GTT GAT CCT TTG GAT ACC CTG G AAT ACT TTTTTT-C6-NH2- ; SEQ ID NO.3: -GGG CCG TCG AAC ACG AGC ATG GTG CGT GGA CCT AGG ATGACC TGA GTA CTG TCC AAT ACT TTTTTT-C6-NH2- ; SEQ ID NO.4: -GCA CTC CTC CCT AAC ATC TCA AGC TTT TTT TTT TTT TTTAGT ATT CAG GCC- ; SEQ ID NO.5: -GCA CTC CTC CCT AAC ATC TCA AGC TTT TTT TTT TTT TTTAGT ATT CCA GGG T- ; SEQ ID NO.6: -GCA CTC CTC CCT AAC ATC TCA AGC TTT TTT TTT TTT TTTAGT ATT GGA CAG T- .

[0026] S2: Preparation of FTO / BiVO4 / NiFe-LD / DNA4 photoelectrode interface: FTO / BiOI was prepared by electrodeposition using a mixture of 0.01 mmol Bi(NO3)3·5H2O and 0.01 mmol KI as the electrolyte. FTO / BiVO4 was then prepared by adding vanadium acetylacetonate and calcining. FTO / BiVO4 was used as the working electrode, with a concentration of 0.125 mmol·L⁻¹. -1FTO / BiVO4 / NiFe-LDH heterojunctions were prepared by electrodeposition using a mixture of Ni(NO3)2·6H2O and 0.125 mmol Fe(NO3)3·9H2O as the electrolyte; hairpin DNA4 was finally fixed by covalent fixation with 3-aminopropyltriethoxysilane and carboxyl groups.

[0027] Carboxylated DNA4 has the nucleotide sequence shown in SEQ ID NO.10, and a hairpin structure was obtained by heating to 95 °C and then allowing it to cool naturally. SEQ ID NO.10: -HOOC-GATGGC GGTAATAT AAA GCGTACGA CGAAT TCGTACGCAAA ATATTA- .

[0028] S3: Preparation of upstream catalytic hairpin self-assembly DNA circuit solution: Mix the solution with a volume ratio of 1:1:1 and a concentration of 5 µmol / L. -1 DNA1, DNA2, and DNA3 strands were each heated to 95 °C, allowed to cool naturally, and then mixed in equal concentrations. The DNA1, DNA2, and DNA3 strands have the nucleotide sequences shown in SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, i.e. SEQ ID NO.7: -GCT TGA GAT GTT AGG GAG GAG TGC CCC AAT CAC AAC GCACTC CTC CCT ATA TTA CCA ACA TC-3; SEQ ID NO.8: -AGG GAG GAG TGC GTT GTG ATT GGG AAC ATC TCA AGC CCCAAT CAC AAC ATA TTA CCG CAC TC-3; SEQ ID NO.9: -GTT GTG ATT GGG GCT TGA GAT GTT GCA CTC CTC CCT AACATC TCA AGC ATA TTA CCC CCA AT-3.

[0029] S4: Preparation of DNA5-CdTe QDs: Aminated DNA5 has the nucleotide sequence shown in SEQ ID NO.11, and is heated to 80-95℃ and naturally cooled to obtain hairpin-structured DNA5. Subsequently, a certain concentration of activated carboxyl-containing CdTe QDs is added, and covalently ligated at a certain temperature to obtain hairpin DNA5-CdTe QDs. SEQ ID NO.11: -ATAT TTT GCGTAC GAA TTC GTC GTA CGC AAA-C6-NH2- .

[0030] S5: Connect the signal converter, photoelectrode interface, upstream liquid phase catalytic hairpin self-assembly module, and downstream electrode interface DNA walker walking module to the reference electrode, counter electrode, and light source to obtain a photoelectrochemical sensor.

[0031] The detection process of tumor cells using the photoelectrochemical sensor provided by this invention is as follows: Photoelectrochemical sensor based on sequential amplification DNA circuit and breast cancer cell typing detection: 30 µL of tumor cells were added to 10 µL of magnetic capture probe and incubated at 37 °C for 30 minutes. After magnetic separation, the supernatant containing the trigger strand was collected, and the signal from the tumor cell surface protein was successfully converted into a DNA strand signal.

[0032] 40 µL of DNA strand signal was added to an unamplified upstream catalytic hairpin self-assembly DNA circuit containing an equal volume of hairpin one, hairpin two, and hairpin three DNA mixture. The mixture was incubated at 25 °C for 4 hours, and a Y-type DNA walker was formed by triggering the amplification of the cyclic CHA signal.

[0033] 40 µL of Y-type DNA walker solution and 10 µL of hairpin DNA 5-CdTe QDs were dropped onto the FTO / BiVO4 / NiFe-LDH / DNA4 photoelectrode and incubated at 37 °C in a humid environment for 3 hours to realize the EDC DNA walker, thereby constructing the FTO / BiVO4 / NiFe-LDH / DNA 4 / 5-CdTe QDs working electrode.

[0034] Finally, a photoelectrochemical sensor was assembled using an FTO / BiVO4 / NiFe-LDH / DNA 4 / 5-CdTe QD working electrode, a saturated Ag / AgCl electrode as the reference electrode, a platinum electrode as the counter electrode, and an LED lamp as the moving light source. The CHI760E electrochemical workstation recorded data in PBS buffer (0.01 mol·L⁻¹). -1The photocurrent signal change (ΔI) was measured in a solution of FTO / BiVO4 / NiFe-LDH / DNA 4 / 5-CdTe QDs and FTO / BiVO4 / NiFe-LDH / hairpin tetra. This difference reflects the amount of protein on the surface of tumor cells and is used for tumor cell subtype analysis.

[0035] The present invention also provides a photoelectrochemical sensor prepared by a method for preparing a photoelectrochemical sensor based on a sequentially amplified DNA circuit, comprising: a working electrode, a reference electrode, a counter electrode, and a light source.

[0036] The working electrode includes: Signal transducers: consist of a protein-specific aptamer nucleic acid chain and a signal triggering chain that hybridizes with it, forming a chain protection structure.

[0037] Photoelectrode interface: including photoelectrode and BiVO4 / NiFe-LDH heterojunction and carboxylated hairpin DNA4 orbital chain sequentially modified on photoelectrode.

[0038] Upstream liquid-phase catalytic hairpin self-assembly module: includes three stable DNA hairpin structures. The target trigger DNA strand opens one of the corresponding hairpins and triggers a cascade hybridization reaction, ultimately self-assembling in the liquid phase to form a three-legged Y-shaped DNA walker; used to respond to the trigger DNA strand generated by the target in solution and construct a three-legged Y-shaped DNA walker to achieve signal amplification.

[0039] Downstream electrode interface DNA walker module: includes a hairpin DNA4 orbital strand fixed on the photoelectrode interface, a three-legged Y-shaped DNA walker that recognizes and hybridizes with the hairpin DNA4 orbital strand, and walks along the orbital strand under the drive of hairpin DNA5-CdTe quantum dots, for signal amplification at the photoelectrode interface.

[0040] The technical solution of the present invention will be further described below with reference to the embodiments: Example: A method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits, such as... Figure 1 As shown: S1: Three magnetic capture probes were prepared and mixed with three different signal-transforming DNA double strands to obtain three magnetic capture probes for specifically recognizing EpCAM, MUC1, and HER2 proteins on the surface of tumor cells. The recognition events of each protein were uniformly converted into the release of the trigger chain (TS). The recognition process and signal transduction characterization are as follows: Figure 2 As shown in Figure A.

[0041] S2: Prepare an FTO / BiVO4 / NiFe-LD / DNA4 photoelectrode interface as a photoelectric conversion material to generate substrate photocurrent under light source irradiation.

[0042] S3: Preparation of upstream catalytic hairpin self-assembled DNA circuit: DNA1, DNA2, and DNA3 chains of certain concentrations were each heated to 95 °C and allowed to cool naturally to obtain hairpin DNA1, DNA2, and DNA3. These were then mixed in equal concentrations and volumes to obtain an unscaled catalytic hairpin self-assembled DNA circuit, as shown below. Figure 2 As shown in B.

[0043] S4: Prepare DNA 5-CdTe QDs.

[0044] S5: Connect the signal converter, photoelectrode interface, upstream liquid phase catalytic hairpin self-assembly module, and downstream electrode interface DNA walker walking module to the reference electrode, counter electrode, and light source to obtain a photoelectrochemical sensor.

[0045] A photoelectrochemical sensor based on a sequentially amplified DNA circuit was used for the detection of tumor surface proteins and the typing of breast cancer cells: 30 µL of tumor cells were added to 10 µL of a magnetic capture probe and incubated at 37 °C for 30 minutes. After magnetic separation, the supernatant containing the trigger strand was collected, and the signal from the tumor cell surface protein was successfully converted into a DNA strand signal.

[0046] 40 µL of DNA strand signal was added to an unamplified upstream catalytic hairpin self-assembly DNA circuit containing an equal volume of hairpin one, hairpin two, and hairpin three DNA mixture. The circuit was incubated at 25 °C for 4 hours. This triggered cyclic CHA signal amplification, forming a Y-type DNA walker. Figure 2 As shown in B.

[0047] 40 µL of Y-type DNA walker solution and 10 µL of hairpin DNA 5-CdTe QDs were dropped onto the FTO / BiVO4 / NiFe-LDH / DNA4 photoelectrode and incubated at 37 °C for 3 hours to realize the EDC DNA walker, thus constructing the FTO / BiVO4 / NiFe-LDH / DNA4 / 5-CdTe QDs working electrode. Figure 2 C and Figure 3 As shown.

[0048] Finally, a photoelectrochemical sensor was assembled using an FTO / BiVO4 / NiFe-LDH / DNA 4 / 5-CdTe QD working electrode, a saturated Ag / AgCl electrode as the reference electrode, a platinum electrode as the counter electrode, and an LED lamp as the moving light source. The sensor was recorded by the CHI760E electrochemical workstation via the switching of the LED, in PBS buffer (0.01 mol·L⁻¹). -1 The photocurrent signal change (ΔI) was measured in a solution of FTO / BiVO4 / NiFe-LDH / DNA 4 / 5-CdTe QDs and FTO / BiVO4 / NiFe-LDH / hairpin DNA4, and was linearly correlated with the amount of protein on the surface of tumor cells. MCF-7 cell MUC1 protein: ΔI (µA) = 22.89 lg C (cell mL) -1 - 15.80; EpCAM protein: ΔI (µA) = 23.33 lg C (cell mL) -1 - 9.92; HER2 protein ΔI (µA) = 4.38 lg C (cell mL) -1 ) - 4.63.

[0049] MUC1 protein in SK-BR-3 cells: ΔI (µA) = 22.42 lg C (cell mL) -1 -20.81; EpCAM protein: ΔI (µA) = 23.87 lg C (cell mL) -1 ) - 21.13; HER2 protein ΔI (µA) = 27.48 lg C (cell mL) -1 ) - 17.40. MUC1 protein in MDA-MB-231 cells: ΔI (µA) = 17.14 lg C (cell mL) -1 ) - 17.19; EpCAM protein: ΔI (µA) = 5.04 lg C (cell mL) -1 - 6.06; HER2 protein ΔI (µA) = 3.93 lg C (cell mL) -1 ) - 4.68.

[0050] Furthermore, the expression distribution and differences of three proteins—MUC1, EpCAM, and HER2—on the cell surfaces of different breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231) and the control group (MCF-10A) were analyzed. The results showed that SK-BR-3 exhibited high HER2 expression, while EpCAM and MUC1 expression were similar to those in MCF-7; MUC1 expression in MDA-MB-231 was stable, but HER2 and EpCAM expression were lower. Based on this, the sensor achieved highly sensitive detection and accurate differentiation of breast cancer subtypes, such as... Figure 4 As shown.

[0051] This research, based on sequential amplification DNA circuits, overcomes the drawbacks of severe signal crosstalk when detecting various trace tumor markers by combining the liquid-phase interface of DNA circuits with the photoelectrochemical sensing interface. It also realizes the detection and subtyping analysis of tumor surface proteins. This detection method has extremely important application prospects and sustainable scientific research value for clinical precision oncology, and is suitable for widespread use.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits, characterized in that, It includes the following steps: S1: Preparation of signal converter: Ferric chloride hexahydrate, sodium acetate and polyethylene glycol are added to ethylene glycol and heated to 200℃ to synthesize ferric oxide magnetic material by hydrothermal method; A concentration of 100 µmol·L -1 Amino-functionalized capture strand DNA solution and concentration of 100 µmol·L -1 The trigger strand DNA solution was mixed in equal volumes and heated to 95 °C, cooled to room temperature, and repeated three times to obtain three signal-converting DNA double strands. Two mL of oxalic anhydride solution and magnetic material were mixed and then mixed with three different signal-transforming DNA double strands to obtain three magnetic capture probes. These probes were used to specifically recognize EpCAM, MUC1 and HER2 proteins on the surface of tumor cells and to convert the recognition events of each protein into the release of the trigger strand. S2: Preparation of photoelectrode interface: FTO / BiOI was prepared by electrodeposition using a mixture of Bi(NO3)3·5H2O and KI as electrolyte, and FTO / BiVO4 was prepared by adding vanadium acetylacetonate and calcining. Using FTO / BiVO4 as working electrode, FTO / BiVO4 / NiFe-LDH heterojunction was prepared by electrodeposition using a mixture of Ni(NO3)2·6H2O and Fe(NO3)3·9H2O as electrolyte. FTO / BiVO4 / NiFe-LDH / DNA4 was prepared by covalently fixing hairpin DNA4 with 3-aminopropyltriethoxysilane and carboxyl groups. S3: Preparation of upstream liquid-phase catalytic hairpin self-assembly module: Heat DNA1, DNA2 and DNA3 chains to 95°C respectively, cool naturally and mix hairpin DNA1, DNA2 and DNA3 in equal concentrations; S4: Preparation of the downstream electrode interface DNA walker module: Aminated DNA5 was heated to 95℃ and naturally cooled to obtain hairpin structured DNA5. 5 µmol·L⁻¹ was added... -1 CdTe QDs with activated carboxyl groups were covalently linked at a set temperature to obtain hairpin DNA 5-CdTe QDs; S5: The working electrode is synthesized by combining the signal converter, photoelectrode interface, upstream liquid phase catalytic hairpin self-assembly module, and downstream electrode interface DNA walker walking module. The working electrode, reference electrode, and counter electrode are electrically connected through an electrochemical workstation, and a light source is installed to obtain a photoelectrochemical sensor.

2. The method for fabricating the photoelectrochemical sensor based on sequentially amplified DNA circuits as described in claim 1, characterized in that: In S1, the amounts of ferric chloride hexahydrate, sodium acetate, polyethylene glycol, and ethylene glycol are 1.35 g, 3.6 g, 1 g, and 30 mL, respectively. The three signal transduction pathways for obtaining DNA double strands are as follows: When the capture strand DNA solution is SEQ ID NO.1 and the trigger strand DNA solution is SEQ ID NO.4, DNA double strand one is obtained; when the capture strand DNA solution is SEQ ID NO.2 and the trigger strand DNA solution is SEQ ID NO.5, DNA double strand two is obtained; when the capture strand DNA solution is SEQ ID NO.3 and the trigger strand DNA solution is SEQ ID NO.6, DNA double strand three is obtained.

3. The method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits as described in claim 1, characterized in that: In S2, the amounts of Bi(NO3)3·5H2O, KI, Ni(NO3)2·6H2O, and Fe(NO3)3·9H2O were 0.01 mmol, 0.01 mmol, and 0.125 mmol·L, respectively. -1 And 0.125 mmol.

4. The method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits as described in claim 1, characterized in that: In S3, the volume ratio of DNA1, DNA2, and DNA3 is 1:1:1, and the concentration of each is 5 µmol / L. -1 .

5. The method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits as described in claim 1, characterized in that: In S4, the set temperature for covalent bonding is 80-95 ℃.

6. The method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits as described in claim 1, characterized in that: In S5, the reference electrode is a saturated Ag / AgCl electrode, the counter electrode is a platinum electrode, and the light source is an infrared laser pointer.

7. The method for fabricating a photoelectrochemical sensor based on sequentially amplified DNA circuits as described in claim 1, characterized in that: In S5, the sequential amplification methods of DNA circuits at different interfaces include: Upstream liquid-phase catalytic hairpin self-assembly interface, the trigger DNA strand released by the target trigger signal conversion DNA double strand is added to the metastable hairpin DNA1, hairpin DNA2 and hairpin DNA3 solution, and incubated at 25 ℃ for 4 hours, triggering the amplification of the cyclic CHA signal to form a Y-shaped DNA walker; The downstream photoelectrode interface of the DNA walker involves adding the fully reacted Y-type DNA walker and hairpin DNA 5-CdTe quantum dots to FTO / BiVO4 / NiFe-LDH / DNA4 and incubating in a humid environment at 37 °C for 3 hours to realize the EDC DNA walker, thereby constructing the working electrode of FTO / BiVO4 / NiFe-LDH / DNA4 / 5-CdTe QDs.

8. A photoelectrochemical sensor prepared by the method for preparing a photoelectrochemical sensor based on a sequentially amplified DNA circuit according to any one of claims 1-7, characterized in that, include: A light source for providing illumination, an electrochemical workstation, and a working electrode, a reference electrode, and a counter electrode that are electrically connected to the electrochemical workstation, respectively; The working electrode includes: Signal transducers: consist of a chain protection structure comprising a protein-specific aptamer nucleic acid chain and a signal triggering chain that hybridizes with it; Photoelectrode interface: including photoelectrode and BiVO4 / NiFe-LDH heterojunction and carboxylated hairpin DNA4 orbital chain sequentially modified on photoelectrode; Upstream liquid-phase catalytic hairpin self-assembly module: used to trigger DNA strands generated in response to target substances in solution and construct a three-legged Y-shaped DNA walker to amplify the signal; Downstream electrode interface DNA walker walking module: used for signal amplification at the photoelectrode interface.

9. The photoelectrochemical sensor as described in claim 8, characterized in that: The upstream liquid-phase catalytic hairpin self-assembly module includes three stable DNA hairpins. The target triggers the DNA strand to open one of the corresponding hairpins and triggers a cascade hybridization reaction, which ultimately self-assembles in the liquid phase to form a three-legged Y-shaped DNA walker. The downstream electrode interface DNA walker module includes a hairpin DNA4 orbital strand fixed on the photoelectrode interface. The three-legged Y-shaped DNA walker recognizes and hybridizes with the hairpin DNA4 orbital strand and walks along the orbital strand under the drive of the hairpin DNA5-CdTe quantum dots.

10. The application of the photoelectrochemical sensor as described in claim 8 in the highly sensitive detection of various protein markers.

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