Signal reversal type photoelectrochemical biosensor for detecting cancer markers and preparation method and application thereof

Abstract

The present invention provides a method for preparing a signal reversal type photoelectrochemical biosensor for detecting cancer markers, comprising the steps of preparing an indium tin oxide / titanium dioxide electrode, preparing an indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode, preparing a conjugate Ab1-Fe3O4@Au-DNA, preparing a conjugate Ab2-AuNPs-DNA, constructing an intermediate probe, and preparing a sensor. The present invention also provides a biosensor prepared using the above method and its application. The above biosensor for detecting cancer markers is based on the direct contact photocurrent direction reversal strategy, the targeted induced three-dimensional dual-support DNA walker cyclic signal amplification technology, and the adsorption characteristics of metal organic framework materials. The photocurrent signal reversal design is performed. Therefore, the above biosensor can realize environmentally friendly detection of cancer markers, and has the advantages of high sensitivity, high selectivity, good reproducibility, high stability, etc.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a signal flipping type photoelectrochemical biosensor for detecting cancer markers, and a preparation method and application thereof. Background Art

[0002] Soluble CD146 (sCD146) is closely related to the occurrence and development of cancer. As a molecular marker, it plays an important role in the early diagnosis and treatment of cancer. The advantages of photoelectrochemical biosensors in detecting molecular markers have attracted much attention, but most of the reported sensors are built based on a unidirectional signal response strategy, which is not conducive to eliminating the adverse effects of interferences in actual samples on the detection anti-interference ability and sensitivity. False positive or false negative errors may occur, which seriously limits its application and development in sCD146 analysis and detection.

[0003] Signal reversal photoelectrochemical biosensors have recently become popular due to their advantages of high selectivity and high sensitivity. As an emerging signal response strategy, the signal reversal mechanism and application research of photocurrent direction reversal still need to be further explored. As the core element of the photocurrent direction reversal system, the photoelectric active material directly affects its reversal efficiency. The two photoelectric active materials in the existing reversal system usually require biological elements (such as antibodies, nucleic acids, and peptides) to connect without direct contact, resulting in an increase in the electron transfer path and a decrease in the photoelectric conversion efficiency. At the same time, the reversal systems constructed based on cadmium sulfide, cadmium telluride, copper oxide, etc. are also limited in their practical applications due to their toxicity or poor photostability. Therefore, the development of a high-performance new photocurrent direction reversal sensing platform is a meaningful and worthy research work. Summary of the invention

[0004] In view of this, it is necessary for the present invention to provide a signal reversal type photoelectrochemical biosensor for detecting cancer markers and a preparation method and application thereof to solve the above problems.

[0005] Specifically, the present invention provides a method for preparing a signal reversal type photoelectrochemical biosensor for detecting cancer markers, comprising the steps of:

[0006] Preparation of Indium Tin Oxide / Titanium Dioxide Electrode:

[0007] Carbonizing the metal organic framework material to obtain titanium dioxide polyhedrons; using the titanium dioxide polyhedrons as a solute to prepare a titanium dioxide polyhedron suspension; dropping the titanium dioxide polyhedron suspension onto an indium tin oxide electrode to obtain an indium tin oxide / titanium dioxide electrode;

[0008] Preparation of ITO / TiO2 / Single-chain Auxiliary Probe Electrode:

[0009] Adsorbing the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide / titanium dioxide electrode to block the active sites on the titanium dioxide polyhedron, and washing to obtain an indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode;

[0010] Preparation of conjugate Ab1-Fe3O4@Au-DNA:

[0011] The aminated Fe3O4 magnetic beads and gold nanoparticles are shaken to make the aminated Fe3O4 magnetic beads and the gold nanoparticles bind to each other through covalent bonds to form Fe3O4@Au particles, thereby obtaining a Fe3O4@Au solution; the first target capture antibody (Ab1) and the support DNA probe (Support DNA) are sequentially added to the Fe3O4@Au solution, and the solution is shaken and incubated at room temperature and subjected to magnetic separation treatment to make the Ab1, the support DNA probe and the Fe3O4@Au particles bind to each other through covalent bonds, thereby obtaining an Ab1-Fe3O4@Au-DNA conjugate solution; wherein the conjugate Ab1-Fe3O4@Au-DNA can be described as: a Fe3O4@Au particle conjugate modified by the first target capture antibody Ab1 and the support DNA probe;

[0012] Preparation of conjugate Ab2-AuNPs-DNA:

[0013] First, a second target capture antibody (Ab2) is added to a gold nanoparticle solution with a pH of 8 to 9 for reaction, and then a supporting DNA probe and a walking DNA probe (DNA walker) are added to form a mixed solution. The mixed solution reacts so that the Ab2, the supporting DNA probe and the walking DNA probe are combined with the gold nanoparticles in the gold nanoparticle solution through covalent bonds to obtain an Ab2-AuNPs-DNA conjugate solution; wherein the conjugate Ab2-AuNPs-DNA can be described as: a gold nanoparticle conjugate modified by the second target capture antibody Ab2, the supporting probe and the walking probe;

[0014] Construct an intermediate probe:

[0015] First, adding different concentrations of cancer markers to the Ab1-Fe3O4@Au-DNA conjugate solution for incubation and magnetic separation, then adding the Ab2-AuNPs-DNA conjugate solution for incubation and magnetic separation, and then adding restriction endonuclease Nt.BsmAl for reaction and magnetic separation to obtain a supernatant containing an intermediate probe for a targeted induced three-dimensional dual-support DNA walker cyclic signal amplification reaction;

[0016] Prepare the sensor:

[0017] The indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode is mixed and incubated with the intermediate probe in the supernatant, so that the intermediate probe forms a specific double strand with the single-stranded auxiliary DNA probe and the single-stranded auxiliary DNA probe is released from the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode, exposing the active sites on the titanium dioxide polyhedron in the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode to obtain an indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode with active sites; carbon nitride quantum dots are adsorbed on the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode to form an indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe / carbon nitride quantum dot composite electrode, so that the titanium dioxide and carbon nitride quantum dots are in direct contact, so as to prepare a direct contact signal flipping type biosensor.

[0018] Preferably, the metal organic framework material is MIL-125.

[0019] Based on the above preparation method, the steps of preparing the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode include: annealing the single-stranded auxiliary DNA probe at 95°C for 5 minutes, and after cooling to room temperature, dropping the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide / titanium dioxide electrode for adsorption treatment, and washing to obtain the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode.

[0020] Based on the above preparation method, the cancer marker is sCD146, and different protein cancer markers can be selected by replacing the corresponding antibodies.

[0021] Based on the above preparation method, the carbon nitride quantum dots are synthesized in one step by microwave method. Specifically, the preparation method of the carbon nitride quantum dots comprises: using citric acid and urea as raw materials, dissolving them in ultrapure water and subjecting them to microwave treatment to form a dark brown solid; cooling the dark brown solid to room temperature and then adding ultrapure water to dissolve it to obtain a dark brown solution; centrifuging the dark brown solution and taking the supernatant to obtain the carbon nitride quantum dots.

[0022] The present invention also provides a signal reversal type photoelectrochemical biosensor for detecting cancer markers, which is mainly prepared by the preparation method of the signal reversal type photoelectrochemical biosensor for detecting cancer markers.

[0023] The present invention also provides an application of the signal reversal type photoelectrochemical biosensor for detecting cancer markers in detecting cancer markers.

[0024] Based on the above application, the biosensor for detecting cancer markers is placed in a Tril-HCl buffer solution containing 0.1M ascorbic acid with a pH of 7.4, and a bias voltage of -0.2V is applied for PEC detection to detect the photocurrent signal of the cancer marker at different concentrations; and a linear equation between the concentration of the cancer marker and the photocurrent signal is constructed based on the detection results.

[0025] Based on the above application, the time for adsorbing the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide / titanium dioxide electrode is 30 to 120 minutes, and the time for adsorbing the carbon nitride quantum dots onto the active sites exposed by the titanium dioxide polyhedron in the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode is 30 to 120 minutes. Preferably, the time for adsorbing the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide / titanium dioxide electrode is 90 minutes, and the time for adsorbing the carbon nitride quantum dots onto the surface of the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode with active sites is 60 minutes.

[0026] Based on the above application, when the concentration range of sCD146 is 10-5000000 fg / mL, the constructed linear regression equation is I = -174.34logC sCD146 -1314.00(R 2 =0.9977), the detection limit is 2.1 fg / mL; wherein I is the photocurrent signal detected by the biosensor for detecting cancer markers, C sCD146 represents the concentration of sCD146.

[0027] Therefore, the biosensor for detecting cancer markers provided by the present invention is a photoelectrochemical biosensor, which belongs to a direct contact titanium dioxide polyhedron / / carbon nitride quantum dot environmentally friendly photocurrent direction reversal system, which overcomes the problems of long electron transfer path or toxicity in the existing photocurrent direction reversal system, and at the same time expands the application and development of photocurrent direction reversal strategy in photoelectrochemical biosensors.

[0028] The above-mentioned biosensor for detecting cancer markers is based on the organic combination of direct contact photocurrent direction reversal strategy, targeted induced three-dimensional dual-support DNA walker cyclic signal amplification technology and metal organic framework material adsorption characteristics, and performs photocurrent signal reversal design, which not only amplifies the readout of the photocurrent signal, but also shortens the electron transfer path between the reversal materials, improves the photocurrent direction reversal efficiency, makes the biosensor detection results more accurate, and improves the detection sensitivity; in addition, the biosensor provided by the present invention can only generate a photocurrent signal in the opposite direction when identifying the target based on the photocurrent direction reversal strategy, so different interfering proteins have no obvious interference with this detection system; therefore, the above-mentioned biosensor provided by the present invention can realize environmentally friendly detection of cancer markers, and has the advantages of high sensitivity, high selectivity, good reproducibility, high stability, etc., and can also realize accurate low-concentration cancer marker detection, and photoelectrochemical bioanalysis provides a new approach with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of the preparation of a signal reversal type photoelectrochemical sensor for detecting cancer markers provided in Example 1 of the present invention.

[0030] Figure 2 This is a representation diagram of the titanium dioxide polyhedron used in the biosensor provided in Example 1 of the present invention.

[0031] Among them, Figure A in the figure is a scanning electron microscope image of MIL-125. Figure B is a scanning electron microscope image of a titanium dioxide polyhedron. Figure C is an X-ray diffraction spectrum of MIL-125 (a) and a titanium dioxide polyhedron (b). Figure D is a titanium 2p X-ray photoelectron spectrum of a titanium dioxide polyhedron. Figure E is an oxygen 1s X-ray photoelectron spectrum of a titanium dioxide polyhedron. Figure F is a nitrogen isothermal adsorption-desorption curve of MIL-125. Figure G is a nitrogen isothermal adsorption-desorption curve of a titanium dioxide polyhedron. Figure H is a UV-visible-near infrared absorption spectrum of a titanium dioxide polyhedron.

[0032] Figure 3 This is a characterization diagram of the carbon nitride quantum dots used in the biosensor provided in Example 1 of the present invention.

[0033] Among them, Figure A in the figure is a transmission electron microscope image of carbon nitride quantum dots. Figure B is an X-ray diffraction spectrum of carbon nitride quantum dots. Figure C is a UV-visible absorption spectrum of carbon nitride quantum dots. Figure D is a Zeta potential image of titanium dioxide polyhedron (a) and carbon nitride quantum dots (b).

[0034] Figure 4 This is a diagram verifying the mechanism of the biosensor provided in Example 1 of the present invention.

[0035] Among them, Figure A in the figure is a photocurrent response curve diagram of three electrodes (curve a represents indium tin oxide / titanium dioxide electrode, curve b represents indium tin oxide / carbon nitride quantum dot electrode, and curve c represents indium tin oxide / titanium dioxide / carbon nitride quantum dot composite electrode). Figures B to D are respectively the Mott-Schottky curves of indium tin oxide / titanium dioxide electrode, indium tin oxide / carbon nitride quantum dot electrode, and indium tin oxide / titanium dioxide / carbon nitride quantum dot composite electrode. Figures E and F are respectively the Tauc plots of titanium dioxide polyhedron and carbon nitride quantum dots.

[0036] Figure 5 It is a representation diagram of the assembly steps of the biosensor provided in Example 1 of the present invention.

[0037] Figure A in the figure is a photocurrent response diagram of different modified electrodes. Figure B is an AC impedance diagram of different modified electrodes. The curves in each figure represent the following modified electrodes: curve a indium tin oxide / titanium dioxide, curve b indium tin oxide / titanium dioxide / single-chain auxiliary probe, curve c indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe, and curve d indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot composite electrode.

[0038] Figure 6 This is a diagram of optimized preparation conditions of the biosensor provided in Example 1 of the present invention.

[0039] Figure A is a graph showing the relationship between the adsorption time of the single-stranded auxiliary DNA probe and the photocurrent during the preparation of the biosensor provided in Example 1 of the present invention. Figure B is a graph showing the relationship between the adsorption time of carbon nitride quantum dots on titanium dioxide polyhedron and the photocurrent during the preparation of the biosensor provided in Example 1 of the present invention.

[0040] Figure 7 This is a linear relationship diagram of the biosensor provided in Example 2 of the present invention for detecting sCD146.

[0041] Figure A in the figure is a photocurrent response diagram of the biosensor detecting different concentrations of sCD146, and Figure B is a linear fitting diagram of the biosensor detecting sCD146 concentration and photocurrent response.

[0042] Figure 8 This is a diagram of the selective experimental results of the detection method constructed using the biosensor provided in Example 2 of the present invention.

[0043] Fig. 9 A diagram showing the results of a stability experiment of a detection method constructed using the biosensor provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0045] The present invention mainly designs a new direct-contact titanium dioxide polyhedron / / carbon nitride quantum dot flipping system based on the advantage of photocurrent direction flipping; combines the targeted induced three-dimensional dual-support DNA walker cyclic amplification technology and the adsorption characteristics of metal-organic framework materials to design photocurrent signal flipping, and constructs a low-cost, highly selective, and ultra-sensitive environmentally friendly photoelectrochemical biosensor for detecting cancer markers. The level of cancer markers in serum is measured to verify the detection accuracy and sensitivity, providing technical support for studying the role of cancer markers in the occurrence and development of cancer.

[0046] The following uses sCD146 as a cancer marker to further illustrate the photoelectrochemical biosensor for detecting cancer markers provided by the present invention, as well as its preparation method and application.

[0047] The raw materials and reagents used in the embodiments of the present invention: sCD146 and its antibodies Ab1 and Ab2 are from Beijing Sino Biological Technology Co., Ltd.; bovine serum albumin, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), and chloroauric acid are all from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Nt.BsmAl nuclease (Nt.BsmAl) was purchased from New England Biotechnology Co., Ltd.

[0048] Terephthalic acid, titanium isopropoxide, N,N-dimethylformamide, citric acid, urea, anhydrous methanol, trisodium citrate, ferric chloride hexahydrate, ethylene glycol, sodium acetate, 3-aminopropyltriethoxysilane, ascorbic acid, potassium ferrocyanide and potassium ferrocyanide, sodium carbonate, and sodium chloride were all analytical grade AR and were obtained from Sinopharm Chemical Reagent Co., Ltd.

[0049] Nucleic acid sequence (5'→3'): DNA walker: SH-(CH2)6-T 60 -AGT GTG CGA GACGGT ATA TTT; Support DNA probe (Support DNA): NH2-(CH2)6-TTT TAT ACC GTC TCG CAC ACTTTC GCT ACT CTG TTT; Single-stranded auxiliary DNA probe: AAA CAG AGT AGC GAA AGT GTG.

[0050] The experimental instruments used in the embodiments of the present invention are:

[0051] All aqueous solutions were prepared using ultrapure water from a Milli-Q filtration system (Millipore Corp., USA). UV-visible spectra were measured using a DS5 UV-visible spectrophotometer (DS5, UK). Transmission electron micrograph images were characterized by a JEOL JEM 2100F (Japan).

[0052] X-ray diffraction was obtained on an X-ray diffractometer (Bruker D8 Advance) using monochromatized Cu Kα radiation. X-ray photoelectron spectra were obtained on a versatile imaging electron spectrometer (Thermo ESCALAB 250XI).

[0053] A xenon lamp (PLS-SXE 300) equipped with a 420 nm cutoff filter was used as the light source (wavelength > 420 nm), and the photoelectrochemical and electrochemical impedance spectroscopy measurements were performed by a CHI 660E electrochemical workstation with an indium tin oxide electrode (5.6 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the counter electrode. EIS measurements were performed in 5 mM (1:1) [Fe(CN)6] 3- / 4- The electrochemical and photoelectrochemical tests used in the embodiments of the present invention all use a three-electrode system: a 5.6 mm diameter indium tin oxide (Zhuhai Kaiwei Optoelectronics Technology Co., Ltd.) conductive glass as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the auxiliary electrode.

[0054] Example 1

[0055] See also Figure 1 Embodiment 1 of the present invention provides a signal flipping photoelectrochemical biosensor for detecting cancer markers. The sensor is mainly prepared by the following steps, and the preparation steps include preparing an indium tin oxide / titanium dioxide electrode, preparing an indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode, preparing a conjugate Ab1-Fe3O4@Au-DNA, preparing a conjugate Ab2-AuNPs-DNA, constructing an intermediate probe, and preparing a sensor.

[0056] 1. Preparation of Indium Tin Oxide / Titanium Dioxide Electrode

[0057] Will be like Figure 2 The metal organic framework material MIL-125 shown in A was carbonized in a tube furnace at 380℃ for 4h to obtain Figure 2The titanium dioxide polyhedron shown in B; the titanium dioxide polyhedron is dispersed in ultrapure water to prepare a titanium dioxide polyhedron suspension with a concentration of 1 mg / mL; 25 μL of the 1 mg / mL titanium dioxide polyhedron suspension is dropped onto a cleaned indium tin oxide working electrode to prepare an indium tin oxide / titanium dioxide electrode.

[0058] from Figure 2 A shows that the MIL-125 polyhedron has a pancake-like structure with an average size of about 600nm and a depth of about 280nm. After carbonization in air, Figure 2 The TiO2 polyhedrons shown in B maintain a similar morphology, but the average size of the TiO2 polyhedrons is reduced. Figure 2 The X-ray powder diffraction pattern shown in C further confirms the successful preparation of titanium dioxide polyhedrons. Figure 2 C can be seen that curve a shows all the diffraction peaks of the MIL-125 polyhedron; after carbonization, the diffraction peaks of the MIL-125 polyhedron completely disappear, as shown in curve b, which corresponds to the crystal plane of the anatase phase structure of titanium dioxide (JCPDSno.73-1764), indicating that anatase titanium dioxide was successfully synthesized.

[0059] X-ray photoelectron spectroscopy was used to analyze the elemental composition and chemical state of titanium dioxide polyhedrons. Figure 2 D can be obtained, and the titanium 2p spectrum is attributed to the titanium 2p of titanium (IV) 3 / 2 and Ti 2p 1 / 2 , where the binding energies are 458.8 and 464.7 eV, respectively. Figure 2 The O1s spectrum shown in E can be decomposed into three peaks at 530.02, 531.4 and 532.8 eV, corresponding to the metal oxide Ti-O, part of C=O or CO and -OH, respectively. This indicates the presence of Ti-OC bonds and -COOH groups. In order to further explore the specific surface area and porous structure of titanium dioxide polyhedron, nitrogen adsorption and desorption isotherm tests were carried out. The results are shown in Figure 2. Figure 2 As shown in E and 2F, the BET specific surface area of ​​the titanium dioxide polyhedron is calculated to be 102.3 m 2 / g.

[0060] in addition, Figure 2 The BJH pore size distribution curve in the inset of F shows that the average pore size of the titanium dioxide polyhedron is 5.1 nm, which is conducive to the rapid mass transfer and adsorption of small molecules. Titanium dioxide polyhedrons with high specific surface area and porous structure may have good photoelectrochemical properties and can provide sufficient active sites. Figure 2H shows the UV-visible-near infrared absorption spectrum of titanium dioxide polyhedron. The maximum absorption band of titanium dioxide polyhedron appears at about 580nm, which is significantly red-shifted compared with conventional titanium dioxide nanomaterials (380nm), which is roughly attributed to its porous structure. The visible absorption of titanium dioxide polyhedron is significantly enhanced and has a wide absorption range, indicating that they are suitable as photoelectric active materials under visible light (wavelength>420nm), which is conducive to the construction of photoelectrochemical biosensors.

[0061] 2. Preparation of Indium Tin Oxide / Titanium Dioxide / Single-chain Auxiliary Probe Electrode

[0062] 10 μM single-stranded auxiliary DNA probe was annealed at 95°C for 5 min, and after cooling to room temperature, 20 μL of the annealed single-stranded auxiliary DNA probe was added dropwise to the surface of the indium tin oxide / titanium dioxide electrode. The single-stranded auxiliary probe was combined with the porous structure and electrostatic adsorption of the titanium dioxide polyhedron on the surface of the indium tin oxide / titanium dioxide electrode to adsorb the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide / titanium dioxide electrode to block the active sites on the titanium dioxide polyhedron. After adsorption for 30 to 120 min, the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode was obtained by washing.

[0063] 3. Preparation of Ab1-Fe3O4@Au-DNA conjugate

[0064] Fe3O4 nanoparticles: Take 0.6g of ferric chloride hexahydrate and dissolve it in 20mL of ethylene glycol to form a clear solution. Then add 1.5g of sodium acetate and stir vigorously for 30min. Then transfer it to a 50mL reactor and place it at 200℃ for 8h. After cooling to room temperature, wash it with anhydrous ethanol and ultrapure water several times, collect the solid by magnetic separation and freeze-dry it to obtain brown-black powder Fe3O4 nanoparticles.

[0065] Au nanoparticles: First, heat 150 mL of 1 mM HAuCl4 to boiling, then quickly add 15 mL of 38.8 mM sodium citrate solution. After vigorously stirring for 10 min, place the flask in an ice water bath to quickly terminate the reaction. Collect the solution and place it at 4 °C for use to obtain Au nanoparticles.

[0066] Preparation of Fe3O4@Au solution: 30 mg of the above Fe3O4 nanoparticles and 1 mL of 97% 3-aminopropyltriethoxysilane were dispersed in 20 mL of anhydrous ethanol, ultrasonicated for 5 min, and shaken at room temperature for 6 h. 1.5 mg / mL of aminated Fe3O4 nanoparticles were obtained by magnetic separation and redispersed in 15 mL of ultrapure water. 1 mL of the above solution was taken, 10 mL of the above prepared Au nanoparticles and 1 mL of ultrapure water were added, and shaken for 3 h to allow the aminated Fe3O4 nanomagnetic beads and Au nanoparticles to bind through covalent bonds. Fe3O4@Au particles were obtained after magnetic separation, and dispersed in 1 mL of water to obtain Fe3O4@Au solution.

[0067] Preparation of Ab1-Fe3O4@Au-DNA: 30 μL of Ab1 was added to 1 mL of the Fe3O4@Au solution, reacted at room temperature for 1 h, and then 100 μL of supporting DNA probe (10 μM) was added, and shaken at room temperature for 12 h to allow Ab1, the supporting DNA probe and the Fe3O4@Au particles to form an Ab1-Fe3O4@Au-DNA conjugate complex through covalent bonds. After magnetic separation, the obtained Ab1-Fe3O4@Au-DNA conjugate complex was redispersed in a PBS buffer solution to obtain an Ab1-Fe3O4@Au-DNA conjugate solution, which was stored for later use.

[0068] 4. Preparation of Ab2-AuNPs-DNA Conjugate

[0069] 30 μL of sodium carbonate solution was added to 1 mL of the Au nanoparticles prepared above until the pH of the solution was in the range of 8 to 9, and then 30 μL of Ab2 was added, and the reaction was carried out at room temperature for 1 hour, and then 10 μL of walking DNA probe and 100 μL of supporting DNA probe were added to form a mixed solution, and the mixed solution was placed at 4°C and reacted for 18 hours, so that Ab2, supporting DNA probe and walking DNA probe were combined with gold nanoparticles through covalent bonds to form Ab2-AuNPs-DNA conjugate complexes. Finally, the unbound nucleic acid chains and antibodies were removed by centrifugation at 15000 rpm, 30 min, and 4°C, and then dispersed in PBS buffer containing 1% bovine serum albumin (PBS buffer solution containing 1% bovine serum albumin and 1% polyethylene glycol 20000) to obtain Ab2-AuNPs-DNA conjugate solution, and stored at 4°C in the dark.

[0070] 5. Preparation of intermediate probe

[0071] Add 10 μL of the target sCD146 to 100 μL of the Ab1-Fe3O4@Au-DNA conjugate solution, react at 37°C for 90 min, remove the unbound target by magnetic separation and washing, add 100 μL of the Ab2-AuNPs-DNA conjugate solution, shake at 37°C for 90 min, separate magnetically again and disperse in 100 μL PBS solution. Then add 5U of restriction endonuclease Nt.BsmAl, react at 37°C for 2h, and take the supernatant after magnetic separation. The supernatant contains the intermediate probe of the target-induced three-dimensional dual-support DNA walker cyclic signal amplification reaction.

[0072] 6. Preparation of Sensors

[0073] The steps of preparing the sensor include: preparing carbon nitride quantum dots, preparing indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe electrodes, and preparing indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot composite electrodes.

[0074] 5.1 The step of preparing carbon nitride quantum dots comprises: synthesizing carbon nitride quantum dots by a microwave one-step method, dissolving 0.5 g of citric acid and 0.5 g of urea in 25 mL of ultrapure water, microwave at medium-high heat for 7 min, observing that the colorless liquid turns into a dark brown solid to form carbon nitride quantum dots, after cooling to room temperature, adding 5 mL of ultrapure water to dissolve to obtain a dark brown solution, centrifuging at 10000 rpm for 5 min, taking the supernatant, obtaining a carbon nitride quantum dot solution, and storing it at 4°C for later use.

[0075] The carbon nitride quantum dots prepared in this step were characterized by transmission electron microscopy for their morphology and size. Figure 3 As shown in A, from Figure 3 In A, it can be observed that the carbon nitride quantum dots are quasi-spherical and evenly dispersed. Figure 3 The high-resolution transmission image in the inset of A shows that the interplanar spacing is about 0.32 nm, which corresponds to the (002) lattice plane of the hexagonal g-C3N4, indicating that the step successfully prepared carbon nitride quantum dots.

[0076] Figure 3 B tested the X-ray diffraction spectrum of carbon nitride quantum dots. The 2θ value of carbon nitride quantum dots showed an obvious characteristic peak at 27.1°, which is the stacking of conjugated aromatic rings on the (002) crystal plane. In addition, Figure 3 C further characterized the UV-visible spectrum of carbon nitride quantum dots and observed that it had an obvious absorption peak at 345nm and a shoulder peak around 445nm, which is basically consistent with existing literature reports.

[0077] In addition, the Zeta potential analysis Figure 3It can be seen from D that the surface of the titanium dioxide polyhedron is positively charged, with a potential of about +23.3 mV; while the carbon nitride quantum dots are negatively charged, about -19.5 mV; this further illustrates that the titanium dioxide polyhedron and the carbon nitride quantum dots can be combined through electrostatic adsorption.

[0078] 5.2 The steps of preparing the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode include: adding the supernatant containing the intermediate probe containing the targeted induced three-dimensional dual-supported DNA walker cyclic signal amplification reaction to the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode, incubating at room temperature for 90 minutes to allow the intermediate probe to specifically bind to the single-stranded auxiliary DNA probe on the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode, forcing the single-stranded auxiliary DNA probe to be released from the titanium dioxide polyhedron, exposing the active sites on the titanium dioxide polyhedron, and obtaining an indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode with titanium dioxide polyhedron active sites.

[0079] 5.3 The steps of preparing the indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot composite electrode include: cleaning the indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe electrode; diluting the carbon nitride quantum dot solution prepared by the microwave one-step method by 10 times to obtain a diluted carbon nitride quantum dot solution; adding 20 μL of the diluted carbon nitride quantum dot solution to the cleaned indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe electrode for adsorption and incubation at room temperature for 60 minutes so that the carbon nitride quantum dots are modified on the surface of the titanium dioxide polyhedron through the porous structure and electrostatic adsorption of the titanium dioxide polyhedron, forming an indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot composite electrode, thereby preparing a direct contact signal flipping sensor.

[0080] (1) Research on the mechanism of direct contact signal flipping sensor

[0081] 1) Experimental conditions and methods for photocurrent signal detection

[0082] The prepared titanium dioxide polyhedron and carbon nitride quantum dot solutions were directly added to the indium tin oxide electrode, and dried overnight to obtain the indium tin oxide / titanium dioxide electrode and the indium tin oxide / carbon nitride quantum dot electrode; in addition, the carbon nitride quantum dot solution was added to the indium tin oxide / titanium dioxide electrode, and incubated for 30 to 120 minutes to obtain the indium tin oxide / titanium dioxide / carbon nitride quantum dot composite electrode. Then, using the electrochemical workstation three-electrode system, the prepared electrode was placed in a Tril-HCl buffer solution (pH 7.4, 0.1M) containing 0.1M ascorbic acid to test the it curve and obtain the photocurrent signal. The results are as follows Figure 4 As shown in A.

[0083] from Figure 4 It can be seen from A that the anodic photocurrent signal of the indium tin oxide / titanium dioxide electrode shown in curve a is 2.124μA, and curve b shows that the indium tin oxide / carbon nitride quantum dot electrode shows a relatively weak anodic photocurrent signal. It can be seen from curve c that after the indium tin oxide / titanium dioxide electrode is incubated with carbon nitride quantum dots, the indium tin oxide / titanium dioxide / carbon nitride quantum dot composite modified electrode formed not only produces a relatively large cathode photocurrent of -3.579μA, but also has the opposite direction to the photocurrent of the indium tin oxide / titanium dioxide electrode shown in curve a. This shows that when the carbon nitride electron dots are in direct contact with the titanium dioxide polyhedron, the carbon nitride electron dots can act as a signal flipping agent to achieve the conversion of the photocurrent direction.

[0084] 2) Mott-Schottky curve test conditions and methods Using the electrochemical workstation three-electrode system, the prepared indium tin oxide / titanium dioxide electrode, indium tin oxide / carbon nitride quantum dot electrode, indium tin oxide / titanium dioxide / carbon nitride quantum dot electrode were placed in Tril-HCl buffer solution (pH 7.4, 0.1M) Na2SO4 solution (pH 6.8, 0.2M) to test the impedance-voltage curve, and then converted to obtain the Mott-Schottky curve. The results are shown in Figure 4 As shown in B~4D.

[0085] from Figure 4 B and Figure 4 It can be seen from C that the ITO / CNO electron dot electrode and the ITO / TiO2 electrode both show positive slopes, corresponding to n-type semiconductor characteristics, while the ITO / TiO2 / CNO quantum dot composite electrode shows a negative slope, corresponding to p-type semiconductor characteristics. This result is consistent with Figure 4 The photocurrent signal shown in A is consistent with the research results.

[0086] 3) Test conditions and methods corresponding to the Tauc plot The UV-visible spectra of titanium dioxide polyhedron solution and carbon nitride quantum dot solution were tested using a DS5 UV-visible spectrophotometer (DS5, UK), and then converted to obtain the Tauc plot. The results are shown in Figure 4 E and Figure 4 As shown in F.

[0087] See also Figure 4 E, the synthesized titanium dioxide polyhedron has a bandgap of 2.52 eV, and its conduction band and valence band are -0.73 V and 1.79 V relative to the normal hydrogen electrode (NHE), respectively. Figure 4 F shows that carbon nitride quantum dots have a band gap of 2.55 eV, and their conduction band and valence band are -0.18 V and 2.37 V respectively relative to the ordinary hydrogen electrode NHE.

[0088] So, from Figure 4It can be concluded that the photocurrent direction reversal mechanism of the present invention is: under the irradiation of visible light (wavelength greater than 420nm), the titanium dioxide polyhedron is excited, and photogenerated electrons / holes are formed on the conduction band / valence band, respectively. Subsequently, the photogenerated electrons are transferred from the conduction band of the titanium dioxide polyhedron -0.73V to the indium tin oxide electrode. At the same time, ascorbic acid, as an electron donor, has an oxidation potential of 0.15V, which is located between the conduction band and valence band of titanium dioxide and can be oxidized by the photogenerated holes of titanium dioxide, resulting in more photogenerated electrons occupying the conduction band of the titanium dioxide polyhedron and transferring to the indium tin oxide electrode, generating a larger anode photocurrent.

[0089] When carbon nitride quantum adsorbs titanium dioxide polyhedrons and modifies them onto indium tin oxide electrodes to form indium tin oxide / titanium dioxide / carbon nitride quantum dot composite modified electrodes, both titanium dioxide polyhedrons and carbon nitride quantum dots are easily excited and form corresponding photogenerated electrons / holes in the conduction band / valence band, respectively. Based on the energy level matching, the photogenerated electrons at -0.73V of the titanium dioxide polyhedron conduction band are transferred to -0.18V of the carbon nitride quantum dot conduction band for electrochemical reduction of oxygen in the electrolyte, where the reduction potential of oxygen is -0.046V as an electron acceptor; the photogenerated holes at 2.37V of the valence band of the carbon nitride quantum dots are transferred to 1.79V of the conduction band of the titanium dioxide polyhedron. In addition, part of the photogenerated electrons in the conduction band of the carbon nitride quantum dots return to the valence band, resulting in the recombination of the photogenerated electron / hole pairs. In this process, ascorbic acid effectively oxidizes and removes holes on the conduction band, inhibits the recombination of photogenerated electron / hole pairs, and promotes the transfer of photogenerated electrons, thereby enhancing the cathode photocurrent of the ITO / TiO2 / CNT quantum dot composite modified electrode. Therefore, based on the excellent matching horizontal energy bands between carbon nitride quantum dots and titanium dioxide polyhedrons, a titanium dioxide polyhedron / / carbon nitride quantum dot photocurrent direction reversal system is formed. Carbon nitride quantum dots can promote the separation and transfer of photogenerated electrons / holes in titanium dioxide polyhedrons, inducing the photocurrent direction reversal of titanium dioxide polyhedrons.

[0090] (2) Characterization of photoelectrochemical biosensors

[0091] Experimental conditions: Under a bias voltage of -0.2 V, the indium tin oxide / titanium dioxide electrode, indium tin oxide / titanium dioxide / single-chain auxiliary probe electrode, indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe electrode, indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot electrode used in the embodiments of the present invention were used as electrodes and placed in a Tril-HCl buffer solution (pH 7.4, 0.1 M) containing 0.1 M ascorbic acid for photoelectrochemical testing and placed in a 5 mM (1:1) [Fe(CN)6] 3- / 4- The impedance test was performed in a solution containing 0.1M KCl, with a frequency range of 0.1Hz to 100kHz and an amplitude of 5mV. Figure 5 shown.

[0092] Due to the consumption of ascorbic acid, the photocurrent decays with the increase of illumination time. Therefore, the present invention displays the photoelectrochemical test results by using the maximum photocurrent value generated by different modified electrodes. Figure 5 It can be seen from A that the indium tin oxide / titanium dioxide electrode shown in curve a has an obvious anode photocurrent of 2.124μA. Curve b shows that the photocurrent of the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode modified with the single-stranded auxiliary DNA probe is weakened, and its value is 1.010μA. This is because the single-stranded auxiliary DNA probe adsorbed on the surface of the electrode has poor conductivity, which hinders the generation of photogenerated electrons, thereby weakening the anode photocurrent signal.

[0093] Curve c shows that when the single-stranded auxiliary DNA probe combines with the intermediate probe to form a double strand, the content of the single-stranded auxiliary DNA probe on the electrode surface decreases, so the anode photocurrent signal of the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode is partially restored (1.304μA). Curve d shows that after the carbon nitride quantum dot solution is added and incubated, due to the introduction of carbon nitride quantum dots and the energy level relationship between titanium dioxide polyhedrons and carbon nitride quantum dots, the polarity of the photoelectric signal is converted, so the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe / carbon nitride quantum dot composite electrode forms a large cathode photocurrent of -2.987μA.

[0094] from Figure 5 B shows that: Curve a shows the R ct The value is 35.73Ω; due to the poor conductivity of nucleic acid chains, the fixation of single-stranded auxiliary DNA probes on the surface of ITO / TiO2 electrode effectively hinders the [Fe(CN)6] 3- / 4- The electron transfer process between ions and electrodes leads to an increase in the resistance of charge transfer. Therefore, curve b shows the R of the ITO / TiO2 / single-chain auxiliary probe electrode. ct The value is 69.86Ω; when the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode is incubated with the nucleic acid chain intermediate probe released by enzyme cleavage, the single-stranded auxiliary probe and the intermediate probe form a double strand due to base pairing and are then washed away. Therefore, curve c shows that the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode obtains a reduced R ctWhen the indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe electrode is immersed in the carbon nitride quantum dot solution for incubation, the single-chain auxiliary probe on the electrode is combined with the intermediate probe, and the active sites on the titanium dioxide polyhedron are released, so that the carbon nitride quantum dots can be directly fixed to the electrode by the pore size and electrostatic adsorption of the titanium dioxide polyhedron, promoting the transfer of electrons. Therefore, curve d shows that the indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot electrode obtains a reduced R ct The value is 55.88Ω. ct The expected change in value is basically the same as Figure 5 The results are consistent with those shown in Figure A, indicating that the photoelectrochemical biosensor for detecting cancer markers has been successfully prepared.

[0095] There are two main factors that affect the photocurrent signal of the signal flipping type photoelectrochemical biosensor for detecting cancer markers provided by the embodiment of the present invention: one is the adsorption time of the single-chain auxiliary probe in the step of preparing the indium tin oxide / titanium dioxide / single-chain auxiliary probe electrode, and the other is the adsorption time of the carbon nitride quantum dots in the process of preparing the indium tin oxide / titanium dioxide / carbon nitride quantum dot electrode in the step of preparing the sensor. The following is a further analysis of these two factors.

[0096] Effect of single-stranded auxiliary probe adsorption time

[0097] Experimental conditions: Basically referring to the method provided in the above-mentioned embodiment 1, the corresponding sensors were prepared according to the different adsorption times of the single-chain auxiliary probe, wherein in the step of "preparing indium tin oxide / titanium dioxide / single-chain auxiliary probe electrode", the adsorption times of the single-chain auxiliary probe were 0, 30, 60, 90, and 120 min, respectively, and in the step of "preparing the sensor", the adsorption time of the carbon nitride quantum dots was 60 min. The other steps were the same as in the embodiment 1. The prepared sensor was used to perform photoelectrochemical tests according to the experimental conditions in the above-mentioned "(2) Characterization of photoelectrochemical biosensors". The test results are shown in FIG. Figure 6 As shown in A.

[0098] from Figure 6It can be seen from A that when the single-stranded auxiliary DNA probe adsorption time is 0 to 90 minutes, the photoelectric response of the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode gradually weakens. This may be because the longer the single-stranded auxiliary DNA probe adsorption reaction time is, the more single-stranded auxiliary DNA probes are fixed on the electrode, thereby enhancing the hindrance to the generation and transfer of photogenerated electrons and holes on the titanium dioxide polyhedron. However, the experiment found that after the adsorption time exceeds 90 minutes, the trend of the photoelectric response weakening gradually tends to be gentle, which means that the amount of single-stranded auxiliary probes that can be fixed on the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode has basically reached saturation. Therefore, 90 minutes is preferably used as the adsorption reaction time of the single-stranded auxiliary DNA probe on the indium tin oxide / titanium dioxide electrode.

[0099] Effect of adsorption time on carbon nitride quantum dots

[0100] Experimental conditions: The adsorption time of carbon nitride quantum dots in the "sensor preparation" step is 0, 30, 60, 90, and 120 min. The experimental structure is as follows Figure 6 As shown in B.

[0101] from Figure 6 It can be seen from Figure B that when the carbon nitride quantum dot adsorption time is 0 to 60 minutes, the cathode photocurrent generated by the indium tin oxide / titanium dioxide / carbon nitride quantum dot composite electrode increases with the extension of the carbon nitride quantum dot adsorption time, and the current response change almost tends to be flat after 60 minutes. Therefore, 60 minutes is preferred as the optimal adsorption time of carbon nitride quantum dots on the indium tin oxide / titanium dioxide electrode.

[0102] Example 2

[0103] This embodiment provides an application of the biosensor for detecting cancer markers provided in Embodiment 1 in detecting cancer markers.

[0104] Specifically, in the preparation process of the above-mentioned biosensor provided in Example 1, the adsorption reaction time of the single-chain auxiliary probe on the indium tin oxide / titanium dioxide electrode is 90 minutes, and the adsorption time of the carbon nitride quantum dots on the indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe electrode is 60 minutes; the biosensor is placed in a 0.1M ascorbic acid Tril-HCl buffer solution (pH 7.4, 0.1M), a -0.2V bias is applied, and a photoelectrochemical test is performed to detect the photocurrent signals of sCD146 at concentrations of 10, 100, 200, 1000, 10000, 50000, 100000, 1000000 and 5000000 fg / mL, respectively; a linear equation between the concentration of sCD146 and the photocurrent signal is constructed based on the detection results, as shown in FIG. Figure 7 shown.

[0105] from Figure 7 It can be seen that as the sCD146 concentration increases from 10 to 5000000fg / mL, the cathode photocurrent generated by the polarity reversal of the indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot electrode increases, and has good linearity with the sCD146 concentration in the range of 10-5000000fg / mL. The linear regression equation is I = -174.34logC sCD146 -1314.00(R 2 =0.9977); wherein I is the photocurrent signal detected by the biosensor for detecting cancer markers, C sCD146 represents the concentration of sCD146.

[0106] Detection limit

[0107] Experimental method or conditions The prepared sensor was used to detect the photocurrent signal of the blank solution 10 times, and the detection limit was calculated based on the detection limit = 3σ / S (where σ is the standard deviation of the blank sample; S is the slope of the calibration curve). The detection limit of sCD146 concentration was calculated to be 2.1 fg / mL.

[0108] Selective experiment

[0109] In the present embodiment, PSA and CEA in serum were selected as interfering proteins in the sCD146 selective experiment. The prepared sensor was used to test the photocurrent signal of the interfering protein solution. The test results are shown in FIG. Figure 8 shown.

[0110] from Figure 8 It can be seen that the photocurrent response caused by the interfering protein PSA and CEA solutions is in the same direction as that of the blank solution, and the current response value is similar. However, the experiment found that when the above interfering proteins were mixed with the target at the same concentration ratio of 100:1, that is, C PAS :C sCD146 500ng / mL:5ng / mL, C CEA :C sCD146 are 500ng / mL:5ng / mL; both produced photocurrent responses with the same direction and similar magnitude as the target sCD146, indicating that the method for detecting sCD146 using the above-mentioned biosensor for detecting cancer markers provided by an embodiment of the present invention has good selectivity.

[0111] Reproducibility experiment

[0112] Five groups of biosensors were prepared under the same experimental conditions, and 200 fg / mL sCD146 solution was tested to obtain photocurrent signals, and the relative standard deviations of the five groups of photocurrents were calculated.

[0113] Five groups of biosensors were prepared under the same experimental conditions and tested 200 fg / mL sCD146 solution to obtain five groups of photocurrent signals, and the relative standard deviation was calculated to be 2.7%.

[0114] Stability test

[0115] The biosensor provided in the embodiment of the present invention was placed in a 4°C refrigerator for 7, 14 and 21 days, and then tested with a 10000 fg / mL sCD146 solution to obtain a photocurrent signal. The test results are shown in Figure 2. Fig. 9 shown.

[0116] The experimental results are as follows Fig. 9 As shown, the photocurrent of the biosensor can maintain 99.7% of the initial signal within 3 weeks.

[0117] Therefore, the application of the signal flipping sensor provided by the embodiment of the present invention in sCD146 detection has the following advantages:

[0118] 1) Super sensitivity: The embodiments of the present invention organically combine the direct contact photocurrent direction reversal strategy, the targeted induced three-dimensional dual-support DNA walker cyclic signal amplification technology, and the adsorption characteristics of metal organic framework materials, which not only amplifies the readout of the photocurrent signal, but also accelerates the electron transfer path between the reversal materials, improves the photocurrent direction reversal efficiency, makes the sensor detection more accurate, and improves the detection sensitivity. Under the optimal experimental conditions, the experiment shows that the linear range of the sensor is 10-5000000fg / mL, and the detection limit is as low as 2.1fg / mL.

[0119] 2) High selectivity: The sensor developed based on the photocurrent direction reversal strategy in the embodiment of the present invention can only generate a photocurrent signal in the opposite direction when the target is recognized; different interfering proteins have no obvious interference with this detection system.

[0120] 3) Good reproducibility: Five groups of biosensors were prepared under the same experimental conditions to detect 200 fg / mL sCD146, and the relative standard deviation was 2.7%.

[0121] 4) High stability: After the biosensor provided in the embodiment of the present invention was placed in a 4° C. refrigerator for 7, 14 and 21 days, the photocurrent detected by sCD146 still maintained about 99% of its initial photocurrent response.

[0122] Therefore, the photoelectrochemical biosensor for detecting cancer markers provided in the embodiments of the present invention integrates a photocurrent direction reversal strategy, a three-dimensional dual-support DNA walker cyclic amplification technology, and the adsorption characteristics of metal-organic framework materials to perform a direct contact photocurrent signal reversal design, thereby developing a new environmentally friendly photoelectrochemical method for low-cost, highly selective, and ultra-sensitive detection of sCD146, improving the photocurrent direction reversal efficiency, and solving the problem that conventional photoelectrochemical biosensors are unable to accurately and ultra-sensitively detect sCD146 due to interferences and sensitivity limitations, providing a new technical strategy for the analysis and research of sCD146, and promoting the early diagnosis and prognosis evaluation of cancer.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A method for preparing a signal reversal type photoelectrochemical biosensor for detecting cancer markers, comprising the steps of: Preparation of Indium Tin Oxide / Titanium Dioxide Electrode: Carbonizing the metal organic framework material to obtain titanium dioxide polyhedrons; using the titanium dioxide polyhedrons as a solute to prepare a titanium dioxide polyhedron suspension; dropping the titanium dioxide polyhedron suspension onto an indium tin oxide electrode to obtain an indium tin oxide / titanium dioxide electrode; Preparation of ITO / TiO2 / Single-chain Auxiliary Probe Electrode: Adsorbing the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide / titanium dioxide electrode to block the active sites on the titanium dioxide polyhedron, and washing to obtain an indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode; Preparation of conjugate Ab1-Fe3O4@Au-DNA: The aminated Fe3O4 nanoparticles and gold nanoparticles are shaken to form Fe3O4@Au particles by covalent bonding, thereby obtaining a Fe3O4@Au solution; the first target capture antibody and the supporting DNA probe are sequentially added to the Fe3O4@Au solution, and the solution is shaken and incubated at room temperature and subjected to magnetic separation treatment to form the first target capture antibody, the supporting DNA probe and the Fe3O4@Au particles by covalent bonding, thereby obtaining an Ab1-Fe3O4@Au-DNA conjugate solution; wherein, The first target capture antibody is represented by Ab1; Preparation of conjugate Ab2-AuNPs-DNA: First, a second target capture antibody is added to a gold nanoparticle solution with a pH of 8 to 9 for reaction, and then a supporting DNA probe and a walking DNA probe are added to form a mixed solution. The mixed solution reacts so that the second target capture antibody, the supporting DNA probe and the walking DNA probe are combined with the gold nanoparticles in the gold nanoparticle solution through covalent bonds to obtain an Ab2-AuNPs-DNA conjugate solution; wherein the second target capture antibody is represented by Ab2; Construct an intermediate probe: First, adding different concentrations of cancer markers to the Ab1-Fe3O4@Au-DNA conjugate solution for incubation and magnetic separation, then adding the Ab2-AuNPs-DNA conjugate solution for incubation and magnetic separation, and then adding restriction endonuclease Nt.BsmAl for reaction and magnetic separation to obtain a supernatant containing an intermediate probe for a targeted induced three-dimensional dual-support DNA walker cyclic signal amplification reaction; Prepare the sensor: The indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode is mixed and incubated with the intermediate probe in the supernatant, so that the intermediate probe forms a specific double strand with the single-stranded auxiliary DNA probe and the single-stranded auxiliary DNA probe is released from the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode, exposing the active sites on the titanium dioxide polyhedron in the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode, thereby obtaining an indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode with active sites; The carbon nitride quantum dots are adsorbed on the indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe electrode to form an indium tin oxide / titanium dioxide / single-chain auxiliary probe / intermediate probe / carbon nitride quantum dot composite electrode, so that the titanium dioxide and the carbon nitride quantum dots are in direct contact to prepare a direct contact signal reversal type biosensor.

2. The method for preparing a biosensor according to claim 1, wherein: The steps of preparing the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode include: annealing the single-stranded auxiliary DNA probe at 95° C. for 5 min, and after cooling to room temperature, dropping the single-stranded auxiliary DNA probe onto the surface of the indium tin oxide / titanium dioxide electrode for adsorption treatment, and washing to obtain the indium tin oxide / titanium dioxide / single-stranded auxiliary probe electrode.

3. The method for preparing a biosensor according to claim 1, characterized in that: The cancer marker is sCD146.

4. The method for preparing a biosensor according to claim 1, characterized in that: The carbon nitride quantum dots are synthesized in one step by a microwave method.

5. A biosensor for detecting cancer markers, which is mainly prepared by the preparation method of the biosensor for detecting cancer markers according to any one of claims 1 to 4.

6. Use of the signal reversal type photoelectrochemical biosensor for detecting cancer markers according to claim 5 in detecting cancer markers.

7. The use according to claim 6, characterized in that The method includes placing the biosensor for detecting cancer markers in a Tril-HCl buffer solution containing 0.1 M ascorbic acid at pH 7.4, and applying a bias voltage of -0.2 V for photoelectrochemical detection to detect the photocurrent signal of the cancer marker at different concentrations; and constructing a linear equation between the concentration of the cancer marker and the photocurrent signal based on the detection results.

8. The use according to claim 7, characterized in that The single-stranded auxiliary DNA probe is adsorbed onto the surface of the indium tin oxide / titanium dioxide electrode for 30 to 120 minutes, and the carbon nitride quantum dots are adsorbed onto the active sites exposed by the titanium dioxide polyhedron in the indium tin oxide / titanium dioxide / single-stranded auxiliary probe / intermediate probe electrode for 30 to 120 minutes.

9. The use according to claim 8, characterized in that When the concentration range of sCD146 was 10-5000000 fg / mL, the linear regression equation constructed was I =-174.34 logC sCD146 -1314.00 (R 2 =0.9977), the detection limit is 2.1 fg / mL; among them, I is a photocurrent signal detected by the biosensor for detecting cancer markers, C sCD146 represents the concentration of sCD146.

Images