Preparation method of photoelectrochemical biological aptamer sensor for detecting alpha fetoprotein based on binary doped composite material

Abstract

The invention discloses a preparation method of a photoelectrochemical biological aptamer sensor for detecting alpha fetoprotein based on a binary doped composite material, and relates to a preparation method of a photoelectrochemical biological aptamer sensor for detecting alpha fetoprotein. The technical problems that an existing electrochemical biosensor for detecting alpha fetoprotein is low in sensitivity, narrow in detection range, low in detection speed and poor in stability are solved. The preparation method comprises the following steps: 1, preparing TiO2 NRA / FTO; (2) preparing a Sn-TiO2 NRA / FTO electrode; (3) preparing a BiVO4 / Sn-TiO2 NRA / FTO (fluorine-doped tin oxide) electrode; and 4, preparing the photoelectrochemical biological aptamer sensor. The linear detection range of alpha fetoprotein detected by the sensor is 0.001 ng / mL-500 ng / mL, the detection limit is as low as 0.75 ng / mL, and excellent gt is shown; the method has long-term stability in 850 seconds, low detection limit and short detection time, and can be used in the field of alpha fetoprotein detection.

Description

Technical Field

[0001] This invention belongs to the field of sensor technology, specifically relating to an optochemical bioaptamer sensor for detecting alpha-fetoprotein and its preparation method. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the main type of primary liver cancer and one of the leading causes of cancer-related deaths worldwide. Due to its insidious early symptoms, most patients are diagnosed at an advanced stage, making early and accurate detection crucial for improving prognosis. In clinical diagnosis, alpha-fetoprotein (AFP) is the most widely used and recognized serological biomarker. Normally, AFP levels in adult serum are extremely low; however, its concentration abnormally increases when hepatocellular carcinoma develops malignant changes. Therefore, highly sensitive and specific quantitative detection of AFP is irreplaceable for early screening, diagnosis, and efficacy evaluation of HCC. Currently, while routine AFP detection methods (such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay) are widely available, they still have limitations such as cumbersome operation, long processing time, high cost, and potential radioactive contamination. Furthermore, the sensitivity and interference resistance of these methods are increasingly insufficient to meet the urgent need for ultra-early detection of trace amounts of AFP.

[0003] In recent years, photoelectrochemical (PEC) biosensors have attracted widespread attention as an emerging technology. They combine the advantages of optical excitation and electrochemical detection: using light as the excitation source, they quantify analytes by measuring changes in photocurrent caused by biorecognition events. This technology boasts significant advantages such as low background signal, high sensitivity, simple equipment, and ease of miniaturization. By combining nucleic acid aptamers with high affinity and specificity as molecular recognition elements with a PEC platform, a photoelectrochemical bioaptamer sensor is formed. This fusion technology combines the high selectivity of molecular recognition with the high sensitivity of PEC detection, providing a promising innovative solution for developing next-generation, rapid, sensitive, and low-cost AFP detection methods.

[0004] Chinese patent CN120629075A discloses a fiber optic microstructure biosensor for detecting alpha-fetoprotein (AFP) and its fabrication method. The sensor mainly consists of a microstructure sensing unit fabricated from a single-mode optical fiber and a gold nanorod composite modification layer. A long-period grating structure is fabricated on a microconical optical fiber using a CO2 laser, and then sequentially modified with MPTMS, GNRs-PEG-COOH, and AFP monoclonal antibody (mAb) to construct a highly sensitive biosensing interface. However, as a purely electrochemical sensor, it is inherently susceptible to interference from electroactive substances in serum, affecting its detection specificity.

[0005] Chinese patent CN108802145A discloses an electrochemical biosensor for detecting alpha-fetoprotein (AFP) and its preparation method. The sensor is constructed based on Au@Ag core-shell nanoparticles and an AFP aptamer. The preparation steps mainly include: synthesizing Au@Ag core-shell nanoparticles with tunable electrochemical activity, modifying the nanoparticle surface with the AFP aptamer, and functionalizing a bare glassy carbon electrode modified with aminobenzenesulfonic acid. However, its use of an antibody as the recognition element results in poor stability, high cost, and a complex electropolymerization process, leading to poor reproducibility. Summary of the Invention

[0006] The present invention aims to address the technical problems of low sensitivity, narrow detection range, slow detection speed, and poor stability of existing electrochemical biosensors for detecting alpha-fetoprotein (AFP), and provides a method for preparing a highly stable photoelectrochemical bioaptamer sensor for detecting AFP based on binary doped composite materials.

[0007] The present invention provides a method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on binary doped composite materials, comprising the following steps:

[0008] I. Preparation of TiO2NRA / FTO:

[0009] TiO2 NRA was deposited on an FTO glass slide using a hydrothermal method to obtain TiO2 NRA / FTO;

[0010] II. Preparation of Sn-TiO2 NRA / FTO electrode:

[0011] Tin tetrachloride was dissolved in water and stirred evenly to obtain a precursor solution. TiO2 NRA / FTO was immersed in the precursor solution for 1-3 hours. The FTO glass slide was then removed, rinsed with deionized water, and dried. The slide was then calcined at 480-520 °C for 0.5-1.5 hours to obtain the Sn-TiO2 NRA / FTO electrode.

[0012] III. Preparation of BiVO4 / Sn-TiO2 NRA / FTO Electrode

[0013] Bismuth nitrate pentahydrate and sodium metavanadate were dissolved separately in hot water, stirred evenly, and then mixed to obtain a mixed solution. The Sn-TiO2 NRA / FTO electrode was immersed in the mixed solution and reacted at a temperature of 160~190℃ for 8~12h. After being removed, it was rinsed with deionized water and dried, and then calcined at a temperature of 480~520℃ for 0.5~1.5h to obtain the BiVO4 / Sn-TiO2 NRA / FTO electrode.

[0014] IV. Fabrication of photoelectrochemical bioaptamer sensors using BiVO4 / Sn-TiO2 NRA / FTO electrodes.

[0015] Preferably, the specific steps for preparing TiO2 NRA / FTO by hydrothermal method in step one are as follows: Add tetrabutyl titanate dropwise to a hydrochloric acid solution with a mass percentage concentration of 18%~20% at a mass percentage of 1.5%, and stir until homogeneous to obtain a mixed solution; place a cleaned FTO glass slide tilted in the inner liner of the reaction vessel, then pour in the mixed solution, immersing the cleaned FTO glass slide in the mixed solution; then react at 170~190℃ for 1~3 hours; after the reaction is complete, rinse with deionized water and air dry to obtain TiO2 NRA / FTO.

[0016] Preferably, the molar concentration of the tin tetrachloride aqueous solution in the precursor solution in step two is 100 mM.

[0017] Preferably, the molar concentration of bismuth nitrate in the mixture described in step three is 1 mM, and the concentration of sodium vanadate is 1.5 mM.

[0018] Preferably, the method for preparing the photoelectrochemical bioaptamer sensor using the BiVO4 / Sn-TiO2 NRA / FTO electrode in step four is as follows:

[0019] A 0.1%–0.15% (w / w) chitosan solution was drop-coated onto a BiVO4 / Sn-TiO2 NRA / FTO electrode. After drying, the electrode was immersed in glutaraldehyde aqueous solution for 1–3 h. Next, an amino-modified alpha-fetoprotein aptamer was drop-coated onto the surface of the BiVO4 / Sn-TiO2 NRA / FTO electrode and incubated for 3–6 h. The electrode was then rinsed with PBS solution at pH 7.4 to obtain an Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrode. The Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrode was then incubated in bovine serum albumin solution for 0.5–1 h, followed by cleaning with PBS solution at pH 7.4 to obtain a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein, denoted as BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO.

[0020] The base sequence of the amino-modified alpha-fetoprotein aptamer of the present invention is: 5'NH2-C6-GTG-ACG-CTC-CTA-ACG-CTG-ACT-CAG-GTG-CAG-TTC-TCG-ACT-CGG-TCT-TGA-TGT-GGG-TCC-TGT-CCG-TCC-GAA-CCA-ATC-3'.

[0021] More preferably, the mass concentration of the glutaraldehyde aqueous solution is 2.5% to 3.0%.

[0022] More preferably, the mass concentration of the bovine serum albumin solution is 1% to 3%.

[0023] The method for quantitative detection of alpha-fetoprotein (AFP) using a photoelectrochemical bioaptamer sensor based on binary doped composite materials is the standard curve method, and the specific steps are as follows:

[0024] 1. Place the BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO sensor in 0.001 ng / mL to 500 ng / mL alpha-fetoprotein standard solutions for 1 h, then rinse the electrode with PBS solution at pH 7.4 to obtain the AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrode;

[0025] 2. On an electrochemical workstation, equipped with a 500W xenon lamp source and a 400nm cutoff filter, a three-electrode system was used with AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The electrolyte was a PBS buffer solution with pH=7.4. It tests were performed under an applied bias voltage of 0.3V to obtain the photoelectric signals corresponding to different concentrations of alpha-fetoprotein. A standard curve was plotted with the logarithm of the alpha-fetoprotein concentration as the abscissa and the corresponding photoelectric signal as the ordinate.

[0026] 3. The BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO sensor was placed in the alpha-fetoprotein (AFP) solution to be tested and kept for 1 hour. Then, the electrode was rinsed with PBS solution at pH 7.4 to obtain the test electrode. On an electrochemical workstation equipped with a 500W xenon lamp source and a 400nm cutoff filter, a three-electrode system was used, with the test electrode as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The electrolyte was PBS buffer solution at pH 7.4. It was tested under an applied bias voltage of 0.3V to obtain the photoelectric signal. The AFP concentration corresponding to the photoelectric signal was then determined from the standard curve, thus achieving the purpose of detecting AFP.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The alpha-fetoprotein photoelectrochemical bioaptamer sensor of this invention employs a simple solvent impregnation method to dope Sn onto TiO2 NRA, followed by a hydrothermal method to load BiVO4 onto Sn-TiO2 NRA, resulting in a BiVO4 / Sn-TiO2 NRA / FTO binary doped composite material. Sn doping effectively modulates the band structure of TiO2, narrowing its bandgap, and simultaneously forms a highly efficient step-type (Type-II) heterojunction with the narrow-bandgap semiconductor BiVO4. This heterojunction structure establishes an ideal built-in electric field at the interface, greatly promoting the directional separation and rapid migration of photogenerated electron-hole pairs and significantly suppressing carrier recombination. Furthermore, the oxygen vacancies introduced by Sn doping and the tight coupling between the modified TiO2 and BiVO4 collectively broaden the absorption range of the composite material in the visible and even near-infrared regions, thereby comprehensively improving the light capture efficiency and photoelectric conversion performance of the photoactive material. Based on this high-performance photoelectric material, the aptamer sensor provides a foundation for highly sensitive detection due to its enhanced photoelectric signal. When the AFP aptamer on the sensor surface specifically captures the target alpha-fetoprotein, the resulting biocomplex effectively hinders interfacial electron transport and causes a significant decrease in photocurrent. This change is quantitatively related to the AFP concentration within a certain range. The superior photoelectric synergistic effect of the BiVO4 / Sn-TiO2 NRA heterojunction not only provides the sensor with a stable and strong initial photocurrent but also amplifies the signal changes caused by biometric events, thereby achieving ultrasensitive detection of AFP with a lower detection limit and a wider linear detection range.

[0029] The sensor prepared in this invention exhibits excellent stability with almost no signal attenuation during a continuous 850-second photocurrent response test. This is mainly attributed to the robust heterojunction structure formed by Sn-TiO2 and BiVO4, and the oxygen vacancies introduced by Sn doping effectively suppress photocorrosion, ensuring the reliability of the sensor during long-term or repeated detection.

[0030] The aptamer recognition element introduced in this invention reduces interference from other antibiotics, improves the specific recognition capability of the photoelectrochemical sensor, and achieves specific and sensitive detection of alpha-fetoprotein (AFP). Furthermore, the detection of AFP by the photoelectrochemical bio-aptamer sensor is based on the photocurrent change generated by the aptamer capturing AFP oxidized by semiconductor holes. The linear detection range is 0.001 ng / mL to 500 ng / mL, with a detection limit as low as 0.75 ng / mL, exhibiting advantages such as low detection limit, short detection time, high stability, and simple operation. This invention modifies the surface of a BiVO4 / Sn-TiO2 NRA electrode with an amino-modified AFP aptamer to enhance the sensor's specific recognition capability. The photoelectrochemical bio-aptamer sensor constructed in this invention has high sensitivity, good selectivity, and a low detection limit, which is beneficial for achieving accurate detection of AFP and has significant implications for the in-depth application of sensors in clinical medical diagnosis. Attached Figure Description

[0031] Figure 1 Here is a SEM image of the TiO2 NRA prepared in Example 1;

[0032] Figure 2 These are SEM images of Sn-TiO2 NRA prepared in Example 1;

[0033] Figure 3 Here is a SEM image of BiVO4 / Sn-TiO2 NRA from Example 1;

[0034] Figure 4 This is an elemental mapping image of the BiVO4 / Sn-TiO2 NRA prepared in Example 1;

[0035] Figure 5 These are XPS energy dispersive spectra of the electrodes prepared in Example 1. A is the full spectrum, B is the high-resolution spectrum of Ti, C is the high-resolution spectrum of O, D is the high-resolution spectrum of Sn, E is the high-resolution spectrum of Bi, and F is the high-resolution spectrum of V.

[0036] Figure 6 The images show the AC impedance spectra of the electrodes prepared in Example 1, where a is the TiO2 NRA electrode, b is the Sn-TiO2 NRA electrode, c is the BiVO4 / Sn-TiO2 NRA electrode, d is the Aptamer / BiVO4 / Sn-TiO2 NRA electrode, e is the BSA / Aptamer / BiVO4 / Sn-TiO2 NRA electrode, and f is the AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA electrode.

[0037] Figure 7The graphs are time-current test curves of the electrodes prepared in Example 1, where a is the TiO2 NRA electrode, b is the Sn-TiO2 NRA electrode, c is the BiVO4 / Sn-TiO2 NRA electrode, d is the Aptamer / BiVO4 / Sn-TiO2 NRA electrode, e is the BSA / Aptamer / BiVO4 / Sn-TiO2 NRA electrode, and f is the AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA electrode.

[0038] Figure 8 This is a graph showing the stability test results of the electrode prepared in Example 1;

[0039] Figure 9 The current response curves of the photoelectrochemical bioaptamer sensor prepared in Example 1 for detecting alpha-fetoprotein at different concentrations are shown.

[0040] Figure 10 Linear curves of different concentrations of alpha-fetoprotein (AFP) detected by the photoelectrochemical bioaptamer sensor prepared in Example 1.

[0041] Figure 11 The image shows the anti-interference test pattern of the photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein prepared in Example 1 against different interfering substances. Prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), immunoglobulin G (IgG) and ascorbic acid (AA) were selected as interfering antigens. Detailed Implementation

[0042] The beneficial effects of the present invention will be verified using the following examples.

[0043] Example 1: The preparation method of the photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on binary doped composite materials in this example is carried out according to the following steps:

[0044] I. Preparation of TiO2NRA / FTO:

[0045] An FTO glass slide with a length × width of 2cm × 1cm was placed in a mixed solution of acetone, ethanol and deionized water in a volume ratio of 1:1:1 and ultrasonically cleaned for 15 minutes, then dried for later use.

[0046] An equal volume of ultrapure water was added to a 36% hydrochloric acid solution, followed by the dropwise addition of tetrabutyl titanate. After stirring until homogeneous, a mixed solution was obtained; the mass concentration of tetrabutyl titanate in the mixed solution was 1.5%.

[0047] The cleaned FTO glass slide was placed at an angle into the inner liner of the reactor, and then the above mixed solution was poured in. The reactor was then placed in a drying oven and kept at 180°C for 2 hours to carry out the reaction. After the reaction was completed, the mixture was dried at room temperature to obtain TiO2 NRA, which is represented as TiO2 NRA / FTO.

[0048] II. Preparation of Sn-TiO2 NRA / FTO electrode:

[0049] SnCl4·5H2O was dissolved in ultrapure water and stirred for 30 min to obtain a precursor solution; the molar concentration of tin tetrachloride in the precursor solution was 100 mM; TiO2 NRA / FTO was immersed in the precursor solution for 2 h, and then the FTO glass slide was taken out, rinsed with deionized water and dried, and calcined at 500 ℃ for 1 h to obtain the Sn-TiO2 NRA / FTO electrode;

[0050] III. Preparation of BiVO4 / Sn-TiO2 NRA / FTO electrode:

[0051] Bismuth nitrate pentahydrate (Bi(NO3)3•5H2O) and sodium metavanadate (NaVO3) were dissolved separately in hot water, stirred evenly, and then mixed to obtain a mixed solution. The molar concentration of bismuth nitrate in the mixed solution was 1 mM, and the molar concentration of sodium metavanadate was 1.5 mM. The Sn-TiO2 NRA / FTO electrode was immersed in the mixed solution and reacted at 180℃ for 10 h. After being removed, it was rinsed with deionized water and dried. Then it was calcined at 500 ℃ for 1 h to obtain the BiVO4 / Sn-TiO2 NRA / FTO electrode.

[0052] IV. Fabrication of photoelectrochemical-biological aptamer sensors using BiVO4 / Sn-TiO2 NRA / FTO electrodes:

[0053] 10 μL of a 0.1% (w / w) chitosan (CS) solution was drop-coated onto a BiVO4 / Sn-TiO2 NRA / FTO electrode. After drying, the electrode was immersed in 5 ml of a 2.5% (w / w) glutaraldehyde solution for 1 h. Next, an amino-modified alpha-fetoprotein aptamer was drop-coated onto the surface of the BiVO4 / Sn-TiO2 NRA / FTO electrode and incubated for 3 h. The electrode was then rinsed with PBS solution at pH 7.4 to obtain Aptamer / BiVO4 / Sn-TiO2 NRA / FTO. The Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrode was incubated in 5 ml of a 3% (w / w) bovine serum albumin (BSA) solution for 1 h, and then washed with PBS solution at pH 7.4 to obtain a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material, denoted as BSA / Aptamer / BiVO4 / Sn-TiO2. NRA / FTO. The amino-modified alpha-fetoprotein adapted to the following base sequence is: 5'NH2-C6-GTG-ACG-CTC-CTA-ACG-CTG-ACT-CAG-GTG-CAG-TTC-TCG-ACT-CGG-TCT-TGA-TGT-GGG-TCC-TGT-CCG-TCC-GAA-CCA-ATC-3'.

[0054] The scanning electron microscope image of TiO2 NRA / FTO obtained in step one of Example 1 is as follows: Figure 1 As shown, where Figure 1 A is a SEM image of the TiO2 NRA cross-section. Figure 1 B is a SEM image of TiO2 NRA top, from Figure 1 As can be seen, TiO2 NRA is tightly bonded to FTO, and the rod-shaped TiO2 NRA is neatly and orderly covered on the FTO surface, which provides sufficient load space for subsequent material loading and plays a supporting role.

[0055] The SEM image of Sn-TiO2 NRA / FTO prepared in step two of Example 1 is shown below. Figure 2 As shown, where Figure 2 A is a SEM image of the Sn-TiO2 NRA cross-section. Figure 2 B is the SEM image at the top of the Sn-TiO2 NRA, from Figure 2 It can be seen that the structure of Sn-TiO2 NRA does not change significantly, but the surface of Sn-TiO2 NRA becomes rougher and exhibits more granular features.

[0056] The SEM image of BiVO4 / Sn-TiO2 NRA / FTO obtained in step three of Example 1 is shown below. Figure 3 As shown, from Figure 3 As can be seen, BiVO4 particles are coated on top of Sn-TiO2 NRA. To further verify this, elemental mapping analysis was performed on the BiVO4 / Sn-TiO2 NRA nanocomposite material, and the resulting elemental mapping images are shown below. Figure 4 As shown, from Figure 4 It can be seen that the five elements Ti, O, Sn, Bi and V are evenly distributed.

[0057] X-ray photoelectron spectroscopy (XPS) was performed on the BiVO4 / Sn-TiO2 NRA composite material prepared in Example 1. Figure 5 As shown, A is the full NRA spectrum of BiVO4 / Sn-TiO2, indicating that the material contains five elements: Ti, O, Sn, Bi, and V. Calibration was performed using the standard C 1s peak at 284.8 eV, and specific analysis of each element in the BiVO4 / Sn-TiO2 NRA was conducted. B is the high-resolution spectrum of Ti, showing three characteristic peaks at 458.7 eV, 463.9 eV, and 466.3 eV, corresponding to Ti... 4 Ti 2p3 / 2 in the ⁺ state, Ti 3 ⁺-state 2p1 / 2 spin orbitals and Ti 4 The high-resolution spectrum of Ti in the ⁺ state shows two characteristic peaks at 529.9 eV and 531.7 eV, corresponding to Ti-O and VO in the O²⁻ state, the -OH group, and the vacant oxygen (Ov). The high-resolution spectrum of Sn in the D state shows two characteristic peaks at 486.8 eV and 495.2 eV, corresponding to the ⁺ state and VO, the -OH group, and the vacant oxygen (Ov), respectively. 4 The Sn 3d5 / 2 and Sn 3d3 / 2 orbitals in the ⁺ state. E is the high-resolution spectrum of Bi, with two characteristic peaks at 159.2 eV and 164.5 eV, corresponding to Bi, respectively. 3+ Bi 4f7 / 2 and Bi 4f 5 / 2 Spin orbitals. The high-resolution spectrum of F for V shows two characteristic peaks at 516.8 eV and 524.2 eV, corresponding to V and V respectively. 5+ The material contains V 2p3 / 2 and V 2p1 / 2 spin orbitals in the Ti³⁺ state. XPS results confirm the successful preparation of BiVO₄ / Sn-TiO₂ NRA. Notably, a distinct characteristic peak was observed at 463.9 eV, which can be attributed to the Ti 2p1 / 2 spin orbitals in the Ti³⁺ state. The presence of Ti³⁺ indicates the presence of oxygen vacancies (Ov) in the material, as the formation of oxygen vacancies leads to local charge imbalance, thereby causing some Ti… 4 ⁺ is reduced to Ti³⁺ to maintain electroneutrality. Ti³⁺ / Ti 4The presence of mixed valence states serves two purposes: firstly, it acts as an additional electron donor, further enhancing the n-type conductivity and carrier concentration of the material; secondly, this defect structure facilitates the formation of localized states, promoting visible light absorption and suppressing the recombination of photogenerated electron-hole pairs. This result corroborates the oxygen vacancy signal at 531.7 eV in the O 1s spectrum, jointly confirming the successful introduction of abundant oxygen vacancies and defect structures during Sn doping and composite material preparation. This provides a crucial microstructural explanation for the significantly enhanced photoelectric activity and long-term stability of the BiVO4 / Sn-TiO2 NRA heterojunction.

[0058] The electrode prepared in Example 1 was subjected to AC impedance testing and current-time testing, and the obtained AC impedance curve is shown below. Figure 6 As shown, the time-current curve is as follows: Figure 7 As shown, where a is TiO2 NRA, b is Sn-TiO2 NRA, c is BiVO4 / Sn-TiO2 NRA, d is Aptamer / BiVO4 / Sn-TiO2 NRA, e is BSA / Aptamer / BiVO4 / Sn-TiO2 NRA, and f is AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA. Figure 5 As can be seen, the semicircle diameter of the curve reflects the magnitude of the resistance encountered by electrons. The smaller the diameter, the less resistance electrons encounter during transfer, and the greater the electron transfer rate. Curve c has the smallest semicircle diameter, corresponding to a larger current, indicating that the photoelectric performance of BiVO4 / Sn-TiO2 NRA is stronger than that of TiO2 NRA (curve a) and Sn-TiO2 NRA (curve b). However, after adding alpha-fetoprotein aptamer and BSA, the semicircle diameters of curves d and e gradually increase, while the corresponding currents gradually decrease. This is because the aptamer and BSA are insulating biomolecules that hinder electron movement. This series of curve changes demonstrates the successful fabrication of a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on binary doped composite materials.

[0059] Sensor stability refers to the ability of a sensor to maintain a relatively stable output signal over a certain period of time. The photoelectrochemical bioaptamer sensor BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO based on a binary doped composite material for detecting alpha-fetoprotein, prepared in Example 1, underwent 21 cycles of switching excitation within 850 s. Figure 8 As shown, the photocurrent intensity of the aptamer sensor remains relatively stable without significant change, indicating that the aptamer sensor has excellent stability.

[0060] To quantitatively detect the content of alpha-fetoprotein (AFP), the photoelectrochemical bioaptamer sensor based on binary doped composite material prepared in Example 1 was used to detect AFP. The standard curve method was employed, and the specific operating steps are as follows:

[0061] 1. Prepare standard solutions of alpha-fetoprotein (AFP) with concentrations of 0.001 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 500 ng / mL. Immerse the photoelectrochemical bioaptamer sensor for detecting AFP based on binary doped composite material prepared in Example 1 into the AFP standard solutions of different concentrations and incubate for 1 h. After the reaction, rinse thoroughly with PBS buffer solution at pH 7.4 and air dry. The obtained electrode is labeled AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO.

[0062] II. On an electrochemical workstation equipped with a 500W xenon lamp source and a 400nm cutoff filter, a three-electrode system was used: AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The electrolyte was 0.1M PBS buffer solution at pH 7.4. It measurements were performed under an applied bias voltage of 0.3V to obtain the photoelectric signals corresponding to different concentrations of alpha-fetoprotein (AFP). A standard curve was plotted with the logarithm of AFP concentration as the x-axis and the corresponding photoelectric signals as the y-axis. The photocurrents corresponding to different concentrations of AFP solutions were measured as follows: Figure 9 The concentrations of AFP, from a to l, are as follows: 0.001 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 500 ng / mL; Figure 8 As can be seen, a standard curve was plotted with ∆I (ΔI=I0-I, where I0 and I represent the photocurrent values ​​of the aptamer sensor before and after AFP incubation, respectively) as the ordinate and the logarithm of the AFP concentration as the abscissa. The resulting standard curve is shown below. Figure 10 Show. From Figure 10 It can be seen that within the linear range of 0.001 ng / mL to 500 ng / mL, ΔI is linearly related to the logarithm of AFP concentration. The obtained linear equation is ΔI(μA) = 7.70538lg(C AFPThe correlation coefficient was 0.997, and the detection limit was 0.75 ng / mL (3S / N). This indicates that the sensor has a wide detection range and a low detection limit for detecting alpha-fetoprotein.

[0063] 3. Place the prepared BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO into the target analyte solution of the actual sample. Based on the detected photoelectric response signal, calculate the ΔI value using the method in step 2. Determine the concentration of AFP in the target analyte based on the standard curve to complete the detection of alpha-fetoprotein.

[0064] To investigate the specificity and selectivity of the photoelectrochemical aptamer sensor for detecting alpha-fetoprotein (AFP), five independent working electrodes were prepared according to the method in Example 1, and the same concentration of AFP was detected under identical experimental conditions. The specific operation process is as follows:

[0065] Five independent photoelectrochemical bioaptamer sensors based on binary doped composite materials for detecting alpha-fetoprotein (AFP) were incubated in 100 nM standard AFP solution for 1 h, then cleaned with PBS solution at pH 7.4 and dried. The resulting electrodes were designated AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrodes. An electrochemical workstation equipped with a 500 W xenon lamp and a 400 nm cutoff filter was used. A three-electrode system was established, with AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The electrolyte was 0.1 M PBS buffer solution at pH 7.4. It tests were performed under an applied bias voltage of 0.3 V. The reproducibility of the sensor was evaluated by calculating the relative standard deviation (RSD) of the five independent electrodes using the photocurrent response signal. The calculated RSD was 1.80%. This demonstrates that the photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on binary doped composite materials prepared in Example 1 has good reproducibility.

[0066] To verify the specificity and selectivity of the photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein (AFP) based on binary doped composite materials prepared in Example 1, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), immunoglobulin G (IgG), and ascorbic acid (AA) were selected as interfering agents (the concentration of AFP and the interfering agents was 100 ng / mL). It was measured on an electrochemical workstation using a 500W xenon lamp light source equipped with a 400nm cutoff filter and an applied bias voltage of 0.3V. The test results are as follows: Figure 11 As shown, from Figure 11As can be seen from the results, the photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein (AFP) prepared in Example 1 only shows a significant photocurrent response to samples containing AFP, while the response signal to other mixed interfering substances is relatively weak. These results confirm that the photoelectrochemical bioaptamer sensor for detecting AFP based on binary doped composite materials has high selectivity for AFP.

Claims

1. A method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on binary doped composite materials, characterized in that, The method includes the following steps: I. Preparation of TiO2NRA / FTO: TiO2 NRA was deposited on an FTO glass slide using a hydrothermal method to obtain TiO2 NRA / FTO; II. Preparation of Sn-TiO2 NRA / FTO electrode: Tin tetrachloride was dissolved in water and stirred evenly to obtain a precursor solution. TiO2 NRA / FTO was immersed in the precursor solution for 1-3 hours. The FTO glass slide was then removed, rinsed with deionized water, and dried. The slide was then calcined at 480-520 °C for 0.5-1.5 hours to obtain the Sn-TiO2 NRA / FTO electrode. III. Preparation of BiVO4 / Sn-TiO2 NRA / FTO Electrode Bismuth nitrate pentahydrate and sodium metavanadate were dissolved separately in hot water, stirred evenly, and then mixed to obtain a mixed solution. The Sn-TiO2 NRA / FTO electrode was immersed in the mixed solution and reacted at a temperature of 160~190℃ for 8~12h. After being removed, it was rinsed with deionized water and dried, and then calcined at a temperature of 480~520℃ for 0.5~1.5h to obtain the BiVO4 / Sn-TiO2 NRA / FTO electrode. IV. Fabrication of photoelectrochemical bioaptamer sensors using BiVO4 / Sn-TiO2 NRA / FTO electrodes.

2. The method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material according to claim 1, characterized in that, The specific steps for preparing TiO2 NRA / FTO by the hydrothermal method in step one are as follows: Add tetrabutyl titanate dropwise to a hydrochloric acid solution with a mass percentage concentration of 18%~20% at a mass percentage of 1.5%, and stir until homogeneous to obtain a mixed solution; place a cleaned FTO glass slide tilted in the inner liner of the reaction vessel, then pour in the mixed solution to immerse the cleaned FTO glass slide in the mixed solution; then react at 170~190℃ for 1~3 hours; after the reaction is complete, rinse with deionized water and air dry to obtain TiO2 NRA / FTO.

3. The method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material according to claim 1 or 2, characterized in that, The molar concentration of the tin tetrachloride aqueous solution in the precursor solution in step two is 100 mM.

4. A method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material according to claim 1 or 2, characterized in that, In step three, the molar concentration of bismuth nitrate in the mixture is 1 mM, and the concentration of sodium vanadate is 1.5 mM.

5. A method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material according to claim 1 or 2, characterized in that, The method for fabricating the photoelectrochemical bioaptamer sensor using the BiVO4 / Sn-TiO2 NRA / FTO electrode in step four is as follows: A 0.1%–0.15% (w / w) chitosan solution was drop-coated onto a BiVO4 / Sn-TiO2 NRA / FTO electrode. After drying, the electrode was immersed in glutaraldehyde aqueous solution for 1–3 h. Next, an amino-modified alpha-fetoprotein aptamer was drop-coated onto the surface of the BiVO4 / Sn-TiO2 NRA / FTO electrode and incubated for 3–6 h. The electrode was then rinsed with PBS solution at pH 7.4 to obtain an Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrode. The Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrode was then incubated in bovine serum albumin solution for 0.5–1 h, followed by cleaning with PBS solution at pH 7.4 to obtain a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein, denoted as BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO.

6. The method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material according to claim 5, characterized in that, The base sequence of the amino-modified alpha-fetoprotein aptamer is: 5'NH2-C6-GTG-ACG-CTC-CTA-ACG-CTG-ACT-CAG-GTG-CAG-TTC-TCG-ACT-CGG-TCT-TGA-TGT-GGG-TCC-TGT-CCG-TCC-GAA-CCA-ATC-3'.

7. A method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material according to claim 5 or 6, characterized in that, The mass concentration of the glutaraldehyde aqueous solution is 2.5%~3.0%.

8. A method for preparing a photoelectrochemical bioaptamer sensor for detecting alpha-fetoprotein based on a binary doped composite material according to claim 5 or 6, characterized in that, The mass concentration of the bovine serum albumin solution is 1% to 3%.

9. The method for quantitatively detecting alpha-fetoprotein (AFP) using the photoelectrochemical bioaptamer sensor based on binary doped composite materials as described in claim 1, characterized in that, This method is the standard curve method.

10. The method for quantitatively detecting alpha-fetoprotein (AFP) using a photoelectrochemical bioaptamer sensor based on binary doped composite materials according to claim 9, characterized in that, The specific operation of the standard curve method is as follows:

1. Place the BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO sensor in 0.001 ng / mL to 500 ng / mL alpha-fetoprotein standard solutions for 1 h, then rinse the electrode with PBS solution at pH 7.4 to obtain the AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO electrode; 2. On an electrochemical workstation, equipped with a 500W xenon lamp source and a 400nm cutoff filter, a three-electrode system was used with AFP / BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The electrolyte was a PBS buffer solution with pH=7.

4. It tests were performed under an applied bias voltage of 0.3V to obtain the photoelectric signals corresponding to different concentrations of alpha-fetoprotein. A standard curve was plotted with the logarithm of the alpha-fetoprotein concentration as the abscissa and the corresponding photoelectric signal as the ordinate.

3. The BSA / Aptamer / BiVO4 / Sn-TiO2 NRA / FTO sensor was placed in the alpha-fetoprotein (AFP) solution to be tested and kept for 1 hour. Then, the electrode was rinsed with PBS solution at pH 7.4 to obtain the test electrode. On an electrochemical workstation equipped with a 500W xenon lamp source and a 400nm cutoff filter, a three-electrode system was used, with the test electrode as the working electrode, a platinum sheet electrode as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. The electrolyte was PBS buffer solution at pH 7.

4. It was tested under an applied bias voltage of 0.3V to obtain the photoelectric signal. The AFP concentration corresponding to the photoelectric signal was then determined from the standard curve, thus achieving the purpose of detecting AFP.

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