Photoelectrochemical aptamer sensor for detecting alpha fetoprotein and preparation method thereof

Abstract

The invention provides a photoelectrochemical aptamer sensor for detecting alpha fetoprotein and a preparation method of the photoelectrochemical aptamer sensor, and relates to the technical field of sensors. According to the invention, a solvothermal method and a chemical reduction method are combined to gradually load BiPO4 nanosheets and Ag NPs onto TiO2NRA, so that the Ag / BiPO4 / TiO2NRA ternary composite material is obtained. Through the synergistic effect of a Type II type n-n heterojunction formed by TiO2NRA and BiPO4 and the surface plasma resonance (SPR) effect of Ag, the utilization rate of a photoelectric material to visible light is increased, and the photoelectric property of the photoelectric material is improved, so that the sensitivity of a photoelectrochemical biosensor is improved, and the detection range of alpha fetoprotein to be detected is expanded. In addition, a Type II type n-n heterojunction constructed by BiPO4 and TiO2NRA improves the transfer rate of electrons, the SPR effect of metal Ag is combined with the Type II type n-n heterojunction structure, a visible light source is utilized to a great extent, the photoelectric property of the photoelectrochemical aptamer sensor is improved, the sensitivity of the alpha fetoprotein photoelectrochemical aptamer sensor is further improved, and the detection limit is lower.

Description

Technical Field

[0001] The invention provides a photoelectrochemical aptamer sensor for detecting alpha-fetoprotein and a preparation method thereof, belonging to the technical field of sensors. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and one of the leading causes of cancer-related deaths worldwide. Early and accurate detection of HCC can effectively treat cancer and improve cancer survival rates. Alpha-fetoprotein (AFP) is the most widely used serum marker for clinical diagnosis of HCC. AFP is a glycoprotein synthesized by the liver in the early fetus, and the synthesis of AFP is quickly inhibited after birth. If malignant lesions occur in liver cells or gonadal embryonic tissues in adulthood, the AFP content in the blood will increase significantly. Therefore, the development of a simple and sensitive method for detecting the AFP concentration in the blood is of great clinical significance. At present, the commonly used methods for detecting AFP concentration are: electrochemical detection method, fluorescent immunoassay method, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunoelectrophoresis, etc. However, these methods have a series of problems such as high analysis cost, complex operation, and slow detection speed, and have not been widely used in the detection of clinical samples.

[0003] Therefore, it is urgent to develop a simple, efficient and low-cost method for the detection of AFP. Photoelectrochemical (PEC) aptamer biosensor is a newly developed biomolecular detection technology that combines the advantages of optical and electrochemical methods, using the photoelectric properties of nanomaterials, modulating light as the input signal of the detection system, and detecting the output photocurrent signal, which reflects the characteristics of high sensitivity, low detection limit, fast detection speed and high signal-to-noise ratio of biological detection.

[0004] The invention with publication number CN119178795A discloses a biosensor reagent combination including a substrate material and a platinum-coated gold nanorod probe, wherein the substrate material includes NH 2 -MXene@AuNPs and biosynthesized quantum dots. Reagent combinations for biosensors, biosensors and their applications in cancer marker detection, used to detect target proteins, can effectively improve detection sensitivity, accuracy and speed, effectively reduce detection difficulty, and are of great significance to the in-depth application of biosensors in medical clinical diagnosis.

[0005] The invention of publication number CN118604080A is based on CuO-CuInS 2 A signal "on-off" PEC aptamer sensor for sensitive detection of streptomycin was constructed using the composite material as the substrate and ferrocene (Fc) as the signal amplifier. 2It provides a stable initial photocurrent for the photocathode active material. SH-Apt is fixed to ITO through Cu-S bonds, and ferrocene-labeled DNA forms double-stranded DNA with SH-Apt through base complementary pairing, increasing the photocurrent and expanding the sensor's detection range for streptomycin. This signal "on-off" type PEC aptamer sensor can achieve sensitive detection of streptomycin and provides new ideas for the rational design of efficient PEC aptamer sensors to detect other analytes. Summary of the invention

[0006] In view of this, the present invention aims to solve the technical problems of low sensitivity, narrow detection range, slow detection speed, etc. in the existing detection of alpha-fetoprotein, and provides a method for preparing and using a photoelectrochemical aptamer sensor constructed for detecting alpha-fetoprotein.

[0007] The preparation method of the photoelectrochemical aptamer sensor for detecting alpha-fetoprotein of the present invention comprises the following steps:

[0008] (1)TiO 2 Preparation of NRA / FTO

[0009] TiO was prepared by hydrothermal method 2 NRA was deposited on FTO glass to obtain TiO 2 NRA / FTO;

[0010] (2)BiPO 4 / TiO 2 Preparation of NRA / FTO electrodes

[0011] Dissolve bismuth nitrate in a mixture of glycerol and water, stir well, and then add NaH 2 PO 4 Continue stirring until the solution is completely dissolved and becomes milky white to obtain a precursor solution;

[0012] TiO 2 The NRA / FTO was immersed in the precursor solution and reacted at a temperature of 150-180° C. for 15-24 hours. After the reaction was completed, the FTO glass sheet was taken out and rinsed with deionized water to obtain BiPO 4 / TiO 2 NRA / FTO electrode;

[0013] (3) Ag / BiPO 4 / TiO 2 Preparation of NRA / FTO electrodes

[0014] BiPO 4 / TiO 2The NRA / FTO electrode was immersed in an ascorbic acid aqueous solution for 10 to 14 hours. After being taken out, it was rinsed with deionized water and dried, and then placed in an AgNO 3 Soak in the aqueous solution for 10 to 14 hours; after the reaction is completed, take out the electrode, wash it with deionized water and dry it to obtain Ag / BiPO 4 / TiO 2 NRA / FTO electrode;

[0015] (4) Preparation of photoelectrochemical aptamer sensors.

[0016] Preferably, step (1) is to drop butyl titanate into a hydrochloric acid solution, stir evenly to obtain a mixed solution; place a cleaned FTO glass sheet in an inner container of a reaction kettle at an angle, pour the mixed solution into the inner container, and immerse the cleaned FTO glass sheet in the mixed solution; then react at 150-160° C. for 5-7 hours; after the reaction is completed, take out the FTO glass sheet and calcine it at a temperature of 450-480° C. for 1.5-2 hours to deposit TiO on the FTO glass sheet. 2 NRA, using TiO 2 NRA / FTO said.

[0017] Further preferably, the mass concentration of hydrochloric acid in step (1) is 36%; and the mass concentration of butyl titanate in the mixed solution is 1.5%.

[0018] Preferably, the mass concentration of glycerol in the mixed solution of glycerol and water in step (2) is 5-8%; the molar concentration of bismuth nitrate in the precursor solution is 2mM; the molar concentration of NaH 2 PO 4 The molar concentration is 1.5 mM.

[0019] Preferably, the molar concentration of ascorbic acid in the ascorbic acid aqueous solution in step (3) is 0.1 M; the AgNO 3 The concentration of the aqueous solution is 0.05~0.3M.

[0020] Preferably, step (4) is to drop-coat the chitosan solution onto the Ag / BiPO 4 / TiO 2 After drying, the NRA / FTO electrode was immersed in a glutaraldehyde aqueous solution for 1 to 3 hours. Then, the amino-modified alpha-fetoprotein aptamer was drop-coated on the Ag / BiPO 4 / TiO 2 The NRA / FTO electrode surface was incubated for 3-6 hours; then the electrode was rinsed with a PBS solution with pH = 7.4 to obtain Aptamer / Ag / BiPO 4 / TiO 2NRA / FTO electrode; then Aptamer / Ag / BiPO 4 / TiO 2 The NRA / FTO electrode was placed in a bovine serum albumin solution and incubated for 0.5 to 1 h, and then cleaned with a PBS solution at pH 7.4 to obtain a photoelectrochemical aptamer sensor for detecting alpha-fetoprotein, which was denoted as BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA / FTO.

[0021] More preferably, the mass concentration of the chitosan solution in step (4) is 0.1% to 0.15%. The base sequence of the amino-modified alpha-fetoprotein aptamer of the present invention is: 5'NH 2 -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'.

[0022] More preferably, the mass concentration of the glutaraldehyde aqueous solution in step (4) is 2.5% to 3.0%.

[0023] More preferably, the mass concentration of the bovine serum albumin solution in step (4) is 1% to 3%.

[0024] The present invention also provides a method for quantitatively detecting alpha-fetoprotein by a photoelectrochemical aptamer sensor for detecting alpha-fetoprotein, which is a standard curve method, specifically performed in the following steps:

[0025] 1. BSA / Aptamer / Ag / BiPO 4 / TiO 2 The NRA / FTO sensor was placed in a standard solution of 0.001 ng / mL to 500 ng / mL of alpha-fetoprotein for 1 h, and then the electrode was rinsed with a PBS solution at pH = 7.4 to obtain AFP / BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA / FTO electrode;

[0026] 2. On the electrochemical workstation, equipped with a 500W xenon lamp light source and a 400nm cutoff filter, use AFP / BSA / Aptamer / Ag / BiPO 4 / TiO 2NRA / FTO was used as the working electrode, platinum electrode as the counter electrode, saturated calomel electrode (SCE) as the reference electrode in a three-electrode system, and the electrolyte was a PBS buffer solution with a pH of 7.4. The It test was performed under an external bias of 0.3 V to obtain the photoelectric signals corresponding to different concentrations of alpha-fetoprotein. A standard curve was drawn with the logarithm of the alpha-fetoprotein concentration as the horizontal axis and the corresponding photoelectric signal as the vertical axis.

[0027] 3. BSA / Aptamer / Ag / BiPO 4 / TiO 2 The NRA / FTO sensor was placed in the alpha-fetoprotein solution to be tested for 1 hour, and then the electrode was rinsed with a PBS solution with a pH of 7.4 to obtain a test electrode; on an electrochemical workstation, a 500W xenon lamp light source and a 400nm cutoff filter were equipped, a three-electrode system with the electrode to be tested as the working electrode, a platinum electrode as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode, and the electrolyte was a PBS buffer solution with a pH of 7.4. An It test was performed under an external bias of 0.3V to obtain a photoelectric signal; then the alpha-fetoprotein concentration corresponding to the photoelectric signal was found on the standard curve, thereby achieving the purpose of detecting alpha-fetoprotein.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The alpha-fetoprotein photoelectrochemical aptamer sensor of the present invention is a simple solvent thermal method combined with a chemical reduction method to convert BiPO 4 Nanosheets and Ag NPs were gradually loaded onto TiO 2 On NRA, Ag / BiPO 4 / TiO 2 NRA ternary composite materials. Because TiO 2 、BiPO 4 The three are closely combined with Ag, TiO 2 NRA and BiPO 4 The formed Type II nn heterojunction and the surface plasmon resonance (SPR) effect of Ag work synergistically to improve the utilization rate of visible light by optoelectronic materials and improve their optoelectronic properties, thereby increasing the sensitivity of photoelectrochemical biosensors and expanding the detection range of alpha-fetoprotein to be measured. 4 With TiO 2 The Type Ⅱnn heterojunction constructed by NRA increases the electron transfer rate. The SPR effect of metal Ag combined with the Type Ⅱnn heterojunction structure makes full use of the visible light source, improves the photoelectric performance of the photoelectrochemical aptamer sensor, and further improves the sensitivity of the alpha-fetoprotein photoelectrochemical aptamer sensor, making the detection limit lower.

[0030] The aptamer recognition element introduced in the present invention weakens the interference of other antibiotics, improves the specific recognition ability of the photoelectrochemical sensor, and realizes the specific and sensitive detection of alpha-fetoprotein. Moreover, the detection of alpha-fetoprotein by the photoelectrochemical aptamer sensor is based on the change in photocurrent generated by the aptamer capturing the alpha-fetoprotein oxidized by semiconductor holes. The linear detection range is 0.001ng / mL to 500ng / mL, and the detection limit is as low as 0.53ng / mL. It has the advantages of low detection limit, short detection time, simple operation, etc.

[0031] The present invention modifies the amino-modified alpha-fetoprotein aptamer to Ag / BiPO 4 / TiO 2 The NRA electrode surface is used to improve the specific recognition ability of the sensor. The photoelectrochemical aptamer sensor constructed by the present invention has high sensitivity, good selectivity and low detection limit, which is conducive to the accurate detection of alpha-fetoprotein and is of great significance to the in-depth application of the sensor in medical clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The TiO prepared in Example 1 2 SEM images of NRA;

[0033] Figure 2 The BiPO prepared in Example 1 4 / TiO 2 SEM images of NRA;

[0034] Figure 3 Example 1 Ag / BiPO 4 / TiO 2 SEM images of NRA;

[0035] Figure 4 The Ag / BiPO prepared in Example 1 4 / TiO 2 Elemental mapping photos from NRA;

[0036] Figure 5 is the AC impedance spectrum of the electrode prepared in Example 1, a is TiO 2 NRA electrode, b is BiPO 4 / TiO 2 NRA electrode, c is Ag / BiPO 4 / TiO 2 NRA electrode, d is Aptamer / Ag / BiPO 4 / TiO 2 NRA electrode, e is BSA / Aptamer / Ag / BiPO 4 / TiO2 NRA electrode, f is AFP / BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA electrode;

[0037] Figure 6 is the time-current test curve of the electrode prepared in Example 1, a is the TiO 2 NRA electrode, b is BiPO 4 / TiO 2 NRA electrode, c is Ag / BiPO 4 / TiO 2 NRA electrode, d is Aptamer / Ag / BiPO 4 / TiO 2 NRA electrode, e is BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA electrode, f is AFP / BS A / Aptamer / Ag / BiPO 4 / TiO 2 NRA electrode;

[0038] Figure 7 This is a current response curve diagram of the photoelectrochemical aptamer sensor for detecting alpha-fetoprotein prepared in Example 1 when detecting alpha-fetoprotein of different concentrations;

[0039] Figure 8 A linear curve diagram of the photoelectrochemical aptamer sensor for detecting alpha-fetoprotein prepared in Example 1 detecting alpha-fetoprotein of different concentrations;

[0040] Fig. 9 This is an anti-interference test diagram of the photoelectrochemical aptamer sensor for detecting alpha-fetoprotein prepared in Example 1 against different interferents, and prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), immunoglobulin G (IgG) and ascorbic acid (AA) were selected as interfering antigens. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present invention clearer, the preferred embodiments of the present invention are further described in detail in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] A method for preparing a photoelectrochemical aptamer sensor for detecting alpha-fetoprotein, comprising the following steps:

[0044] (1)TiO 2 Preparation of NRA / / FTO:

[0045] A FTO glass sheet with a length × width = 2 cm × 1 cm was placed in a mixed solution (equal volumes of acetone, ethanol and deionized water) and ultrasonically cleaned for 15 min, and then dried for later use;

[0046] Butyl titanate is added dropwise to a hydrochloric acid solution with a mass concentration of 36%, and the mixture is stirred evenly to obtain a mixed solution; the mass concentration of the butyl titanate in the mixed solution is 1.5%.

[0047] The washed FTO glass piece was placed in an inclined manner in the inner tank of the reactor, and then the mixed solution was poured in. The reactor was then placed in a drying oven and kept at 150°C for 6 hours for reaction. After the reaction was completed, the FTO glass piece with the film was placed in a muffle furnace and calcined at 450°C for 1.5 hours to obtain TiO 2 NRA, using TiO 2 NRA / FTO said;

[0048] (2)BiPO 4 / TiO 2 Preparation of NRA / FTO electrodes

[0049] Bi(NO 3 ) 3 ·5H 2 O was dissolved in ultrapure water containing 5.5 wt% glycerol and stirred vigorously for 30 min before adding NaH 2 PO 4 12H 2 O, continue stirring for 30 minutes, stirring until the solution is completely dissolved and becomes milky white, to obtain a precursor solution; the molar concentration of bismuth nitrate in the precursor solution is 2mM; the NaH 2 PO 4 The molar concentration is 1.5 mM.

[0050] Pour the precursor solution into the TiO 2 The NRA / FTO reactor was placed in a muffle furnace and reacted for 17 hours at 180°C. After the reaction, the FTO glass substrate with the film was rinsed with deionized water to obtain BiPO 4 / TiO 2 NRA / FTO electrode;

[0051] (3) Ag / BiPO 4 / TiO 2 Preparation of NRA / FTO electrodes

[0052] BiPO 4 / TiO 2 The NRA / FTO electrode was immersed in a 0.1 M ascorbic acid solution for 12 h. After being taken out, it was rinsed with deionized water and dried, and then placed in a 0.1 M AgNO 3 The solution was immersed for 12 h. After the reaction was completed, the electrode was taken out, washed with deionized water and dried to obtain Ag / BiPO 4 / TiO 2 NRA / FTO electrode;

[0053] (4) Preparation of photoelectrochemical alpha-fetoprotein aptamer sensor:

[0054] 10 μL of 0.1 wt% chitosan (CS) solution was drop-coated on the Ag / BiPO 4 / TiO 2 After drying, the NRA / FTO electrode was placed in 5 ml of 2.5 wt% glutaraldehyde solution and soaked for 1 h. Then, the amino-modified alpha-fetoprotein aptamer was drop-coated on the Ag / BiPO 4 / TiO 2 The NRA / FTO electrode surface was incubated for 3 h; then the electrode was rinsed with a PBS solution with a pH of 7.4 to obtain Aptamer / Ag / BiPO 4 / TiO 2 NRA / FTO; Aptamer / Ag / BiPO 4 / TiO 2 The NRA / FTO electrode was placed in 5 ml of 3 wt% bovine serum albumin (BSA) solution and incubated for 1 h, and then cleaned with a pH = 7.4 PBS solution to obtain a photoelectrochemical aptamer sensor for detecting alpha-fetoprotein, which was recorded as BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA / FTO. The amino-modified alpha-fetoprotein aptamer base sequence is: 5'NH 2 -C 6 -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'.

[0055] The TiO obtained in step (1) of Example 1 2 SEM photos of NRA / FTO are shown in Figure 1 As shown, Figure 1 A is TiO 2 SEM image of NRA cross section, Figure 1 B is TiO 2 SEM image of the top of the NRA, from Figure 1 It can be seen that TiO 2 NRA is tightly combined with FTO and the rod-shaped TiO 2 NRA covers the FTO surface neatly and orderly, which provides sufficient loading space for the subsequent material loading and plays a supporting role.

[0056] The BiPO prepared in step (2) of Example 1 4 / TiO 2 SEM images of NRA / FTO are shown in Figure 2. Figure 2 As shown, from Figure 2 It can be seen that the smooth cubic nanostructured BiPO 4 Covered with TiO 2 NRA top.

[0057] The Ag / BiPO obtained in step (2) of Example 1 4 / TiO 2 SEM images of NRA / FTO are shown in Figure 2. Figure 3 As shown, from Figure 3 It can be seen that with the increase of AgNO 3 With the addition of Ag nanoparticles, the 4 To further verify, the Ag / BiPO 4 / TiO 2 NRA nanocomposite materials were subjected to element mapping test and analysis, and the element mapping photos obtained were as follows: Figure 4 As shown, from Figure 4 It can be seen that the five elements Ti, O, Bi, P and Ag are evenly distributed.

[0058] The electrode prepared in Example 1 was subjected to an AC impedance test and a current-time test, and the obtained AC impedance curve is as follows: Figure 5 As shown, the time-current curve is Figure 6 As shown, where a is TiO 2 NRA, b is BiPO 4 / TiO 2 NRA, c is Ag / BiPO 4 / TiO 2 NRA, d is Aptamer / Ag / BiPO 4 / TiO 2 NRA, e is BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA, f is AFP / BSA / Aptamer / Ag / BiPO4 / TiO 2 NRA, from Figure 5 It can be seen that the semicircle diameter of the curve reflects the size of the resistance encountered by the electron. The smaller the diameter, the smaller the resistance encountered by the electron in the transfer process, and the faster the electron transfer rate. The semicircle diameter of curve c is the smallest, and the corresponding current is larger, indicating that Ag / BiPO 4 / TiO 2 The photoelectric performance of NRA is stronger than that of TiO 2 NRA (curve a) and BiPO 4 / TiO 2 Photoelectric performance of NRA (curve b). However, after adding AFP aptamer and BSA, the semicircle diameters of curves d and e gradually increased, while the corresponding currents gradually decreased. This is because the aptamer and BSA are insulating biomacromolecules that hinder the movement of electrons. This series of curve changes indicates the successful preparation of the photoelectrochemical aptamer sensor for detecting AFP.

[0059] In order to quantitatively detect the content of alpha-fetoprotein, the photoelectrochemical aptamer sensor prepared in Example 1 was used to detect alpha-fetoprotein, and the standard curve method was adopted. The specific operation steps are as follows:

[0060] 1. Prepare alpha-fetoprotein standard solutions, the concentrations of which are 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, respectively; immerse the photoelectrochemical aptamer sensor for detecting alpha-fetoprotein in the alpha-fetoprotein standard solutions of different concentrations and incubate for 1 hour, rinse with PBS buffer solution with a pH of 7.4 after the reaction, dry it, and record the obtained electrode as AFP / BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA / FTO;

[0061] 2. On the electrochemical workstation, equipped with a 500W xenon lamp light source and a 400nm cutoff filter, use AFP / BSA / Aptamer / Ag / BiPO 4 / TiO 2The three-electrode system with NRA / FTO as the working electrode, platinum electrode as the counter electrode, and saturated calomel electrode (SCE) as the reference electrode was used as the electrolyte. The electrolyte was 0.1M PBS buffer solution with pH = 7.4. The It test was carried out under an external bias voltage of 0.3V to obtain the photoelectric signals corresponding to different concentrations of alpha-fetoprotein. A standard curve was drawn with the logarithm of the alpha-fetoprotein concentration as the horizontal axis and the corresponding photoelectric signal as the vertical axis. The photocurrent corresponding to the alpha-fetoprotein solution of different concentrations was measured as follows: Figure 7 As shown, the concentrations of AFP from a to l are: 0.001ng / mL, 0.005ng / mL, 0.01ng / mL, 0.05ng / mL, 0.1ng / mL, 0.5ng / mL, 1ng / mL, 5ng / mL, 10ng / mL, 50ng / mL, 100ng / mL, 500ng / mL; from a to l Figure 7 It can be seen that with ΔI (ΔI = I 0 -I, where I 0 The standard curve was drawn with the logarithm of the AFP concentration as the vertical axis, and the obtained standard curve was shown in FIG. Figure 8 As shown. Figure 8 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)=9.25105lg(C AFP ng / mL)+67.37811, the correlation coefficient is 0.995, and the detection limit is 0.53ng / mL (3S / N), which shows that the sensor has a wide detection range and a low detection limit for the detection of alpha-fetoprotein;

[0062] 3. Prepared BSA / Aptamer / Ag / BiPO 4 / TiO 2 The NRA is placed in the target test solution of the actual sample, and the ΔI value is obtained according to the detected photoelectric response signal using the calculation method in step 2. The concentration of AFP in the target is obtained according to the standard curve to complete the detection of alpha-fetoprotein.

[0063] Example 2

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

[0065] Five independent photoelectrochemical aptamer sensors for detecting alpha-fetoprotein were placed in a 100 nM standard alpha-fetoprotein solution for incubation for 1 h, and then washed with a pH = 7.4 PBS solution. The electrodes obtained after drying were recorded as AFP / BSA / Aptamer / Ag / BiPO 4 / TiO 2 NRA / FTO electrode. The electrochemical workstation was equipped with a 500W xenon lamp light source and a 400nm cutoff filter to measure the AFP / BSA / Aptamer / Ag / BiP O 4 / TiO 2 The three-electrode system with NRA / FTO as the working electrode, platinum electrode as the counter electrode, and saturated calomel electrode (SCE) as the reference electrode, and the electrolyte was 0.1M PBS buffer solution with pH=7.4, was used for the It test under an applied bias voltage of 0.3V. The reproducibility of the sensor was evaluated by calculating the relative standard deviation (RSD) of the five independent electrodes through the response signal of the photocurrent, and the calculated result of RSD was 2.91%. This indicates that the photoelectrochemical aptamer sensor prepared in Example 1 has good reproducibility.

[0066] In order to verify the specific selectivity of the photoelectrochemical aptamer sensor for detecting alpha-fetoprotein prepared in Example 1, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), immunoglobulin G (IgG) and ascorbic acid (AA) were selected as interfering substances (the concentrations of alpha-fetoprotein and interfering substances were both 100 ng / mL). The It test was performed on an electrochemical workstation using a 500W xenon lamp light source, a 400nm cutoff filter, and an applied bias voltage of 0.3V. The test results are shown in Figure 2. Fig. 9 As shown, from Fig. 9 It can be seen that the photoelectrochemical aptamer sensor prepared in Example 1 has an obvious photocurrent response only to the sample containing alpha-fetoprotein, while the response signal to other mixed interferents is relatively weak. These results confirm that the photoelectrochemical aptamer sensor of the present invention has a high selectivity for alpha-fetoprotein.

[0067] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing a photoelectrochemical aptamer sensor for detecting alpha-fetoprotein, characterized in that: The following steps are involved: (1) Preparation of TiO2 NRA / FTO TiO2 NRA was deposited on FTO glass sheet by hydrothermal method to obtain TiO2 NRA / FTO; (2) Preparation of BiPO4 / TiO2 NRA / FTO electrode Dissolve bismuth nitrate in a mixed solution of glycerol and water, stir evenly, add NaH2PO4 and continue stirring until the solution is completely dissolved and turns milky white to obtain a precursor solution; Immerse TiO2 NRA / FTO in the precursor solution and react at a temperature of 150-180° C. for 15-24 hours. After the reaction is completed, take out the FTO glass sheet and rinse it with deionized water to obtain a BiPO4 / TiO2NRA / FTO electrode; (3) Preparation of Ag / BiPO4 / TiO2 NRA / FTO electrode The BiPO4 / TiO2 NRA / FTO electrode is placed in an ascorbic acid aqueous solution and soaked for 10 to 14 hours; after being taken out, it is rinsed with deionized water and dried, and then placed in an AgNO3 aqueous solution and soaked for 10 to 14 hours; after the reaction is completed, the electrode is taken out and washed with deionized water and dried to obtain an Ag / BiPO4 / TiO2 NRA / FTO electrode; (4) Preparation of photoelectrochemical aptamer sensors.

2. The preparation method according to claim 1, characterized in that The step (1) is to drop butyl titanate into a hydrochloric acid solution and stir evenly to obtain a mixed solution; place a cleaned FTO glass sheet in an inner container of a reaction kettle at an angle, pour the mixed solution into the inner container, and immerse the cleaned FTO glass sheet in the mixed solution; then react at 150-160° C. for 5-7 hours; after the reaction is completed, take out the FTO glass sheet and calcine it at a temperature of 450-480° C. for 1.5-2 hours to deposit TiO2NRA on the FTO glass sheet, which is expressed as TiO2NRA / FTO.

3. The preparation method according to claim 2, characterized in that: The mass concentration of hydrochloric acid in step (1) is 36%; the mass concentration of butyl titanate in the mixed solution is 1.5%.

4. The preparation method according to claim 1, characterized in that: The mass concentration of glycerol in the mixed solution of glycerol and water in step (2) is 5-8%; the molar concentration of bismuth nitrate in the precursor solution is 2mM; and the molar concentration of NaH2PO4 in the precursor solution is 1.5mM.

5. The preparation method according to claim 1, characterized in that: The molar concentration of ascorbic acid in the ascorbic acid aqueous solution in step (3) is 0.1M; the concentration of the AgNO3 aqueous solution is 0.05-0.3M.

6. The preparation method according to claim 1, characterized in that: Step (4) is to drop chitosan solution onto the Ag / BiPO4 / TiO2 NRA / FTO electrode, and after drying, soak the electrode in a glutaraldehyde aqueous solution for 1 to 3 hours; then drop amino-modified alpha-fetoprotein aptamer onto the surface of the Ag / BiPO4 / TiO2 NRA / FTO electrode and incubate for 3 to 6 hours; then rinse the electrode with a PBS solution at pH = 7.4 to obtain an Aptamer / Ag / BiPO4 / TiO2 NRA / FTO electrode; then place the Aptamer / Ag / BiPO4 / TiO2 NRA / FTO electrode in a bovine serum albumin solution and incubate for 0.5 to 1 hour, and then clean it with a PBS solution at pH = 7.4 to obtain a photoelectrochemical aptamer sensor for detecting alpha-fetoprotein, recorded as BSA / Aptamer / Ag / BiPO4 / TiO2NRA / FTO.

7. The preparation method according to claim 6, characterized in that: The mass concentration of the chitosan solution in step (4) is 0.1% to 0.15%.

8. The preparation method according to claim 6, characterized in that: The mass concentration of the glutaraldehyde aqueous solution in step (4) is 2.5% to 3.0%.

9. The preparation method according to claim 6, characterized in that: The mass concentration of the bovine serum albumin solution in step (4) is 1% to 3%.

10. A photoelectrochemical aptamer sensor for detecting alpha-fetoprotein, characterized in that: Prepared by any one of the methods of claims 1 to 9.

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