A photoelectrochemical biosensor for detecting Cyfra21-1 and its preparation method

Abstract

The present invention discloses a photoelectrochemical biosensor for detecting Cyfra21-1 and its preparation method. The biosensor comprises a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite fixed to the surface of an FTO glass electrode. A probe sequence is fixed to the surface of the nanocomposite. The probe sequence partially binds to a single-stranded nucleic acid sequence, which is connected to a signal amplification factor via its terminal modification group. The present invention achieves detection of Cyfra21-1 using a photoelectrochemical biosensor, which is simple, highly sensitive, and easy to operate.

Description

Technical Field

[0001] The present invention relates to a photoelectrochemical biosensor for detecting Cyfra21-1 and a preparation method thereof, and more particularly to a photoelectrochemical biosensor based on a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite modified electrode. Background Art

[0002] With the rapid development of science, technology, and the economy, the question of whether environmental pollution may cause cancer has drawn profound attention. According to previous reports, lung cancer is the most common malignant tumor and the leading cause of death worldwide. It is divided into two histological types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is the predominant type of lung cancer, accounting for approximately 80-85% of all lung cancers [Zhang, YY, Wu, TT, Cui, QQ, Qu, ZF, Zhang, Y., Ma, HM, Wei, Q., Biosens. Biology, 2023, 222, 114992.]. Because patients are often diagnosed at an advanced stage, metastasis and recurrence of NSCLC are the main causes of death in lung cancer patients [Wang, YF, Li, YX, Zhuang, XM, Tian, CY, Fu, XL, Luan, F., Biosens. Biology, 2021, 190, 113371.]. As a tumor marker for NSCLC, cytokeratin 21-1 (Cyfra21-1, a fragment of cytokeratin-19) is considered to be the most important biomarker for detecting NSCLC, especially squamous cell carcinoma [Yang, YC, Liu, MH, Yang, SM, Chan, YH, ACS Sens., 2021, 6, 4255-4264.]. To date, although various analytical methods for detecting Cyfra21-1 have been developed, such as electrochemiluminescence, electrochemistry, surface plasmon resonance, and fluorescence, they still have limitations such as limited sensitivity and complex operation. Therefore, the development of a simple, rapid, and highly sensitive serum Cyfra21-1 detection method is of great significance for early clinical diagnosis.

[0003] Photoelectrochemical bioanalysis is an emerging bioanalytical technique that utilizes light-induced electron transfer between the analyte, a photoactive substance, and an electrode. Due to its short detection cycle, low background signal, and high sensitivity, it has been applied to the detection of tumor markers [Wu, TT, Yu, SQ, Dai, L., Feng, JH, Ren, X., Ma, HM, Wang, XY, Wei, Q., Ju, HX, ACSSens., 2022, 7, 1732-1739.]. Based on these characteristics, a new photoelectrochemical bioanalytical method was proposed for the sensitive detection of Cyfra21-1.

[0004] Transition metal carbon / nitrides (MXenes, M = transition metal, X = C, N, C / N, enes = modifying group) are two-dimensional layered materials with tunable optical and electronic band gaps. They have the advantages of high specific surface area, rich active sites, high electron transport efficiency, adjustable physicochemical properties, variable interlayer spacing and excellent optoelectronic properties [Khan, K., Tareen, A. K., Iqbal, M., Ye, Z., Xie, Z. J., Mahmood, A., Mahmood, N., Zhang, H., Small, 2023, 2206147.]. The unique structure of MXenes has attracted considerable attention as photoelectrochemical materials in the field of sensing. First, the internal conductive transition metal carbon / nitride layer enables efficient electron transfer. Second, the abundant transition metal oxides on the surface serve as active sites for rapid redox reactions, enabling efficient photoelectrocatalytic activity. Finally, the inherently large surface area of two-dimensional materials helps address the high detection limit and improve sensitivity in sensing and monitoring applications [Sunantha, G., Chamorn, C., Mohan, G., Dao, J., Food Chem. Toxicol, 2022, 168, 113377]. However, in their natural aqueous state, MXenes are highly susceptible to oxidation, and interlayer van der Waals forces leading to stacking and aggregation limit their application in photoelectrochemical sensing. To overcome these issues and improve the photoelectric conversion efficiency of these materials, they must be functionalized to enhance their application in photoelectrochemical sensing.

[0005] Current literature reports indicate that interfacial heterojunction engineering is crucial for suppressing carrier recombination and improving photoelectric conversion efficiency. Graphite-like carbon nitride is a non-metallic semiconductor photoelectrically active material that has been considered the most promising material in recent years due to its simple preparation, environmental friendliness, and chemical stability [Liu, J., Yu, Y., Qi, RL, Cao, CY, Liu, XY, Zheng, YJ, Song, WG, Appl. Catal. BEnviron., 2019, 244, 459-464.]. Furthermore, coupling with semiconductors is a common approach to achieve better photoelectric performance, effectively reducing the recombination of photogenerated electron-hole pairs. Semiconductor quantum dots not only have strong light absorption but also exhibit red or blue shifts depending on their composition, shape, and size. Among them, CdSe quantum dots have been widely used in the field of photoelectrochemical bioanalysis due to their advantages such as narrow band gap, wide light absorption range, and rapid generation of photogenerated electron-hole pairs [Tang, YY, Chen, JS, Liu, XP, Mao, CJ, Jin, BK, Microchem. J., 2022, 181, 107773.].

[0006] The present invention combines titanium carbide, graphite-like carbon nitride and cadmium selenide quantum dots to prepare a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite modified electrode. This can give full play to the advantages of the nanocomposite material, expand the spectral absorption range, improve the utilization rate of light energy and enhance stability. It is a new strategy for the development of photoelectrochemical bioanalysis and will have broad application prospects in fields such as bioanalysis and clinical diagnosis. Summary of the Invention

[0007] In order to address the deficiencies of the above-mentioned prior art, the present invention provides a photoelectrochemical biosensor for detecting Cyfra21-1 based on titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite materials and a preparation method thereof, so as to achieve simple and rapid detection of Cyfra21-1 using a photoelectrochemical biosensor with high photoelectrochemical current and high biocompatibility.

[0008] The present invention solves the technical problem by adopting the following technical solutions:

[0009] The present invention first discloses a photoelectrochemical biosensor for detecting Cyfra21-1, which is characterized in that: the surface of the photoelectrochemical biosensor is covered with a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material on an FTO glass electrode, a probe sequence is fixed on the surface of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material, the probe sequence can be partially combined with a single-stranded nucleic acid sequence, and the single-stranded nucleic acid sequence is connected to a signal amplification factor through its terminal modification group.

[0010] Furthermore, the preparation method of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material is as follows: pure water is added to the graphite-like carbon nitride to obtain a dispersion, and then the cadmium selenide quantum dot solution and the titanium carbide dispersion are sequentially added, and ultrasonic dispersion is performed to uniformly disperse the mixture, thereby obtaining a dispersion of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material. The specific steps are as follows:

[0011] Step 1, dispersing bulk carbon nitride in pure water and ultrasonically treating the resulting dispersion, high-speed centrifuging the resulting solid, placing it in a reactor and adding concentrated nitric acid, reacting at high temperature, adding pure water and ultrasonically treating the resulting product, and then centrifuging and washing it until neutral to obtain graphite-like carbon nitride, which is then dried for later use;

[0012] Step 2: adding pure water to the graphite-like carbon nitride obtained in step 1 to obtain a dispersion, then adding the cadmium selenide quantum dot solution to the obtained dispersion, ultrasonically homogenizing and leaving it overnight; then adding the titanium carbide dispersion to the obtained uniform mixed solution, ultrasonically homogenizing and leaving it overnight to obtain a dispersion of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material.

[0013] Furthermore, the signal amplification factor is obtained by the following method: silver nitrate and indium nitrate are added to pure water and stirred to dissolve, followed by the addition of mercaptopropionic acid and the adjustment of the pH to 9-10, the addition of sodium sulfide under inert gas protection and reflux at high temperature, and after the reaction is completed, the product is allowed to settle overnight and centrifuged and washed to obtain the signal amplification factor. The amount ratio of silver nitrate, indium nitrate, pure water, mercaptopropionic acid, and sodium sulfide is 0.1 mmol: 0.4 mmol: 50 mL: 0.1 mmol: 0.6 mmol.

[0014] Furthermore, the high-temperature reflux temperature is 100° C. and the reaction time is 2 h.

[0015] Furthermore, the probe sequence can recognize both the single-stranded nucleic acid sequence and Cyfra21-1; the single-stranded nucleic acid sequence will base-pair with the probe sequence, and its binding ability is weaker than that of Cyfra21-1. Specifically, in the present invention, the probe sequence is 5'-GAAGGGAGGAATGGTGTCAGGGGCGAGGGTTTTTT-SH-3', and the single-stranded nucleic acid sequence is 5'-NH2-CGCCCCTGACACCATTCCT-3'.

[0016] The preparation method of the photoelectrochemical biosensor for detecting Cyfra21-1 of the present invention is carried out according to the following steps:

[0017] Step 1: ultrasonically clean the FTO glass electrode with acetone, water, and ethanol in sequence, and then dry it for later use;

[0018] Step 2, evenly adding a dispersion of titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material onto the surface of the FTO glass electrode cleaned in step 1, and drying the mixture at room temperature to obtain a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material modified electrode;

[0019] Step 3, adding the probe sequence to the surface of the modified electrode prepared in step 2, incubating at low temperature and then rinsing with phosphate buffer solution; then adding the single-stranded nucleic acid sequence to its surface, incubating at a constant temperature and then rinsing with phosphate buffer solution; activating the signal amplification factor at room temperature and then adding it to the electrode surface under constant temperature conditions to connect it with the single-stranded nucleic acid sequence through a group, and rinsing with phosphate buffer solution to obtain a photoelectrochemical biosensor for detecting Cyfra21-1.

[0020] Furthermore, in step 3, the low-temperature incubation temperature is 4° C. and the time is 12-13 h, and the constant-temperature incubation temperature is 37° C. and the time is 70-80 min.

[0021] The method for detecting Cyfra21-1 using the above biosensor is as follows:

[0022] Step A, adding a Cyfra21-1 sample to be tested to the surface of the photoelectrochemical biosensor and incubating at a constant temperature, then removing the sample and rinsing it with a phosphate buffer solution to obtain a biosensor electrode to be tested; Cyfra21-1 has a stronger specific recognition ability with the probe sequence and can competitively cleave the single-stranded nucleic acid sequence modified with a signal amplification factor;

[0023] Step B: Perform a photoelectrochemical test on the biosensor electrode to be tested obtained in step 1 in a phosphate buffer solution containing 0.1 mol / LAA to obtain the photocurrent intensity of the Cyfra21-1 sample to be tested, and use the standard relationship curve between the photocurrent intensity and the concentration of the Cyfra21-1 sample to determine the concentration of the Cyfra21-1 sample to be tested.

[0024] Furthermore, the standard relationship curve is obtained by performing photoelectrochemical tests on a biosensor electrode to be tested, which is made of a series of standard Cyfra21-1 samples with concentrations ranging from 0.001 to 100 pM, obtaining the photocurrent intensity corresponding to each concentration of Cyfra21-1 sample, and then fitting the curve with the logarithm of the concentration of the Cyfra21-1 sample as the horizontal axis and the photocurrent intensity as the vertical axis. Figure 1 As shown: The concentrations corresponding to the lines from left to right are: 0.001pM, 0.01pM, 0.1pM, 1pM, 10pM, 100pM; the standard relationship curve is I (μA) = 15.0047-2.3906logC Cyfra21-1The test showed that when the Cyfra21-1 sample concentration was in the range of 0.001-100 pM, the photocurrent intensity decreased with the increase of Cyfra21-1 sample concentration, and was linearly related to the concentration, with the detection limit reaching 0.73 fM.

[0025] The photoelectrochemical test is a three-electrode system with the biosensor electrode to be tested as the working electrode, the Pt electrode as the counter electrode, and the saturated silver chloride electrode as the reference electrode. A 250W xenon lamp is used as the light source with a wavelength range of 280-1000nm and an applied voltage of 0V. The current changes are recorded using a CHI660D electrochemical workstation.

[0026] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0027] 1. The present invention realizes the detection of Cyfra21-1 by photoelectrochemical biosensor, which has a simple method, a wide detection range, high sensitivity and easy operation;

[0028] 2. The detection method of Cyfra21-1 of the present invention requires a small amount of sample and has low detection cost;

[0029] 3. The present invention prepares a photoelectrochemical biosensor by using titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material, which has high photoelectrochemical current, good biocompatibility and excellent stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the test results of photoelectrochemical conversion of Cyfra21-1 standard samples with a concentration of 0.001-100 pM in the present invention, and the inset is the standard relationship curve.

[0031] Figure 2 The graphite-like carbon nitride of the present invention is characterized by transmission electron microscopy (TEM).

[0032] Figure 3 These are the transmission electron microscopy (TEM) characterization results of the graphite-like carbon nitride / tin disulfide quantum dot nanocomposite material of the present invention.

[0033] Figure 4 This is the high-resolution transmission electron microscopy (HRTEM) characterization result of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material in the present invention.

[0034] Figure 5 This is the photocurrent diagram of titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material and a composite material of one or two of the three. It can be seen from the figure that the photocurrent after the combination of the three materials has a significant advantage over other combinations. DETAILED DESCRIPTION

[0035] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, the following detailed description of specific embodiments of the present invention is provided in conjunction with the accompanying drawings. The following is merely an example and illustration of the concept of the present invention. Any modification, supplement, or substitution of the described specific embodiments by a person skilled in the art without departing from the concept of the invention or exceeding the scope defined by the claims shall fall within the scope of protection of the present invention.

[0036] The preparation method of the dispersion of titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material used in the following examples is as follows:

[0037] Step 1: Disperse bulk carbon nitride in pure water and ultrasonically treat for 12 hours. The obtained dispersion is centrifuged at 10,000 rpm for 15 minutes, and the obtained solid is dried at 60°C for 12 hours. Take 150 mg of the obtained solid and place it in a reactor and add 450 μL of concentrated nitric acid. After reacting at 140°C for 4 hours, the obtained product is added to 150 mL of pure water and ultrasonically treated for 4 hours. Then, it is centrifuged and washed until neutral to obtain a graphite-like phase carbon nitride. It is dried at 60°C for 12 hours and retained for later use.

[0038] Step 2: add pure water to the graphite-like carbon nitride obtained in step 1 and ultrasonically treat to obtain a 1 mg / mL dispersion; take 1 mL of the obtained dispersion and add 200 μL of cadmium selenide quantum dot solution; ultrasonically homogenize the obtained mixed solution and place it at 4°C for 12 hours; take 1 mL of the obtained mixed solution and add 0.75 mg of titanium carbide solid; ultrasonically homogenize and place it at 4°C for 12 hours to obtain a dispersion of titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material. Among them, the cadmium selenide quantum dot solution is prepared according to the method in the article "A label-free photoelectrochemical immunosensor for carcinoembryonic antigen detection based on g-C3N4 / CdSenano composite" (XPLiu, JSChen, CJMao, BKJin, A label-free photoelectrochemical immunosensor for carcinoembryonic antigen detection based on g-C3N4 / CdSenano composite, Analyst, 2021, 146, 146-155.), and the obtained product solution is centrifuged and washed, and the collected solid material is dissolved to 15 mL to obtain the required cadmium selenide quantum dot solution.

[0039] The signal amplifier used in the following examples was obtained as follows: silver nitrate (0.1 mmol) and indium nitrate (0.4 mmol) were added to pure water (50 mL) and stirred to dissolve. Mercaptopropionic acid (0.1 mmol) was then added and the pH adjusted to 9-10. Sodium sulfide (0.6 mmol) was added under inert gas and refluxed at 100°C for 2 h. After the reaction, the product was allowed to settle for 12 h and washed by centrifugation. The resulting target product was evenly dispersed in 10 mL of pure water and stored for later use. The signal amplifier was activated at room temperature by adding 500 μL of a mixture of EDC (20 mg / mL) and NHS (10 mg / mL) to 200 μL of the signal amplifier, shaking evenly, and then allowing to stand at room temperature for 1 h.

[0040] The phosphate buffer solution (0.1 mol / L, pH 7.4) and the probe sequences and single-stranded nucleic acid sequences used in the following examples were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0041] The 0.1 mol / L phosphate buffer solution containing 0.1 mol / L ascorbic acid (AA) at a pH of 7.4 used in the following examples was prepared as follows: 1.1496 g of Na2HPO4, 0.2964 g of NaH2PO4·2H2O, 0.8 g of NaCl, and 1.7613 g of AA were weighed and prepared into 100 mL of aqueous solution.

[0042] Example 1

[0043] In this example, the photoelectrochemical biosensor for detecting Cyfra21-1 was first prepared according to the following steps:

[0044] Step 1: Ultrasonic clean the FTO glass electrode with acetone, water and ethanol in sequence, then dry it at 60°C for 12 hours and set aside.

[0045] Step 2: Evenly add 30 μL of the dispersion of titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material onto the surface of the FTO glass electrode cleaned in step 1. After drying at room temperature, a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material modified electrode is obtained.

[0046] Step 3: Add 30 μL of 1 μM probe sequence to the surface of the modified electrode prepared in step 2, incubate at 4°C for 12 hours, remove and rinse with phosphate buffer solution; then add 30 μL of 1 μM single-stranded nucleic acid sequence to the surface, incubate at 37°C for 70 minutes and rinse with buffer solution; take 30 μL of the signal amplification factor after room temperature activation and add it to the electrode surface, incubate at 37°C for 70 minutes and connect it to the single-stranded nucleic acid sequence through the group, and rinse with phosphate buffer solution to obtain a photoelectrochemical biosensor for detecting Cyfra21-1.

[0047] Using the biosensor of this embodiment, the method for detecting Cyfra21-1 is as follows:

[0048] Step A, adding a Cyfra21-1 sample to be tested to the surface of the photoelectrochemical biosensor, incubating at 37° C. for 60 minutes, then removing the sample and rinsing it with a phosphate buffer solution to obtain a biosensor electrode to be tested;

[0049] Step B: Perform a photoelectrochemical test on the biosensor electrode to be tested obtained in step 1 in a phosphate buffer solution containing 0.1 mol / LAA to obtain the photocurrent intensity of the Cyfra21-1 sample to be tested, and use the standard relationship curve between the photocurrent intensity and the concentration of the Cyfra21-1 sample to determine the concentration of the Cyfra21-1 sample to be tested.

[0050] To verify the feasibility of the method of this embodiment, Cyfra21-1 samples with known concentrations of 0.001 pM, 0.01 pM, 0.1 pM, 1 pM, 10 pM, and 100 pM were taken. The biosensor of this embodiment was used to detect and calculate the concentrations of each sample according to the above method, which were 0.0010 pM, 0.0099 pM, 0.0995 pM, 1.34 pM, 10.14 pM, and 92.9 pM, respectively. It can be seen that the prepared biosensor has rapid, sensitive, accurate, and efficient detection of the target Cyfra21-1.

[0051] Example 2

[0052] In this example, the step 3 of the photoelectrochemical biosensor preparation method in Example 1, "adding 30 μL of 1 μM single-stranded nucleic acid sequence," was changed to "adding 30 μL of 1.5 μM single-stranded nucleic acid sequence." The remaining conditions and steps were the same as in Example 1. The resulting biosensor had similar morphology and properties to those of the biosensor obtained in Example 1. Similar biological detection results were obtained by testing the same Cyfra21-1 sample.

[0053] Example 3

[0054] In this example, the step 3 of the photoelectrochemical biosensor preparation method in Example 1, "adding 30 μL of 1 μM single-stranded nucleic acid sequence, incubating at 37°C for 70 min, and then rinsing with buffer solution" was changed to "adding 30 μL of 1 μM single-stranded nucleic acid sequence, incubating at 37°C for 80 min". The remaining conditions and steps were the same as in Example 1. The resulting biosensor had similar morphology and properties to the biosensor obtained in Example 1. Similar biological detection results were obtained by detecting the same test substance Cyfra21-1.

[0055] Example 4

[0056] In this example, the step 3 of the photoelectrochemical biosensor preparation method in Example 1, "taking 30 μL of the signal amplification factor after activation at room temperature and incubating at a constant temperature of 37°C for 70 min" was changed to "taking 30 μL of the signal amplification factor after activation at room temperature and incubating at a constant temperature of 37°C for 80 min." The remaining conditions and steps were the same as in Example 1. The resulting biosensor had similar morphology and properties to the biosensor obtained in Example 1. Similar biological detection results were obtained by testing the same Cyfra21-1 sample to be tested.

[0057] Example 5

[0058] In this example, the step A of the method for detecting Cyfra21-1 in Example 1, "incubating the Cyfra21-1 sample at a constant temperature of 37°C for 60 minutes" was changed to "incubating the Cyfra21-1 sample at a constant temperature of 37°C for 70 minutes". The remaining conditions and steps were the same as those in Example 1. The resulting biosensor had similar morphology and properties to the biosensor obtained in Example 1. Similar biological detection results were obtained by detecting the same Cyfra21-1 sample to be tested.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A photoelectrochemical biosensor for detecting Cyfra21-1, characterized in that: The photoelectrochemical biosensor comprises a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material covered on the surface of an FTO glass electrode, and a probe sequence fixed on the surface of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material; the probe sequence partially binds to a single-stranded nucleic acid sequence, and the single-stranded nucleic acid sequence is connected to a signal amplification factor via a terminal modification group.

2. The photoelectrochemical biosensor for detecting Cyfra21-1 according to claim 1, characterized in that: The preparation method of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material comprises the following steps: adding pure water to graphite-like carbon nitride to obtain a dispersion liquid; then sequentially adding a cadmium selenide quantum dot solution and a titanium carbide dispersion liquid; and uniformly dispersing the mixture by ultrasonication to obtain a dispersion liquid of the titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material.

3. The photoelectrochemical biosensor for detecting Cyfra21-1 according to claim 1, characterized in that: The probe sequence can be recognized by the single-stranded nucleic acid sequence and Cyfra21-1; the single-stranded nucleic acid sequence will be complementary to the probe sequence base pairing, and its binding ability is weaker than Cyfra21-1.

4. The photoelectrochemical biosensor for detecting Cyfra21-1 according to claim 1, characterized in that: The signal amplification factor is obtained as follows: Take silver nitrate and indium nitrate and add pure water and stir to dissolve, then add mercaptopropionic acid and adjust the pH to 9-10, add sodium sulfide under inert gas protection and reflux at high temperature, after the reaction is completed, let the product settle overnight and centrifuge and wash to obtain the signal amplification factor.

5. The photoelectrochemical biosensor for detecting Cyfra21-1 according to claim 4, characterized in that: The usage ratio of silver nitrate, indium nitrate, pure water, mercaptopropionic acid and sodium sulfide is 0.1mmol:0.4mmol:50mL:0.1mmol:0.6mmol.

6. The photoelectrochemical biosensor for detecting Cyfra21-1 according to claim 4, characterized in that: The temperature of the high-temperature reflux is 100° C. and the reaction time is 2 h.

7. A method for preparing the photoelectrochemical biosensor according to any one of claims 1 to 6, characterized in that: The steps include: Step 1: ultrasonically clean the FTO glass electrode with acetone, water, and ethanol in sequence, and then dry it for later use; Step 2, evenly adding a dispersion of titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material onto the surface of the FTO glass electrode cleaned in step 1, and drying the mixture at room temperature to obtain a titanium carbide / graphite-like carbon nitride / cadmium selenide quantum dot nanocomposite material modified electrode; Step 3, adding the probe sequence to the surface of the modified electrode prepared in step 2, incubating at low temperature and then rinsing with phosphate buffer solution; then adding the single-stranded nucleic acid sequence to its surface, incubating at a constant temperature and then rinsing with phosphate buffer solution; activating the signal amplification factor at room temperature and then adding it to the electrode surface under constant temperature conditions to connect it with the single-stranded nucleic acid sequence through a group, and rinsing with phosphate buffer solution to obtain a photoelectrochemical biosensor for detecting Cyfra21-1.

8. The preparation method according to claim 7, characterized in that: In step 3, the low-temperature incubation temperature is 4° C. and the time is 12-13 hours, and the constant-temperature incubation temperature is 37° C. and the time is 70-80 minutes.

9. A method for detecting Cyfra21-1 using the photoelectrochemical biosensor according to any one of claims 1 to 6, characterized in that: The steps include: Step A, adding a Cyfra21-1 sample to be tested to the surface of the photoelectrochemical biosensor and incubating it at a constant temperature, then removing it and rinsing it with a phosphate buffer solution to obtain a biosensor electrode to be tested; Step B: Perform a photoelectrochemical test on the biosensor electrode to be tested obtained in step 1 in a phosphate buffer solution containing 0.1 mol / LAA to obtain the photocurrent intensity of the Cyfra21-1 sample to be tested, and use the standard relationship curve between the photocurrent intensity and the concentration of the Cyfra21-1 sample to determine the concentration of the Cyfra21-1 sample to be tested.

10. The method according to claim 9, characterized in that: The standard relationship curve is obtained by performing photoelectrochemical testing on the biosensor electrode to be tested, which is made of a series of standard Cyfra21-1 samples with concentrations ranging from 0.001 to 100 pM, obtaining the photocurrent intensity corresponding to each concentration of Cyfra21-1 sample, and then fitting the curve with the logarithm of the concentration of the Cyfra21-1 sample as the horizontal axis and the photocurrent intensity as the vertical axis.

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