Preparation method of PEC immunosensor for cancer marker SCCA

Abstract

The invention discloses a preparation method of a PEC immunosensor for a cancer marker SCCA, and relates to the technical field of photoelectrochemical immunosensing. The electrode material of the PEC immunosensor is Hamp; c, Hamp is carried out; and C is a composite material of HHOF-101 and CdS-QDs (quantum dots). The present invention is directed to a method for preparing a novel Hamp; the construction of the C heterojunction material provides a preparation method and application of a rapid and sensitive PEC immunosensor.

Description

Technical Field

[0001] The present invention belongs to the technical field of photoelectrochemical immunosensing, and particularly relates to a preparation method of a PEC immunosensor for cancer biomarker SCCA. Background Art

[0002] Squamous cell carcinoma antigen (SCCA), as an important tumor biomarker, plays a key role in the diagnosis, condition monitoring, and prognosis evaluation of various cancers. As a serine protease inhibitor, it was initially isolated from cervical squamous cell carcinoma tissue and was subsequently found to be abnormally elevated in the sera of patients with various squamous cell carcinomas such as lung, esophagus, head and neck cancers. Clinically, by detecting the content change of SCCA in patients, it can provide an important basis for the early diagnosis of cancer, which is conducive to doctors formulating treatment plans in a timely manner and improving the cure rate and survival rate of patients. At the same time, during the treatment process, the dynamic monitoring of SCCA levels can help doctors evaluate the treatment effect, judge whether the disease recurs or metastasizes, and thus adjust the treatment strategy.

[0003] However, traditional SCCA detection methods have many limitations. For example, enzyme-linked immunosorbent assay (ELISA) has high sensitivity and specificity, but the operation process is cumbersome, requiring multiple washing and incubation steps, with a long detection time and difficult to achieve rapid detection; radioimmunoassay (RIA) has high sensitivity, but there is a problem of radioactive contamination, which poses potential hazards to operators and the environment; chemiluminescence immunoassay (CLIA) has a relatively fast detection speed, but the instrument and equipment are expensive and the maintenance cost is high, which limits its wide application in primary medical institutions. Against this background, photoimmunosensor technology has emerged. A photoimmunosensor is a new type of biosensor that combines immunological principles with photoelectric detection technology. It uses the specific binding reaction between antigen and antibody to convert biological signals into easily detectable optical signals or electrical signals, thereby realizing the quantitative analysis of the target substance. This sensor has the advantages of high sensitivity, fast response speed, simple operation, and low cost, and can meet the clinical requirements for rapid and accurate detection of SCCA.

[0004] As a metal-organic framework material, HOF-101 has a high specific surface area, a tunable pore structure, and good chemical stability, which can provide abundant active sites for the immobilization of biomolecules. CdS quantum dots (CdS-QDs), on the other hand, have unique optical and electrical properties, and their size effect and quantum confinement effect endow them with excellent optoelectronic conversion performance. Combining the two to construct an H&C heterojunction can, on the one hand, give full play to the structural advantages of HOF-101, increase the active area of the sensor, and improve the immobilization amount of antibodies; on the other hand, the introduction of CdS quantum dots can optimize the optoelectronic conversion process and enhance the optoelectrochemical response ability of the sensor. The sensing interface constructed in this way can achieve efficient photoelectrochemical detection of SCCA based on the antigen-antibody specific immunorecognition reaction, significantly improving the sensitivity and detection accuracy of the sensor.

[0005] Therefore, in this invention, CdS-QDs were first prepared by the magnetic heating stirring method and then compounded with HOF-101 to prepare the HOF-101 / CdS-QDs (H&C) composite material. The results showed that the prepared 0D / 1D heterostructure exhibited extremely excellent performance. In terms of the optoelectronic conversion efficiency, H&C was significantly improved through the sensitization effect of CdS. In addition, in the presence of SCCA, the photocurrent signal of the proposed PEC sensor was selectively reduced, which might be due to the inherent insulation and steric hindrance of biomacromolecules, thus interrupting the electron transfer process. Therefore, a visible-light-responsive PEC sensor based on the H&C heterostructure was proposed for the quantitative analysis of SCCA in serum samples based on the antigen-antibody (Ag-Ab) specific recognition reaction. This method has high selectivity and sensitivity. Through the good energy level matching between HOF-101 and CdS-QDs, as a recognition probe to detect SCCA, it shows a detection limit as low as 0.6 pg / mL. In particular, the preparation and application of the photoelectrochemical immunosensor for the cancer biomarker SCCA in this invention have important clinical significance and broad application prospects, and have important value for promoting the early diagnosis and precise treatment of cancer. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects of the prior art, develop a method for constructing a new type of H&C heterojunction material, and provide a preparation method and application of a rapid and sensitive PEC immunosensor;

[0007] The purpose of this invention is to use the prepared PEC immunosensor to achieve ultrasensitive detection of the cancer biomarker SCCA, and the detection method is the standard curve method.

[0008] Another purpose of this invention is to provide a mechanism analysis of the efficient photocurrent output signal of the H&C heterojunction based on the Z-type electron transfer mechanism for reference.

[0009] The PEC immunosensor provided by the present invention relies on the specific immune recognition property of antigen-antibody (Ag-Ab) and can achieve accurate quantitative analysis of SCCA. During the detection process, the sensor completely separates the excitation light source from the detection signal, effectively avoiding the influence of background interference. Because of this, PEC immunoassay has higher sensitivity and can accurately determine the tumor marker SCCA, providing strong support for relevant detection work.

[0010] The present invention selects HOF-101 as one of the HOFs. Its large specific surface area can provide more photoactive sites, and its unique advantage of high aspect ratio allows for fast and long-distance charge transfer. The present invention designs and constructs a heterojunction by combining it with cadmium sulfide quantum dots (CdS-QDs). The formed heterojunction has a high visible light utilization rate, thereby improving the efficiency of electron-hole separation. First, CdS-QDs quantum dots are prepared by the magnetic heating stirring method, and then they are combined with HOF-101 to prepare a heterostructured HOF-101 / CdS-QDs (H&C) composite material. The formation of this heterojunction structure enables the material to have a high visible light utilization rate, can fully absorb and utilize the energy in visible light, thereby significantly improving the efficiency of electron-hole separation and further enhancing the performance of the sensor. The results show that the H&C composite material exhibits excellent performance, not only performing well in photoelectrochemical activity, but also effectively improving the detection sensitivity and accuracy of the sensor in the application of PEC immunosensor, providing a more reliable material basis for the accurate detection of the tumor marker SCCA and showing broad application prospects.

[0011] The present invention is realized through the following technical solutions:

[0012] A PEC immunosensor for the cancer marker SCCA, with the H&C / FTO conductive glass modified by Ab and BSA as the working electrode for detecting the cancer marker SCCA in serum. The detection steps are as follows: in the presence of a series of concentration gradients of SCCA standard products, incubate the H&C / FTO conductive glass electrode at 4°C, and the prepared electrode is marked as SCCA / H&C / FTO; perform photoelectric tests using a three-electrode system; record the change in photocurrent using PEC technology to obtain a standard curve; selectively determine SCCA in the actual sample according to the standard curve; repeat the test three times;

[0013] Among them, H&C is a composite material of HOF-101 and CdS-QDs; FTO is fluorine-doped tin oxide conductive glass; the three-electrode system is the standard three-electrode system of a CHI-660E electrochemical workstation, including a working electrode (H&C / FTO conductive glass electrode), an auxiliary electrode (platinum wire), and a reference electrode (Ag / AgCl); during the detection process, a xenon lamp with a 420 nm filter is used as the light source. The PEC test is carried out at room temperature.

[0014] The steps for determining the content of SCCA and plotting the standard curve are as follows:

[0015] ① Prepare different concentrations of SCCA standard solutions with 5 - 10 mL of 0.1 M phosphate buffer solution (PBS) with a pH of 7.4. Drop 20 - 35 μL of SCCA solutions with different concentrations onto the surface of the H&C / FTO electrode successively modified with Ab and BSA. At this time, the electrode is marked as SCCA / H&C / FTO. Incubate at 4 °C for 60 - 80 min, air-dry, and then connect it to the electrochemical workstation. Immerse the electrode in the phosphate buffer solution (PBS) with a pH of 7.4 respectively, turn on the light source, and make it irradiate the surface of the SCCA / H&C / FTO electrode. At this time, measure the magnitude of the photocurrent intensity of SCCA / H&C / FTO.

[0016] ② According to the linear relationship between the obtained current value and the logarithm of the SCCA concentration, plot the working curve.

[0017] ③ Use the working curve method to obtain the concentration of SCCA in the sample to be tested.

[0018] The preparation of the PEC immunosensor includes the following steps:

[0019] Step 1. Synthesis of HOF-101 material: Dissolve 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBAPy) in DMF by ultrasonic waves. Then, quickly pour methanol into the H4TBAPy solution and stir for 12 h. Centrifuge to collect the formed yellow precipitate and wash it, and then dry it under vacuum at room temperature to obtain it.

[0020] Step 2. Synthesis of CdS-QDs: Mix CdCl2·2.5H2O and thiourea in ultrapure water to form a Cd2+ / thiourea precursor fluid; add mercaptoacetic acid (TGA) to ultrapure water and then mix it with the above solution; adjust the pH value of the mixed solution with 1.0 mol·L-1 NaOH, and then add Na2S·9H2O to it, and obtain CdS-QDs by stirring.

[0021] Step 3. Preparation of H&C composite material: Mix HOF-101 and CdS-QDs solution in a certain proportion, and ultrasonicate at room temperature to obtain a uniform H&C solution;

[0022] Step 4. Preparation of HOF-101 / FTO, CdS-QDs / FTO, and H&C / FTO conductive glass electrodes: Drop HOF-101 with DMF as the solvent, CdS-QDs with ultrapure water as the solvent, and the H&C composite material mixed in a certain proportion onto the fixed area of FTO respectively, and dry in an oven at 120 °C to obtain HOF-101 / FTO, CdS-QDs / FTO, and H&C / FTO conductive glass electrodes respectively;

[0023] Step 5. Construction of PEC immunosensor: Drop antibody Ab on the H&C / FTO conductive glass electrode, dry at room temperature and wash with phosphate buffer solution; and add bovine serum albumin solution to block non-specific adsorption sites, incubate to obtain the PEC immunosensor of H&C / Ab / BSA;

[0024] In the said Step 1, m(H4TBAPy):m(DMF):m(methanol) is 20:1:12.

[0025] In the said Step 2, m(CdCl2·2.5H2O):m(thiourea):m(Na2S·9H2O) is 8:3:4; adjust the pH to pH = 10 - 11; the stirring time is 30 - 40 min.

[0026] In the said Step 3, the mass (mg) ratio of HOF-101 to the volume (mL) of CdS is 2 - 3:2 - 4; the ultrasonication time is 5 - 20 min; after ultrasonication, the H&C composite material gradually becomes uniform and stable.

[0027] In the said Step 4, the dropping amounts of HOF-101, CdS-QDs, and H&C composite material are respectively: HOF-101 20 - 40 μL; CdS-QDs 20 - 40 μL; H&C composite material 20 - 40 μL; the drying time is 10 - 15 h.

[0028] In the said Step 5, the amount of Ab introduced into the electrode is 20 - 40 μL, the incubation time is 1 - 1.5 h; the addition amount of BSA is 20 - 40 μL of 0.1 wt%; the incubation time is 1 - 1.5 h.

[0029] For the PEC immunosensor for SCCA, the preparation method is as follows: Different concentrations of SCCA standard solutions are prepared using 5 - 15 mL of 0.1 M phosphate buffer solution with pH = 7.4. 20 - 40 μL of SCCA solutions with different concentrations are respectively drop-coated onto the surface of the H&C / FTO conductive glass electrode sequentially modified with antibody and bovine serum albumin. At this time, the electrode is labeled as SCCA / H&C / FTO, incubated at 4 °C for 1 - 1.5 h, and after air-drying, it is used for PEC testing.

[0030] The detection method of the PEC immunosensor is as follows: The working electrode end of the prepared immunosensor is immersed in the phosphate buffer solution, an external light source is irradiated on the surface of the SCCA / H&C / FTO electrode, and the voltage is adjusted; the photocurrent intensity at this time is displayed on the computer, and the read value corresponds to the concentration of SCCA (0.001 - 10 ng / mL), thereby realizing the rapid and sensitive detection of SCCA.

[0031] A preparation method of a PEC immunosensor based on the HOF-101 and CdS-QDs (H&C) heterojunction is as follows:

[0032] (1) Synthesis of HOF-101 material

[0033] Dissolve 40 mg of H4TBAPy in 2 mL of DMF by ultrasonic wave. Then, quickly pour 24 mL of methanol into the H4TBAPy solution and stir for 12 h. Centrifuge at 8500 r / min for 5 min to collect the formed yellow precipitate and wash it, and then dry it under vacuum at room temperature to obtain the product.

[0034] (2) Synthesis of CdS-QDs

[0035] Mix 0.02 - 0.05 g of CdCl2·2.5H2O and 0.012 - 0.016 g of thiourea in 14 mL of ultrapure water to form a Cd2+ / thiourea precursor fluid; add 38 μL of thioglycolic acid TGA to 20 mL of ultrapure water and mix the two solutions; adjust the pH value of the mixed solution to 10 - 11 with 1.0 mol·L-1 NaOH, and then add 0.02 - 0.03 g of Na2S·9H2O and stir for 30 - 40 min to obtain CdS-QDs;

[0036] (3) Preparation of H&C composite material

[0037] Mix HOF-101 and CdS-QDs at a mass (mg) / volume (mL) ratio of 1.5:3, and perform ultrasonic treatment at room temperature (25 ± 0.5 °C) for 5 - 20 min to obtain the H&C composite material; after ultrasonic treatment, the H&C composite material gradually becomes uniform and stable;

[0038] (4) Preparation of HOF-101 / FTO, CdS / FTO and H&C / FTO

[0039] First, 20 - 40 μL of HOF-101 material was drop-coated on the fixed area of FTO, and then dried in an oven at 120 °C for 10 - 15 h to prepare HOF-101 / FTO; 20 - 40 μL of CdS-QDs with ultrapure water as the solvent was dried in an oven at 120 °C for 10 - 15 h to prepare CdS-QDs / FTO; 20 - 40 μL of the prepared H&C material was drop-coated and dried in an oven at 120 °C for 10 - 15 h to prepare H&C / FTO; the prepared HOF-101 / FTO and CdS-QDs / FTO were used for subsequent study of the photocurrent intensity of H&C / FTO, and the prepared H&C / FTO was used for subsequent construction of a PEC immunosensor and SCCA detection;

[0040] (5) Construction of a PEC immunosensor

[0041] On the H&C / FTO conductive glass electrode, 20 - 40 μL of capture Ab was introduced into the electrode and incubated at 4 °C for 10 - 15 h; after natural drying, the obtained electrode was washed with phosphate buffer solution, and 20 - 40 μL of 0.1 wt% bovine serum albumin was added to block non-specific adsorption sites and incubated for 60 - 80 min; finally, a PEC immunosensor of H&C / Ab / BSA was prepared;

[0042] In the step (4), the FTO is a rectangular thin sheet of 2×2×0.2 cm3, and the fixed area is a circular area of 1×1 cm2, which was washed with acetone, ultrapure water, and ethanol in sequence for 20 - 40 min before use;

[0043] In the step (5), the pH of the phosphate buffer solution is 7.4.

[0044] Preparation of a PEC immunosensor for SCCA detection, the steps are as follows:

[0045] Relying on the specific binding of Ag-Ab dependent on SCCA, a prepared PEC immunosensor was used to detect a series of SCCA standard products with concentration gradients; the H&C / FTO electrode and SCCA samples with different concentrations were incubated at room temperature for a period of time, and the prepared electrode was labeled as SCCA / H&C / FTO; a standard three-electrode system of a CHI-660E electrochemical workstation was used for photocurrent testing, with the H&C / FTO electrode as the working electrode, a platinum wire as the auxiliary electrode, and Ag / AgCl as the reference electrode; a xenon lamp with a 420 nm filter was used as the light source (CEL-HXF300, Beijing); using the PEC technology, the photocurrent changes after different concentrations of SCCA standard products were attached to the H&C / FTO electrode were recorded to obtain a standard curve, and then the SCCA in the actual sample was selectively determined according to the standard curve; all tests were carried out at room temperature and repeated three times;

[0046] The steps for measuring the content of SCCA and plotting the standard curve are as follows:

[0047] ① Prepare different concentrations of SCCA standard solutions using 5 - 15 mL of 0.1 M phosphate buffer solution with pH = 7.4. Drop 20 - 40 μL of SCCA solutions with different concentrations onto the surface of the H&C / FTO electrode sequentially modified with antibodies and bovine serum albumin. At this time, the electrode is labeled as SCCA / F&C / FTO. Incubate at 4 °C for 60 - 80 min, air dry, and then connect it to the electrochemical workstation. Immerse the electrode in the phosphate buffer solution with pH = 7.4 respectively, turn on the light source, and make it irradiate the surface of the SCCA / H&C / FTO electrode. At this time, measure the magnitude of the photocurrent intensity of SCCA / H&C / FTO;

[0048] ② According to the linear relationship between the obtained current value and the logarithm of the SCCA concentration, plot the working curve;

[0049] ③ Using the working curve method, obtain the concentration of SCCA in the sample to be tested;

[0050] The detection operation steps of the PEC sensor for SCCA are as follows:

[0051] ① Prepare different concentrations of SCCA standard solutions using 5 - 15 mL of 0.1 M phosphate buffer solution with pH = 7.4. Drop 20 - 40 μL of SCCA solutions with different concentrations onto the surface of the H&C / FTO electrode sequentially modified with antibodies and bovine serum albumin. At this time, the electrode is labeled as SCCA / H&C / FTO. Incubate at 4 °C for 60 - 80 min, air dry for later use;

[0052] ② Immerse the working electrode terminal (SCCA / H&C / FTO) in step ① into the phosphate buffer solution, irradiate the surface of the SCCA / H&C / FTO electrode with an external light source, and adjust the voltage.

[0053] ③ Display the photocurrent intensity at this time on the computer terminal, and the read value corresponds to the concentration of SCCA (0.001 - 10 ng / mL), thus realizing the rapid and portable detection of SCCA.

[0054] Compared with the traditional methods for determining SCCA, the present invention has the following advantages:

[0055] The present invention designs a PEC immunosensor based on a novel H&C heterojunction material, which can be accurately used for the rapid and sensitive detection of the cancer biomarker SCCA.

[0056] For the sensor of the present invention, when the volume ratio of the mass (mg) of HOF-101 to the volume (mL) of CdS-QDs is 1.5:3, the usage amount of F&C is 20 - 40 μL, the ultrasonic treatment time of H&C is 10 - 20 min, using a 0.1 M phosphate buffer solution with pH = 7.4 as the base solution, and the co-incubation of antigen and antibody is 60 - 80 min, the performance of the sensor is optimal; the detection linear range of SCCA is 0.001 - 10 ng / mL, and the detection limit is 0.6 ng / mL.

[0057] For the optoelectronic performance of the PEC sensor of the present invention, at +0.3 V, the photocurrent intensity of H&C is improved under the sensitization of CdS-QDs, and the H&C of the present invention is continued to be used as an efficient PEC material.

[0058] Using the PEC immunosensor prepared by the present invention to detect SCCA, the results show that it has a good recovery rate, and it is expected to have an excellent performance in the rapid clinical detection of SCCA. Description of the Drawings

[0059] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0060] Figure 1 It is the construction and SCCA detection flow chart of the PEC immunosensor;

[0061] Figure 2 It is the scanning electron microscope (SEM) image of HOF-101;

[0062] Figure 3 is the scanning electron microscope (SEM) spectrum of H&C;

[0063] Figure 4 is the high-resolution scanning electron microscope (HR-SEM) spectrum of HOF-101;

[0064] Figure 5 is the high-resolution scanning electron microscope (HR-SEM) spectrum of H&C;

[0065] Figure 6 is the transmission electron microscope (TEM) spectrum of HOF-101;

[0066] Figure 7 is the transmission electron microscope (TEM) spectrum of H&C;

[0067] Figure 8 is the energy-dispersive X-ray (EDS) total element distribution spectrum of H&C;

[0068] Figure 9 is the Brunauer-Emmett-Teller (BET) curve of HOF-101 and H&C;

[0069] Figure 10 is the Fourier transform infrared (FT-IR) spectrum of HOF-101, CdS-QDs and H&C;

[0070] Figure 11 is the X-ray diffraction analysis (XRD) spectrum of HOF-101, CdS-QDs and H&C;

[0071] Figure 12 is the X-ray photoelectron (XPS) spectrum of HOF-101, CdS-QDs and H&C;

[0072] Figure 13 is the ultraviolet-visible diffuse reflectance (UV-Vis-DRS) spectrum of HOF-101, CdS-QDs and H&C;

[0073] Figure 14 is the photocurrent comparison of HOF-101, CdS-QDs and H&C;

[0074] Figure 15 is the electrochemical impedance (EIS) spectrum of HOF-101, CdS-QDs and H&C;

[0075] Figure 16 is the result of the condition optimization experiment;

[0076] Figure 17 is the Tauc curve of HOF-101, CdS-QDs and H&C;

[0077] Figure 18 Mott-Schottky (M-S) spectra of HOF-101, CdS-QDs, and H&C;

[0078] Figure 19 Mechanism diagram;

[0079] Figure 20 Linear result diagram;

[0080] Figure 21 Selectivity result diagram;

[0081] Figure 22 Photostability diagram of the constructed H&C.

[0082] The realization, functional features, and advantages of the objectives of the present invention will be further described with reference to the embodiments and the accompanying drawings. Specific Embodiments

[0083] The following will further elaborate on the technical solutions of the present invention in conjunction with the accompanying drawings and specific embodiments, but it does not constitute any limitation to the present invention. Any limited modifications made within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0084] Instrumentation and Reagents

[0085] 1.1 Instruments:

[0086] Electronic analytical balance, KQ-5200 ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., Jiangsu, China), scanning electron microscope (Zeiss sigma 300), Thermo Fisher Nicolet iS20 Fourier transform infrared spectrometer (Thermo Fisher, USA), DF-101S collector type constant temperature heating magnetic stirrer (Gongyi Yuhua Instrument Co., Ltd.), energy dispersive spectrometer (EDS) equipped with an Oxford X-MAX scanning electron microscope for obtaining elemental distribution, transmission electron microscope morphology obtained from a JEM-2100 transmission electron microscope (JEOL, Japan), X-ray diffraction (XRD) obtained using a D8 Advance diffractometer (Bruker, Germany), X-ray photoelectron spectroscopy (XPS) performed on an ESCALAB 250Xi X-ray photoelectron emission spectrometer, UV-500PC ultraviolet-visible spectrophotometer for testing absorbance (Shanghai Yuanxi Instruments Co., Ltd., China), and all photoelectrochemical detection processes were carried out on a photoelectrochemical workstation (CHI-660E, China), with a 500W xenon lamp (CEL-HXF300, Beijing, China) used as the light source.

[0087] 1.2 Reagents

[0088] Bovine serum albumin (Hefei BOSF Biotechnology Co., Ltd.), deionized water (Wahaha Group Co., Ltd.), absolute ethanol (Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.), acetone (Shandong Yuwang Industrial Co., Ltd.), N, N-dimethylformamide (Tianjin Concord Chemical Reagent Co., Ltd.), thiourea (Shanghai Macklin Biochemical Co., Ltd.), 1, 3, 6, 8-tetrakis(4-carboxyphenyl)pyrene (Shanghai Macklin Biochemical Co., Ltd.), SCCA standard product (Shanghai Zeye Biotechnology Co., Ltd.), mercaptoacetic acid (Shanghai Macklin Biochemical Co., Ltd.), sodium sulfide nonahydrate (Shanghai Macklin Biochemical Co., Ltd.), cadmium chloride hemihydrate (Shanghai Macklin Biochemical Co., Ltd.).

[0089] Example 1

[0090] A preparation method of a PEC immunosensor is as follows:

[0091] The overall process is as Figure 1 shown.

[0092] (1) Synthesis and purification of HOF-101 material

[0093] Dissolve 40 mg of H4TBAPy in 2 mL of DMF by ultrasonic wave. Then, quickly pour 24 mL of methanol into the H4TBAPy solution and stir for 12 h. Centrifuge at 8500 r / min for 5 min to collect the formed yellow precipitate and wash it, and then vacuum dry it at room temperature to obtain it.

[0094] (2) Synthesis of CdS-QDs

[0095] Mix 0.04 g of CdCl2·2.5H2O and 0.015 g of thiourea in 14 mL of ultrapure water to form a Cd2+ / thiourea precursor fluid. Add 38 μL of mercaptoacetic acid TGA to 20 mL of ultrapure water, and mix the two solutions. Adjust the pH value of the mixed solution to 10.5 with 1.0 mol·L-1 NaOH, and then add 0.02 g of Na2S·9H2O to it and stir for 30 min to obtain a CdS-QDs solution.

[0096] (3) Preparation of H&C composite material

[0097] Dissolve 1.5 mg of HOF-101 powder in 3 mL of CdS-QDs solution, and mix it evenly by ultrasonic wave to obtain the H&C solution. Obtain the H&C composite material by ultrasonic treatment at room temperature (25 ± 0.5 °C) for 15 min. After ultrasonic treatment, the H&C solution gradually becomes uniform and stable.

[0098] (4) Preparation of PEC immunosensor

[0099] First, clean the FTO conductive glass electrode (2×2×1 cm3) successively with acetone, absolute ethanol, and deionized water. Drop 30 μL of 2 mg·mL-1 HOF-101 material (using DMF as the solvent) onto the FTO, and then dry it in an oven at 120 °C for 12 h to prepare HOF-101 / FTO; directly drop 30 μL of CdS-QDs aqueous solution onto the FTO and dry it in an oven at 120 °C for 12 h to prepare CdS-QDs / FTO; drop 30 μL of the prepared H&C material and dry it in an oven at 120 °C for 12 h to prepare H&C / FTO. The prepared HOF-101 / FTO, CdS-QDs / FTO, and H&C / FTO are used for subsequent photocurrent intensity studies, and H&C / FTO is used for subsequent PEC immunosensor construction and SCCA detection.

[0100] On the H&C / FTO conductive glass electrode, drop 30 μL of Ab and coat it on the electrode, incubate at 4 °C for 12 h. After natural drying at 4 °C, wash the obtained electrode with phosphate buffer solution. Finally, drop 30 μL of 0.1 wt% bovine serum albumin and incubate at 4 °C for 1 h to block non-specific binding sites, and the PEC immunosensor of H&C / Ab / BSA is successfully constructed.

[0101] Characterization and detection:

[0102] ① Morphological characterization of the prepared HOF-101, CdS-QDs, and H&C materials

[0103] Characterize the size, morphology, and surface element distribution of the materials by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDS) spectroscopy, and specific surface area (BET) measurement.

[0104] The morphological characteristics of HOF-101, CdS-QDs, and H&C were evaluated in detail using SEM and TEM. Figure 2The SEM image of HOF-101 with a blocky morphology is shown, and the surface has a certain roughness. This blocky structure enables light to undergo multiple reflections and refractions on the material surface, thereby increasing the optical path of light within the material. This is beneficial for light absorption because as the propagation path of light in the material becomes longer, there are more opportunities for the material to absorb light, thus increasing the generation probability of photo-generated carriers. After being combined with CdS-QDs, it can be observed that the quantum dots are distributed on the surface of HOF-101 or embedded therein, and the size of the quantum dots is relatively small. The presence of these quantum dots changes the light absorption mode. Due to the quantum confinement effect of CdS quantum dots, they can absorb light of specific wavelengths, and due to their small size and distribution in the material, the light absorption in the material becomes more uniform, avoiding the situation of overly strong or overly weak local light absorption, and improving the light absorption efficiency of the entire material system( Figure 3 ). In addition, the morphologies shown in the HR-SEM images of the prepared HOF-101 and H&C are as Figure 4 , 5. HOF-101 presents a relatively complex blocky and flaky structure, with an uneven surface. This structure can increase the scattering of light and prolong the propagation path of light within the material, thereby improving the light absorption efficiency and generating more photo-generated carriers. However, the electron transport channels within HOF-101 are relatively limited and irregular, and the electron transport process is prone to being hindered, which is not conducive to the efficient transport of electrons. The surface of the H&C heterojunction consists of a large number of fine particles. Compared with HOF-101, CdS quantum dots are evenly distributed on the surface of HOF-101, forming more electron transport channels. These channels provide a smoother transport path for electrons, helping electrons to quickly migrate to the electrode and improving the overall electron transport efficiency and optoelectronic performance. Figure 6 shows that HOF-101 shows a relatively uniform and delicate microstructure. This structure may contribute to the orderly propagation of light within the material. The uniform structure may endow light absorption with a certain stability, and to a certain extent, ensure the generation of photo-generated carriers. In the H&C heterojunction, an obvious interface is formed between HOF-101 and CdS quantum dots. It provides a more efficient transport path for electrons, facilitating electrons to quickly reach the electrode to participate in the reaction and improving the overall photoelectrochemical performance( Figure 7 ). The energy-dispersive diffraction (EDS) spectrum of H&C( Figure 8 ) indicates the presence of elements C, H, Cd, N, O, and S in the formed H&C, and further proves the successful preparation of H&C. The pore structures of the HOF-101 and H&C composite materials were characterized using N2 adsorption-desorption isotherms( Figure 9)。Through BET analysis, the BET surface area of HOF-101 is 2.09 m2·g-1, the average pore diameter is 2.50 nm, and the pore volume is 0.00086 cm3·g-1. The BET surface area of H&C is 1.79 m2·g-1, the average pore diameter is 1.65 nm, and the pore volume is 0.000069 cm3·g-1. The change in BET parameters further confirms that CdS quantum dots are filled or attached to HOF-101, occupying the pore space and resulting in a significant decrease in pore volume.

[0105] ② Characterization of the optical properties of HOF-101, CdS-QDs and H&C materials

[0106] The optical properties of the materials were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction analysis (XRD) spectroscopy, X-ray photoelectron (XPS) spectroscopy, high-resolution X-ray photoelectron (HR-XPS) spectroscopy, and ultraviolet-visible diffuse reflectance (UV-Vis-DRS) spectroscopy.

[0107] Infrared spectroscopy is an analytical technique used to study the molecular structure and chemical bonds of materials. Figure 10 The results show that for HOF-101, 1691.19 cm-1 and 1604.56 cm-1 may correspond to the stretching vibration of the carbonyl group (C=O). 1425.40 cm-1 and 1280.15 cm-1 may correspond to the C-C stretching vibration of the aromatic ring. 858.60 cm-1 and 810.11 cm-1 may correspond to the vibration of the metal-ligand. For CdS quantum dots, 2116.89 cm-1 and 2144.66 cm-1 may correspond to the stretching vibration of the Cd-S bond. 1642.07 cm-1 and 1447.31 cm-1 may correspond to the stretching vibration of the S-H or C-H bond. 1136.87 cm-1 and 1103.20 cm-1 may correspond to the bending vibration of the Cd-S bond. In H&C, 3393.78 cm-1 and 3414.93 cm-1 may correspond to the stretching vibration of the O-H or N-H bond. 2944.18 cm-1 and 2947.84 cm-1 may correspond to the stretching vibration of the C-H bond. 1591.53 cm-1 and 1401.05 cm-1 may correspond to the C=C stretching vibration of the aromatic ring. 1001.30 cm-1 and 970.16 cm-1 may correspond to the stretching vibration of the C-O or C-N bond. The above analysis results indicate that the carboxyl functional group of HOF-101 may form a stable interface with CdS through hydrogen bonds, further promoting the separation and transfer of charges.

[0108] The XRD patterns of the three materials Figure 11)Analysis shows that multiple distinct diffraction peaks appear in the XRD spectra of HOF-101 and CdS quantum dots, and the positions of these peaks correspond to their specific crystal structures. The presence of these peaks indicates that HOF-101 has good crystallinity. The characteristic diffraction peaks of HOF-101 and CdS appear in the XRD spectrum of H&C, indicating that H&C is a composite heterojunction structure composed of HOF-101 and CdS. The intensity and width of the diffraction peaks of H&C may be between those of HOF-101 and CdS, indicating that the formation of the composite heterojunction structure has an impact on the crystal structures of both.

[0109] The XPS spectrum contains photoelectron peaks of different orbitals such as C1s, O1s, N1s, Cd3d, and S2p, indicating the presence of elements such as carbon (C), oxygen (O), nitrogen (N), cadmium (Cd), and sulfur (S) in the sample. The photoelectron peaks of different elements appear at specific binding energy positions, which is the basis for elemental qualitative analysis ( Figure 12 ). H&C shows peak shifts in Cd 3d and S2p, which confirms the formation of an active interface of the Z-type heterostructure between CdS-QDs and HOF-101. This interface can generate an internal electric field, which helps to confirm the formation of an active interface of the Z-type heterostructure between CdS-QDs and HOF-101. This interface can generate an internal electric field, which helps to guide the efficient transport of photo-generated carriers along a specific direction and avoid energy loss caused by disordered diffusion. The BE spectrum of CdS-QDs shows that the BE spectra of C, O, Cd, and S elements all shift to higher values, indicating the outflow of charge carriers, thus leading to a decrease in the electron cloud density around them. In contrast, the BE spectrum of HOF-101 shows that the BE spectra of C and O elements both shift to lower values, indicating the inflow of charge carriers, thus leading to an increase in the electron cloud density around them. These changes in BE indicate that significant charge transfer occurs at the active interface of the Z-type heterostructure formed between CdS-QDs and HOF-101. This dynamic balance of charge transfer not only optimizes the electronic structure of the material but also promotes the further improvement of the separation and migration efficiency of photo-generated carriers.

[0110] The light absorption capabilities of HOF-101, CdS-QDs, and H&C were evaluated using UV-Vis DRS spectra. Figure 13 It shows that the absorption spectrum of H&C covers the partial absorption ranges of HOF-101 and CdS respectively, having absorption in both the ultraviolet region and showing a certain absorption ability in the visible light region. This means that H&C combines the light absorption advantages of the two single materials and broadens the light response range.

[0111] ③Characterization of the optoelectronic properties of HOF-101, CdS-QDs, and H&C materials

[0112] PEC analysis can be used to evaluate the changes in optoelectronic properties after the surface modification or modification of materials. Therefore, the PEC properties of HOF-101, CdS-QDs, and H&C were further analyzed. The results showed that when an external voltage of 0.3 V was applied, the photocurrent of H&C was significantly improved after being modified with CdS-QDs( Figure 14 ). At the same time, the EIS diagram( Figure 15 ) also showed that the interfacial charge transfer resistance (Rct) of H&C was between CdS-QDs and HOF-101.

[0113] ④ Optimization of experimental conditions

[0114] The photocurrent intensity of the PEC biosensor is affected by the ultrasonic time of material mixing, the amount of drop coating, and the incubation time of Ag-Ab( Figure 16 ). In order to make the PEC sensor have better performance, a series of experimental conditions were optimized. After screening, when 20 mg of HOF-101 was mixed evenly with 1.5 mL of CdS-QDs solution, H&C had the highest photocurrent intensity. When the ultrasonic mixing time of H&C was 15 min and the drop coating volume of H&C was 30 μL, the FTO modified with H&C showed the best photocurrent effect. Considering the best effect of the photocurrent, 0.1 M PBS solution (pH = 7.4) was selected as the best buffer solution for testing. In addition, the optimal incubation time of Ag-Ab was determined to be 70 min.

[0115] ⑤ Analysis of experimental mechanism

[0116] The Tauc curves of the three materials were obtained using the UV-Vis DRS spectra, and their band gaps (Eg) were analyzed. In addition, the valence band potential (EVB) and conduction band potential (ECB) of CdS-QDs and HOF-101 were studied in combination with the Mott-Schottky (M-S) diagram (M-S) curve. As Figure 17 shown, the Eg values of HOF-101, CdS-QDs, and H&C were calculated to be 2.06, 1.92

[0117] and 1.74 eV respectively by the following formula: (αhυ)1 / n = A(hυ - Eg)(1)

[0118] (α is the absorption index, h represents Planck's constant, υ represents frequency, A is a constant, Eg is the semiconductor band gap, and the exponent n is directly related to the semiconductor type.)

[0119] The M-S curve( Figure 18)Among them, the intersection point of the straight line and the x-axis is the flat-band potential of HOF-101, CdS-QDs, and H&C, and the slopes of the straight-line parts of all three are positive values, which are typical characteristics of n-type semiconductors. Generally speaking, for n-type semiconductors, their flat-band potential is 0.1 eV higher than the conduction-band potential. Therefore, for the standard hydrogen electrode (NHE), the conduction-band potentials (ECB) of HOF-101 and CdS-QDs are -1.03 and -0.71 eV respectively. According to the following calculation formula: EVB = ECB + Eg (2)

[0120] (where ECB is the potential of the conduction-band (CB) edge, EVB represents the potential of the valence-band (VB) edge; Eg represents the band-gap energy.)

[0121] It can be further analyzed that the valence-band potentials (EVB) of HOF-101 and CdS-QDs are 1.03 and 1.21 eV respectively.

[0122] Figure 19 It shows that due to the different energy-band positions of the two, after forming a heterojunction, when irradiated with light (hv), both CdS-QDs and HOF-101 generate photo-generated carriers. Under the action of the internal electric field, the free electrons are redistributed until their Fermi levels are the same. Therefore, there is a potential "Z" electron-transport pathway between CdS-QDs and FJU-200. Under the irradiation of the excitation light, the narrow-band-gap CdS-QDs absorb energy, and the photo-generated e- of CdS-QDs transfers from the VB to the CB, then from the CB of CdS-QDs to the VB of HOF-101, and finally reaches the CB of HOF-101. Among them, HOF-101 plays the role of an electron-transfer bridge, transporting the e- to FTO finally to form an anodic photocurrent.

[0123] Example 2

[0124] The detection of SCCA using the PEC immunosensor is carried out as follows:

[0125] For the PEC determination of SCCA, first, SCCA with different concentrations was prepared using 0.1 M PBS solution with pH = 7.4. Different concentrations of SCCA (20 μL) were respectively drop-coated on the surface of the H&C / FTO electrode sequentially modified with antibody (Ab) and bovine serum albumin (BSA), incubated at 4 °C for 70 min, and then washed with PBS solution. Analysis was carried out in PBS solution (0.1 M, pH = 7.4), and the current-time (I-t) curve was recorded. All electrochemical tests were performed on a CHI-660E electrochemical workstation, using a traditional three-electrode system: the H&C-modified FTO as the working electrode, the Ag / AgCl electrode as the reference electrode, and a platinum sheet (1.0 × 1.0 cm2) as the counter electrode. The electrolyte solution was 0.1 M PBS (pH = 7.4) buffer solution.

[0126] Under the optimal experimental conditions, the photocurrent intensities of different concentrations of SCCA were obtained ( Figure 20 ). The results showed that the photocurrent gradually decreased with the increase in SCCA concentration. It is speculated that this is mainly due to the accumulation of SCCA, which seriously hindered the electron transfer at the interface of HOF-101 and CdS-QDs, resulting in a gradual decrease in photocurrent. At the same time, the detection linear range of SCCA was 0.001 - 10 ng / mL, and the detection limit was 0.6 pg / mL.

[0127] The calculated linear relationship expression was I = 42.5298 - 2.7846[C], and R2 was 0.9910. Compared with some previously reported detection methods for SCCA, the proposed PEC immunosensor showed a wider linear range and higher sensitivity. The results indicated that the constructed PEC immunosensor could accurately and rapidly detect SCCA.

[0128] Selectivity, stability, and reproducibility of the PEC immunosensor:

[0129] Several representative samples were selected from actual samples, and these samples might contain indicators such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and prostate-specific antigen (PSA). Using these substances as interfering substances, a specificity test was carried out to evaluate the accuracy and reliability of the experiment. The results of the selectivity investigation of H&C are as Figure 21 shown, indicating that H&C has good selectivity.

[0130] When evaluating the performance of materials and sensors, stability is a crucial indicator. To explore its stability, taking H&C as the object, the on-off light irradiation operation was repeated within a time range of 300 s, and its changes during this process were investigated in detail. The results showed ( Figure 22) After 300 s of light illumination, the photocurrent intensity of the PEC photoanode modified with H&C could still maintain 83.14% of the initial value. In addition, the prepared materials and immunosensors were stored in a refrigerator (4 °C) for 25 d. They were taken out for measurement every 5 d. After 25 d, the photocurrent intensity of the prepared materials could still reach more than 96.3% of the previously measured value. Next, its reproducibility was evaluated. Under the same conditions, six electrodes modified with H&C materials and PEC sensors modified with antibodies (incubated with 3 ng / mL SCCA) were prepared. The test results showed that the RSD value of the six electrodes modified with H&C materials was 3.1%, indicating that the constructed H&C materials had good reproducibility.

[0131] Detection of SCCA:

[0132] To verify the feasibility of the established method, this study investigated serum as an actual sample. Under the optimal experimental conditions, the accuracy and precision of this analytical method were evaluated by measuring the recovery rate and relative standard deviation (RSD) values of spiked serum samples. As can be seen from the data in Table 1, in actual serum samples, the detection recovery rate of SCCA was between 100.0% and 110.3%, and the RSD did not exceed 2.3%. The above results fully demonstrated that the constructed PEC immunosensor could be effectively used for the detection of SCCA in complex biological matrices.

[0133] Table 1 PEC determination of SCCA in human serum samples (n = 3)

[0134]

[0135] The above are only the preferred embodiments of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a PEC immunosensor for cancer biomarker SCCA, characterized in that, The electrode material of the PEC immunosensor is H&C, and the H&C is a composite material of HOF-101 and CdS-QDs.

2. The preparation method of a PEC immunosensor for cancer marker SCCA according to claim 1, characterized in that, It consists of a three-electrode system with an H&C / FTO conductive glass electrode modified with Ab and BSA as the working electrode, a platinum wire as the auxiliary electrode, and Ag / AgCl as the reference electrode, for detecting the cancer biomarker SCCA; Among them, Ab is an antibody, BSA is bovine serum albumin, and FTO is fluorine-doped tin oxide conductive glass.

3. The preparation method of a PEC immunosensor for cancer biomarker SCCA according to any one of claims 1 to 2, characterized in that, It includes the following steps: Step 1. Synthesis of HOF-101 material: Dissolve 1,3,6,8-tetra(4-carboxyphenyl)pyrene (H4TBAPy) in DMF by ultrasonic wave; then, quickly pour methanol into the H4TBAPy solution and stir for 12 h; Centrifuge to collect the formed yellow precipitate and wash it, and then dry it under vacuum at room temperature to obtain it; Step 2. Synthesis of CdS-QDs: Mix CdCl2·2.5H2O and thiourea in ultrapure water to form a Cd2+ / thiourea precursor fluid; Add mercaptoacetic acid (TGA) to ultrapure water and then mix it with the above solution; Adjust the pH value of the mixed solution with 1.0 mol·L-1 NaOH, and then add Na2S·9H2O to obtain CdS-QDs by stirring; Step 3. Preparation of H&C composite material: Mix HOF-101 and CdS-QDs solution in a certain proportion and ultrasonicate at room temperature to obtain a uniform H&C solution; Step 4. Preparation of HOF-101 / FTO, CdS-QDs / FTO, and H&C / FTO conductive glass electrodes: Drop HOF-101 with DMF as the solvent, CdS-QDs with ultrapure water as the solvent, and the H&C composite material mixed in a certain proportion on the fixed area of FTO respectively, and dry them in an oven at 120 °C to obtain HOF-101 / FTO, CdS-QDs / FTO, and H&C / FTO conductive glass electrodes respectively; Step 5. Construction of the PEC immunosensor: Drop the antibody Ab on the H&C / FTO conductive glass electrode, dry it at room temperature and wash it with phosphate buffer solution; And add bovine serum albumin solution to block non-specific adsorption sites, incubate to obtain the PEC immunosensor of H&C / Ab / BSA.

4. The preparation method of a PEC immunosensor for cancer marker SCCA according to claim 3, characterized in that, In the said Step 1, m(H4TBAPy):m(DMF):m(methanol) is 20:1:

12.

5. The preparation method of a PEC immunosensor for cancer biomarker SCCA according to claim 3, wherein In the said Step 2, m(CdCl2·2.5H2O):m(thiourea):m(Na2S·9H2O) is 8:3:4; Adjust the pH to pH = 10-11; The stirring time is 30-40 min.

6. The preparation method of a PEC immunosensor for cancer biomarker SCCA according to claim 3, wherein, In the said Step 3, the mass (mg) ratio of HOF-101 to the volume (mL) of CdS is 2-3:2-4; The ultrasonic time is 5-20 min; After ultrasonic treatment, the H&C composite material gradually becomes uniform and stable.

7. The preparation method of a PEC immunosensor for cancer biomarker SCCA according to claim 3, characterized in that, In the step 4, the dropping amounts of HOF-101, CdS-QDs and H&C composite material are respectively: 20 - 40 μL of HOF-101; 20 - 40 μL of CdS-QDs; 20 - 40 μL of H&C composite material; and the drying time is 10 - 15 h.

8. The preparation method of a PEC immunosensor for cancer marker SCCA according to claim 3, characterized in that, In the step 5, the amount of Ab introduced into the electrode is 20 - 40 μL, and the incubation time is 1 - 1.5 h; the addition amount of BSA is 20 - 40 μL of 0.1 wt%; and the incubation time is 1 - 1.5 h.

9. The preparation method of a PEC immunosensor for cancer biomarker SCCA according to claim 1, wherein, The method for detecting cancer marker SCCA is as follows: Different concentrations of SCCA standard solutions are prepared by using 5 - 15 mL of 0.1 M phosphate buffer solution with pH = 7.

4. 20 - 40 μL of SCCA solutions with different concentrations are respectively dropped onto the surface of the H&C / FTO conductive glass electrode sequentially modified by antibody and bovine serum albumin. At this time, the electrode is marked as SCCA / H&C / FTO, and incubated at 4°C for 1 - 1.5 h. After air drying, it is used for PEC testing; among them, during the detection process, a xenon lamp with a 420 nm filter is used as the light source, and the test is carried out at room temperature.

10. The preparation method of a PEC immunosensor for cancer biomarker SCCA according to claim 9, characterized in that, The method for detecting cancer marker SCCA is as follows: The PEC immunosensor detection method is as follows: The prepared working electrode end is immersed in the phosphate buffer solution, and an external light source is irradiated on the surface of the SCCA / H&C / FTO electrode to adjust the voltage; the photocurrent intensity at this time is displayed on the computer, and the read value corresponds to the concentration of SCCA (0.001 - 10 ng / mL), so as to realize the rapid and sensitive detection of SCCA.

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