Photoelectrochemical immunosensor based on SnCdBi composite electrode material and application of photoelectrochemical immunosensor in colorectal cancer diagnosis

Abstract

The invention relates to the technical field of photoelectrochemical immunodetection, in particular to a detection method based on Snamp; cd < amp >; the invention discloses a photochemical immunosensor of a Bi composite electrode material and application of the photochemical immunosensor in colorectal cancer diagnosis. The composite electrode material is of a ternary heterojunction structure constructed by introducing cadmium sulfide and bismuth sulfide into tin disulfide. The composite electrode material is prepared by the following steps: sequentially adding cadmium acetate dihydrate, tin chloride pentahydrate, thiourea and bismuth nitrate pentahydrate into deionized water, ultrasonically stirring at room temperature until powder is dissolved, and heating an obtained white colloidal solution for synthesis reaction; and cooling the reaction solution to room temperature in air, centrifugally filtering, precipitating, washing, heating and drying. Cadmium sulfide and bismuth sulfide are introduced into tin disulfide to construct a ternary heterojunction, the light absorption range of the ternary heterojunction is expanded, the electron mobility is improved, and the photoelectric property is greatly improved compared with that of a single material. The constructed photochemical immunosensor is used for detecting CEA in serum.

Description

Technical Field

[0001] The present invention relates to the technical field of photoelectrochemical immunoassay, and in particular to a preparation method of a photoelectrochemical (PEC) immunosensor platform constructed based on a ternary heterojunction Sn&Cd&Bi composite material and its application in colorectal cancer (CRC) detection. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract. Its incidence is on the rise, increasing by 2% per year on average, and it is the second leading cause of cancer death worldwide. In recent years, the incidence of colorectal cancer has increased at an extremely fast rate, and it is estimated that by 2030, the global burden of CRC will increase to 60%. Lifestyle and diet are considered to be two important factors that induce CRC. Due to the disorder of the biological clock of most young people, the incidence of CRC in young people is currently showing an alarming upward trend. Studies have shown that the concentration of carcinoembryonic antigen (CEA) is closely related to the incidence of CRC. The expression level of CEA in serum is currently the most commonly used and most valuable tumor marker for preoperative diagnosis, staging, treatment guidance and prognosis evaluation of CRC. Due to its low specificity and sensitivity, it is often combined with other markers in clinical practice to improve its diagnostic efficacy. Generally speaking, the level of CEA in a normal human body should be less than 5.0 ng·mL -1 If the concentration exceeds this range, you need to pay attention to whether there is a risk of cancer.

[0003] Therefore, it is the general trend to establish a simple, rapid and sensitive CEA detection method. At present, many methods for CEA detection have been proposed in clinical practice, such as electrochemiluminescence, fluorescence assay, colorimetry, enzyme-linked immunosorbent assay and photoelectrochemical (PEC) immunoassay, but some methods also have the disadvantages of being time-consuming, labor-intensive, complicated to operate and high in analytical cost. Among them, PEC immunoassay has attracted widespread attention due to its advantages of low cost, simple operation and high sensitivity. This method achieves the purpose of detection through the process of photoelectric signal conversion and recognition. It has applications in the fields of biological analysis, environmental monitoring, food safety and industrial production. In recent years, it has been often used to detect biomarkers and is a very promising new sensing technology.

[0004] In order to construct a PEC immunosensor with high sensitivity and high performance, it is essential to find an efficient photoactive material. Photoactive materials can convert light signals into electrical signals, thereby generating photocurrents, and changes in photocurrents will greatly affect the sensitivity of the analysis. In recent years, with the rapid development and progress of optoelectronic technology, nanomaterials have been widely used in the construction of PEC immunosensors, including carbon-based nanomaterials, metal oxides / sulfides, metal organic frameworks (MOFs), etc. Among them, metal sulfides have stable and excellent photoelectric properties, suitable energy band structures, and excellent light absorption capabilities. In addition, metal sulfides are of various types and have adjustable structures, and are considered to be an ideal photoelectric active material. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and provide a preparation method and application of a PEC immunosensor platform constructed based on a ternary heterojunction Sn&Cd&Bi composite material. The PEC immunosensor platform has a fast response capability and higher sensitivity, and exhibits excellent photoelectric analysis performance, which provides more valuable significance for the future clinical joint diagnosis and prognosis evaluation of CRC.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0007] The invention provides a PEC immunosensor composite electrode material. The composite electrode material is a ternary heterojunction structure constructed by introducing tin disulfide SnS2, cadmium sulfide CdS and bismuth sulfide Bi2S3.

[0008] In the above technical solution, further, the method for preparing the composite electrode material comprises the following steps:

[0009] 1) Cadmium acetate dihydrate (C4H6CdO4·2H2O), tin chloride pentahydrate (SnCl4·5H2O), thiourea (CH4N2S) and bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) are sequentially added into deionized water, and stirred in ultrasound at room temperature until the powder is dissolved to obtain a white colloidal solution;

[0010] 2) heating the white colloidal solution obtained in step 1) for synthesis reaction;

[0011] 3) The reaction solution obtained in step 2) is cooled to room temperature in air, centrifuged and filtered, and the precipitate is washed with deionized water and anhydrous ethanol respectively, and then heated and dried.

[0012] In the above technical solution, further, in step 1), the addition ratio of cadmium acetate dihydrate, tin chloride pentahydrate, thiourea and bismuth nitrate pentahydrate is: 2.3-2.9 mmol: 0.6-0.12 mmol: 5.7-6.3 mmol: 0-0.10 g.

[0013] In the above technical solution, further, in step 2), the temperature of the synthesis reaction is 180-200° C., and the synthesis time is 12-16 hours.

[0014] In the above technical solution, further, in step 3), the centrifugation is carried out by centrifuging at 3000-5000 r·m -1 Centrifuge at a speed of 10 to 20 minutes to obtain a precipitate.

[0015] The present invention provides a PEC immunosensor electrode, wherein the electrode is prepared from the above-mentioned composite electrode material.

[0016] The present invention provides a PEC immunosensor for detecting CEA, wherein the sensor is prepared from the above-mentioned composite electrode material.

[0017] In the above technical solution, further, the preparation method of the PEC immunosensor comprises the following steps:

[0018] 1) The composite electrode material is dissolved in water and dispersed uniformly by ultrasonication to obtain a composite material dispersion;

[0019] 2) Punch holes in the insulating adhesive and stick it on the conductive layer of FTO;

[0020] 3) The composite electrode material dispersion is evenly dripped into the holes of the insulating glue and dried overnight at 40-80°C to obtain Sn&Cd&Bi electrodes;

[0021] 4) CEA antibody was evenly applied on the Sn&Cd&Bi electrode prepared in step 3) and incubated for 60-120 min, and then 1% wt bovine serum albumin BSA was applied on the electrode surface and incubated for 90 min; the incubation temperature was 2-6°C.

[0022] In the above technical solution, further, each time the electrode is modified, it is necessary to thoroughly wash the electrode with a PBS buffer solution to remove unreacted free protein.

[0023] In this study, a new photoelectrochemical immunosensing platform was successfully constructed based on the Sn&Cd&Bi ternary heterojunction. The synthesized composite material Sn&Cd&Bi exhibited excellent photoelectric properties due to the introduction of CdS and Bi2S3, with efficient photocurrent response, and was able to achieve selective detection of CEA, which is of great significance for the joint diagnosis and prognosis evaluation of clinical cancer.

[0024] The present invention provides the use of the aforementioned PEC immunosensor in a colorectal cancer diagnostic device; or its use in detecting the CEA expression level in serum.

[0025] In the above technical scheme, CEA in serum is further detected: 20 μL of CEA antigens of different concentrations are incubated with PEC immunosensors at 4°C for 90 minutes to allow the antigen to fully bind to the antibody; after washing the immunosensor with PBS buffer solution to remove unbound antibody antigens, the photocurrent intensity is recorded, and a standard curve of photocurrent intensity versus CEA concentration is drawn; the prepared immunosensor is fully reacted with the pretreated sample to be tested, and the photocurrent intensity is measured, and the concentration of CEA in the sample to be tested can be obtained after substituting it into the standard curve for calculation.

[0026] The above experiments all used CHI 660E electrochemical workstation to record the photocurrent intensity, using a traditional three-electrode system: Ag / AgCl electrode as the reference electrode, platinum electrode as the counter electrode, and the prepared new Sn&Cd&Bi electrode as the working electrode. A 250W xenon lamp was used as the excitation light, the bias voltage was set to 0V, the light source was switched on and off every 10s, and the photocurrent-time (It) curve was recorded. The electrolyte solution tested was 0.1M PBS buffer solution (pH=7.4), and AA was additionally added as an electron donor during the It curve test.

[0027] In this study, the relationship between the photocurrent response and the logarithm of CEA concentration was -6 ~1ng·mL -1 Within this range, there is a good linear relationship between the photocurrent and the logarithm of the concentration. Compared with other previously reported detection methods for CEA, the prepared PEC immunosensor has higher sensitivity and a wider linear range, which meets the clinical detection requirements for CEA and has certain potential and value.

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

[0029] 1) The electrode material of the present invention uses tin disulfide (SnS2) with a two-layer structure, which has good stability, high sensitivity, non-toxicity and low cost.

[0030] 2) The present invention introduces cadmium sulfide (CdS) and bismuth sulfide (Bi2S3) into tin disulfide (SnS2) to construct a ternary heterojunction, which expands the light absorption range of the ternary heterojunction, improves the electron mobility, and greatly improves the photoelectric performance compared with a single material.

[0031] 3) The PEC immunosensor provided by the present invention has a short detection time, the detection can be completed quickly within 3 minutes, and can be operated at room temperature.

[0032] 4) The PEC immunosensor of the present invention exhibits strong selectivity for CEA and a wide linear range of 10 -6 ~1ng·mL -1 , detection limit as low as 0.00018pg·ml -1 , which well meets the clinical requirements for CEA testing.

[0033] 5) The PEC immunosensor provided by the present invention has a simple preparation process and adopts a simple solvothermal method. It has high sensitivity and good stability. By optimizing the combination of photoelectric active materials, the sensitivity and specificity of detection are improved, providing a more efficient detection method for the diagnosis, staging, treatment guidance and prognosis evaluation of CRC in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 are scanning electron microscope images; A is a scanning electron microscope morphology image of SnS2, B is a scanning electron microscope morphology image of CdS, C is a scanning electron microscope morphology image of Bi2S3, D is a scanning electron microscope morphology image of Sn&Cd&Bi ternary heterojunction, E is a projection electron microscope image of Sn&Cd&Bi ternary heterojunction, and F is a high-resolution transmission electron microscope image of Sn&Cd&Bi ternary heterojunction;

[0035] Figure 2 X-ray diffraction patterns; A shows the X-ray diffraction patterns of pure SnS2, CdS and Bi2S3, and B shows the X-ray diffraction patterns of SnS2&CdS, Bi&Sn&Cd;

[0036] Figure 3 It is the performance diagram of specific surface area, pore volume and pore size; A is the adsorption / desorption curve of Sn&Cd&Bi, and B is the pore volume distribution of SnS2, SnS2&CdS, Sn&Cd&Bi;

[0037] Figure 4 Spectra of composite materials; A is the full scan spectrum of Sn&Cd&Bi, B is the C1s high resolution X-ray photoelectron spectrum, C is the O1s high resolution X-ray photoelectron spectrum, D is the Sn 3d high resolution X-ray photoelectron spectrum, E is the Cd 3d high resolution X-ray photoelectron spectrum, and F is the Bi 4f-s2p high resolution X-ray photoelectron spectrum;

[0038] Figure 5 UV-visible diffuse reflectance spectra of SnS2, CdS, Bi2S3, SnS2&CdS and Sn&Cd&Bi;

[0039] Figure 6 are the photocurrent response and EIS Nyquist diagram; A is the photocurrent response diagram of SnS2, SnS2&CdS, Sn&Cd&Bi; B is the photocurrent response diagram of FTO (a), FTO&Sn&Cd&Bi (b), FTO&Sn&Cd&Bi&Ab (c), FTO&Sn&Cd&Bi&Ab&BSA (d), FTO&Sn&Cd&Bi&Ab&BSA&Ag (e); C is the EIS Nyquist diagram of FTO, SnS2, SnS2&CdS, Sn&Cd&Bi; D is the EIS Nyquist diagram of Sn&Cd&Bi&Ab, Sn&Cd&Bi&BSA, Sn&Cd&Bi&Ab&BSA&Ag;

[0040] Figure 7 The material band gap energy diagram (Tauc diagram) calculated according to the Tauc plot method: A. SnS2, B. CdS (B), C. Bi2S3, D. SnS2&CdS, E. Sn&Cd&Bi;

[0041] Figure 8 Mott-Schottky (MS) diagram: A. SnS2, B. CdS, C. Bi2S3;

[0042] Fig. 9 It is the ultraviolet photoelectron spectrum (UPS) diagram: A. SnS2, B. CdS, C. Bi2S3;

[0043] Fig.10 The photocurrent formation process of the PEC immunosensor;

[0044] Fig.11 Optimization results for the preparation of PEC immunosensor: A. Bi ratio in the material, B. Solution pH, C. Effect of AA concentration;

[0045] Fig.12 is the photocurrent response and linear curve; A is different CEA concentrations (10 -6 -1ng·mL -1 , n = 3), B is the linear curve of the photocurrent response and the logarithmic value of CEA concentration;

[0046] Fig.13 The experimental results of Example 6 are shown in Figure A. The specific detection of CEA by the PEC sensor, where: (a) blank, (b) 10 ng·mL -1 CYFRA21-1, (c) 10 ng mL -1 AFP, (d) 10 ng·mL -1PSA, (e) 10 ng·mL -1 BSA, (f) 10 ng mL -1 ALP, (g) 10 ng·mL -1 CEA; B is the photostability of Sn&Cd&Bi; C is the long-term storage stability of the PEC sensor; D is the repeatability of the PEC immunosensor. DETAILED DESCRIPTION

[0047] The following is a further detailed and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention, but it does not constitute any limitation to the present invention. Any limited modifications made within the scope of protection of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0048] Example 1

[0049] Synthesis of Sn&Cd&Bi ternary heterojunction.

[0050] The Sn&Cd&Bi ternary heterojunction was synthesized by a simple solvothermal method with reference to the literature. First, 2.6mmol cadmium acetate dihydrate (C4H6CdO4·2H2O), 0.9mmol tin chloride pentahydrate (SnCl4·5H2O), 6.0mmol thiourea (CH4N2S) and 0.02g bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) were added to 33mL deionized water in sequence and stirred in ultrasound at room temperature for 30min until the powder dissolved. Then the prepared white colloidal solution was transferred to a 50mL Teflon-lined autoclave and heated at 180℃ for 16h. After the reaction was completed, it was cooled to room temperature in air. The centrifuge was used at 4200r·s -1 The sample was centrifuged at a speed of , and the supernatant was removed. The precipitate was washed with deionized water and ethanol for 3 times, each time for 5 minutes, and then the sample was dried in an oven at 60°C overnight. The final black product was the Sn&Cd&Bi composite material.

[0051] The preparation methods of SnS2, CdS, Bi2S3 and SnS2 / CdS electrodes are as follows.

[0052] SnS2 / CdS nanocomposites were prepared by a one-step hydrothermal method: First, 2.6mmol cadmium acetate dihydrate (C4H6CdO4·2H2O), 0.9mmol tin chloride pentahydrate (SnCl4·5H2O), and 6.0mmol thiourea (CH4N2S) were added to 33mL ultrapure water in sequence and stirred for 30min until the powder dissolved. The prepared white colloidal solution was then added to a 50mL Teflon-lined autoclave and heated at 180℃ for 16h. At the end of the reaction, the reactor was cooled to room temperature in air. After washing and centrifugation, the final product was obtained and dried at 60℃ overnight. (0.1g glucose was added as carbon dots).

[0053] SnS2: 0.9mmol tin chloride pentahydrate (SnCl4·5H2O), and 6.0mmol thiourea were added to 33mL ultrapure water in sequence and stirred for 30min until the powder dissolved. Then the prepared white colloidal solution was added to a 50mL Teflon-lined autoclave and heated at 180℃ for 16h. After the reaction, the reactor was cooled to room temperature in air. After washing and centrifugation, the final product was obtained and dried at 60℃ overnight.

[0054] CdS: 2.6mmol cadmium acetate dihydrate (C4H6CdO4·2H2O), 6.0mmol thiourea were added to 33mL ultrapure water in sequence and stirred for 30min until the powder was dissolved. Then the prepared white colloidal solution was added to a 50mL Teflon-lined autoclave and heated at 180℃ for 16h. After the reaction, the reactor was cooled to room temperature in air. After washing and centrifugation, the final product was obtained and dried at 60℃ overnight.

[0055] Bi2S3: 0.02g bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) and 6.0mmol thiourea were added to 33mL ultrapure water in sequence and stirred for 30min until the powder dissolved. Then the prepared white colloidal solution was added to a 50mL Teflon-lined autoclave and heated at 180℃ for 16h. After the reaction, the reactor was cooled to room temperature in air. After washing and centrifugation, the final product was obtained and dried at 60℃ overnight.

[0056] Various optical characterization results of Sn&Cd&Bi ternary heterojunction are as follows:

[0057] Scanning electron microscopy characterization: Figure 1 From A, B, and C, it can be clearly observed that SnS2 is a regular hexagonal nanosheet, CdS presents a unique starfish-like structure, and Bi2S3 is a nanowire structure. Figure 1In the composite material D, it can be seen that the hexagonal nanosheets of SnS2 are evenly distributed and embedded in the starfish-shaped structure of CdS, which greatly increases the active sites of the photoelectric reaction, indicating the successful combination of SnS2 and CdS. Bi2S3 is difficult to be clearly observed because it exhibits an extremely fine nanowire structure and accounts for a small proportion in the composite material. Figure 1 E is the transmission electron microscopy characterization image of the Sn&Cd&Bi ternary heterojunction, which also proves the successful preparation of the nanocomposite material. In addition, we also give the high-resolution transmission electron microscopy (HRTEM) image of the composite material. Figure 1 A series of lattice fringe spacings of the composite material can be clearly observed in F. Among them, the lattice fringe spacing of 0.289nm corresponds to the (101) plane of SnS2 nanosheets, 0.334nm corresponds to the (100) plane of CdS, and 0.354nm is attributed to the (130) plane of Bi2S3, indicating the effective combination of the three single materials and good crystallinity. The energy dispersive diffraction (EDS) spectrum of the composite material reveals the elemental composition of the material and the proportion of each element. The figure clearly reflects the presence of Sn, Cd, S, Bi, and O elements, further proving the successful preparation of the composite material.

[0058] The lattice structure of the material was characterized by XRD patterns. Figure 2 A shows the XRD spectra of pure SnS2, CdS and Bi2S3. It can be seen that the main diffraction peaks of SnS2 correspond to the (001), (100), (101), (102), (110), (111), (103) and (201) crystal planes, which are well pointed to the (JCPDS No.23-0677) standard card of hexagonal SnS2. The crystal planes corresponding to the CdS diffraction peaks are mainly (002), (100), (101), (110), (103), (112) and (201), belonging to (JCPDS No.41-1049), while a series of crystal planes of Bi2S3 are consistent with (JCPDS No.17-0720). From Figure 2 It can be observed in B that it well contains the diffraction peaks of three single materials, which has an obvious correspondence with the standard card, which also strongly confirms the successful synthesis of the material.

[0059] We tested the specific surface area, pore volume and pore size of the material to explore the performance of the material. From the N2 adsorption / desorption isotherm of the material ( Figure 3 A) It can be seen that the material is close to the IV type curve. The amount of adsorbed gas gradually increases with the increase of pressure at a lower relative pressure, and an H3 type adsorption hysteresis loop appears in the middle section, which shows that it is a mesoporous material. Figure 3B is the pore volume and pore size distribution diagram of the material. It can be seen that the pore size of the composite material is mostly below 5nm, mainly mesopores, with an average pore size of 41.8230nm and a pore volume of 0.0368cm 3 ·g -1 Compared with a single material, it shows a larger specific surface area, which can provide more active adsorption sites and has good adsorption performance. At the same time, due to the coverage and embedding of SnS2 nanosheets and linear Bi2S3 on CdS, the pore volume and pore size of the composite material have been significantly reduced, and the number of pores has also changed, which also proves the successful combination of materials.

[0060] The XPS spectra of the composite materials are shown in Figure 4 As shown in A, the presence of C, O, Sn, Cd, S and Bi elements is strongly confirmed. In the high-resolution spectrum of C1s ( Figure 4 B), we can see three characteristic peaks at 284.68, 285.79, and 288.08 eV, which belong to CC / C=C, CO, and C=NC structures, respectively. The two peaks of O 1s at 531.2 and 532.8 eV correspond to C=O and -OH structures, respectively. Figure 4 C) The spectrum of Sn is shown in Figure 4 D, shows two absorption peaks at 487.1 and 495.5 eV, corresponding to Sn 3d 5 / 2 and Sn 3d 3 / 2 . Figure 4 In E, the peaks of Cd 3d were observed at the binding energies of 405.2 and 412.0 eV, which were attributed to Cd 3d 3 / 2 and Cd3d 5 / 2 The peaks centered at 157.90, 162.88 eV are attributed to Bi 3+ Bi 4f 5 / 2 and Bi 4f 7 / 2 structure, while 159.14 and 161.44 eV are attributed to S2p 3 / 2 and S2p 1 / 2 (See Figure 4 F). The above results demonstrate the successful preparation of the Sn&Cd&Bi ternary heterojunction.

[0061] The optical properties of the materials were evaluated using UV-Vis DRS spectroscopy. Figure 5It can be clearly seen that after SnS2 is combined with CdS, the light absorption capacity is significantly enhanced. As a narrow bandgap semiconductor, Bi2S3 has excellent light absorption performance. It can be seen from the figure that its absorption edge is almost close to 1000nm, which can absorb the entire visible light range. Therefore, after Bi2S3 is added to the material, the absorption band of the composite material has a significant red shift, and the absorption edge is widened from the original 600nm to 900nm. The light absorption range is widened, which greatly improves the material's utilization of light and shows excellent optical properties.

[0062] Example 2

[0063] Preparation of PEC immunosensor.

[0064] First, the FTO conductive glass was ultrasonically cleaned in acetone, anhydrous ethanol and deionized water at room temperature for 30 minutes each time, and then dried in a 60°C oven. A small hole with a diameter of 10 mm was punched on a small square piece of insulating glue with a round puncher, and the FTO conductive glass was pasted on the conductive layer of FTO after the surface was completely dried. An appropriate amount of the above-obtained Sn&Cd&Bi black powder was dissolved in deionized water and dispersed evenly in ultrasound to obtain a concentration of 2 mg·mL -1 The Sn&Cd&Bi composite material dispersion is evenly applied to the circular hole with 100μL Sn&Cd&Bi dispersion, so that it can be fully exposed to light in a fixed area in subsequent tests. After the dispersion is dried at 60°C overnight, a new Sn&Cd&Bi electrode can be obtained. The preparation method of SnS2, CdS, and Bi2S3 electrodes is the same.

[0065] 20 μL of CEA antibody was evenly applied to the prepared Sn&Cd&Bi electrode and incubated for 90 minutes, and then 20 μL of bovine serum albumin (BSA, 1% wt) was incubated on the electrode surface for 90 minutes to block nonspecific binding sites. Thus, the PEC immunosensor of CEA was successfully constructed. During the entire construction process, the incubation steps were all carried out at 4°C, and each time the electrode was modified, the electrode needed to be thoroughly washed with PBS buffer solution to remove unreacted free protein.

[0066] Various optical characterization results of the PEC immunosensor are as follows:

[0067] We studied the photocurrent response during the step-by-step construction of the sensor. Figure 6As shown in A, the bare FTO electrode exhibits a very weak photocurrent. After SnS2 is modified on its surface, the photocurrent is enhanced due to the strong light absorption ability of SnS2. After further modification with CdS, the photocurrent is significantly enhanced because it hinders the recombination of photogenerated electron-hole pairs of SnS2 and has a high light absorption coefficient. With the formation of the Sn&Cd&Bi ternary heterojunction, the introduction of Bi2S3 greatly improves the mobility of carriers, the photocurrent is almost increased to 5 times the original, and the photoelectric efficiency is greatly improved. Subsequently, during the detection process, with the layer-by-layer incubation of CEA antibodies, BSA and CEA antigens, the photocurrent of the material gradually decreased. This may be due to the large volume of CEA antibodies, BSA and CEA antigens that create spatial hindrances to the material, affecting the process of electron transfer to the electrode surface ( Figure 6 B).

[0068] Figure 6 C and D are EIS tests of PEC immunosensors. The step-by-step construction process of the sensor is studied by constructing an equivalent circuit diagram. Generally speaking, the equivalent circuit diagram mainly includes solution resistance (Rs), electron transfer resistance (Ret), ohmic impedance (Zω) and double-layer capacitance (CdL). The Ret value corresponds to the diameter of the semicircle in the curve. It can be observed from the figure that the bare FTO electrode shows a very small semicircle. With the gradual modification of SnS2, CdS and Bi2S3, the diameter of the semicircle of the curve gradually decreases, the Ret value decreases, and the charge transfer efficiency of the material is greatly improved. However, with the introduction of CEA antibody, BSA and CEA antigen, their steric hindrance hinders the electron transfer process, and electrons and holes cannot contact the electrode surface well. The Ret value also gradually increases accordingly, which is also consistent with the test results of the It curve mentioned above, which means that the PEC immunosensor is successfully constructed.

[0069] The PEC sensing mechanism is as follows

[0070] We also studied the band gap of the material based on the Tauc plot method, by constructing (αhν) 2 The band gap energy (Eg) is calculated from the relationship between hν and hν. Figure 7 It can be concluded that the band gap energies of single materials SnS2, CdS and Bi2S3 are 2.08, 2.18 and 1.28 eV respectively. After SnS2 is combined with CdS, the band gap of the material is significantly reduced. After the introduction of Bi2S3, the band gap energy is further reduced, which greatly increases the migration speed of electrons and effectively improves the photoelectric properties of the material.

[0071] Figure 8From the MS test of the materials, it can be seen that the slopes of the three curves are all positive, indicating that the three single materials all have the characteristics of typical n-type semiconductors. From the intersection of the oblique line and the x-axis, it can be concluded that their flat band potentials are -0.66, -0.71 and -0.79 eV relative to the Ag / AgCl electrode, respectively. According to the formula E(NHE)=E(Ag / AgCl)+0.197, the flat band potentials of the materials relative to the standard hydrogen electrode (NHE) are -0.46, -0.51 and -0.60 eV. The flat band potential of the n-type semiconductor is 0.1 eV higher than the conduction band potential, and the conduction band potentials (ECB) of SnS2, CdS and Bi2S3 relative to NHE are calculated to be -0.56, -0.61 and -0.70 eV, respectively. Combined with the obtained Eg value, the valence band potentials (EVB) of the materials can be further concluded to be 1.52, 1.57 and 0.58 eV, respectively, according to the following formula.

[0072] EVB=ECB+Eg

[0073] (EVB is the valence band (VB) potential value, ECB is the conduction band (CB) potential value, and Eg is the band gap energy.)

[0074] Fig. 9 The work function (φ) and Fermi level (E Fermi ), the relationship between the two is: Φ = hν-(E Fermi -E cutoff )(where hv represents the ultraviolet light energy of 21.22 eV; E Fermi represents the Fermi level; E cut off is the cutoff edge after linear fitting), so we can infer that the Φ of SnS2, CdS and Bi2S3 are 7.28, 7.26 and 7.49 eV respectively, and according to the difference between the vacuum energy level and the standard hydrogen electrode potential of 4.5 eV, we can further infer that the Fermi levels of the three are 2.78, 2.76 and 2.99 eV respectively. After the three contact to form a heterojunction, due to the E Fermi There is a difference between the two, the carriers will continue to migrate, and the free electrons will be redistributed until the Fermi level is the same. Fig.10It can be seen that due to the high electron mobility of Bi2S3 and CdS and the extremely narrow band gap of Bi2S3, electrons can quickly jump from VB to CB in Bi2S3, and then continue to transfer from the CB of Bi2S3 to the CdS closest to it, and then to SnS2, while the photogenerated holes are transported from VB in the opposite direction, thus forming a transfer path similar to the "III" type between the ternary heterojunctions, which effectively suppresses the recombination of photogenerated electron-hole pairs and improves the mobility of carriers. Finally, the electrons are transferred to the surface of the FTO electrode, and in the environment where the electron donor AA continuously outputs free electrons, a stable and efficient anode photocurrent is generated.

[0075] Example 3

[0076] We optimized a series of test conditions including material ratio, AA concentration and pH value of buffer solution, so that the prepared PEC immunosensor can achieve its optimal performance. Photoelectric active material is an important factor in determining the intensity of photocurrent. Bi2S3 has a narrow band gap and high photoelectric conversion efficiency. A small amount of introduction can well enhance the photocurrent response of the material. During the experiment, we also found that the dispersibility of Bi2S3 is poor, so we first optimized its doping amount. Fig.11 As shown in A, according to the preparation method of Example 1, by changing the different Bi(NO3)3·5H2O doping amounts, when only 0.02g of Bi(NO3)3·5H2O is added, the photocurrent intensity can be enhanced to 5 to 6 times of the original. When the Bi doping ratio is further increased, the photocurrent intensity decreases instead. Therefore, we choose 0.02g Bi(NO3)3·5H2O as the optimal doping amount.

[0077] In addition, we also studied the pH value of the buffer solution. The acid-base environment of the buffer solution will directly affect the overall performance of the PEC immunosensor. The pH values ​​of the buffer solution were adjusted to 5, 6, 7, 7.4, 8, and 9, respectively, and used as electrolyte solutions in the experimental process. Fig.11 As shown in Figure B, in a buffer solution environment with a pH of 5 to 9, the photocurrent intensity shows a trend of first increasing and then decreasing, reaching a maximum value at a pH of 7.4, so we choose PBS with a pH of 7.4 as the buffer solution. In a long-term test environment, the occurrence of photocorrosion is inevitable, and AA, as an excellent electron donor, can effectively avoid the occurrence of this process. Considering the test process, along with the extension of the test time and the impact of photocorrosion on the material, we added AA as an electron donor to the buffer solution. Fig.11As can be seen in C, after adding different concentrations of AA to the buffer solution, it was found that the photocurrent intensity reached the maximum when the AA concentration was 0.2M. The photocurrent intensity did not change significantly when the concentration continued to increase, and the solubility would also deteriorate when AA was added in excess. Finally, we chose PBS (pH=7.4) containing 0.2M AA as the buffer solution for subsequent photocurrent tests.

[0078] Example 4

[0079] Under the optimal experimental conditions in Example 3, the detection capability of the PEC immunosensor prepared in Example 2 for a series of different CEA concentrations was tested. -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1ng·mL -1 CEA is tested. Fig.12 It can be observed in A that with the increase of CEA concentration, the charge transfer process is hindered, resulting in a gradual decrease in the photocurrent value. Fig.12 B shows the relationship between the photocurrent response and the logarithmic value of CEA concentration. -6 ~1ng·mL -1 In the range, there is a good linear relationship between the photocurrent and the logarithm of the concentration, and the fitting linear relationship equation is I = 23.1824-15.9670lgc, R 2 =0.9946, according to the formula 3σ / S, the detection limit of CEA can be as low as 0.00018pg·ml -1 , where σ is the standard deviation of 11 measurements in the blank sample, and S is the slope of the linear regression equation. Compared with other previously reported detection methods for CEA, the prepared PEC immunosensor has higher sensitivity and a wider linear range.

[0080] Table 1 Comparison of different CEA detection methods

[0081]

[0082] [1]Yang H,Xu Y,Hou Q,et al.Magnetic antifouling material basedratiometric electrochemical biosensor for the accurate detection of CEA inclinical serum[J].Biosensors&Bioelectronics,2022,208:114216.

[0083] [2]Wu Y,Chen X,Luo X,et al.Bimetallic organic framework Cu / UiO-66mediated"fluorescence turn-on"method for ultrasensitive and rapid detectionof carcinoembryonic antigen(CEA)[J].Analytica Chimica Acta,2021,1183:339000.

[0084] [3]Li H,Shi L,Sun D-e,et al.Fluorescence resonance energy transferbiosensor between upconverting nanoparticles and palladium nanoparticles forultrasensitive CEA detection[J].Biosensors&Bioelectronics,2016,86:791-798.

[0085] [4]Wang X,Liao X,Mei L,et al.An immunosensor using functionalizedCu2O / Pt NPs as the signal probe for rapid and highly sensitive CEA detectionwith colorimetry and electrochemistry dual modes[J].Sensors and Actuators B-Chemical,2021,341:130032.

[0086] [5]Yang W, Huang T, Zhao M, et al. High peroxidase-like activity of ironand nitrogen co-doped carbon dots and its application in immunosorbent assay[J]. Talanta, 2017,164:1-6.

[0087] [6]Wang J, Bei J, Guo X, et al. Ultrasensitive photoelectrochemicalimmunosensor for carcinoembryonic antigen detection based on pillar 5arene-functionalized Au nanoparticles and hollow PANI hybrid BiOBr heterojunction[J]. Biosensors&Bioelectronics, 2022, 208: 114220.

[0088] [7]Li H, Li Y, Zhang

[0089] Example 5

[0090] In order to investigate the feasibility and practicality of the prepared PEC immunosensor in clinical practice, we used healthy adult serum as actual sample and determined the CEA content in human serum by standard addition method. -1The sample was centrifuged at a speed of 10 minutes, and the obtained serum supernatant was diluted 3 times with 0.1M PBS solution to obtain the blank sample for the final experiment. Different concentrations of CEA were added to it to determine the recovery rate. As can be seen from Table 2, the recovery rate of serum was between 98.0 and 103.1%, and the RSD value was less than 4%. The results show that the prepared PEC immunosensor can well realize the sensitive detection of CEA in clinical practice.

[0091] Table 2 Determination of CEA in actual samples using PEC sensor (n=3)

[0092]

[0093] Example 6

[0094] like Fig.13 As shown in A, we measured the photocurrent changes of the PEC immunosensor in the presence of other interfering substances to investigate its specificity. -1 In the presence of prostate-specific antigen (PSA), α-fetoprotein (AFP), bovine serum albumin (BSA), alkaline phosphatase (ALP) and cytokeratin-19 fragment (CYFRA21-1) interfering substances, the photocurrent intensity was very stable and did not change significantly. After CEA was added to the mixed solution of these interfering substances, the photocurrent dropped significantly, indicating that the prepared PEC immunosensor can well realize the exclusive determination of CEA.

[0095] Good stability is a necessary prerequisite for evaluating the analytical precision and accuracy of the sensor. Fig.13 As shown in Figure B, the prepared PEC immunosensor was continuously illuminated for 400 seconds, with a cycle of 20 seconds. As a result, no significant change in the photocurrent was observed, indicating that it can continuously and stably output photocurrent signals for a certain period of time under illumination conditions. In order to better evaluate the performance of the sensor, we also evaluated its long-term stability. The prepared PEC immunosensor was stored at 4°C and its photocurrent was tested every other day. After two weeks, its photocurrent response could still reach more than 90% of the original ( Fig.13 C). The above results show that the constructed PEC immunosensor can show good stability and has certain storage significance.

[0096] Repeatability is crucial to avoid errors in the entire experimental process. Therefore, we also prepared 5 identical independent electrodes under the same environment to test the repeatability of the sensor. Under the optimal experimental conditions, the repeatability of the sensor was 1 ng·ml -1 CEA antigen was incubated ( Fig.13D). The test results show that the RSD of the photocurrent values ​​of the five identical electrodes is 3.66%, which proves that the prepared PEC immunosensor has good repeatability to a certain extent.

[0097] The above embodiments are only for the convenience of ordinary technicians in the technical field to understand and use the invention, but not to limit it; people familiar with the technology in this field can still modify and optimize the specific implementation methods in the new embodiments or replace some technical features with equivalents; and apply the general principles described here to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A PEC immunosensor composite electrode material, characterized in that: The composite electrode material is a ternary heterojunction structure constructed by introducing cadmium sulfide CdS and bismuth sulfide Bi2S3 into tin disulfide SnS2.

2. The composite electrode material according to claim 1, characterized in that The preparation method of the composite electrode material comprises the following steps: 1) Cadmium acetate dihydrate (C4H6CdO4·2H2O), tin chloride pentahydrate (SnCl4·5H2O), thiourea (CH4N2S) and bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) are sequentially added into deionized water, and stirred in ultrasound at room temperature until the powder is dissolved to obtain a white colloidal solution; 2) heating the white colloidal solution obtained in step 1) for synthesis reaction; 3) The reaction solution obtained in step 2) is cooled to room temperature in air, centrifuged and filtered, and the precipitate is washed with deionized water and anhydrous ethanol respectively, and then heated and dried.

3. The composite electrode material according to claim 2, characterized in that In step 1), the addition ratio of cadmium acetate dihydrate, tin chloride pentahydrate, thiourea and bismuth nitrate pentahydrate is: 2.3-2.9 mmol: 0.6-0.12 mmol: 5.7-6.3 mmol: 0-0.10 g.

4. The composite electrode material according to claim 2, characterized in that: In step 2), the temperature of the synthesis reaction is 180-200° C., and the synthesis time is 12-16 hours.

5. The composite electrode material according to claim 2, characterized in that: In step 3), the centrifugation is carried out by centrifuging at 3000-5000 rpm -1 Centrifuge at a speed of 10 to 20 minutes to obtain a precipitate.

6. A PEC immunosensor electrode, characterized in that: The electrode is prepared from the composite electrode material according to any one of claims 1 to 6.

7. A PEC immunosensor for detecting CEA, characterized in that: The sensor is prepared from any one of the composite electrode materials 1 to 6.

8. The PEC immunosensor according to claim 8, characterized in that: The preparation method of the PEC immunosensor comprises the following steps: 1) The composite electrode material is dissolved in water and dispersed uniformly by ultrasonication to obtain a composite material dispersion; 2) Punch holes in the insulating adhesive and stick it on the conductive layer of FTO; 3) The composite electrode material dispersion is evenly dripped into the holes of the insulating glue and dried overnight at 40-80°C to obtain Sn&Cd&Bi electrodes; 4) CEA antibody was evenly applied on the Sn&Cd&Bi electrode prepared in step 3) and incubated for 60-120 min, and then 1% wt bovine serum albumin BSA was applied on the electrode surface and incubated for 90 min; the incubation temperature was 2-6°C.

9. Use of the PEC immunosensor according to claim 7 in a colorectal cancer diagnostic device; or in detecting the expression level of CEA in serum.

10. The use according to claim 9, characterized in that: Detection of CEA in serum: incubate different concentrations of CEA antigen with PEC immunosensor to allow antigen and antibody to fully bind; after washing the immunosensor with PBS buffer solution, record the photocurrent intensity and draw a standard curve of photocurrent intensity versus CEA concentration; After the prepared immunosensor is fully reacted with the pretreated sample to be tested, the photocurrent intensity is measured, and the concentration of CEA in the sample to be tested can be obtained by substituting it into the standard curve and calculating.

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