A carbon dot-covalent organic framework-based photocathode material and a preparation method and application thereof

Abstract

The present application relates to a kind of carbon dot-based covalent organic framework-based photocathode material and its preparation method and application, belong to photoelectrochemical biosensor technical field.A kind of carbon dot-based covalent organic framework-based photocathode material, the photocathode material is CD on the electrically conductive substrate in situ growth T-D-COF film, carbon dot in the CD T-D-COF film is uniformly distributed in the COF channel formed by TAPA and DHNDA condensation.In addition, the present application is based on CD T-D-COF constructs a kind of photoelectrochemical immunosensor array that can accurately identify trace A beta 42 in human plasma, it is in sensitivity, specificity and practicability all achieve significant breakthrough, especially suitable for reliable detection of extremely low concentration marker in complex biological matrix, it shows broad prospects in clinical conversion application.

Description

Technical Field

[0001] This invention relates to a photocathode material based on a carbon dot@covalent organic framework, its preparation method and application, belonging to the field of photoelectrochemical biosensor technology. Background Technology

[0002] Alzheimer's disease (AD) is a significant neurodegenerative disease whose complex pathophysiological processes pose a major challenge to global public health. To date, only a limited number of disease-modifying therapies are available; however, timely diagnosis can help healthcare professionals implement early treatment, thereby slowing disease progression. The pathological features of AD primarily include the abnormal accumulation of amyloid-β protein and neurofibrillary tangles, leading to neuronal loss and brain atrophy. Aβ peptides, neurotoxic peptides composed of 39 to 43 amino acids, are centrally characterized by the abnormal aggregation of amyloid plaque deposition and neuroinflammatory plaque formation. Studies have shown that the level of intrabrain accumulation of the amyloid-β protein 1–42 fragment (Aβ42) is closely related to the pathological processes of Alzheimer's disease, and changes in Aβ42 concentration are considered a potential biomarker for early diagnosis of AD.

[0003] In the early stages of Alzheimer's disease (AD), patients often experience few or no symptoms, making it difficult for healthcare providers to identify the disease in a timely manner. Therefore, most patients are diagnosed only in the later stages of the disease, when symptoms become more pronounced and the problems are more severe. Early screening and identification of potential patients, along with early intervention or treatment, would improve patients' quality of life, reduce the burden of social care, and raise the nation's overall health level. Currently, existing diagnostic methods are often costly and not readily available.

[0004] Imaging techniques such as cranial magnetic resonance imaging (MRI) and positron emission tomography (PET) are widely used in the diagnosis of Alzheimer's disease (AD), but their high cost and limited accessibility hinder early and timely detection. While cerebrospinal fluid (CSF)-based assays can quantify Aβ peptides, Aβ oligomers, and tau protein, CSF collection is invasive, requires hospitalization, and carries procedural risks. Therefore, there is an urgent need for affordable, non-invasive diagnostic alternatives to facilitate early identification and intervention. Blood-based biomarker assays offer a less invasive, cost-effective, and highly scalable approach suitable for longitudinal monitoring of disease progression and treatment response. Blood contains a variety of biomolecules, including proteins, peptides, lipids, and metabolites, which dynamically reflect central nervous system activity, offering significant potential for clinical diagnostics and large-scale population screening.

[0005] The concentration of Aβ in blood is typically one to two orders of magnitude lower than that in CSF, ranging from fg / mL to pg / mL. This low concentration is significantly affected by the complexity of the blood matrix, posing a major challenge to the sensitivity, specificity, and anti-interference capabilities of analytical detection. Therefore, there is an urgent need for a rapid, accurate, and cost-effective method to simultaneously quantify Aβ42 and Aβ40 to advance the early diagnosis of Alzheimer's disease. Photoelectrochemical (PEC) biosensing is an innovative detection technology that combines high sensitivity, ease of miniaturization, and potential for instrument simplification. However, traditional PEC sensors typically operate in a single-channel mode, limiting their multi-target detection capabilities and analytical efficiency. With the increasing demand for precision diagnosis, the development of PEC immunosensors with higher throughput and accuracy is particularly urgent. To this end, researchers have proposed various strategies based on multi-channel detection. Based on the structural design of the substrate electrode, existing multi-channel PEC immunosensors can be mainly divided into two categories: single-electrode mode and electrode array mode. The single-electrode mode achieves independent recognition of multiple targets by introducing multiple enzyme labeling strategies or using photoactive materials that respond to specific wavelengths. Nevertheless, this mode still falls short of meeting the demands of truly high-throughput detection. In contrast, the electrode array mode, combining multiple independent electrodes with a multi-channel electrochemical workstation, possesses the capability to achieve high-throughput detection. However, it still faces the technical challenge of inconsistent responses between electrodes in practical applications.

[0006] As key components of PEC biosensors, photoactive materials—such as quantum dots, heterojunctions, nanocomposites, or hybrid materials, as well as covalent organic frameworks (COFs)—play a central role in achieving efficient signal conversion and detection. COFs, in particular, are crystalline porous materials formed by tunable organic molecular units linked by strong covalent bonds, possessing an extended π-conjugated system and a highly ordered pore structure. Their structural stability stems from the strong and rigid covalent bonds formed between light elements such as hydrogen, carbon, nitrogen, and oxygen, typically constructed through Schiff base condensation reactions. This intrinsic stability, combined with extensive π-electron delocalization, narrow bandgap characteristics, and abundant functionalizable sites on the surface, gives COFs significant advantages in constructing photoelectro-responsive biosensors. However, their practical applications are still limited by the rapid recombination of photogenerated electron-hole pairs and energy losses during excited-state charge transport, resulting in relatively low overall photoelectric conversion efficiency. Therefore, developing high-performance COF-based PEC biosensors remains a significant challenge. To address these challenges, researchers have proposed several effective strategies: introducing photosensitizers into the COF framework to enhance light absorption; precisely controlling the band structure through ligand engineering; and constructing donor-acceptor (DA) conjugated systems within the framework to promote charge separation and migration. These strategies synergistically optimize the material's photoresponse range, carrier separation efficiency, and charge transport kinetics, thereby significantly improving the sensitivity and response performance of COF-based biosensors in photoelectrochemical detection. Summary of the Invention

[0007] This invention provides a photocathode material based on carbon dots@covalent organic frameworks, its preparation method, and its application. First, an ultrathin TAPA-DHNDA-COF (TD-COF) film is constructed in situ on a fluorine-doped tin oxide (FTO) conductive substrate to build a sensing interface. This COF material is formed by the condensation of tris(4-aminophenyl)amine (TAPA) and 2,6-dihydroxynaphthalene-1,5-dicarboxaldehyde (DHNDA). The resulting TD-COF has a highly ordered porous structure and exhibits strong adhesion to the FTO substrate, which is beneficial for improving the structural stability and interfacial charge transport efficiency of the material. To further optimize its photoelectric performance, carbon dots (CDs) are introduced into the COF channels to construct a CDs@TAPA-DHNDA-COF (CDs@TD-COF) nanocomposite material. This composite structure outperforms the original COF material in both light absorption and electronic conductivity. An Aβ42 capture antibody (Ab(Aβ42)) was immobilized on the electrode surface as a recognition element. As the Aβ42 antigen gradually binds, the cathodic photocurrent of the CDs@TD-COF / FTO electrode decreases in a gradient, indicating that the photocurrent intensity is negatively correlated with the Aβ42 concentration.

[0008] The first objective of this invention is to provide a photocathode material based on carbon dots@covalent organic frameworks, wherein the photocathode material is a CDs@TD-COF thin film grown in situ on a conductive substrate, wherein the carbon dots (CDs) in the CDs@TD-COF thin film are uniformly distributed in the COF channels formed by the condensation of tris(4-aminophenyl)amine (TAPA) and 2,6-dihydroxynaphthalene-1,5-dicarboxaldehyde (DHNDA).

[0009] Further, the CDs are prepared by the following method: citric acid and ethylenediamine are added to ultrapure water and reacted at 180~220℃ for 4~6 h. After cooling to room temperature, the reaction solution is filtered through a filter membrane and freeze-dried to obtain the CDs. The mass-volume ratio of citric acid, ethylenediamine and ultrapure water is 0.4 g : 0.3~0.6 mL : 8~12 mL.

[0010] Furthermore, the thickness of the CDs@TD-COF film is 0.1~0.3 mm.

[0011] The second objective of this invention is to provide a method for preparing the aforementioned CDs@TD-COF photocathode material, comprising the following steps: dissolving TAPA and DHNDA in a mixed solvent prepared by mixing ethanol, thiobenzene, and acetic acid to obtain a TAPA solution and a DHNDA solution, respectively; dissolving CDs in an ethanol solution to obtain a CDs solution; mixing the above three solutions and drop-coating them onto the surface of a conductive substrate, incubating at room temperature, washing, and drying to obtain a CDs@TD-COF thin film, which is the photocathode material.

[0012] Furthermore, the volume ratio of ethanol, thiol, and acetic acid in the mixed solvent is 3~6 : 3~6 : 1.

[0013] Further, the volume ratio of the TAPA solution, DHNDA solution, and CDs solution is 3~5 : 3 : 1~3, wherein the concentration of the TAPA solution and DHNDA solution is 1.5~2 mg / mL, and the concentration of the CDs solution is 30~50 mg / mL.

[0014] Furthermore, the room temperature incubation time is 10-20 minutes.

[0015] Furthermore, the conductive substrate is a fluorine-doped tin oxide (FTO) conductive substrate.

[0016] A third objective of this invention is to provide a method for preparing an immune sensing array based on the aforementioned photocathode material, comprising the following steps:

[0017] (1) Dissolve TAPA and DHNDA in a mixed solvent prepared by mixing ethanol, thiol and acetic acid to obtain TAPA solution and DHNDA solution respectively; dissolve CDs in ethanol solution to obtain CDs solution; mix the above three solutions and drop them onto the circular cutouts on the surface of the conductive substrate with the circular cutout sticker, the amount of drop coating on each circular cutout is 30-60 μL, incubate at room temperature, wash and dry to obtain multiple independent circular detection points with photocathode material; (2) Drop 40-60 μL of Aβ42 capture antibody with a concentration of 10-25 μg / mL onto the surface of each circular detection point of the above photocathode material, incubate at 37°C for 60-80 min, wash with PBS at pH 7.4, and air dry; then drop 30-60 μL of bovine serum albumin solution with a concentration of 1-3 wt% onto the surface of each circular detection point, incubate at 37°C for 30-60 min to block the sites on the surface where no antibody is bound, and air dry to obtain the final product.

[0018] A fourth objective of this invention is to provide an immune sensing array prepared by the above method.

[0019] The sensor array described in this invention preferably consists of seven detection points arranged in a circle, with each detection point physically isolated from the others by insulating stickers to ensure the independence of the signal regions and the accuracy of the measurements. The system employs a sequential illumination strategy, using a single light source to excite each detection point in turn, achieving continuous, accurate signal acquisition with high throughput potential. The first detection point is set as a self-calibration reference point to correct background interference in real time and compensate for signal fluctuations caused by fabrication differences between electrodes.

[0020] A fifth object of the present invention is to provide a method for detecting Aβ42 using the immunosensor array obtained above, comprising the following steps: (1) Solution preparation: Prepare blank buffer and Aβ42 standard sample solutions with different concentration gradients; (2) Plotting the Aβ42 standard curve: Add blank buffer to the first circular detection point on the above-mentioned immunosensor array as a control, and add Aβ42 standard sample solutions of different concentrations to the remaining detection points in sequence. Incubate at 37℃ for 60~80 min as the working electrode. Then, use the Ag / AgCl electrode as the reference electrode and the platinum sheet electrode as the counter electrode to form a three-electrode system. Using a sequential illumination strategy, each detection point is excited sequentially using a single light source. The It method is used to detect the change in photocurrent value before and after the light source is switched on and off at each detection point under the illumination of the light source. Plot the standard curve with the change in photocurrent value as the vertical axis and the logarithm of the Aβ42 concentration as the horizontal axis. (3) Detection of Aβ42: Replace the Aβ42 standard sample solution in step (2) with the test solution, and perform the detection in the rest according to the method described in step (2). Based on the photocurrent intensity and the standard curve, the concentration of Aβ42 in the test solution can be obtained.

[0021] Preferably, the concentrations of the Aβ42 standard sample solutions are 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, and 100 pg / mL, respectively.

[0022] Furthermore, in step (2), in the three-electrode system, the electrolyte solution is a PBS buffer solution containing ascorbic acid.

[0023] Furthermore, in step (2), the excitation light source is a 20 W ultraviolet lamp with an ultraviolet wavelength of 405 nm.

[0024] Furthermore, in step (2), the detection time range of the It method is 0~120 s, the power switch interval is once every 10 s, and the bias voltage is 0 V.

[0025] The beneficial effects of this invention are as follows: This invention successfully constructs a high-throughput photoelectrochemical immunosensor array based on COF heterojunction material, achieving efficient and sensitive detection of Aβ42, a biomarker related to Alzheimer's disease (AD). By introducing carbon dots (CDs) into TAPA-DHDNA-COF (TD-COF), the photogenerated carrier generation and transfer efficiency of TD-COF is significantly enhanced, and a photoelectrochemical immunosensor array capable of accurately identifying trace amounts of Aβ42 in human plasma is constructed based on CDs@TD-COF. Experimental results not only verify the key role of this sensor array in the early diagnosis of AD, but also further confirm the important value of plasma biomarkers, represented by Aβ42, in the timing of disease intervention. The constructed sensor array exhibits excellent analytical performance, achieving the detection of Aβ42 in the range of 1 fg / mL to 100 pg / mL. This invention introduces an optically addressed detection mechanism and a self-calibration strategy, effectively reducing signal differences between electrodes and improving the reproducibility and accuracy of detection, thereby achieving high-throughput detection of different samples. By optimizing key parameters such as buffer pH and antigen incubation time, the reaction efficiency and overall response stability of the sensing interface were further improved. Specificity experiments showed that the sensor exhibits high selectivity for target analytes in complex biological samples; long-term stability tests showed that it maintained over 93.7% of its initial response after 30 days of continuous use. The relative standard deviation (RSD) of the sensor arrays prepared in multiple batches was less than 0.43%, confirming its good reproducibility and practical application feasibility. In summary, the Aβ42 photoelectrochemical immunosensor array provided by this invention represents a significant breakthrough in sensitivity, specificity, and practicality, and is particularly suitable for the reliable detection of extremely low concentrations of biomarkers in complex biological matrices, showing broad prospects for clinical translational applications. This invention provides a replicable technical path for developing novel biosensing technologies for neurodegenerative diseases and is expected to promote the development of non-invasive, dynamic monitoring technologies for various major disease biomarkers. Attached Figure Description

[0026] Figure 1 Characterization of CDs@TD-COF obtained in Example 1: A, SEM image of FTO electrode; B, SEM image of TD-COF / FTO; C, SEM image of CDs@TD-COF / FTO; D, XRD patterns of TD-COF and CDs@TD-COF; E, XPS full spectrum of CDs@TD-COF; F, Carbon spectrum of CDs@TD-COF; G, Nitrogen spectrum of CDs@TD-COF.

[0027] Figure 2 The FI-IR spectrum of CDs@TD-COF obtained in Example 1 is shown.

[0028] Figure 3 The UV-vis absorption spectra of CDs, TD-COF, and CDs@TD-COF in Example 1 are shown.

[0029] Figure 4 The fluorescence emission spectra of TD-COF and CDs@TD-COF in Example 1 are shown.

[0030] Figure 5 The image shows the detection results of different concentrations of Aβ42 by the photocathode-type PEC immunosensor array obtained in Example 2; A, detection performance (a: 1 fg / mL, b: 10 fg / mL, c: 100 fg / mL, d: 1 pg / mL, e: 10 pg / mL, f: 100 pg / mL), B, calibration curve on logarithmic coordinates, concentration range: 1 fg / mL to 100 pg / mL.

[0031] Figure 6 The image shows the specificity evaluation results of the photocathode-type PEC immunosensor array obtained in Example 2, where the concentration of the interfering substance is 100 pg / mL and the concentration of the target substance is 1 pg / mL.

[0032] Figure 7 The graph shows the repeatability (n=8) results of the photocathode-type PEC immunosensor array obtained in Example 2 for detecting 1 pg / mL Aβ42.

[0033] Figure 8 The following figures show the stability test results of the photocathode-type PEC immunosensor array obtained in Example 2: A, short-term stability test; B, long-term stability test. Detailed Implementation

[0034] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0035] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0036] Example 1 A method for preparing a CDs@TAPA-DHNDA-COF (CDs@TD-COF) photocathode material includes the following steps: 0.42 g of citric acid and 0.536 mL of ethylenediamine were added to 10 mL of ultrapure water and heated in a vacuum drying oven at 200 °C for 5 h. After cooling to room temperature, the reaction solution was filtered through a 0.22 μm filter membrane to obtain an orange CDs solution. The CDs solution was lyophilized and reconstituted with 10 mL of ethanol for later use.

[0037] A mixed solvent was prepared by mixing ethanol, trimethylbenzene, and acetic acid in a volume ratio of 5:5:1. 1 mg of TAPA and 1 mg of DHNDA were dissolved separately in 550 μL of the mixed solvent. 300 μL of the TAPA solution, 300 μL of the DHNDA solution, and 100 μL of the reconstituted CDs solution were mixed. This mixture was then drop-coated onto the surface of an FTO conductive substrate and incubated at room temperature for 10 min. Finally, the CDs@TD-COF / FTO electrode was immersed in dichloromethane to elute excess monomer, and then dried in air to obtain a rose-red CDs@TD-COF thin film attached to the conductive substrate, which is the CDs@TD-COF photocathode material.

[0038] Characterization of CDs@TD-COF photocathode material The surface morphology of FTO, TD-COF / FTO (a photocathode material without carbon dots), and CDs@TD-COF / FTO was characterized using scanning electron microscopy (SEM). The results are shown in the figure. Figure 1 It can be seen that the original FTO surface exhibits a clear crystal structure. Figure 1 A), while TD-COF / FTO and CDs@TD-COF / FTO samples showed typical TD-COF polymer layered structures ( Figure 1 B and Figure 1 C), indicating that the material was successfully loaded onto the conductive substrate. To further reveal its crystal structure characteristics, X-ray diffraction (XRD) analysis was performed. Multiple significant diffraction peaks appeared in the XRD pattern of TD-COF, indicating its high crystallinity; among them, the weak reflection peak and the broad diffraction peak at approximately 25° belonging to the (001) crystal plane further confirmed the ordered assembly of TD-COF in a two-dimensional layered crystal framework. The XRD pattern of the CDs@TD-COF nanocomposite was highly consistent with that of pure TD-COF, indicating that the intrinsic crystal structure of TD-COF remained intact and well-preserved after the introduction of CDs. Figure 1 D). Furthermore, the elemental composition and chemical state of CDs@TD-COF were analyzed by X-ray photoelectron spectroscopy (XPS), and characteristic signals of C, N, and O were detected at 284.8 eV, 400.0 eV, and 532.0 eV, respectively. Figure 1 E). The high-resolution C 1s spectrum can be fitted to the peak positions corresponding to C=N, C=C, and C=O bonds, indicating the presence of abundant conjugated structures and polar functional groups in the material. Figure 1 F); the N 1s spectrum showed the presence of C=N, CN, and -NH bonds, further verifying the successful construction of the TD-COF skeleton (F). Figure 1 G).

[0039] The Fourier transform infrared (FT-IR) spectra of this material are shown below. Figure 2 The results showed that a Schiff base structure exists in TD-COF, which is formed by the condensation reaction between an aldehyde group and an amine group. This is particularly evident at 1620 cm⁻¹. - The C=N stretching vibration peak appearing at position ¹ clearly confirms the successful construction of the imine bond. Figure 2 The optical properties of the material were further characterized by UV-Vis diffuse reflectance spectroscopy and fluorescence spectroscopy, and the results are shown in [Figure number missing]. Figure 3 and Figure 4 It is noteworthy that carbon dots exhibit significant absorption characteristics in the 300–400 nm range. Introducing them into TD-COF enhances the overall light absorption capacity of the composite material, which is beneficial for promoting the generation and separation efficiency of photogenerated carriers. Figure 3 The fluorescence test results of TD-COF and CDs@TD-COF provide strong evidence for this. Generally, stronger fluorescence emission implies a higher photogenerated carrier recombination rate. When the excitation wavelength is 300 nm, the fluorescence intensity of CDs@TD-COF at 690 nm is significantly lower than that of pure TD-COF. This obvious fluorescence quenching phenomenon indicates that the introduction of carbon dots effectively suppresses the electron-hole recombination process and plays a key role in improving charge separation efficiency. Figure 4 ).

[0040] Example 2 A method for preparing a photocathode-type PEC immune sensing array based on the photocathode material described in Example 1 includes the following steps: 0.42 g of citric acid and 0.536 mL of ethylenediamine were added to 10 mL of ultrapure water and heated in a vacuum drying oven at 200 °C for 5 h. After cooling to room temperature, the reaction solution was filtered through a 0.22 μm filter membrane to obtain an orange CDs solution. The CDs solution was lyophilized and reconstituted with 10 mL of ethanol for later use.

[0041] A mixed solvent was prepared by mixing ethanol, thiol, and acetic acid in a volume ratio of 5:5:1. Circular perforated insulating stickers, each with a diameter of 10 mm, were adhered to the surface of an FTO conductive substrate in a straight line. 1 mg of TAPA and 1 mg of DHNDA were dissolved separately in 550 μL of the above mixed solvent. 300 μL of TAPA solution, 300 μL of DHNDA solution, and 100 μL of reconstituted CDs solution were mixed. This mixture was then drop-coated onto the circular perforations on the FTO electrode surface, 40 μL per perforation, and incubated at room temperature for 10 min. Finally, the CDs@TD-COF / FTO electrode was immersed in dichloromethane to elute excess monomer, and then dried in air to obtain a rose-red CDs@TD-COF thin film adhered to the conductive substrate, resulting in multiple independent circular detection points with photocathode material.

[0042] 40 μL of p-tau217 capture antibody at a concentration of 10 μg / mL was dropped onto the surface of each circular detection point of the photocathode material obtained above. The mixture was incubated at 37°C for 60 min, and each detection point was thoroughly washed with PBS buffer at pH 7.4 and allowed to air dry. After antibody fixation, 40 μL of 1 wt% BSA solution was dropped onto the surface of each circular detection point and incubated at 37°C for 30 min to block the sites on the surface where no antibody was bound. The mixture was then allowed to air dry to obtain the CDs@TD-COF / FTO immunosensor array.

[0043] A method for detecting amyloid β-like protein fragments 1-42 based on the above-obtained photocathode-type PEC immunosensor array includes the following steps: (1) Solution preparation: Prepare blank buffer and Aβ42 standard sample solutions with different concentration gradients. The Aβ42 antigen concentration gradients are 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL and 100 pg / mL, respectively.

[0044] (2) Plotting the Aβ42 standard curve: 40 μL of blank buffer was added to the first circular detection point on the obtained immunosensor array as a control, and 40 μL of Aβ42 standard sample solution of different concentrations was added to the remaining detection points in sequence. The mixture was incubated at 37℃ for 60 min to form the DT-COF@P5FIn+Ab(p-tau217)+BSA+p-tau217 immune complex, which was used as the working electrode. Then, the Ag / AgCl electrode was used as the reference electrode and the platinum electrode was used as the counter electrode. The three electrodes formed a three-electrode system. 30 mL of PBS buffer solution containing 30 mM ascorbic acid (AA) was used as the electrolyte solution. A sequential illumination strategy was adopted, and each detection point was excited in sequence using a single light source (20 W UV lamp with a UV wavelength of 405 nm). The It method was used to record the current response change of each detection point under the light source under a bias voltage of 0 V. The light source was switched on and off alternately every 10 s. The standard curve was plotted with the photocurrent intensity as the ordinate and the logarithm of the Aβ42 concentration as the abscissa. Photocurrent curves of the working electrode under both illuminated and unilluminated conditions were collected using an electrochemical workstation. A cycle was defined as the UV lamp switching on and off every 10 seconds, and the difference between the highest and lowest points on the ordinate was taken as the photocurrent intensity. The resulting standard curve is shown below. Figure 5 The regression equation is Photocurrent(μA) = 0.20197 log C Aβ42 + 0.05584 (R 2 = 0.993). After calibration, batch-to-batch variability was significantly reduced. The difference between the calibration point and the detection signal was linearly related to the logarithm of the p-tau217 concentration. The corrected regression equation was Photocurrent (μA) = 0.05694 log C Aβ42 – 0.19504 (R 2 = 0.993). The detection limit of this method reaches 0.15 fg / mL, demonstrating extremely high sensitivity.

[0045] (3) Detection of Aβ42: Replace the Aβ42 standard sample solution in step (2) with the test solution, and perform the detection in the rest according to the method described in step (2). Based on the photocurrent intensity and the standard curve, the concentration of Aβ42 in the test solution can be obtained.

[0046] The Ag / AgCl electrode, used as a reference electrode, ensured the reproducibility of the experiment. The platinum sheet electrode, used as the counter electrode, ensured the current flow on the working electrode, guaranteeing that the studied reaction occurred on the working electrode. Adding AA to the PBS buffer solution as an electrode donor facilitated the timely consumption of electrons and holes on the electrode during the reaction, promoting its continuation. Adopting these parameters resulted in better photocurrent performance.

[0047] Example 3 Using the CDs@TD-COF / FTO immunosensor array prepared in Example 2, a specific recognition experiment was performed on Aβ42 that may be present in human plasma. The test solutions incubated on the photocathode-type PEC immunosensor array consisted of 100 pg / mL of interfering agents and 1 pg / mL of Aβ42. The interfering agents were phosphorylated tau protein 217 (p-tau217), carcinoembryonic antigen (CEA), cardiac troponin (cTnT), alpha-fetoprotein (AFP), dopamine (DA), human serum albumin (HSA), and bovine serum albumin (BSA). After incubation, the working electrode was thoroughly cleaned with PBS buffer, and the photocurrent intensity was measured using the same procedure as in Example 2. The results are as follows: Figure 7 As shown, the photocurrent decreases significantly in the presence of Aβ42. In the absence of Aβ42, incubation of the interfering material does not cause a significant change in the photocurrent on the electrodes. This indicates that the CDs@TD-COF / FTO array exhibits excellent selectivity and specificity for Aβ42.

[0048] Example 4 Eight CDs@TD-COF / FTO arrays were prepared according to the method for preparing photocathode-type PEC immunosensor arrays in Example 2. Aβ42 at a concentration of 1 pg / mL was detected to evaluate the repeatability of the sensor arrays. The results are as follows: Figure 8 As shown, the photocurrent intensities measured by the eight CDs@TD-COF / FTO arrays are similar, with a relative standard deviation (RSD) of 0.43%, indicating that the array has good repeatability.

[0049] Example 5 The CDs@TD-COF / FTO array prepared in Example 2 was used to detect Aβ42 at a concentration of 1 pg / mL. Multiple on / off light cycles were applied over a period of 485 s, and the photocurrent response was monitored. The results are as follows: Figure 8 As shown in Figure A, it can be seen that the photocurrent intensity remains basically stable under repeated light switching cycles, indicating that the array has good short-term stability.

[0050] Example 6 The CDs@TD-COF / FTO array prepared in Example 2 was used to detect Aβ42 at a concentration of 1 pg / mL. The change in the photocurrent response of the array to Aβ42 detection after four weeks of storage was investigated. The results are as follows: Figure 8 As shown in Figure B, it can be seen that after four weeks of storage, the photocurrent response signal still maintains 93.7% of the initial value, indicating that the sensor array has good long-term storage stability.

[0051] Example 7 The feasibility and applicability of the CDs@TD-COF / FTO array prepared in Example 2 for detecting Aβ42 concentration in human plasma were evaluated. Human plasma samples containing different concentrations of Aβ42 were used as analytes and incubated on the CDs@TD-COF / FTO array. Human plasma was diluted 200-fold with PBS buffer at pH 7.4, and three concentration levels of Aβ42 standard solutions (low (sample 1), medium (sample 2), and high (sample 3)) were added to form spiked samples, which were then measured according to the detection method described in Example 2. The results are shown in Table 1. It can be seen that the average recovery rate of Aβ42 in diluted human plasma ranged from 98.9% to 103.2%, with a relative standard deviation (RSD) not exceeding 4.3%. These results indicate that the CDs@TD-COF / FTO array constructed using this method can be used for the determination of Aβ42 in human plasma, possessing good accuracy and precision, and high detection sensitivity. It has potential application value and promotion prospects in the fields of bioanalysis and clinical medicine.

[0052] Table 1. Experimental results of CDs@TD-COF / FTO array for detecting Aβ42 in human plasma (n = 3)

Claims

1. A photocathode material based on carbon dots@covalent organic frameworks, characterized in that: The photocathode material is a CDs@TD-COF thin film grown in situ on a conductive substrate. In the CDs@TD-COF thin film, carbon dots (CDs) are uniformly distributed in the COF channels formed by the condensation of tris(4-aminophenyl)amine (TAPA) and 2,6-dihydroxynaphthalene-1,5-dicarboxaldehyde (DHNDA).

2. The photocathode material according to claim 1, characterized in that: The CDs are prepared by the following method: citric acid and ethylenediamine are added to ultrapure water and reacted at 180~220℃ for 4~6 h. After cooling to room temperature, the reaction solution is filtered through a filter membrane and freeze-dried to obtain the CDs. The mass-volume ratio of citric acid, ethylenediamine and ultrapure water is 0.4 g : 0.3~0.6 mL : 8~12 mL.

3. The photocathode material according to claim 1, characterized in that: The thickness of the CDs@TD-COF film is 0.1~0.3 mm.

4. The method for preparing the photocathode material according to any one of claims 1 to 3, characterized in that: TAPA and DHNDA were dissolved in a mixed solvent prepared by mixing ethanol, thiobenzene, and acetic acid to obtain TAPA solution and DHNDA solution, respectively. CDs were dissolved in an ethanol solution to obtain CDs solution. The above three solutions were mixed and drop-coated onto the surface of a conductive substrate, incubated at room temperature, washed, and dried to obtain CDs@TD-COF thin film, which is the photocathode material.

5. The preparation method according to claim 4, characterized in that: The volume ratio of ethanol, thiol, and acetic acid in the mixed solvent is 3-6: 3-6: 1; the volume ratio of TAPA solution, DHNDA solution, and CDs solution is 3-5: 3: 1-3, wherein the concentration of TAPA solution and DHNDA solution is 1.5-2 mg / mL, and the concentration of CDs solution is 40-60 mg / mL.

6. The preparation method according to claim 4, characterized in that: The room temperature incubation time is 10~20 min; the conductive substrate is a fluorine-doped tin oxide (FTO) conductive substrate.

7. A method for preparing an immune sensing array based on the photocathode material of claim 1, characterized in that: Includes the following steps: (1) Dissolve TAPA and DHNDA in a mixed solvent prepared by mixing ethanol, thiol and acetic acid to obtain TAPA solution and DHNDA solution respectively; dissolve carbon dots in ethanol solution to obtain CDs solution; mix the above three solutions and drop them onto the circular cutouts on the surface of the conductive substrate with the circular cutout sticker, the amount of drop coating on each circular cutout is 30-60 μL, incubate at room temperature, wash and dry to obtain multiple independent circular detection points with photocathode material; (2) Drop 40-60 μL of Aβ42 capture antibody with a concentration of 10-25 μg / mL onto the surface of each circular detection point of the above photocathode material, incubate at 37°C for 60-80 min, wash with PBS at pH 7.4, and air dry naturally; Next, drop 30-60 μL of 1-3 wt% bovine serum albumin solution onto the surface of each circular detection point, incubate at 37°C for 30-60 min to block the sites on the surface where no antibody has bound, and then air dry naturally to obtain the final product.

8. A method for detecting Aβ42 using an immunosensor array prepared according to claim 7, characterized in that: Includes the following steps: (1) Solution preparation: Prepare blank buffer and Aβ42 standard sample solutions with different concentration gradients; (2) Plotting the Aβ42 standard curve: Add blank buffer to the first circular detection point on the immunosensing array described in claim 7 as a control, and add Aβ42 standard sample solutions of different concentrations to the remaining detection points in sequence. Incubate at 37°C for 60~80 min as the working electrode. Then, use the Ag / AgCl electrode as the reference electrode and the platinum sheet electrode as the counter electrode. The three form a three-electrode system. A sequential illumination strategy was adopted, using a single light source to excite each detection point in turn. The It method was used to detect the change in photocurrent value before and after the light source was switched on at each detection point under illumination. A standard curve was plotted with the change in photocurrent value as the ordinate and the logarithm of Aβ42 concentration as the abscissa. (3) Detection of Aβ42: Replace the Aβ42 standard sample solution in step (2) with the test solution, and perform the detection in the rest according to the method described in step (2). Based on the photocurrent intensity and the standard curve, the concentration of Aβ42 in the test solution can be obtained.

9. The detection method according to claim 8, characterized in that: In step (2), in the three-electrode system, the electrolyte solution is a PBS buffer solution containing ascorbic acid.

10. The detection method according to claim 8, characterized in that: In step (2), the excitation light source is a 20 W ultraviolet lamp with an ultraviolet wavelength of 405 nm; the detection time range of the It method is 0~120 s, the power switch interval is once every 10 s, and the bias voltage is 0 V.

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