Photoelectrochemical sandwich immunosensor and sensing test paper for detecting Alzheimer's disease biomarker starch beta-like protein as well as preparation and application of photoelectrochemical sandwich immunosensor and sensing test paper

Abstract

The invention belongs to the technical field of photoelectrochemical PEC biosensors, and particularly relates to a PEC sandwich immunosensor for detecting a starch beta-like protein 1-42 fragment A beta 1-42 of an AD biomarker of Alzheimer's disease, sensing test paper and preparation and application of the PEC sandwich immunosensor and the sensing test paper. The sensor or the sensing test paper takes a U-coated C composite material as an immune matrix, is combined with a capture antibody Ab1, and seals uncombined recognition sites by using bovine serum albumin (BSA), so that A beta1-42 is specifically captured and recognized; pDA / P-Z is taken as a signal substance of the immune probe, and is combined with a detection antibody Ab2, and then A beta 1-42 is specifically captured and recognized, so that a U-C-Ab1-BSA-(A beta 1-42)-Ab2-PDA / P-Z sandwich immune complex is formed, and a detection signal is enhanced; the PEC sandwich immunosensor and the PEC-LFIA test strip constructed by the invention can accurately detect trace Abeta1-42 in artificial cerebrospinal fluid and human plasma, and show good linearity and reproducibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of photoelectrochemical biosensors, and specifically relates to a photoelectrochemical sandwich immunosensor for detecting the Alzheimer's disease biomarker amyloid β-like protein, a sensor paper, and their preparation and application. Background Art

[0002] Data from the National Bureau of Statistics for 2023 shows that my country has 280 million people aged 60 and over, accounting for approximately 19.84% of the total population, and 210 million people aged 65 and over, accounting for approximately 14.86% of the total population, indicating that my country has rapidly entered a deeply aging society. Alzheimer's disease (AD) is the most common type of dementia and a common condition that causes loss of daily living abilities in the elderly. The "China Alzheimer's Disease Report 2024" reveals that there are over 16.99 million cases of AD and other dementias in my country, with the prevalence and mortality rates of AD and other dementias in my country slightly higher than the global average. AD and related dementias are the fifth leading cause of death in my country and have become one of the most expensive, lethal, and burdensome medical conditions, seriously impacting public health and sustainable social development in my country. However, the diagnosis and treatment rates for AD in my country remain low, with a shortage of specialized physicians and insufficient awareness of the disease. In response to the call of the "Healthy China Action (2019-2030)", preventing or delaying the occurrence and development of AD, reducing the disease burden, improving the national health level, strengthening the prevention and diagnosis and treatment of AD, slowing the occurrence and development of AD, and reducing the social and family burden are public health issues that need to be addressed urgently. It is also an important part of the national strategy to actively respond to population aging.

[0003] The application of validated biomarkers has become a key tool for early diagnosis and progression monitoring of AD. In July 2023, the Alzheimer's Association announced new draft diagnostic criteria for Alzheimer's disease, which divided biomarkers into three categories. The latest framework also added blood biomarker recommendations. AD biomarkers include amyloid beta-like protein, phosphorylated tau protein, glial fibrillary acidic protein, and neurofilament light chain. Amyloid beta-like protein and phosphorylated tau protein can be used for early screening of high-risk populations, while phosphorylated tau protein, glial fibrillary acidic protein, and neurofilament light chain are often used to assess disease progression. "ATN" is a biological framework for AD research proposed by the National Institute on Aging and the Alzheimer's Association of the United States, where "A" stands for Aβ, "T" stands for Tau protein, and "N" stands for biomarkers associated with neurodegeneration. Currently, clinical testing for amyloid-β and phosphorylated Tau protein is performed by extracting cerebrospinal fluid using positron emission tomography (PET) combined with radioactive tracers or lumbar puncture. However, these tests are limited by their high cost and the need for a professional nursing environment, and subjects have a psychological resistance to invasive examinations. In contrast, peripheral blood-based biomarker testing provides a promising alternative. The collection process of this method is widely available and has advantages over PET scanning and lumbar puncture in terms of feasibility. More importantly, blood tests are more accessible to non-professionals and can be rapidly promoted to cope with the increasing number of AD cases. To this end, this project proposes to use plasma as the main biological sample and amyloid-β protein 1-42 fragment (Aβ1-42) as a representative AD marker to study AD detection technology with high sensitivity and strong accuracy.

[0004] The concentration of AD biomarkers in plasma is typically ten times lower than in cerebrospinal fluid, making efficient screening for AD biomarkers in plasma a significant challenge. Recent studies have demonstrated that ultrasensitive detection methods are crucial for effective analysis of plasma biomarkers. Photoelectrochemical (PEC) sandwich immunosensors show promising application in this regard. This technology enhances the PEC phenomenon through the interaction between a modified substrate on an electrode and a labeled conjugate attached to a secondary antibody or DNA strand. Two specific antibodies are used to detect the same antigen, enhancing selectivity through a dual recognition mechanism: the capture antibody is immobilized on a solid phase and binds to the target antigen, while the detection antibody is attached to a different site. This design effectively minimizes nonspecific binding and background noise, thereby improving signal clarity. PEC sandwich immunosensors perform well in complex biological samples such as serum and urine, providing accurate results without the need for sample purification. Their high sensitivity enables detection of target antigens even at low concentrations, making them a reliable antigen detection method. The advantage of this technology lies in its ability to rapidly and accurately identify AD biomarkers, providing important support for early diagnosis and intervention. Summary of the Invention

[0005] To construct an ultrasensitive PEC sandwich immunosensor, the present invention used cadmium telluride quantum dot (CdTe-QDs)-sensitized UiO-66 (U@C) as the PEC immunomatrix and a composite heterojunction of polydopamine (PDA)-modified PCN-224 and indium zinc sulfide (ZnIn2S4, ZIS) (PDA / P@Z) as the PEC immunoprobe label. Subsequently, an amyloid β-like protein 1-42 fragment (Aβ1-42) capture antibody (Ab1) and the PEC immunoprobe were integrated into a lateral flow immunoassay strip. A custom-made dual-working electrode printed electrode was then combined with the lateral flow immunoassay strip to create a PEC-based lateral flow immunoassay strip (PEC-LFIA strip). The prepared PEC sandwich immunosensor and PEC-LFIA strip demonstrated the potential to recognize Aβ1-42 in the fg / mL to ng / mL range in samples such as PBS buffer, artificial cerebrospinal fluid, and human plasma. In addition, the classification learning K-nearest neighbor algorithm was used to predict the Aβ1-42 concentration under three environments. By analyzing and integrating a large amount of experimental data, an AI prediction model for Aβ1-42 concentration was constructed, which provides an important research basis for early screening of AD and the preparation of large-scale instruments and equipment.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a PEC sandwich immunosensor. The surface of an FTO electrode is covered with a U@C composite material as a PEC immune matrix, which is then combined with a capture antibody (Ab1) and bovine serum albumin (BSA) to block unbound recognition sites, thereby specifically capturing and identifying Aβ1-42. PDA / P@Z is then used as a signal substance of the immune probe and combined with a detection antibody (Ab2) to form a U@C-Ab1-BSA-(Aβ1-42)-Ab2-PDA / P@Z sandwich immune complex, which again specifically captures and identifies Aβ1-42 to enhance the detection signal.

[0008] The U@C composite material is UiO-66 sensitized by cadmium telluride quantum dots CdTe-QDs; the PDA / P@Z is a composite heterojunction of PCN-224 modified with polydopamine (PDA) and indium zinc sulfide (ZnIn2S4, ZIS).

[0009] In the above technical solution, in the U@C composite material, the mass ratio of UiO-66 and CdTe-QDs is 4-6:6-4;

[0010] The amount of U@C composite material covered on the surface of the FTO electrode is 1 mg / mL, 35 to 50 μL, wherein the size of the FTO electrode is 20×20×2.2 mm (length×width×thickness);

[0011] The amount of capture antibody Ab1 bound by the U@C composite material is 10-20 μg / mL, 40 μL, wherein the U@C composite material is 1 mg / mL, 45 μL;

[0012] The amount of the PDA / P@Z combined with the detection antibody Ab2 is 1.5 to 2.5 mg, wherein the amount of the detection antibody Ab2 is 25 μg / ml, 2 mL.

[0013] In the above technical solution, the preparation of U@C includes: dispersing UiO-66 and CdTe-QDs in a water-ethanol mixed solution, and ultrasonicating to obtain a U@C suspension;

[0014] The preparation of the PDA / P@Z comprises: in situ growing ZIS on the surface of PCN-224 by a hydrothermal method to obtain a P@Z heterojunction, and then modifying P@Z with PDA to obtain PDA / P@Z.

[0015] In the above technical solution, the preparation of the U@C composite material includes the following steps:

[0016] (1) Preparation of UiO-66: Dissolve zirconium chloride (ZrCl4) and terephthalic acid (TA) in a mixture of DMF and acetic acid, and heat to react; after cooling, centrifuge to collect the precipitate, wash with DMF, ethanol, and deionized water, dry, and calcine to obtain UiO-66;

[0017] (2) Preparation of CdTe-QDs: Dissolve hydrated cadmium chloride (CdCl2·2.5H2O) and 3-mercaptopropionic acid (MPA) in deionized water to obtain a cadmium precursor solution, adjust the pH and set aside; Dissolve tellurium powder (Te powder) and sodium borohydride (NaBH4) in deionized water, heat and stir under a nitrogen atmosphere, and then add them to the cadmium precursor solution to obtain a CdTe-QDs solution for heating reaction; Then, dialyze the obtained solution and freeze-dry it to obtain CdTe-QDs powder;

[0018] (3) UiO-66 and CdTe-QDs were added to a water-ethanol solution and ultrasonically treated to obtain a U@C suspension; the U@C suspension was freeze-dried to obtain a U@C freeze-dried powder for later use;

[0019] The preparation of the PDA / P@Z comprises the following steps:

[0020] (1) Zirconium chloride octahydrate (ZrOCl2·8H2O), tetracarboxyphenylporphyrin (H2TCPP), and benzoic acid (BA) were dissolved in DMF. The resulting mixture was stirred under heating to promote the formation of coordination bonds within the MOF. The product was washed and dried to obtain PCN-224 powder.

[0021] (2) The P@Z heterojunction is obtained by in situ growth of ZIS on the surface of PCN-224 by a hydrothermal method. PCN-224 is added to an aqueous solution containing zinc chloride (ZnCl2), indium chloride tetrahydrate (InCl3·4H2O) and sodium thiosulfate (TAA), stirred and mixed, and the mixed solution is heated to react. The product is washed and dried to obtain a P@Z heterojunction.

[0022] (3) Dopamine (DA) was dispersed in Tris-HCl, and then isopropanol was added. After magnetic stirring, the obtained dark brown suspension was centrifuged to collect the product, washed with deionized water, and freeze-dried for later use; P@Z and PDA were dispersed in deionized water, magnetically stirred, and washed with deionized water multiple times to obtain PDA / P@Z.

[0023] In the above technical solution, in the preparation steps of the U@C composite material:

[0024] In the step (1), the molar ratio of ZrCl4 and TA is 1:1, the volume ratio of the mixture of DMF and acetic acid is 9-13.5 mL:1-1.5 mL, the amount of ZrCl4 and TA and the ratio of the DMF and acetic acid mixture are 0.2 g:10-15 mL, the temperature of the heating reaction is 110-130°C, the reaction time is 20-25 hours, the temperature of the calcination is 100-200°C, and the calcination time is 10-18 hours;

[0025] In the step (2), the molar ratio of tellurium powder (Te powder) to sodium borohydride (NaBH4) is 5-10 mg:75-150 mg, the ratio of the amount of Te powder to NaBH4 and deionized water is 5-10 mg:5-10 mL, the reaction temperature is 50-70°C, and the reaction time is 40-70 min; the pH of the cadmium precursor solution is adjusted to 10-12 using 1M NaOH, and the obtained CdTe-QDs solution is reacted at 130-150°C for 2-3 h;

[0026] In the step (3), the mass ratio of UiO-66 and CdTe-QDs composite is 4-6:6-4; the volume of water-ethanol is 10-20 mL; and in the water-ethanol solution, the volume ratio of water to ethanol is 3:1.

[0027] In the preparation steps of the PDA / P@Z:

[0028] In the step (1), the mass ratio of ZrOCl2·8H2O, H2TCPP, and BA is 280-300 mg:80-100 mg:2.8-2.9 g, the ratio of the amount of ZrOCl2·8H2O, H2TCPP, BA to DMF is 3.16-3.3 g:100-140 mL, the reaction temperature is 80-95°C, and the reaction time is 4-6 h;

[0029] In the step (2), the mass ratio of PCN-224, ZnCl2, InCl3·4H2O, and TAA is 15-20 mg: 8-10 mg: 36-40 mg: 36-40 mg, and the concentrations of ZnCl2, InCl3·4H2O, and TAA in the aqueous solution are 0.4-0.5 mg / mL, 0.9-1 mg / mL, and 0.9-1 mg / mL, respectively; the reaction temperature is 140-170°C, and the reaction time is 10-12 h;

[0030] In the step (3), the mass ratio of P@Z to PDA is 1:1, and the ratio of the amount of P@Z to PDA to water is 20 mg:10-20 mL.

[0031] The present invention also provides a method for preparing the aforementioned PEC sandwich immunosensor. The method includes ultrasonically washing the FTO electrode with acetone, a mixture of ethanol and NaOH, and ultrapure water, respectively. A hole is punched in the center of the insulating tape and placed on the conductive surface of the dried FTO electrode. The FTO electrode is then ultrasonically cleaned for 20 minutes using a mixture of acetone, a 1M NaOH and 50% ethanol solution (1:1 volume ratio), and ultrapure water. The cleaned FTO electrode is then dried at 60°C for 5 hours. Ultrasonic cleaning of the FTO electrode in acetone is beneficial for cleaning fat-soluble impurities, cleaning in a mixed solution of NaOH and ethanol-water is for modifying the FTO electrode, and cleaning in ultrapure water is beneficial for washing away the acetone or NaOH residues in the first two steps. The preparation method comprises the following steps: dispersing a U@C suspension on the surface of the FTO electrode, drying it, then drop-coating Ab1, incubating, rinsing unbound Ab1, and then adding BSA; adding a solution of Aβ1-42 to be tested, incubating it, adding a PDA / P@Z-Ab2 immune probe, incubating it again, and then rinsing uncoupled PDA / P@Z-Ab2 to complete the U@C+Ab1+BSA+(Aβ1-42)+Ab2+P@Z sandwich immune reaction;

[0032] The PDA / P@Z-Ab2 immunoprobe is a conjugation of PDA / P@Z and Ab2, reacted at 4°C for 12 hours, washed to remove unconjugated Ab2, and then redispersed in a PBS buffer solution;

[0033] The incubation temperature is 37°C (the optimum temperature for protein, which is conducive to the binding of biomacromolecules); the drying temperature is 60°C;

[0034] The drop coating volume of Ab1, BSA, Aβ1-42, and P@Z-Ab2 is 40 μL. The drop coating volume of 40 μL is selected because, on the one hand, it is easy to dry, and on the other hand, 40 μL is sufficient to cover the electrode surface. The concentration is selected to ensure the maximum photocurrent intensity while obtaining accurate results and saving antibody solution.

[0035] The present invention also designs a PEC-LFIA sensor test strip, which includes a PVC plate, a sample plate, a nitrocellulose membrane (NC membrane), and an absorption plate arranged in sequence on one side of the PVC plate, with the NC membrane placed between the sample plate and the absorption plate; the test strip also includes a screen-printed electrode; the detection line (T line) of the NC membrane is located at the coated Ab1, and the quality control line (C line) of the NC membrane is located at the coated goat anti-mouse IgG antibody; the PDA / P@Z-Ab2 immunoprobe is fixed on the sample plate, and the T line and C line of the NC membrane are aligned with the two working electrodes of the screen-printed electrode; the sample plate is prepared by immersing a glass fiber membrane in a PBS buffer containing 1% BSA, 1% sucrose, and 1% Tween-20;

[0036] The U@C composite material is the aforementioned U@C composite material; the PDA / P@Z is the aforementioned PDA / P@Z.

[0037] The present invention also provides the use of the aforementioned PEC sandwich immunosensor or PEC-LFIA test strip in detecting the Aβ1-42 content; detecting the Aβ1-42 concentration in an unknown solution: incubating the unknown solution on the prepared PEC immunosensor or PEC-LFIA test strip sample plate, detecting the photocurrent intensity of the sensor or test strip T line, and combining it with the standard curve to obtain the Aβ1-42 concentration in the unknown solution.

[0038] The present invention also provides use of the aforementioned PEC sandwich immunosensor or the aforementioned PEC-LFIA test strip in the preparation of a medical device for diagnosing AD.

[0039] The present invention also provides a method for detecting Aβ1-42 concentration, comprising the following steps: based on the aforementioned sensor or the aforementioned test strip, obtaining the photocurrent value, detection time and detection environment of the sensor or the test strip, wherein the detection environment is PBS buffer, artificial cerebrospinal fluid or human plasma; inputting the photocurrent value, detection time and detection environment of the sensor or the test strip into a trained Aβ1-42 concentration detection model, wherein the Aβ1-42 concentration detection model models the concentration of Aβ1-42 based on the K-nearest neighbor algorithm, selects Euclidean distance as the distance metric, and sets the value of K to 1, inputs the photocurrent value, detection time and detection environment, and outputs the Aβ1-42 concentration corresponding to the photocurrent value, detection time and detection environment; and obtaining the output of the trained Aβ1-42 concentration detection model as a detection result.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This paper proposes the application of photosensitive MOFs in PEC sandwich immunoassay for the detection of Aβ1-42, an early AD biomarker. We used CdTe-QDs-sensitized UiO-66 as a PEC immunomatrix and PDA / P@Z-Ab2 as a PEC immunoprobe, significantly improving the detection sensitivity of Aβ1-42. The constructed PEC sandwich immunosensor and PEC-LFIA test strip can accurately detect trace amounts of Aβ1-42 in artificial cerebrospinal fluid and human plasma, showing good linearity and reproducibility. On this basis, we used a classification learning algorithm to perform machine learning operations on these two detection platforms and established an Aβ1-42 concentration prediction model. The model can accurately predict Aβ1-42 concentration under all three detection environments with an accuracy rate of over 94%. This platform demonstrates great potential in the diagnosis of early AD and provides an effective method for early screening of high-risk AD populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is an investigation into the effects of different mass percentages of UiO-66 and CdTe-QDs on the photocurrent intensity of U@C.

[0043] Figure 2 This is an investigation of the effect of different U@C drop coating volumes on the U@C photocurrent intensity.

[0044] Figure 3 This is an investigation of the effects of different concentrations of capture antibodies on the photocurrent intensity of the U@C-Ab1-Aβ1-42-Ab2-P@Z immune complex.

[0045] Figure 4 This study aimed to investigate the effect of the amount of P@Z added during antibody coupling on the photocurrent intensity of the U@C-Ab1-Aβ1-42-Ab2-P@Z immune complex.

[0046] Figure 5 Investigation of the PEC immunosensor for detecting different concentrations of Aβ1-42; A. Detection performance, B. Logarithmic calibration graph, concentration range: 10 fg / mL-100 ng / mL (ah).

[0047] Figure 6 This is the specificity investigation of the PEC immunosensor, where the concentration of the interfering substance is 100 pg / mL and the concentration of the target substance is 1 pg / mL.

[0048] Figure 7 The repeatability of the PEC immunosensor in detecting 100 pg / mL Aβ1-42 (n=5).

[0049] Figure 8 The short-term stability of the PEC immunosensor within 260 s.

[0050] Figure 9 Results of the PEC immunosensor detecting Aβ1-42 in artificial cerebrospinal fluid and human plasma; A. Linear response of the PEC sensor to Aβ1-42 in artificial cerebrospinal fluid; B. Logarithmic calibration graph of the PEC sensor for Aβ1-42 in artificial cerebrospinal fluid; C. Linear response of the PEC sensor to Aβ1-42 in 200-fold diluted human plasma; D. Logarithmic calibration graph of the PEC sensor for Aβ1-42 in 200-fold diluted human plasma.

[0051] Figure 10 Results of PEC-LFIA test strips detecting different concentrations of Aβ1-42; A. Linear response; B. Logarithmic calibration graph.

[0052] Figure 11Results of the PEC-LFIA test strip immunosensor detecting Aβ1-42 in artificial cerebrospinal fluid and human plasma; A. Linear response of the PEC-LFIA test strip to Aβ1-42 in artificial cerebrospinal fluid; B. Logarithmic calibration graph of the PEC-LFIA test strip for Aβ1-42 in artificial cerebrospinal fluid; C. Linear response of the PEC-LFIA test strip to Aβ1-42 in 200-fold diluted human plasma; D. Logarithmic calibration graph of the PEC-LFIA test strip for Aβ1-42 in 200-fold diluted human plasma.

[0053] Figure 12 Characterization of U@C; SEM image of A.UiO-66, SEM image of BU@C, TEM image of CU@C, TEM-EDS image of DJ.U@C.

[0054] Figure 13 TEM image of CdTe-QDs.

[0055] Figure 14 Characterization of P@Z; A. SEM image of PCN-224, B. SEM image of ZnIn2S4, CD. SEM image of P@Z, EH. SEM-EDS image of P@Z.

[0056] Figure 15 Schematic diagram of the PEC-LFIA test strip structure.

[0057] Figure 16 The fitting model of the PEC sandwich immunosensor (A), the fitting model of the PEC-LFIA test strip (B), and the Aβ1-42 concentration prediction platform constructed based on the two models.

[0058] Figure 17 Schematic diagram of PEC sandwich immunosensor and PEC-LFIA test strip detection. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0060] Example 1 Optimization of the ratio of UiO-66 to CdTe-QDs

[0061] UiO-66 and CdTe-QDs were immersed in a 3:1 water-ethanol solution (V:V) and ultrasonicated for 30 minutes to obtain a U@C suspension. The U@C suspension was freeze-dried to obtain a U@C freeze-dried powder for use. Among them, the mass ratios of UiO-66 and CdTe-QDs were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively, and the U@C powders obtained contained 10wt% UiO-66, 90wt% CdTe-QD, 20wt% UiO-66, 80wt% CdTe-QDs, 30wt% UiO-66, 70wt% CdTe-QDs, and 40wt% U UiO-66, 60wt% CdTe-QDs, containing 50wt% UiO-66, 50wt% CdTe-QDs, containing 60wt% UiO-66, 40wt% CdTe-QDs, containing 70wt% UiO-66, 30wt% CdTe-QDs, containing 80wt% UiO-66, 20wt% CdTe-QDs, containing 90wt% UiO-66, 10wt% CdTe-QDs.

[0062] The photocurrent signals of the U@C suspensions prepared in different proportions were tested, and the experimental results are as follows: Figure 1 As shown in Figure 3, as the mass percentage of CdTe-QDs gradually increases and the mass percentage of UiO-66 gradually decreases, the photocurrent value shows an upward trend. However, when the proportion of CdTe-QDs exceeds 60%, the stability of the photocurrent signal decreases significantly. Considering the photoelectric value and the stability of the composite material, the mass percentage of CdTe-QDs is set to 60% and the mass percentage of UiO-66 is set to 40%.

[0063] Example 2 Optimization of U@C suspension droplet coating amount

[0064] A U@C suspension was prepared with a mass ratio of UiO-66 to CdTe-QDs of 4:6. This suspension was then drop-coated on a FTO electrode and incubated at 37°C for 1 hour before PEC testing. The concentration of the U@C suspension was 1 mg / mL, and the drop-coated amounts were 30 μL (0.03 mg), 35 μL (0.035 mg), 40 μL (0.04 mg), 45 μL (0.04 mg), and 50 μL (0.05 mg), respectively.

[0065] The PEC test shows the effect of different U@C suspension volumes on the U@C photocurrent intensity. Figure 2 When the drop volume of the U@C composite suspension was 45 μL, the photocurrent response changed most significantly; therefore, 45 μL was selected as the optimal amount of the U@C composite.

[0066] Example 3 Optimization of the amount of capture antibody added

[0067] About 38 μL of the capture antibody stock solution with a concentration of 2.63 mg / mL was taken and diluted to obtain capture antibody solutions with concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL, respectively. 40 μL of the capture antibody Ab1 solution was drop-coated on the surface of the U@C composite material. After incubation at 37°C, a PEC sandwich immunosensor was prepared to study the effects of different capture antibody concentrations on the photoelectric signal of the formed sandwich immune complex (U@C+Ab1+BSA+Aβ1-42+Ab2+P@Z). Figure 3 The results show that when the capture antibody concentration is less than 10 μg / mL, the amount of capture antibody added is too small, and the amount of sandwich immune complex formed is also too small. However, when the capture antibody is added too much, the photoelectric signal decreases because the capture antibody can inhibit the photoelectric signal. Therefore, choosing 10 μg / mL of capture antibody can maximize the photoelectric signal of the sandwich immune complex.

[0068] Example 4 Optimizing the amount of secondary antibody coupling material PDA / P@Z added

[0069] 10 mg of P@Z and 10 mg of PDA were dispersed in 10 mL of deionized water. The mixture was magnetically stirred for 24 h and washed with deionized water several times to obtain PDA / P@Z.

[0070] PDA / P@Z was combined with 1 mL of 50 μg / mL Ab2, and unconjugated sites were blocked by adding 50 μL of 1 wt% BSA. The mixture was shaken at 4°C for 12 hours and then washed with 7.4% PBS to remove unconjugated Ab2. The resulting PDA / P@Z-Ab2 was redispersed in 2 mL of 7.4% PBS. The added amounts of PDA / P@Z were 0.5 mg, 1 mg, 1.5 mg, 2 mg, and 2.5 mg, respectively.

[0071] The study examined the effect of different P@Z addition amounts on the photocurrent intensity of the sandwich immune complex (U@C+Ab1+BSA+Aβ1-42+Ab2+P@Z) formed during antibody coupling. Figure 4 The results show that the optimal addition amount of PDA / P@Z is about 2 mg.

[0072] Example 5 Detection of Aβ1-42 Concentration in Different Samples Using Photocurrent Intensity

[0073] Preparation of PEC immunosensor, including the following steps:

[0074] (1) Preparation of PEC immune matrix:

[0075] 0.5 mmol ZrCl4 and 0.5 mmol TA were added to a mixed solution of 13.5 mL DMF and 1.5 mL acetic acid and sonicated for 30 minutes. The mixture was then transferred to a polytetrafluoroethylene reactor and heated at 120°C for 24 hours. The resulting precipitate was washed three times with DMF, ethanol, and deionized water, respectively. The product was dried at 60°C for 12 hours to obtain UiO-66 powder. To completely remove any remaining unreacted material, the resulting yellow-white solid product was calcined in a vacuum oven at 120°C for 12 hours to obtain the final sample.

[0076] 0.04 mmol Te powder and 2 mmol NaBH4 were added to 4 mL of deionized water and magnetically stirred at 60°C under nitrogen for 70 min. Then, 0.23 mmol CdCl2·2.5H2O and 50 μL MPA were added to 50 mL of deionized water, and the pH was adjusted to 11 with 1 M NaOH. The solution obtained in the first step (2 mL) was injected into the cadmium precursor solution. The resulting CdTe-QDs solution was reacted at 140°C for 2 h. Subsequently, the CdTe-QDs solution was dialyzed using a dialysis bag with a molecular weight of approximately 500 Da for 24 h, freeze-dried, and the powdered CdTe-QDs were collected.

[0077] 6 mg of UiO-66 and 4 mg of CdTe-QDs powder were dispersed in a water-ethanol (volume ratio 3:1) mixture and sonicated for 30 min to obtain a U@C suspension. The U@C suspension was freeze-dried to obtain U@C lyophilized powder for subsequent use.

[0078] (2) Preparation of PEC immunoprobe signal substance P@Z

[0079] 300 mg of ZrOCl2·8H2O, 50 mg of H2TCPP, and 3 g of benzoic acid were added to 100 mL of DMF and stirred magnetically at 90°C for 5 h. The resulting product was washed three times with DMF and dried at 60°C for 5 h to obtain PCN-224 powder.

[0080] A P@Z heterojunction was obtained by in situ growth of ZIS on the surface of PCN-224 via a hydrothermal method. 15 mg of PCN-224 was added to 20 mL of water containing 27 mg of ZnCl2, 120 mg of InCl3·4H2O, and 120 mg of TAA. The reaction was heated at 160°C for 12 hours. The product was washed with ethanol and water and dried at 60°C.

[0081] Dissolve 100 mg of DA in 100 mL of Tris-HCl (8.8%), then add 50 mL of isopropanol and stir magnetically for 24 hours. Wash the product with deionized water, freeze-dry, and set aside. Add 10 mg of P@Z and 10 mg of PDA to 10 mL of deionized water and stir magnetically for 24 hours. Finally, couple 2 mg of the treated PDA / P@Z with 50 μg of Ab2 and react at 4°C for 12 hours. Wash with PBS (7.4%) to remove unconjugated Ab2, and redisperse the resulting P@Z-Ab2 in 2 mL of PBS (7.4%) for later use.

[0082] Characterization of PEC immune matrix and PEC immune probe signaling

[0083] The synthesized nanomaterials were preliminarily analyzed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Figure 12 and Figure 13 shown. Figure 12 A shows that UiO-66 presents a consistent hexahedral morphology with an average particle size of approximately 200 nm. Figure 12 In Figure B, U@C maintains a similar hexahedral morphology, but its surface is significantly rougher and dotted with fine particles compared to UiO-66, providing preliminary evidence for the attachment of CdTe-QDs to the UiO-66 surface. Subsequently, transmission electron microscopy (TEM) verified the successful synthesis of CdTe-QDs and their integration into UiO-66. Figure 12 C shows the TEM image of U@C, which clearly shows the core-shell structure. Energy spectrum analysis further elucidates the elemental composition of the sample. Figure 12 As shown in DJ, Zr was found inside U@C, while Cd and Te were observed on its surface, further confirming the successful synthesis of U@C. Figure 13 AC shows the smooth spherical morphology, uniform size, excellent dispersion, high crystallinity, and monodispersity of CdTe-QDs.

[0084] exist Figure 14 In AB, PCN-224 presents a nanoparticle morphology with an average particle size of about 80 nm, while ZIS presents a flower-like structure composed of nanosheets. Figure 14 In C, ZIS formed on the surface of PCN-224 to obtain a composite material P@Z with an average particle size of about 150nm. The presence and distribution of Zr, Zn, In and S were confirmed by SEM-EDS. This once again proved the growth of ZIS on the surface of PCN-224 ( Figure 14 DH).

[0085] (3) Preparation of PEC immunosensor

[0086] First, a FTO electrode was sonicated for 20 minutes using a mixture of acetone, 50% ethanol, and 1M NaOH, and then ultrapure water. After drying, a centrally perforated insulating tape was applied to the FTO electrode surface to form a sensing area. 45 μL of a U@C suspension was dispersed on the FTO electrode surface and dried at 60°C for 12 hours. Then, 40 μL of 10 μg / mL Ab1 was drop-coated on the U@C surface and incubated at 37°C for 1 hour. Unbound Ab1 was then rinsed with PBS 7.4. 40 μL of 1 wt% BSA was added as a nonspecific binding blocker.

[0087] To detect the antigen Aβ1-42, 40 μL of Aβ1-42 solution was added to the FTO electrode and incubated at 37°C for 1 hour to form the U@C+Ab1+BSA+Aβ1-42 immunoconjugate. Finally, 40 μL of a 1 mg / mL PDA / P@Z-Ab2 PEC immunoprobe was added and incubated at 37°C. Unconjugated P@Z-Ab2 was rinsed with PBS 7.4 to complete the sandwich immunoreaction (U@C+Ab1+BSA+Aβ1-42+Ab2+P@Z). The concentrations of Aβ1-42 antigen added to the modified electrode were: 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL.

[0088] Detection of Aβ1-42 includes the following steps:

[0089] Three-electrode system: the prepared PEC immunosensor was used as the working electrode; the Ag / AgCl electrode was used as the reference electrode; and the platinum electrode was used as the counter electrode.

[0090] At a concentration of 0.1 mol·L -1 Ascorbic acid (AA) was added to the PBS buffer as an electron donor.

[0091] The excitation light source used was a 20W UV lamp with a UV wavelength of 405nm, which was switched on and off every 10s and an external voltage of 0.0V was applied.

[0092] An electrochemical workstation was used to collect the photocurrent curves of the working electrode under illumination and non-illumination conditions. The UV lamp was switched on and off for 10 seconds as one cycle, and the difference between the highest and lowest points on the ordinate was taken as the photocurrent intensity.

[0093] Draw a standard curve about Aβ1-42 concentration: Use the photocurrent intensity of the sensor as the ordinate and the Aβ1-42 concentration as the abscissa to draw a standard curve about Aβ1-42 concentration, and obtain a standard curve graph ( Figure 5 ).

[0094] The logarithm of the antigen concentration and the photocurrent intensity were linearly fitted to obtain the calibration equation. The obtained regression equation was: Photocurrent values ​​(nA) = 52.7 Log C Aβ1-42 +680.7(R 2 =0.9921). Because the immune complex formed on the electrode surface has a strong photocurrent, the photocurrent signal increases with increasing Aβ1-42 concentration in the range of 10 fg / mL-100 ng / mL, showing a good linear relationship. The limit of detection (LOD) is calculated to be 1.1 fg / mL, demonstrating good sensitivity and accuracy.

[0095] Detection of Aβ1-42 concentration in unknown solution: The unknown solution was incubated into the prepared PEC immunosensor, and the photocurrent intensity of the sensor was detected. Combined with the standard curve, the concentration of Aβ1-42 in the unknown solution could be obtained.

[0096] An Ag / AgCl electrode was used as a reference electrode to ensure experimental reproducibility. A platinum electrode served as a counter electrode to ensure that the current at the working electrode flowed smoothly, ensuring that the reaction under investigation occurred there. The addition of ascorbic acid (AA) to the PBS buffer solution as an electrode donor facilitated the timely consumption of electron holes on the electrode during the reaction, promoting the continuation of the reaction. These parameters were employed to achieve optimal photocurrent results.

[0097] Example 6

[0098] The PEC immunosensor prepared in Example 5 was used to perform specific recognition experiments for biomarkers potentially present in cerebrospinal fluid and human plasma. The PEC immunosensor was incubated with interfering substances at a concentration of 100 pg / mL and Aβ1-42 at a concentration of 1 pg / mL. These analytes included carcinoembryonic antigen (CEA), cardiac troponin (cTnT), alpha-fetoprotein (AFP), dopamine (DA), human serum albumin (HSA), and bovine serum albumin (BSA). After incubation, the modified electrode was thoroughly rinsed with PBS buffer, and the photocurrent intensity was measured.

[0099] The results are as follows Figure 6 As shown in the figure, in the absence of Aβ1-42, incubation with interfering substances did not result in a significant change in the photocurrent on the electrode. However, in the presence of Aβ1-42, no significant photocurrent fluctuations were observed after incubation of the above-mentioned interfering substances on the electrode. Therefore, the presence of interfering proteins did not significantly affect the photocurrent response of the PEC immunosensor, indicating that the PEC immunosensor has excellent selectivity and specificity for Aβ1-42.

[0100] Example 7

[0101] Five PEC immunosensors prepared in Example 5 were used to detect Aβ1-42 at a concentration of 100 pg / mL, and the repeatability of the constructed sensors was tested.

[0102] The results are as follows Figure 7 As shown, the photocurrent intensities of the five PEC immunosensors measured were similar, and the calculated relative standard deviation (RSD) value was 4.5%, demonstrating the good repeatability of the sensors.

[0103] Example 8

[0104] The PEC immunosensor prepared in Example 5 was used to detect Aβ1-42 at a concentration of 100 pg / mL. The constructed sensor was subjected to a short-term stability test, and the intensity change of the photocurrent was observed after multiple on / off irradiation cycles within 260 s.

[0105] The results are as follows Figure 8 As shown, the photocurrent remained unchanged after multiple on / off illumination cycles within 260 s, indicating that the sensor has good short-term stability.

[0106] Example 9

[0107] The feasibility and applicability of the PEC immunosensor prepared in Example 5 were tested for measuring Aβ1-42 concentrations in artificial cerebrospinal fluid (ACSF) and human plasma. The PEC immunosensor was incubated with ACSF samples or human plasma samples containing varying Aβ1-42 concentrations. The human plasma was diluted 200-fold with PBS 7.4 buffer. Solutions of relatively low, medium, and high Aβ1-42 concentrations were sequentially introduced into the ACSF samples and diluted human plasma samples, and the spiked solutions were analyzed using the same procedures and conditions as in Example 5.

[0108] The results are shown in Tables 1 and 2. The average recovery rates of Aβ1-42 in artificial cerebrospinal fluid (ASF) ranged from 94.98% to 99.27%, with relative standard deviations of no more than 5.3%. The average recovery rates of Aβ1-42 in diluted human plasma ranged from 97.38% to 99.77%, with relative standard deviations of no more than 4.9%. These results demonstrate that the PEC immunosensor prepared by this method is feasible for the determination of Aβ1-42 in ASF and human plasma, exhibiting good sensitivity and promising application potential in the fields of biology and medicine.

[0109] Table 1 Recovery test results of Aβ1-42 in artificial cerebrospinal fluid using PEC immunosensor (n=3)

[0110]

[0111] Table 2 Recovery test results of Aβ1-42 in human plasma using PEC immunosensor (n=3)

[0112]

[0113] Example 10 Preparation of PEC-LFIA Test Strips and Detection of Aβ1-42 Concentrations in Different Samples Using Photocurrent Intensity

[0114] Preparation of PEC-LFIA test strips includes the following steps:

[0115] (1) Preparation of PEC immune matrix

[0116] 0.5 mmol of zirconium chloride and 0.5 mmol of terephthalic acid were added to a mixed solution of 13.5 mL of DMF and 1.5 mL of acetic acid and sonicated for 30 minutes. Subsequently, the above mixture was transferred to a polytetrafluoroethylene reactor and heated at 120°C for 24 hours. The resulting precipitate was washed three times with DMF, ethanol, and deionized water, respectively. The product was dried at 60°C for 12 hours to obtain UiO-66 powder. To completely remove the remaining unreacted substances, the yellow-white solid product was calcined in a vacuum oven at 120°C for 12 hours to obtain the final sample.

[0117] 0.04 mmol tellurium powder and 2 mmol NaBH₄ were added to 4 mL of deionized water and magnetically stirred at 60°C under nitrogen for 70 min. Subsequently, 0.23 mmol CdCl₂·2.5H₂O and 50 μL MPA were added to 50 mL of deionized water, and the pH was adjusted to 11 with 1 M NaOH. The solution (2 mL) obtained in the first step was injected into the cadmium precursor solution. The resulting CdTe-QDs solution was reacted at 140°C for 2 h. Subsequently, the CdTe-QDs solution was dialyzed for 24 h using a dialysis bag with a molecular weight of approximately 500 Da and freeze-dried to collect the powdered CdTe-QDs. 6 mg UiO-66 and 4 mg CdTe-QDs powder were dispersed in a water-ethanol (volume ratio 3:1) mixture and sonicated for 30 min to obtain a U@C suspension. The U@C suspension was freeze-dried to obtain U@C freeze-dried powder for subsequent use.

[0118] (2) Preparation of P@Z immunoprobe

[0119] 300 mg of ZrOCl2·8H2O, 100 mg of H2TCPP, and 2.8 g of benzoic acid were added to 140 mL of DMF and magnetically stirred at 90°C for 5 h. The resulting product was washed three times with DMF and dried at 60°C for 5 h to obtain PCN-224 powder.

[0120] A P@Z heterojunction was obtained by in situ growth of ZIS on the surface of PCN-224 via a hydrothermal method. 20 mg of PCN-224 was added to 20 mL of water containing 8 mg of ZnCl2, 36 mg of InCl3·4H2O, and 36 mg of TAA. The reaction was heated at 160°C for 12 hours. The product was washed with ethanol and water and dried at 60°C.

[0121] Dissolve 100 mg of DA in 100 mL of Tris-HCl (8.8%), then add 50 mL of isopropanol and stir magnetically for 24 hours. Wash the product with deionized water, freeze-dry, and set aside. Add 10 mg of P@Z and 10 mg of PDA to 10 mL of deionized water and stir magnetically for 24 hours. Finally, couple 2 mg of the treated PDA / P@Z with 50 μg of Ab2 at 4°C for 12 hours. Wash with PBS (7.4%) to remove unconjugated Ab2, and redisperse the resulting P@Z-Ab2 in PBS buffer for later use.

[0122] (3) Assembling PEC-LFIA test strips

[0123] The screen-printed electrodes (SPCEs) used in PEC-LFIAs are made by printing carbon ink onto a PET sheet. These serve as the working electrode (WE) and the counter electrode (CE). The reference electrode (RE) is made using Ag / AgCl ink. After printing, the homemade electrodes are dried in a 60°C oven for 30 minutes. After printing, the sensing area is covered with an insulating layer for further use.

[0124] PEC-LFIAs consists of five main components ( Figure 15 ), each component has the following dimensions (width × length, mm 2 ): Polyvinyl chloride (PVC) board (5×60mm 2 ), sample plate (5×20mm 2 ), nitrocellulose (NC) membrane (5×30 mm 2 ), screen-printed electrodes and absorption plates (5×12mm 2). The glass fiber membrane was soaked in PBS buffer containing 1% BSA, 1% sucrose and 1% Tween-20, and then dried at 37°C for 1 hour to prepare the sample plate. The sample plate and screen-printed electrode were then attached to the PVC plate. Ab1 (0.1 mg / mL) and goat anti-mouse IgG antibody (5 mg / mL) were sprayed onto two designated areas of the NC membrane, respectively, to complete the establishment of the T line and C line. The T line and C line were aligned with the working electrodes WE1 and WE2 of the screen-printed electrode, respectively. The NC membrane and absorption plate were cut and placed on the electrodes to complete the test strip assembly. Subsequently, 40 μL of PDA / P@Z-Ab2 was drop-coated on the sample plate and incubated at 37°C for 1 hour. The 40 μL solution did not completely infiltrate the sample plate, so it would not contaminate the NC membrane, while ensuring a sufficiently large coupling area between the PDA / P@Z-Ab2 immunoprobe and the antigen.

[0125] Before PEC detection, 40 μL of Aβ1-42 sample was dropped onto the sample plate and incubated at 37°C for 1 hour. Before the test, 100 μL of PBS 7.4 (30 mM AA) was introduced into the sample plate to rinse the bound immune complex (P@Z+Ab2+Aβ1-42) and unbound immune probe (P@Z+Ab2). PEC detection was performed after 5 minutes. The concentrations of Aβ1-42 antigen added to the test strips were 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 1 μg / mL, and 10 μg / mL, respectively.

[0126] The correction equation ( Figure 9 ), the regression equation obtained is Photocurrent values ​​(nA) = 271.4Log CAβ1-42 + 1819.4(R 2 =0.94). Because the immune complex formed on the T-line surface has a strong photocurrent, the photocurrent signal increases with increasing Aβ1-42 concentration in the range of 100 fg / mL-10 ng / mL, showing a good linear relationship. The limit of detection (LOD) is calculated to be 1.1 fg / mL, demonstrating good sensitivity and accuracy.

[0127] Detection of Aβ1-42 concentration in unknown solution: Incubate the unknown solution onto the prepared PEC-LFIA test strip sample plate, detect the photocurrent intensity of the test strip T line, and combine it with the standard curve to obtain the Aβ1-42 concentration in the unknown solution.

[0128] Example 11 PEC-LFIA test strips for determining Aβ1-42 concentrations in artificial cerebrospinal fluid and human plasma

[0129] The feasibility and applicability of the test strips prepared using this method for measuring Aβ1-42 concentrations in artificial cerebrospinal fluid and human plasma were investigated using the methods of Example 35. Unlike Example 35, the test strips were incubated with artificial cerebrospinal fluid samples or human plasma samples containing varying concentrations of Aβ1-42. The human plasma was diluted 200-fold with PBS 7.4 buffer. Solutions of relatively low, medium, and high Aβ1-42 concentrations were sequentially introduced into the artificial cerebrospinal fluid samples and diluted human plasma samples, and the spiked solutions were analyzed using the same procedures and conditions as in Example 35.

[0130] Experimental results:

[0131] The experimental results are shown in Tables 3-4. The average recoveries of Aβ1-42 in artificial cerebrospinal fluid (ASF) ranged from 90.73% to 112.18%, and in diluted human plasma from 95.46% to 108.27%. The relative standard deviations (RSDs) for both assays were no more than 5%. These results demonstrate that the test strips prepared using this method are feasible for the determination of Aβ1-42 in both ASF and human plasma, exhibiting good sensitivity and promising application potential in fields such as biology and medicine.

[0132] Table 3 Recovery test results of Aβ1-42 in artificial cerebrospinal fluid using PEC-LFIA test paper (n=3)

[0133]

[0134] Table 4 Recovery test results of Aβ1-42 in human plasma using PEC-LFIA test paper (n=3)

[0135]

[0136] Example 12 Machine Learning Model for Aβ1-42 Prediction

[0137] The K-nearest neighbor algorithm was used to model the analyte concentration based on the photocurrent values ​​of the sensor and test strip, as well as parameters such as detection time and detection environment. Two sensing systems were constructed in a three-dimensional space, each including two additional factors (detection time and detection environment). In this space, the Euclidean distance was selected as the distance metric, and the value of K was set to 1. The model was trained using 70% of the experimental data and validated using the remaining 30% of the experimental data, achieving a validation accuracy of over 94%. For ease of analysis, the three detection environments were numerically coded using 0, 1, and 2 (where 0 represents PBS buffer, 1 represents artificial cerebrospinal fluid, and 2 represents human plasma). The predicted analyte concentration ranged from 1 pg / mL to 100 ng / mL. The clear classification of each detection environment, high data quality, and minimal outliers indicate the potential of the model for practical application. For Aβ1-42 concentration measurement, the sensor or test strip photocurrent value, detection time, and selected detection environment were input, and the Aβ1-42 concentration was directly output.

Claims

1. A PEC sandwich immunosensor, characterized in that: The FTO electrode surface is covered with a U@C composite material as an immune matrix, which is then bound to Ab1, and unbound recognition sites are blocked with BSA, thereby specifically capturing and recognizing Aβ1-42. PDA / P@Z is then used as the signal of the immune probe and combined with Ab2 to specifically capture and recognize Aβ1-42 again, forming a U@C-Ab1-BSA-(Aβ1-42)-Ab2-PDA / P@Z sandwich immune complex to enhance the detection signal. The U@C composite material is UiO-66 sensitized by cadmium telluride quantum dots (CdTe-QDs); the PDA / P@Z is a composite heterojunction of PCN-224 modified by polydopamine (PDA) and zinc indium sulfide (ZIS).

2. A PEC sandwich immunosensor according to claim 1, characterized in that: In the U@C composite material, the mass ratio of UiO-66 and CdTe-QDs is 4-6:6-4; The amount of U@C composite material covered on the surface of the FTO electrode is 1 mg / mL, 35 to 50 μL, wherein the size of the FTO electrode is 20×20×2.2 mm; The amount of capture antibody Ab1 bound by the U@C composite material is 10-20 μg / mL, 40 μL, wherein the U@C composite material is 1 mg / mL, 45 μL; The amount of the PDA / P@Z combined with the detection antibody Ab2 is 1.5 to 2.5 mg, wherein the amount of the detection antibody Ab2 is 25 μg / ml, 2 mL.

3. The PEC sandwich immunosensor according to claim 2, characterized in that The preparation of U@C comprises: dispersing UiO-66 and CdTe-QDs in a water-ethanol mixed solution, and ultrasonicating to obtain a U@C suspension; The preparation of the PDA / P@Z comprises: in situ growing ZIS on the surface of PCN-224 by a hydrothermal method to obtain a P@Z heterojunction, and then modifying P@Z with PDA to obtain PDA / P@Z.

4. The PEC sandwich immunosensor according to claim 3, characterized in that The preparation of the U@C composite material comprises the following steps: (1) Preparation of UiO-66: Dissolve zirconium chloride (ZrCl4) and terephthalic acid (TA) in a mixture of DMF and acetic acid, heat and react; after cooling, centrifuge and collect the precipitate, wash with DMF, ethanol, and deionized water, dry, and calcine to obtain UiO-66; (2) Preparation of CdTe-QDs: Dissolve hydrated cadmium chloride (CdCl2·2.5H2O) and 3-mercaptopropionic acid (MPA) in deionized water to obtain a cadmium precursor solution, adjust the pH and set aside; Dissolve tellurium powder and sodium borohydride (NaBH4) in deionized water, heat and stir under nitrogen, and add them to the cadmium precursor solution to obtain a CdTe-QDs solution for heating reaction; Then, dialyze the obtained solution and freeze-dry it to obtain CdTe-QDs powder; (3) UiO-66 and CdTe-QDs were added to a water-ethanol solution and ultrasonically treated to obtain a U@C suspension; the U@C suspension was freeze-dried to obtain a U@C freeze-dried powder for later use; The preparation of the PDA / P@Z comprises the following steps: (1) Zirconium chloride octahydrate (ZrOCl2·8H2O), tetracarboxyphenylporphyrin (H2TCPP) and benzoic acid (BA) are dissolved in DMF, the resulting mixture is stirred under heating, the product is washed and dried to obtain PCN-224 powder; (2) PCN-224 is added to an aqueous solution containing zinc chloride (ZnCl2), indium chloride tetrahydrate (InCl3·4H2O) and sodium thiosulfate (TAA), stirred and mixed, the mixed solution is heated to react, the product is washed and dried to obtain a P@Z heterojunction; (3) P@Z and PDA were dispersed in deionized water, magnetically stirred, and washed with deionized water multiple times to obtain PDA / P@Z.

5. The PEC sandwich immunosensor according to claim 4, characterized in that In the preparation steps of the U@C composite material: In the step (1), the molar ratio of ZrCl4 and TA is 1:1, the volume ratio of the mixture of DMF and acetic acid is 9-13.5 mL:1-1.5 mL, the amount of ZrCl4 and TA and the ratio of the DMF and acetic acid mixture are 0.2 g:10-15 mL, the temperature of the heating reaction is 110-130°C, the reaction time is 20-25 hours, the temperature of the calcination is 100-200°C, and the calcination time is 10-18 hours; In the step (2), the molar ratio of Te powder to NaBH4 is 5-10 mg:75-150 mg, the ratio of the amount of Te powder to NaBH4 and deionized water is 5-10 mg:5-10 mL, the reaction temperature is 50-70° C., and the reaction time is 40-70 min; the pH of the cadmium precursor solution is adjusted to 10-12 using 1 M NaOH, and the obtained CdTe-QDs solution is reacted at 130-150° C. for 2-3 h; In the step (3), the mass ratio of UiO-66 and CdTe-QDs composite is 4-6:6-4; the volume of water-ethanol is 10-20 mL; and in the water-ethanol solution, the volume ratio of water to ethanol is 3:

1. In the preparation steps of the PDA / P@Z: In the step (1), the mass ratio of ZrOCl2·8H2O, H2TCPP, and BA is 280-300 mg:80-100 mg:2.8-2.9 g, the ratio of the amount of ZrOCl2·8H2O, H2TCPP, BA to DMF is 3.16-3.3 g:100-140 mL, the reaction temperature is 80-95°C, and the reaction time is 4-6 h; In the step (2), the mass ratio of PCN-224, ZnCl2, InCl3·4H2O, and TAA is 15-20 mg: 8-10 mg: 36-40 mg: 36-40 mg, and the concentrations of ZnCl2, InCl3·4H2O, and TAA in the aqueous solution are 0.4-0.5 mg / mL, 0.9-1 mg / mL, and 0.9-1 mg / mL, respectively; the reaction temperature is 140-170°C, and the reaction time is 10-12 h; In the step (3), the mass ratio of P@Z to PDA is 1:1, and the ratio of the amount of P@Z to PDA to water is 20 mg:10-20 mL.

6. A method for preparing the PEC sandwich immunosensor according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: dispersing a U@C suspension on the surface of an FTO electrode, drying the suspension, then drop-coating Ab1, incubating, washing away unbound Ab1, and then adding BSA; adding a solution of Aβ1-42 to be tested, incubating, adding a PDA / P@Z-Ab2 immune probe, incubating again, and washing away uncoupled PDA / P@Z-Ab2 to complete a U@C / Ab1 / BSA / Aβ1-42+Ab2+P@Z sandwich immune reaction; The PDA / P@Z-Ab2 immunoprobe is a PDA / P@Z coupled with Ab2, reacted at 4°C, washed to remove unconjugated Ab2, and then redispersed in a PBS buffer solution; The incubation temperature is 37°C; the drying temperature is 60°C.

7. A PEC-LFIA test strip, characterized in that: The test strip comprises a PVC plate, on one side of which a sample plate, a nitrocellulose membrane (NC membrane) and an absorption plate are sequentially arranged, with the NC membrane being placed between the sample plate and the absorption plate; the test strip further comprises a screen-printed electrode; the detection line (T line) of the NC membrane is located at the coated Ab1, and the quality control line (C line) of the NC membrane is located at the coated goat anti-mouse IgG antibody; a PDA / P@Z-Ab2 immunoprobe is fixed on the sample plate, and the T line and C line of the NC membrane are aligned with the two working electrodes of the screen-printed electrode; the sample plate is prepared by immersing a glass fiber membrane in a PBS buffer solution containing 1% BSA, 1% sucrose and 1% Tween-20; The U@C composite material is the U@C composite material according to any one of claims 1 to 5; the PDA / P@Z is the PDA / P@Z according to any one of claims 1 to 5.

8. Use of the PEC sandwich immunosensor according to claim 1, the method for preparing the PEC sandwich immunosensor according to claim 6, or the PEC-LFIA test strip according to claim 7 in detecting Aβ1-42 content.

9. Use of the PEC sandwich immunosensor according to claim 1, the method for preparing the PEC sandwich immunosensor according to claim 6, or the PEC-LFIA test strip according to claim 7 in preparing a medical device for diagnosing AD.

10. A method for detecting Aβ1-42 concentration, characterized in that: The method comprises the following steps: based on the sensor of claim 1 or the test strip of claim 7, obtaining the photocurrent value, detection time and detection environment of the sensor or the test strip, wherein the detection environment is PBS buffer, artificial cerebrospinal fluid or human plasma; inputting the photocurrent value, detection time and detection environment of the sensor or the test strip into a trained Aβ1-42 concentration detection model, wherein the Aβ1-42 concentration detection model models the concentration of Aβ1-42 based on a K-nearest neighbor algorithm, selects Euclidean distance as a distance metric, and sets the value of K to 1, inputs the photocurrent value, detection time and detection environment, and outputs the Aβ1-42 concentration corresponding to the photocurrent value, detection time and detection environment; and obtaining the output of the trained Aβ1-42 concentration detection model as a detection result.

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