Preparation method of alzheimer's disease early screening sensor, sensor and early screening method

Abstract

The application discloses a preparation method of an Alzheimer's disease early screening sensor, the sensor and an early screening method. An electrode substrate and a composite precursor liquid can be prepared respectively, the precursor liquid is spin-coated and solidified to form a photoelectric composite channel layer, after the surface is activated by mercaptoacetic acid and an activation liquid, an A beta 42 aptamer is incubated and fixed, non-specific sites are blocked by bovine serum albumin, and finally the target sensor is prepared. The application integrates photoelectrochemistry and transistor sensing technology, realizes the integration of channel layer photosensitivity and biological recognition function, amplifies weak potential signals relying on photoelectric synergistic effect, has low detection limit, fast detection speed and low energy consumption. The application effectively prevents non-specific interference, has excellent detection specificity and sensitivity, has simple preparation process, low cost and easiness in mass production, innovates the A beta 42 detection idea, is suitable for early low-concentration sample screening of Alzheimer's disease, and has extremely high clinical popularization value.

Description

Technical Field

[0001] This invention relates to the field of sensor technology, and in particular to a method for preparing an early screening sensor for Alzheimer's disease, the sensor itself, and the early screening method. Background Technology

[0002] Alzheimer's disease (AD) is a group of central nervous system degenerative diseases with insidious onset and progressive development, and it is also the most common type of dementia in the elderly. In its early stages, the disease has no typical external symptoms. By the time clinical manifestations such as cognitive decline and memory impairment appear, irreversible brain damage has already occurred, resulting in extremely poor intervention outcomes. Numerous pathological studies have confirmed that abnormally elevated concentrations of β-amyloid protein 42 (Aβ42) in peripheral blood and cerebrospinal fluid are the earliest detectable biomarkers of AD. Utilizing Aβ42 for non-invasive, ultra-trace-level early screening is currently a core research direction in the field of Alzheimer's disease prevention and control.

[0003] Currently, the main clinically mature methods for detecting Aβ42 include lumbar puncture cerebrospinal fluid analysis, PET brain imaging, single-molecule immunoassay array (Simoa), and enzyme-linked immunosorbent assay (ELISA). Among these, lumbar puncture is an invasive procedure with low patient tolerance; PET equipment is expensive and carries a high radiation risk, making it unsuitable for large-scale community screening; ELISA has drawbacks such as a high detection limit and weak anti-interference ability; while Simoa has excellent sensitivity, its high instrument costs and cumbersome testing procedures make it difficult to popularize at the grassroots level.

[0004] Field-effect transistor biosensors have become a popular technology for the in vitro detection of labeled objects due to their advantages of being label-free, fast-response, and miniaturizable. Organic electrochemical transistors (OECTs) have excellent signal amplification capabilities by relying on the ion-electron synergistic conduction characteristics.

[0005] However, existing purely electrically driven OECT biosensors still have significant technical shortcomings: both the excitation and readout signals of the device are electrical signals, and a bias voltage is continuously applied throughout the test. In electrolyte systems, baseline drift and 1 / f low-frequency noise are easily generated. The weak interfacial charge changes caused by trace targets in body fluids are easily masked by electrical interference. When faced with ultra-low concentrations of Aβ42 in blood at fg / mL or even ag / mL, the detection sensitivity is difficult to meet the needs of early screening. At the same time, the single conductive polymer channel lacks photoresponse characteristics, cannot amplify the sensing signal with the assistance of an external light source, has a narrow detection linear range, and poor signal-to-noise ratio under complex biological samples.

[0006] In summary, there is currently a lack of a detection process that combines low contact resistance, high photoelectric response, and excellent biological specificity recognition capabilities, which cannot meet the clinical needs for rapid and accurate early screening of trace amounts of Aβ42 in peripheral blood. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the deficiency in the prior art of lacking a detection process that takes into account low contact resistance, high photoelectric response and excellent biological specific recognition ability, which cannot meet the clinical needs of rapid and accurate early screening of peripheral blood trace Aβ42. The present invention provides a method for preparing an Alzheimer's disease early screening sensor, the sensor and the early screening method.

[0008] To solve the above-mentioned technical problems, the present invention provides a method for preparing an early screening sensor for Alzheimer's disease, comprising: Step I, preparing an electrode substrate and a composite precursor solution respectively, wherein the electrode substrate preparation process includes: Step S1, growing a silicon dioxide insulating layer on the surface of a pretreated substrate; Step S2, sputtering and depositing a source electrode and a drain electrode on the surface of the insulating layer, and reserving an exposed channel area to obtain the electrode substrate; the composite precursor solution preparation process includes: Step a, subjecting a mixed solution of cadmium dichloride and thioacetamide to high-pressure isothermal treatment to obtain crude cadmium sulfide; Step b, purifying the crude cadmium sulfide and then reacting it with poly(… A 3,4-ethylenedioxythiophene-poly(styrene sulfonate) aqueous solution was mixed to obtain the composite precursor solution; Step II: The composite precursor solution was spin-coated onto the channel region of the electrode substrate and then vacuum dried and cured to form a photoelectric composite channel layer; Step III: The surface of the photoelectric composite channel layer was rinsed with mercaptoacetic acid solution, and then the channel region was surface activated with an activation mixture; Step IV: An Aβ42 aptamer solution was dropped onto the surface of the activated photoelectric composite channel layer and incubated to allow the Aβ42 aptamer to covalently bind to the channel layer; Step V: Non-specific sites in the channel layer were blocked with bovine serum albumin solution to obtain the target Alzheimer's disease early screening sensor.

[0009] In one embodiment of the present invention, step S1 includes: step S11, placing a p-type silicon substrate with a crystal orientation of (100), a resistivity of less than 0.5 Ω·cm, and a thickness of 500~625 μm in an isopropanol solution for ultrasonic cleaning for 15 min, then placing it in an ethanol solution for ultrasonic cleaning for 15 min, rinsing with deionized water, and drying with nitrogen to obtain a pretreated substrate; step S12, using SiH4 and N2O as precursor gases to alternately vapor-deposit a silicon dioxide insulating layer on the surface of the pretreated substrate, wherein the vapor-phase deposition temperature is 280~320℃, the deposition time is 15~20 min, and the thickness of the insulating layer is 280~320 nm.

[0010] In one embodiment of the present invention, step S2 specifically involves: attaching and fixing a rigid mask to the surface of the insulating layer, and then continuously depositing Ni metal thin films and Au metal thin films in a vacuum cavity by magnetron sputtering. A rectangular cutout window is opened in the middle of the mask, and no metal film is deposited in the window area during the sputtering deposition process, so as to reserve a channel area exposed on the surface of the insulating layer between the two electrodes.

[0011] In one embodiment of the present invention, the Ni metal film has a thickness of 18~22nm, the Au metal film has a thickness of 28~32nm, the channel region has a width of 0.18~0.22mm, and the channel region has a length of 5.8~6.2mm.

[0012] In one embodiment of the present invention, step a includes: step a1, preparing a mixed precursor solution by mixing cadmium dichloride and thioacetamide at a mass ratio of 1:1; step a2, transferring the mixed precursor solution to a high-pressure reactor and reacting it at a constant temperature of 170~200℃ for more than 24 hours, and obtaining crude cadmium sulfide product after cooling. Step b includes: step b1, washing the crude cadmium sulfide product with deionized water, filtering, and drying it sequentially to obtain purified cadmium sulfide; step b2, mixing the purified cadmium sulfide with a poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) aqueous solution at a mass ratio of 1:1 and stirring to obtain CdS-PEDOT:PSS composite precursor solution.

[0013] In one embodiment of the present invention, in step II, the composite precursor liquid is spin-coated to the channel region at a rotation speed of 1400~1600 r / min until the thickness of the photoelectric composite channel layer after curing is 50~100 nm, and then transferred to an environment of 170~200℃ for vacuum drying and curing.

[0014] In one embodiment of the present invention, in step III, after rinsing the photoelectric composite channel layer with a 3 mmol / L mercaptoacetic acid solution, a mixed activation solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is dropped onto the channel region to activate the surface of the channel region.

[0015] In one embodiment of the present invention, step IV specifically involves: adding Aβ42 aptamers to the surface of the activated photoelectric composite channel layer and incubating it at 35-40°C for 1 hour to complete the covalent coupling of the aptamers; step V specifically involves: rinsing the incubated channel region with PBS buffer solution to remove unbound free Aβ42 aptamers, and then adding bovine serum albumin solution to the surface of the channel region to seal the blank active sites of unbound aptamers on the film, thereby obtaining the target Alzheimer's disease early screening sensor.

[0016] The present invention also provides an early screening sensor for Alzheimer's disease, which is fabricated using the above-described method for preparing an early screening sensor for Alzheimer's disease. The sensor comprises: a substrate; an insulating layer deposited on the substrate; a source electrode and a drain electrode symmetrically disposed on the surface of the insulating layer and electrically isolated from each other, with the source electrode and drain electrode forming an exposed rectangular channel region; a photoelectric composite channel layer covering the channel region and respectively overlapping the source electrode and the drain electrode; and a plurality of Aβ42 aptamers connected to specific sites on the photoelectric composite channel layer.

[0017] This invention also provides a method for early screening of Alzheimer's disease, which utilizes the aforementioned Alzheimer's disease early screening sensor. The method includes: sealing the portions of the source and drain electrodes that extend into the test solution using insulating silicone, leaving only the channel layer in direct contact with the solution; placing the sensor and an external Ag / AgCl reference electrode together in a phosphate buffer solution containing Aβ42 as a detectant, applying a gate voltage using the Ag / AgCl reference electrode as an ion gate electrode; fixing the source-drain voltage Vds and the gate voltage Vgs, vertically irradiating the channel layer with a visible light source, recording the source-drain current in both illuminated and dark states, and using the difference between the two, the IDS photoresponse, as a quantitative signal; establishing a standard curve based on the changes in the IDS photoresponse under different Aβ42 detectant concentrations, thereby determining the concentration of Aβ42 in the test solution.

[0018] In one embodiment of the present invention, the source-drain voltage Vds is 0.1 V, the gate voltage Vgs is 0 V, and the visible light source is a laser with a wavelength of 532 nm and a light intensity of 1 mW / cm².

[0019] The technical solution of the present invention has the following advantages compared with the prior art:

[0020] The Alzheimer's disease early screening sensor preparation method, sensor, and early screening method described in this invention combine photoelectrochemistry and transistor sensing, adopting a photoexcitation-electric detection transistor sensing mode. It integrates photosensitive and biometric functions within the same channel, amplifying the signal through the synergistic effect of the hydrothermal cadmium sulfide and PEDOT:PSS complex system. Identification is achieved by relying on the minute potential change on the electrode surface induced by trace amounts of the detectable substance. It requires a small reaction system volume, has a low detection limit, and can rapidly capture low concentrations of Aβ42 biomarkers. The entire device is simple to operate, highly sensitive, and consumes less energy. By covalently immobilizing the aptamer with carboxyl activation and blocking non-specific sites, interference from extraneous proteins is eliminated, and false positive results are avoided. The process is simple, easy to mass-produce, and uses inexpensive raw materials. It opens up a new technical path for Aβ42 detection at the device structure level. The resulting sensor exhibits excellent detection performance and specificity, is suitable for early screening of low-concentration samples for Alzheimer's disease, and has significant clinical application value. Attached Figure Description

[0021] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0022] Figure 1 This is a schematic diagram of the structure of the Alzheimer's disease early screening sensor in a preferred embodiment of the present invention;

[0023] Figure 2 This is a schematic diagram illustrating the function of an early screening method for Alzheimer's disease in another embodiment of the present invention;

[0024] Figure 3 This is a standard curve of the detection results of the Alzheimer's disease early screening sensor at different concentrations of Aβ42 in the embodiments of the present invention.

[0025] Explanation of reference numerals in the accompanying drawings: 100, substrate; 200, insulating layer; 300, source electrode (drain electrode); 400, photoelectric recombination channel layer; 500, Aβ42 aptamer; 600, light source; 700, gate electrode; 800, solution to be tested. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0027] Example 1: This example provides a method for preparing an early screening sensor for Alzheimer's disease, which includes:

[0028] Step I involves preparing the electrode substrate and the composite precursor solution separately. These two processes are independent and can be carried out simultaneously, which can reduce the overall preparation time and improve processing efficiency.

[0029] The electrode substrate fabrication process includes:

[0030] Step S1: A silicon dioxide insulating layer is grown on the surface of the pretreated substrate. The silicon dioxide insulating layer can isolate the conductive silicon substrate from the upper metal electrode and sensing channel, avoid the substrate leakage current from interfering with the detection signal, and ensure the electrical stability of the device. The insulating layer film formed by low temperature vapor deposition is uniform and dense, with stable insulation performance and strong bonding with the silicon substrate.

[0031] Further, step S1 includes:

[0032] Step S11: First, place a p-type silicon substrate with crystal orientation (100), resistivity less than 0.5 Ω·cm and thickness of 550 μm in isopropanol solution for ultrasonic cleaning for 15 min, then place it in ethanol solution for ultrasonic cleaning for 15 min, rinse with deionized water and dry with nitrogen to obtain the pretreated substrate. This pretreatment method can thoroughly remove particulate impurities, organic oil stains and metal contaminants attached to the surface of the silicon substrate, eliminate defects such as pinholes in the insulating layer and interface leakage caused by surface contaminants, and ensure that the surface of the silicon substrate with (100) crystal orientation is flat and uniform, which can ensure the consistency of film growth during subsequent silicon dioxide insulating layer deposition. The low resistivity p-type silicon substrate has excellent mechanical stability. In different implementations, the thickness of 500~625 μm takes into account both the substrate support strength and the device thinning requirements. The complete cleaning process can optimize the interface state of the silicon wafer surface, improve the bonding strength between the subsequently deposited insulating layer and the substrate, avoid sensor signal drift and device performance batch differences caused by impurities from the source, and lay a clean and regular substrate foundation for the stable preparation of subsequent electrodes and photoelectric channel layers.

[0033] Step S12: Using SiH4 and N2O as precursor gases, a silicon dioxide insulating layer is alternately vapor-deposited on the pretreated substrate surface. The vapor-phase deposition temperature is 300℃, the deposition time is 18 min, and the insulating layer thickness is 300 nm. The low-temperature deposition range does not cause thermal damage to the silicon substrate structure. The controllable deposition time of 15~20 min can accurately obtain a uniform, dense, and pinhole-free silicon dioxide film. This insulating layer can completely isolate the bottom conductive silicon substrate from the upper metal electrode and photoelectric composite channel layer, effectively blocking substrate leakage current and avoiding interference from the substrate conductivity characteristics with the weak potential signal generated by biometrics, ensuring the stability of the device's electrical baseline. At the same time, the silicon dioxide film has strong chemical inertness and high flatness, which can not only improve the interface adhesion strength between the subsequent metal electrode and the substrate, but also provide a regular and flat adhesion surface for subsequent mercaptoacetic acid carboxyl modification, eliminating the device sensing performance dispersion problem caused by uneven insulating layer thickness, unifying the electrical insulation performance of different substrates, and significantly reducing the signal detection error of mass-produced sensors. In different implementations, the deposition temperature can be set to 280~320 ℃ according to actual processing requirements, and the deposition thickness of the silicon dioxide insulating layer can be configured to 280~320 nm.

[0034] Step S2: Sputter and deposit source and drain electrodes on the surface of the insulating layer, leaving exposed channel areas to obtain the electrode substrate. In this embodiment, a Ni / Au bilayer source and drain electrode is deposited by magnetron sputtering using a hard mask. The nickel bottom layer enhances the interfacial bonding between the metal thin film and the silicon dioxide insulating layer, preventing electrode detachment. The gold surface layer has excellent conductivity, reducing the electrode's own resistance. The mask's hollow structure precisely reserves exposed channel areas of uniform size, providing standardized forming space for subsequent sensing films, ensuring consistent sensing baselines for different devices, and reducing batch detection errors.

[0035] Specifically, step S2 in this embodiment is as follows: a rigid mask is attached and fixed to the surface of the insulating layer, and then Ni metal thin film and Au metal thin film are continuously deposited in layers in the vacuum cavity by magnetron sputtering. A rectangular cutout window is opened in the middle of the mask. During the sputtering deposition of metal, no metal film is deposited in the window area, so as to reserve a channel area exposed on the surface of the insulating layer between the two electrodes. The rectangular cutout window in the center of the hard mask precisely defines the metal thin film forming area, ensuring that the window area is completely free of metal coverage. This allows for the regular pre-reservation of a standard channel area for exposing the insulating layer between the source and drain electrodes. The layered Ni film significantly improves the interfacial adhesion between the upper Au metal film and the silicon dioxide insulating layer, effectively preventing peeling and detachment of the electrode film. The surface Au film has extremely low intrinsic resistance and excellent chemical stability, which can reduce the signal loss of the electrode itself and is not easily corroded by biological samples. The magnetron sputtering vacuum film deposition process can obtain metal electrodes with high density and uniform thickness. Combined with the hard mask patterning, it can be formed in one step without additional photolithography etching processes, simplifying the processing flow. At the same time, the uniform channel area can ensure that the sensing response area size of each device is consistent, eliminating the detection baseline offset caused by the difference in channel area and improving the performance consistency of mass-produced sensors.

[0036] Furthermore, in this embodiment, the Ni metal film thickness is 20 nm, the Au metal film thickness is 30 nm, the channel region width is 0.2 mm, and the channel region length is 6.0 mm. In different embodiments, the Ni metal film thickness can be set to 18-22 nm, the Au metal film thickness can be set to 28-32 nm, the channel region width can be set to 0.18-0.22 mm, and the channel region length can be set to 5.8-6.2 mm. Among them, the nickel film with a thickness of 18~22 nm can fully play the role of bonding and transition, firmly connecting the silicon dioxide insulating layer and the gold conductive layer, avoiding local adhesion failure due to the nickel layer being too thin and increasing the interface impedance due to the nickel layer being too thick. The gold film with a thickness of 28~32 nm can balance low conductivity loss and material cost. Insufficient gold film thickness will lead to insufficient electrode conductivity and significant signal attenuation, while excessive thickness will result in waste of precious metal raw materials. The rectangular channel with a limited size can precisely control the effective working area of ​​the photoelectric composite sensing film, stabilize the photoexcitation region and the biomarker binding reaction region, unify the charge transport path length of each device, avoid the difference in photoelectric response intensity caused by the fluctuation of channel length and width, and enable the batch-produced sensors to have a consistent signal detection baseline, effectively reducing the deviation of batch detection data. At the same time, this size ratio can balance the signal amplification effect and the overall chip size, achieving device miniaturization while ensuring high sensitivity detection capability.

[0037] In this embodiment, the preparation process of the composite precursor liquid includes:

[0038] Step a: After subjecting the mixed solution of cadmium dichloride and thioacetamide to high-pressure hydrothermal isothermal treatment, crude cadmium sulfide product is obtained. Among them, the hydrothermal method can generate cadmium sulfide particles with high crystallinity and excellent visible light response performance, stronger photosensitivity, controllable reaction conditions, and higher purity of powder product.

[0039] In this embodiment, step a includes:

[0040] Step a1: Prepare a mixed precursor solution by mixing cadmium dichloride and thioacetamide in a 1:1 mass ratio. This ensures that the cadmium source and sulfur source are fully matched, avoiding the generation of by-products and reduction of the purity of cadmium sulfide powder due to excessive use of a single raw material.

[0041] Step a2: The mixed precursor solution is transferred to a high-pressure reactor and reacted at a constant temperature of 180°C for more than 24 hours. After cooling, crude cadmium sulfide is obtained. During the process of obtaining crude cadmium sulfide, the closed high-pressure hydrothermal environment allows for sufficient contact between reactant molecules. In different embodiments, a temperature range of 170~200°C ensures stable crystal growth, and a sufficient reaction time of more than 24 hours improves the integrity of cadmium sulfide crystallization. The resulting cadmium sulfide particles are uniform in size, have few defects, and possess excellent visible light photoelectric response activity. Compared with high-temperature calcination synthesis, the hydrothermal reaction conditions are mild and do not cause severe powder agglomeration. This ensures the performance stability of the film-forming photosensitive component in the subsequent composite precursor solution from the source, providing a high-quality inorganic photosensitive raw material basis for the photoelectric composite channel layer to achieve optical signal amplification and improve the detection sensitivity of markers.

[0042] Step b: Purify the crude cadmium sulfide product to remove residual impurities and prevent them from damaging the conductive system. Then, mix it with an aqueous solution of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) (PEDOT:PSS) to obtain the composite precursor solution. PEDOT:PSS can uniformly disperse cadmium sulfide powder to prevent agglomeration and at the same time endow the slurry with dual ionic and electronic conductivity. A fully functional sensing film can be formed by a single spin coating, eliminating the need for multi-layer step-by-step coating operations.

[0043] Specifically, in this embodiment, step b includes:

[0044] Step b1: After washing, filtering and drying the crude cadmium sulfide product with deionized water, purified cadmium sulfide is obtained. This can fully remove the soluble salts and unreacted precursor impurities remaining from the hydrothermal reaction, avoid the residual impurities from damaging the conductive polymer system and weakening the photosensitivity of cadmium sulfide, and ensure the purity and photoelectric activity of the powder are stable.

[0045] Step b2: After mixing and stirring the purified cadmium sulfide with poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) aqueous solution at a mass ratio of 1:1, a CdS-PEDOT:PSS composite precursor solution is obtained. The equal mass ratio can balance the proportion of inorganic photosensitive materials and organic conductive polymers. The PEDOT:PSS aqueous solution can achieve uniform dispersion of cadmium sulfide powder and prevent particle agglomeration and sedimentation, giving the precursor solution good spin-coating film-forming properties. At the same time, it can allow cadmium sulfide to be uniformly embedded in the conductive polymer network, so that the film has both visible light photoelectric excitation capability and dual ion and electron conduction characteristics. The sensing film with photosensitive and charge transport functions can be formed in one step without multiple coating layers. It reduces the contact impedance of the multilayer film interface, realizes the synergistic signal amplification effect of photoexcitation and electrical detection, simplifies the device processing process, and ensures the uniformity of photoelectric sensing performance of batch devices from the raw material ratio level.

[0046] Step II: After spin-coating the composite precursor liquid onto the channel region of the electrode substrate, vacuum drying and curing are performed. The spin-coating process can precisely control the film thickness, and vacuum drying can quickly remove the solvent, resulting in a pinhole-free, continuous, and uniform photoelectric composite channel layer. Cadmium sulfide and conductive polymers are tightly integrated in the same film, realizing the integration of photosensitive conduction and charge transport, reducing the contact impedance between multilayer films, and amplifying weak biometric signals based on the photoexcitation-electric detection mode, thus significantly improving the detection sensitivity of biomarkers.

[0047] Specifically, in this embodiment, the composite precursor liquid is spin-coated onto the channel region at a speed of 1500 r / min until the thickness of the photoelectric composite channel layer after curing is 80 nm, and then transferred to an environment of 180°C for vacuum drying and curing. In different embodiments, the composite precursor liquid can be spin-coated onto the channel region at a speed of 1400~1600 r / min according to actual needs until the thickness of the photoelectric composite channel layer after curing is 50~100 nm, and then transferred to an environment of 170~200°C for vacuum drying and curing.

[0048] The composite precursor liquid was spin-coated at a speed of 1400-1600 r / min, which allowed for uniform spreading of the slurry in the standardized channel area by relying on centrifugal force. This enabled precise control of the 50-100 nm thickness of the photoelectric composite channel layer after curing. This thickness range ensures sufficient interweaving between the cadmium sulfide photosensitive particles and the PEDOT:PSS conductive polymer, achieving efficient photogenerated charge separation and conduction, while avoiding the problems of charge transport hindrance caused by excessively thick film layers and insufficient photosensitive active sites caused by excessively thin film layers. After spin-coating, the mixture was then transferred to a speed of 170-200... Vacuum drying and curing at ℃ allows for rapid removal of moisture and dispersing solvent from the precursor solution, effectively preventing defects such as bubbles, pinholes, and cracks in the film during the drying process. This curing temperature thoroughly removes residual solvents, improves film density and structural stability, and does not damage the cadmium sulfide crystal photosensitive structure and PEDOT:PSS conductive framework. This allows for a tight integration of the inorganic photosensitive phase and the organic conductive phase, significantly reducing the charge transport impedance within the film layer. It fully leverages the synergistic amplification advantages of photoexcitation and electrosensing, ensuring a high degree of uniformity in the thickness, composition distribution, and conductive photosensitive performance of each batch of channel films, and stabilizing the sensor's detection baseline and signal response intensity.

[0049] Step III: Rinse the surface of the photoelectric composite channel layer with a mercaptoacetic acid solution, and then activate the channel area with an activation mixture. Mercaptoacetic acid can introduce a large number of carboxyl active sites on the film surface, and the activation solution can activate the carboxyl groups, providing binding sites for subsequent covalent fixation of aptamers. Compared with physical adsorption, this modification method has stronger binding force, and the aptamers are not easy to fall off during the detection process, ensuring the long-term detection stability of the sensor.

[0050] In this embodiment, after rinsing the photoelectric composite channel layer with a 3 mmol / L mercaptoacetic acid solution, a mixed activation solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is dropped onto the channel region to activate the surface of the channel region. The addition of this mixed activation solution to the channel region efficiently activates the carboxyl groups on the film surface, significantly improving the covalent coupling efficiency between the carboxyl groups and the amino groups of the Aβ42 aptamer. Compared to methods that rely solely on physical adsorption to immobilize the aptamer, this activation treatment significantly enhances the aptamer binding strength, making it less prone to detachment during sample detection and buffer rinsing, effectively extending the sensor's reusability. Furthermore, the uniform and controllable surface activation treatment ensures a uniform distribution of active sites across the entire channel region, avoiding uneven detection signals caused by differences in local probe modification density. This ensures the sensor's recognition specificity and the stability of detection results from the interface modification level.

[0051] Step IV: After adding an Aβ42 aptamer solution to the surface of the activated photoelectric composite channel layer, incubate the layer to allow the Aβ42 aptamer to covalently bind to the channel layer. The activated carboxyl sites covalently couple with the amino groups of the aptamer, firmly modifying the aptamer onto the surface of the channel film. The aptamer can specifically recognize and bind to Aβ42 markers in the sample. After the marker binds, it will cause a small potential change at the electrode interface. The device can capture this weak electrical signal to complete quantitative detection. It requires less sample volume, has a lower detection limit, and can quickly identify low-concentration markers in early body fluids.

[0052] In this embodiment, step IV specifically involves: after adding Aβ42 aptamer to the surface of the activated photoelectric composite channel layer, incubating it at 37°C for 1 hour to complete the covalent coupling of the aptamer. The mild constant temperature incubation range of 35~40°C ensures that the molecular structure of the aptamer is intact and its biological activity is not destroyed, while also effectively promoting the full covalent cross-linking reaction between the amino group of the aptamer and the activated carboxyl group on the surface of the channel layer. The controllable incubation time of 1 hour ensures that the aptamer is uniformly, densely, and stably fixed on the channel surface, avoiding insufficient recognition sites and decreased sensitivity due to excessively low modification density, or steric hindrance due to excessively dense modification affecting the biomarker binding efficiency. This enables the functionalized channel region to have a stable and specific Aβ42-specific recognition capability, accurately capturing trace amounts of Aβ42 biomarkers in the sample and effectively converting them into detectable interfacial potential changes. This lays the core biosensing foundation for the sensor to achieve low detection limit and high specificity in early screening of Alzheimer's disease.

[0053] Step V: By sealing non-specific sites in the channel layer with bovine serum albumin solution and blocking unused carboxyl sites on the channel layer that have not bound aptamers, the non-specific adsorption of impurities in the sample is blocked from the source, effectively avoiding false positive test results and improving the sensor's specificity for Aβ42 recognition. Finally, a target Alzheimer's disease early screening sensor integrating photosensitivity, biometric recognition, and electrical signal transduction functions is obtained. The entire process is simple to operate and has low energy consumption. Relying on a novel transistor optoelectronic composite sensing structure, it provides a new technical approach for the early detection of Aβ42. The process is compatible with general micro-nano devices, is easy to scale up for mass production, and has outstanding value for clinical screening applications.

[0054] Specifically, step V in this embodiment involves rinsing the incubated channel region with PBS buffer solution to remove unbound free Aβ42 aptamers and eliminate interference from free aptamers on subsequent sample detection signals. Then, bovine serum albumin solution is added to the surface of the channel region to seal the blank active sites of unbound aptamers on the membrane. Bovine serum albumin can occupy all blank active sites of unbound aptamers on the membrane surface, fundamentally blocking the non-specific adsorption of impurities and interfering peptides in the detection sample into the channel layer. This effectively avoids false positive signals caused by non-specific binding and significantly improves the sensor's specificity for Aβ42 biomarkers. After two steps of biopassivation treatment—cleaning and sealing—an Alzheimer's disease early screening sensor with both a stable photosensitive conductive substrate and a highly specific biorecognition interface is finally obtained, ensuring accurate and reliable subsequent clinical body fluid sample detection data.

[0055] Example 2: See Figure 1As shown, this embodiment provides an early screening sensor for Alzheimer's disease, which is fabricated using the method described in Embodiment 1. It includes: a substrate 100; an insulating layer 200 deposited on the substrate 100; a source electrode 300 and a drain electrode 300 symmetrically disposed on the surface of the insulating layer 200 and electrically isolated from each other, forming an exposed rectangular channel region between the source electrode 300 and the drain electrode 300; a photoelectric composite channel layer 400 covering the channel region and overlapping the source electrode 300 and the drain electrode 300; and a plurality of Aβ42 aptamers 500 connected to specific sites on the photoelectric composite channel layer 400. In this embodiment, the substrate 100 serves as the carrier of the entire sensor. A p-type silicon substrate with controllable specifications is selected, providing sufficient mechanical strength to offer a flat and stable support for all upper thin-film structures. Simultaneously, the substrate's material properties are uniform, ensuring consistent substrate conditions for batch devices and mitigating sensing performance deviations caused by substrate defects. The insulating layer 200, deposited on top of the substrate 100, completely isolates the conductive silicon substrate 100 from the upper electrode 300 and the photoelectric composite channel layer 400, blocking leakage current generated by the substrate 100 and preventing the substrate's conductive signal from masking the weak interfacial potential changes generated by the Aβ42 marker bonding. This maintains a stable electrical detection baseline for the device. Furthermore, the flat and inert surface of the insulating layer 200 enhances the adhesion of the upper metal electrode 300. The source electrode 300 and drain electrode 300, symmetrically arranged on the surface of the insulating layer 200 and electrically isolated from each other, constitute the charge transport path of the device. The double-layer metal structure has both adhesion and low-resistance conductivity. The two layers enclose a standardized exposed rectangular channel area, which defines the effective working range of photoelectric sensing, unifies the charge transport distance and photoexcitation reaction area of ​​each device, and eliminates the detection baseline offset caused by size differences. The photoelectric composite channel layer 400, which covers the rectangular channel area and overlaps the source electrode 300 and drain electrode 300 respectively, is composed of cadmium sulfide photosensitive powder and PEDOT:PSS conductive polymer. It can generate photogenerated carriers under illumination to amplify the signal and has dual ion and electron conduction capabilities. It can quickly convert the small interfacial potential changes caused by the Aβ42 aptamer 500 capturing the marker into a readable electrical signal. The integrated thin film structure reduces interlayer contact impedance and improves signal transmission efficiency.Multiple Aβ42 aptamers 500, covalently linked to specific sites on the photoelectric composite channel layer 400, serve as biometric units. They can specifically bind to Aβ42, a core biomarker of Alzheimer's disease, in the sample. After binding, they change the charge distribution at the channel layer interface, inducing detectable potential fluctuations. The specific recognition capability of the aptamers reduces the probability of false positives. Combined with the signal amplification effect of the photoelectric composite channel layer 400, high-sensitivity detection of trace biomarkers is achieved. The fully assembled structure works synergistically from top to bottom, integrating multiple functions such as insulation support, charge conduction, photoelectric signal amplification, and biometric specific recognition, forming an integrated micro-sensor device suitable for early screening of low-concentration samples.

[0056] See Figure 3 As shown, the Alzheimer's disease early screening sensor in this example exhibits good sensing linearity for different concentrations of key Alzheimer's disease biomarkers, demonstrating its practical feasibility.

[0057] Example 3: See Figure 2 As shown, this embodiment provides an early screening method for Alzheimer's disease, which utilizes the early screening sensor for Alzheimer's disease shown in Embodiment 2, and includes:

[0058] By using insulating silicone to seal the portions of the source and drain electrodes that extend into the test solution 800, leaving only the channel layer in direct contact with the solution, this operation can avoid electrochemical corrosion caused by direct contact between the metal electrodes and the electrolyte. At the same time, it can eliminate stray currents and non-Radida background current interference generated at the electrode-solution interface, ensuring that all electrical signals are generated only by the Aβ42 binding reaction on the surface of the channel layer, significantly reducing detection baseline noise and improving the signal-to-noise ratio.

[0059] The sensor and the external Ag / AgCl reference electrode were then placed in a phosphate buffer solution containing the Aβ42 analyte. The Ag / AgCl reference electrode was used as the ion gate electrode 700 to apply the gate voltage. The phosphate buffer solution maintained the pH stability of the detection system, avoiding the impact of pH fluctuations on the specific binding efficiency of the aptamer and Aβ42. The constant potential of the Ag / AgCl reference electrode provided a precise and controllable gate electric field. By regulating the carrier transport inside the channel layer through ion regulation, a stable transistor sensing test system was constructed.

[0060] With fixed source-drain voltage Vds and gate voltage Vgs, the channel layer is vertically irradiated using a visible light source 600, and the source-drain currents in the illuminated and dark states are recorded respectively. The difference between the two, IDS photoresponse, is used as a quantitative signal. Visible light can excite cadmium sulfide in the channel layer to generate a large number of photogenerated carriers. The photoelectric synergistic effect amplifies the current fluctuations caused by the weak interface potential changes induced by Aβ42 binding. The difference between the bright and dark currents can directly eliminate the substrate static current that does not change with illumination, further weakening background interference, amplifying the concentration-related characteristic response signal, effectively reducing the detection limit, and adapting to the early detection of low-concentration biomarkers.

[0061] Furthermore, in this embodiment, the source-drain voltage Vds is 0.1 V, the gate voltage Vgs is 0 V, and the visible light source is a laser with a wavelength of 532 nm and a light intensity of 1 mW / cm². The low source-drain voltage significantly reduces the overall energy consumption of the device while avoiding irreversible electrochemical side reactions induced by high voltage in the channel layer. The 0 V gate voltage relies on the Ag / AgCl reference electrode to achieve ion balance control of the system, without introducing additional electric field interference to the specific binding of the aptamer and Aβ42, ensuring that the interface charge change is only caused by the marker binding behavior. A visible light laser with a wavelength of 532 nm and an intensity of 1 mW / cm² is selected as the excitation source. This wavelength of visible light can accurately match the light absorption range of cadmium sulfide photosensitive particles, which can efficiently excite the generation of photogenerated electron-hole pairs. The mild light intensity of 1 mW / cm² can provide sufficient photogenerated carriers to amplify the signal without causing denaturation of the bioaptamer protein structure or photo-aging degradation of the channel layer polymer matrix due to excessive light intensity. Stable and controllable electrical parameters and optical excitation conditions enable the IDS photoresponse values ​​corresponding to different concentrations of Aβ42 samples to show regular linear changes, reducing parameter fluctuation errors during the detection process and improving the accuracy and repeatability of the standard curve fitting and the final concentration quantification results.

[0062] Finally, a standard curve is established based on the change in IDS photoresponse under gradient concentrations of Aβ42. The actual concentration of Aβ42 in the sample can be accurately deduced by relying on the linear correspondence of the standard curve. The entire detection process is simple to operate, requires a small sample volume, and has low energy consumption. It achieves highly specific and sensitive quantitative screening by relying on the differential current reading of phototransistors, providing a standardized and quantifiable detection method for early non-invasive body fluid screening of Alzheimer's disease.

[0063] In summary, the preparation method, sensor, and early screening method of the Alzheimer's disease early screening sensor described in this invention combine photoelectrochemistry with transistor sensing, adopting a photoexcitation-electric detection transistor sensing mode. It integrates photosensitive and biometric functions within the same channel, amplifies the signal through the synergistic effect of the hydrothermal cadmium sulfide and PEDOT:PSS complex system, and completes identification based on the minute potential change on the electrode surface caused by trace amounts of the detectable substance. It requires a small reaction system volume, has a low detection limit, and can rapidly capture low concentrations of Aβ42 biomarkers. The entire device is simple to operate, highly sensitive, and consumes less energy. By covalently immobilizing the aptamer with carboxyl activation and blocking non-specific sites, it can eliminate interference from extraneous proteins and avoid false positive results. The process is simple, easy to mass-produce, and uses inexpensive raw materials. It opens up a new technical path for Aβ42 detection from the device structure level. The resulting sensor has excellent detection performance and specificity, is suitable for early screening of low-concentration samples for Alzheimer's disease, and has significant clinical application value.

[0064] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing an early screening sensor for Alzheimer's disease, characterized in that: include: Step I: Prepare the electrode substrate and the composite precursor solution separately, wherein, The electrode substrate fabrication process includes: Step S1: Grow a silicon dioxide insulating layer on the surface of the pretreated substrate; Step S2: Sputter and deposit source and drain electrodes on the surface of the insulating layer, leaving exposed channel areas to obtain the electrode substrate; The preparation process of the composite precursor liquid includes: Step a: The mixed solution of cadmium dichloride and thioacetamide is subjected to high pressure and constant temperature treatment to obtain crude cadmium sulfide product. Step b: After purifying the crude cadmium sulfide product, it is mixed with an aqueous solution of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) to obtain the composite precursor solution; Step II: Spin-coating the composite precursor liquid onto the channel region of the electrode substrate and then vacuum drying and curing it to form a photoelectric composite channel layer; Step III: Rinse the surface of the photoelectric composite channel layer with a mercaptoacetic acid solution, and then activate the channel area with an activation mixture; Step IV: After adding an Aβ42 aptamer solution to the surface of the activated photoelectric composite channel layer, incubate the layer to allow the Aβ42 aptamer to covalently bind to the channel layer. Step V: Block non-specific sites in the channel layer with bovine serum albumin solution to obtain the target Alzheimer's disease early screening sensor.

2. The method for preparing the Alzheimer's disease early screening sensor according to claim 1, characterized in that: Step S1 includes: Step S11: First, place a p-type silicon substrate with crystal orientation (100), resistivity less than 0.5 Ω·cm and thickness of 500~625 μm in isopropanol solution for ultrasonic cleaning for 15 min, then place it in ethanol solution for ultrasonic cleaning for 15 min, rinse with deionized water and dry with nitrogen to obtain the pretreated substrate. Step S12: Using SiH4 and N2O as precursor gases, a silicon dioxide insulating layer is alternately vapor-deposited on the surface of the pretreated substrate. The vapor deposition temperature is 280~320℃, the deposition time is 15~20min, and the thickness of the insulating layer is 280~320nm.

3. The method for preparing the Alzheimer's disease early screening sensor according to claim 1, characterized in that: Step S2 specifically involves: attaching and fixing a rigid mask to the surface of the insulating layer, and then continuously depositing Ni and Au metal films in a vacuum chamber by magnetron sputtering. A rectangular cutout window is opened in the middle of the mask. During the metal sputtering deposition process, no metal film is deposited in the window area, so as to reserve a channel area exposed on the surface of the insulating layer between the two electrodes.

4. The method for preparing the Alzheimer's disease early screening sensor according to claim 3, characterized in that: The Ni metal film has a thickness of 18~22nm, the Au metal film has a thickness of 28~32nm, the channel region has a width of 0.18~0.22mm, and the channel region has a length of 5.8~6.2mm.

5. The method for preparing the Alzheimer's disease early screening sensor according to claim 1, characterized in that: Step a includes: Step a1: Prepare a mixed precursor solution by mixing cadmium dichloride and thioacetamide at a mass ratio of 1:1; Step a2: Transfer the mixed precursor solution to a high-pressure reactor and react at a constant temperature of 170~200℃ for more than 24 hours. After cooling, crude cadmium sulfide product is obtained. Step b includes: Step b1: The crude cadmium sulfide product is washed with deionized water, filtered, and dried sequentially to obtain purified cadmium sulfide. Step b2: After mixing and stirring the purified cadmium sulfide with poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) aqueous solution at a mass ratio of 1:1, CdS-PEDOT:PSS composite precursor solution is obtained.

6. The method for preparing the Alzheimer's disease early screening sensor according to claim 1, characterized in that: In step II, the composite precursor liquid is spin-coated onto the channel region at a speed of 1400~1600 r / min until the thickness of the photoelectric composite channel layer after curing is 50~100 nm, and then transferred to an environment of 170~200℃ for vacuum drying and curing.

7. The method for preparing the Alzheimer's disease early screening sensor according to claim 1, characterized in that: In step III, after rinsing the photoelectric composite channel layer with a 3 mmol / L mercaptoacetic acid solution, a mixed activation solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is dropped onto the channel region to activate the surface of the channel region.

8. The method for preparing the Alzheimer's disease early screening sensor according to claim 1, characterized in that: Step IV specifically involves: after adding Aβ42 aptamer to the surface of the activated photoelectric composite channel layer, incubating it at 35~40℃ for 1 hour to complete the covalent coupling of the aptamer; Step V specifically involves rinsing the incubated channel region with PBS buffer solution to remove unbound free Aβ42 aptamers. Then, bovine serum albumin solution is dropped onto the surface of the channel region to block the blank active sites of unbound aptamers on the membrane, thus obtaining the target Alzheimer's disease early screening sensor.

9. An early screening sensor for Alzheimer's disease, characterized in that: The Alzheimer's disease early screening sensor is prepared by the method described in any one of claims 1 to 8, comprising: Substrate; An insulating layer, wherein the insulating layer is deposited on the substrate; The source electrode and the drain electrode are symmetrically disposed on the surface of the insulating layer and electrically isolated from each other. The source electrode and the drain electrode enclose an exposed rectangular channel region. A photoelectric composite channel layer, wherein the photoelectric composite channel layer covers the channel region and is respectively connected to the source electrode and the drain electrode; Multiple Aβ42 aptamers are attached to specific sites on the photoelectric composite channel layer.

10. A method for early screening of Alzheimer's disease, characterized in that: Early screening for Alzheimer's disease using the Alzheimer's disease early screening sensor of claim 9, comprising: Insulating silicone is used to seal the portions of the source and drain electrodes that extend into the solution to be tested, leaving only the channel layer in direct contact with the solution. The sensor and the external Ag / AgCl reference electrode were placed together in a phosphate buffer solution containing Aβ42 detectant, and the Ag / AgCl reference electrode was used as the ion gate electrode to apply the gate voltage. With fixed source-drain voltage Vds and gate voltage Vgs, the channel layer is vertically illuminated by a visible light source, and the source-drain current in the illuminated state and the dark state is recorded respectively. The difference between the two, IDS photoresponse, is used as a quantitative signal. A standard curve was established based on the changes in the IDS photoresponse under different Aβ42 detector concentrations to determine the concentration of Aβ42 in the test solution.

11. The method for early screening of Alzheimer's disease according to claim 10, characterized in that: The source-drain voltage Vds is 0.1 V, the gate voltage Vgs is 0 V, and the visible light source is a laser with a wavelength of 532 nm and a light intensity of 1 mW / cm².

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