Field effect tube biosensor for Alzheimer's disease and preparation method thereof

Abstract

The present disclosure provides a field effect transistor biosensor for Alzheimer's disease and a preparation method. For the detection of serum markers of Alzheimer's disease, the field effect transistor biosensor includes: a substrate layer; a channel layer formed on the substrate layer, a source electrode and a drain electrode formed on the channel layer; a dielectric layer formed in the channel, the channel being the area between the source electrode and the drain electrode on the channel layer; a first passivation layer formed on the source electrode and a second passivation layer formed on the drain electrode; a functionalized molecular connection layer located on the dielectric layer; a nucleic acid aptamer fixed on the functionalized molecular connection layer; and a blocking molecule located above the dielectric layer for blocking redundant sites on the dielectric layer and the functionalized molecular connection layer. The field effect transistor biosensor achieves high-sensitivity detection, high-selectivity detection, and anti-interference detection of Aβ protein aggregates, a marker of AD, in serum, and can directly detect serum plasma samples without complex sample processing.

Description

Technical Field

[0001] The present disclosure relates to a field effect transistor biosensor for Alzheimer's disease and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with high morbidity and mortality. Existing medical diagnosis usually uses cerebrospinal fluid analysis combined with neuroimaging (PET-CT), which causes patients to endure great pain and extremely high testing costs. When patients show symptoms, the disease is already irreversible, so the diagnosis is often not timely. The early asymptomatic diagnosis of Alzheimer's disease is currently recognized as a major basic scientific problem to be solved. With the acceleration of population aging, rapid and large-scale screening of AD at the community level has become an important way to prevent and treat AD. However, the existing diagnostic methods have high testing costs, slow detection speeds, and are invasive. They are not suitable for screening of AD diseases, especially for screening of early asymptomatic AD diseases and tracking of disease progression.

[0003] Aβ protein aggregates (Aβ) are serum protein markers of AD. 42 or Aβ 40 ) can be used as an ideal, highly sensitive, and low-invasive indicator for early detection of AD. Since the content of Aβ protein aggregates, a disease marker of AD, in serum is low, it is easily interfered by a large number of background proteins such as albumin and antibodies in serum. Therefore, a highly sensitive anti-interference detection method is required to accurately detect it.

[0004] At present, the detection technology for the content of Aβ protein aggregates, a serum protein marker of AD, is mainly enzyme-linked immunosorbent assay (EILISA), but the detection sensitivity and accuracy of this method are not enough. The single-molecule immune array technology (Simoa) and immunoprecipitation mass spectrometry (IP-MS) equipment are used, which have high sensitivity, but the detection cost is high, the operation is complicated, the efficiency is low, and the response is slow, which cannot achieve large-scale rapid screening applications. In addition, because the serum environment is a high-ionic strength environment, affected by Debye shielding, field effect transistor biosensors (FET sensors) that use macromolecules such as antibodies as probe molecules cannot work in a high-ionic environment. Therefore, similar FET sensors will not be able to effectively sense the molecular concentration of Aβ protein aggregates, a serum protein marker of AD.

[0005] Therefore, there is a need for a convenient, efficient and highly sensitive detection element and detection technology suitable for the detection of AD serum markers. Summary of the invention

[0006] In order to solve at least one of the above technical problems, the present disclosure provides a field effect transistor biosensor for Alzheimer's disease and a preparation method thereof. The field effect transistor can be used for the detection of Alzheimer's disease serum markers and has high detection sensitivity, anti-interference and selectivity.

[0007] A first aspect of the present disclosure provides a field effect transistor biosensor for Alzheimer's disease, wherein the field effect transistor biosensor is used for detecting serum markers of Alzheimer's disease, and the field effect transistor biosensor comprises:

[0008] substrate layer;

[0009] a channel layer formed on the substrate layer;

[0010] a source electrode and a drain electrode formed on the channel layer;

[0011] a dielectric layer formed in a channel, wherein the channel is a region between the source and the drain on the channel layer;

[0012] a first passivation layer formed on the drain electrode and a second passivation layer formed on the source electrode;

[0013] A functionalized molecular connection layer formed on the dielectric layer, wherein the functionalized molecular connection layer is used to provide functional group sites for connecting nucleic acid aptamers;

[0014] A nucleic acid aptamer fixed on the functionalized molecular connection layer, wherein the nucleic acid aptamer corresponds to an Alzheimer's disease serum protein marker;

[0015] The blocking molecules are located above the medium layer and are used to block the redundant sites on the medium layer and the functionalized molecular connection layer. The blocking molecules include bovine serum albumin, 6-mercapto-1-hexanol and Tween 20.

[0016] In some possible implementations, the dielectric layer is a high-κ dielectric layer.

[0017] In some possible implementations, the dielectric layer is hafnium oxide, yttrium oxide or aluminum oxide.

[0018] In some possible implementations, the functionalized molecular connection layer is one of the following: gold nanoparticles deposited on the dielectric layer; 3-aminopropyltriethoxysilane (APTES) coupled to the dielectric layer;

[0019] Some possible implementations include: fixing the nucleic acid aptamer on the functionalized molecular gold nanoparticle connection layer through a gold-thiol bond; or,

[0020] The nucleic acid aptamer is fixed on the medium layer modified by APTES by amide bond linkage.

[0021] In some possible implementations, the thickness of the functionalized molecular connection layer is 1 nm to 8 nm.

[0022] In some possible implementations, the nucleic acid aptamer is a nucleic acid aptamer of thiol-modified Aβ protein.

[0023] In some possible implementations, the nucleic acid aptamer includes:

[0024] Aβ 42 Aggregate probe nucleic acid sequence: 5′-AGTCTAGGATTCGGCGTGGGT TAATTTTTTGCTGCCTGTGGTGTTGGGGCGGGTGCG-3′;

[0025] Aβ 40 Aggregate probe nucleic acid sequence: 5'-GCTGCCTGTGGTGTTGGGGCGGGTGCG -3'.

[0026] In some possible implementations, the field effect transistor biosensor is specifically used for detecting early-stage Alzheimer's disease.

[0027] A second aspect of the present disclosure provides a method for preparing a field effect transistor biosensor, wherein the field effect transistor biosensor is used for detecting serum markers of Alzheimer's disease, and the method comprises:

[0028] providing a substrate;

[0029] Etching to form a channel layer on the substrate;

[0030] forming a source electrode and a drain electrode on the channel layer;

[0031] Depositing a dielectric layer in the channel between the source electrode and the drain electrode on the channel layer;

[0032] preparing a functionalized molecular connection layer on the dielectric layer;

[0033] Spin coating a first passivation layer on the source electrode and spin coating a second passivation layer on the drain electrode;

[0034] Immobilizing a nucleic acid aptamer on the functionalized molecular connection layer, wherein the nucleic acid aptamer corresponds to an Alzheimer's disease serum protein marker;

[0035] The redundant sites on the medium layer and the functionalized molecular connection layer are sealed by a sealing agent, wherein the sealing agent comprises bovine serum albumin, 6-mercapto-1-hexanol and Tween 20.

[0036] In some possible implementations, the functionalized molecular connection layer is one of the following: gold nanoparticles deposited on the dielectric layer; 3-aminopropyltriethoxysilane (APTES) coupled to the dielectric layer.

[0037] In some possible implementations, the fixing of the nucleic acid aptamer on the functionalized molecular connection layer includes: fixing the nucleic acid aptamer on the gold nanoparticle functional layer on the medium layer through a gold-thiol bond; or fixing the nucleic acid aptamer on the APTES-modified medium layer through an amide bond.

[0038] In some possible implementations, the nucleic acid aptamer is fixed on the gold nanoparticle functional layer on the dielectric layer in the form of gold-thiol bonds, including:

[0039] A nucleic acid aptamer solution of thiol-modified Aβ protein is prepared, wherein the solution environment is pure water, phosphate buffer solution (PBS) or 4-morpholineethanesulfonic acid buffer solution (MES), and the solution pH range is 7-7.8;

[0040] The nucleic acid aptamer solution is dripped onto the gold nanoparticle functional layer region above the dielectric layer for 12-24 hours to allow the gold nanoparticles to fully react with the thiol groups, thereby fixing the nucleic acid aptamer on the functionalized molecular connection layer above the dielectric layer via gold-sulfur bonds.

[0041] In some possible implementations, the nucleic acid aptamer is fixed on the medium layer modified with APTES by amide bond linkage, including: preparing a nucleic acid aptamer solution of Aβ protein with a 5-terminal modified carboxyl group, the solution environment is pure water, PBS or MES, and the solution pH range is 5-7.4;

[0042] The nucleic acid aptamer solution is dripped onto the silane amino-functionalized dielectric layer, and 0.05 mol to 0.1 mol of carbodiimide and 1 mmol to 10 mmol of N-hydroxysuccinimide (EDC / NHS) are added to activate the carboxyl group for 2-4 hours, so that the amino functional group of the functionalized molecular connection layer fully reacts with the carboxyl group of the nucleic acid aptamer to form an amide bond, thereby fixing the nucleic acid aptamer on the functionalized molecular connection layer above the dielectric layer.

[0043] In some possible implementations, the thickness of the functionalized molecular connection layer is 1 nm to 8 nm.

[0044] In some possible implementations, the sealing of the redundant sites on the medium layer and the functionalized molecular connection layer by a sealing agent includes: performing multiple sealing operations on the redundant sites on the medium layer in a predetermined order by using one or two of the bovine serum albumin, 6-mercapto-1-hexanol and Tween 20 for each sealing operation; or, using a mixed sealing agent comprising bovine serum albumin, 6-mercapto-1-hexanol and Tween 20 to seal the redundant sites on the medium layer.

[0045] In some possible implementations, the blocking of the redundant sites on the medium layer and the functionalized molecular connection layer by a blocking agent includes: first using 6-mercapto-1-hexanol as a blocking agent for 0.5-1 hour, and then using Tween 20 and bovine serum albumin for 1.5 hours to 2.5 hours.

[0046] In some possible implementations, the blocking of redundant sites on the medium layer and the functionalized molecular connection layer by a blocking agent includes: using 6-mercapto-1-hexanol, bovine serum albumin, and Tween 20 for blocking in a random order; wherein the blocking time of the 6-mercapto-1-hexanol is 0.5-1 hour, the blocking time of the bovine serum albumin is 1.5-3 hours, and the blocking time of the Tween 20 is more than 0.5 hour.

[0047] In some possible implementations, the nucleic acid aptamer is a nucleic acid aptamer of thiol-modified Aβ protein.

[0048] The present invention adopts small molecule nucleic acid aptamers as probe molecules of functionalized carbon nanotube transistor sensors, which is helpful to achieve high-sensitivity detection of target biological molecules in high ionic strength solution environments such as serum.

[0049] The field effect tube biosensor disclosed in the present invention has been verified by experiments to achieve high sensitivity, high selectivity and anti-interference detection of Aβ protein aggregates and Tau protein, which are AD markers in serum. The detection is real-time, the detection time is less than 5 minutes, and the detection limit can reach 1fM (Fermi), which can meet the sensitivity required by human serum-related indicators and is superior to other current detection methods. In addition, the present invention can directly detect serum plasma samples without complex sample processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings illustrate exemplary embodiments of the present disclosure and together with the description serve to explain the principles of the present disclosure. These drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification.

[0051] Figure 1is a schematic structural diagram of a field effect transistor biosensor for Alzheimer's disease according to some embodiments of the present disclosure;

[0052] Figure 2 is an example diagram of a sequence of a nucleic acid aptamer according to some embodiments of the present disclosure;

[0053] Figure 3 is a three-dimensional schematic diagram of the structure and working principle of a field effect transistor biosensor according to some embodiments of the present disclosure;

[0054] Figure 4 is a schematic diagram of the working principle of nucleic acid aptamers in field effect transistor biosensors according to some embodiments of the present disclosure;

[0055] Figure 5 is a schematic flow chart of a method for preparing a field effect transistor biosensor for Alzheimer's disease according to some embodiments of the present disclosure;

[0056] Figure 6~Figure 10 It is a schematic diagram of the device structure in the process of preparing a field effect transistor biosensor according to some embodiments of the present disclosure.

[0057] Fig.11 is a schematic diagram of a test result of directly detecting a serum plasma sample using the field effect tube biosensor disclosed in the present invention;

[0058] Fig.12 is another schematic diagram of test results of direct detection of serum plasma samples by the field effect tube biosensor disclosed in the present invention;

[0059] Fig.13 It is a schematic diagram of the selectivity test results of the field effect tube biosensor array disclosed in the present invention in 1XPBS, serum environment and control groups with three orders of magnitude higher concentrations;

[0060] Fig.14 The field effect tube biosensor array disclosed in the present invention is mixed with multiple interferents to detect Aβ in a serum environment. 42 Schematic diagram of the test results;

[0061] Fig.15 The field effect tube biosensor array disclosed in the present invention is mixed with multiple interferents to detect Aβ in a serum environment. 40 Schematic diagram of the test results;

[0062] Fig.16 It is a schematic diagram of the detection results of the field effect tube biosensor disclosed in the present invention and other various detection platforms;

[0063] Fig.17 It is a schematic diagram of the detection results of the field effect transistor biosensor using different blocking agents according to some embodiments of the present disclosure.

[0064] Description of Reference Numerals

[0065] 100 Field Effect Transistor Biosensor

[0066] 110 substrate layer

[0067] 111 The first layer of the substrate layer

[0068] 112 The second layer of the substrate layer

[0069] 120 Channel layer

[0070] 130 Source

[0071] 140 Drain

[0072] 150 Dielectric layer

[0073] 161 First passivation layer

[0074] 162 Second passivation layer

[0075] 170 Functionalized Molecular Connector Layer

[0076] 180 Nucleic Acid Aptamers

[0077] 190 Blocked molecules DETAILED DESCRIPTION

[0078] The present disclosure is further described in detail below in conjunction with the accompanying drawings and implementations. It is understood that the specific implementations described herein are only used to explain the relevant content, rather than to limit the present disclosure. It should also be noted that, for ease of description, only the parts related to the present disclosure are shown in the accompanying drawings.

[0079] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in the present disclosure can be combined with each other. The technical solution of the present disclosure will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0080] Unless otherwise specified, the exemplary embodiments / embodiments shown will be understood as providing exemplary features of various details of some ways in which the technical concept of the present disclosure can be implemented in practice. Therefore, unless otherwise specified, the features of the various embodiments / embodiments can be combined, separated, interchanged and / or rearranged without departing from the technical concept of the present disclosure.

[0081] The use of cross-hatching and / or shading in the accompanying drawings is generally used to make the boundaries between adjacent components clear. As such, unless otherwise specified, the presence or absence of cross-hatching or shading does not convey or indicate any preference or requirement for the specific materials, material properties, dimensions, proportions, commonalities between the components shown, and / or any other characteristics, attributes, properties, etc. of the components. In addition, in the accompanying drawings, the sizes and relative sizes of the components may be exaggerated for clarity and / or descriptive purposes. When the exemplary embodiments can be implemented differently, the specific process sequence can be performed in a different order than described. For example, two successively described processes can be performed substantially simultaneously or in an order opposite to the described order. In addition, the same figure numbers represent the same components.

[0082] When a component is referred to as being "on" or "over," "connected to," or "coupled to" another component, the component may be directly on, directly connected to, or directly coupled to the other component, or intervening components may be present. However, when a component is referred to as being "directly on," "directly connected to," or "directly coupled to" another component, there are no intervening components. For this purpose, the term "connected" may refer to a physical connection, an electrical connection, etc., with or without intervening components.

[0083] For descriptive purposes, the present disclosure may use spatially relative terms such as "under," "beneath," "under," "down," "over," "upper," "above," "higher," and "side (e.g., in a "sidewall")," to describe the relationship of one component to another (other) component as shown in the accompanying drawings. The spatially relative terms are intended to encompass different orientations of the device in use, operation, and / or manufacture in addition to the orientation depicted in the accompanying drawings. For example, if the device in the accompanying drawings is turned over, components described as "under" or "beneath" other components or features would subsequently be positioned "over" the other components or features. Thus, the exemplary term "under" can encompass both the "above" and "below" orientations. Furthermore, the device may be otherwise oriented (e.g., rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein should be interpreted accordingly.

[0084] The terms used here are for the purpose of describing specific embodiments, and are not intended to be restrictive. As used here, unless the context clearly indicates otherwise, the singular forms "one (kind, person)" and "said (the)" are also intended to include plural forms. In addition, when the terms "comprise" and / or "include" and their variations are used in this specification, it is explained that there are stated features, integral bodies, steps, operations, parts, assemblies and / or their groups, but it is not excluded that there are or add one or more other features, integral bodies, steps, operations, parts, assemblies and / or their groups. It should also be noted that, as used here, the terms "substantially", "approximately" and other similar terms are used as approximate terms and not as degree terms, so that they are used to explain the inherent deviations of the measured values, calculated values ​​and / or the values ​​provided that will be recognized by those of ordinary skill in the art.

[0085] Figure 1 Schematic diagram of the structure of a carbon nanotube biosensor for Alzheimer's disease according to one embodiment of the present disclosure.

[0086] like Figure 1 As shown, the carbon nanotube biosensor 100 can be used for the detection of serum markers of Alzheimer's disease, such as Figure 1 As shown, the carbon nanotube biosensor 100 may include:

[0087] Substrate layer 100;

[0088] A channel layer 120 formed on the substrate layer;

[0089] A source 130 and a drain 140 formed on the channel layer;

[0090] A dielectric layer 150 formed in the channel, where the channel is a region between the source 130 and the drain 140 on the channel layer 120;

[0091] A first passivation layer 161 formed on the drain electrode 140 and a second passivation layer 162 formed on the source electrode 130;

[0092] A functionalized molecular connection layer 170 formed on the dielectric layer 150, wherein the functionalized molecular connection layer 170 is used to provide functional group sites for connecting nucleic acid aptamers;

[0093] A nucleic acid aptamer 180 fixed on the functionalized molecular connection layer 170;

[0094] The blocking molecules 190 are located above the dielectric layer 150 and are used to block the redundant sites on the dielectric layer 150 and the functionalized molecular connecting layer 170 .

[0095] In some embodiments, the substrate layer 110 may include a first layer 111 formed of a silicon (Si) material and a second layer 112 located on the first layer, and the second layer 112 may be formed of a material such as silicon dioxide (SiO2). In addition, the substrate layer 110 may also be, but not limited to, a separate silicon substrate layer, a silicon dioxide substrate layer, or a flexible substrate, etc. The present disclosure does not limit the material, structure, etc. of the substrate layer 110.

[0096] In some embodiments, the material of the channel layer 120 is carbon nanotubes, which may be, but are not limited to, network-shaped semiconductor carbon nanotube films or aligned semiconductor carbon nanotube films. For example, a disordered network carbon nanotube film may be used to form the channel layer 120, and the thickness of the channel layer 120 is preferably 1 nm to 2 nm.

[0097] In some embodiments, the source 130 and the drain 140 may respectively adopt a stacked metal structure, for example, the stacked metal structure may include a first metal layer as a bottom layer, an ohmic contact layer as an intermediate layer, and a second metal layer as a top layer. In some embodiments, the source 130 and the drain 140 may respectively adopt the following structure: a p-type ohmic contact layer is formed with a metal with a high work function (for example, palladium (Pd)), a 0.3 nm titanium (Ti) metal layer is used below the p-type ohmic contact layer to increase adhesion, and a metal layer of various metals (for example, gold (Au)) may be covered above the p-type ohmic contact layer to increase conductivity.

[0098] In some embodiments, the source electrode 130 and the drain electrode 140 can be formed on the channel layer 120 by methods such as electron beam evaporation. The size of the source electrode 130 and the drain electrode 140 can be freely selected according to the overall size of the device. For example, the thickness of the source electrode 130 can be but not limited to 10nm~100nm, and the thickness of the drain electrode 140 can be but not limited to 10nm~100nm.

[0099] In some embodiments, the dielectric layer 150 may be a high-κ dielectric layer with a κ value greater than 10, so as to improve the efficiency of the gate regulating the channel and enhance the sensitivity of the sensor. For example, the material of the dielectric layer 150 may be, but is not limited to, yttrium oxide (Y2O3), hafnium oxide, aluminum oxide or other oxides.

[0100] In some embodiments, the thickness of the dielectric layer 150 can be determined according to the size of the entire device. For example, the thickness of the dielectric layer 150 can be less than 15 nm and greater than 3 nm to ensure sufficient gate control capability while preventing gate dielectric leakage.

[0101] In some embodiments, the materials of the first passivation layer 161 and the second passivation layer 162 may be, but are not limited to, various electron beam glues, photoresists, silicon nitride (SiNx), silicon oxide, etc. The material of the first passivation layer 161 may be the same as or different from the material of the second passivation layer 162. The thickness of the first passivation layer 161 and the second passivation layer 162 may be, but are not limited to, 2 micrometers (μm).

[0102] The first passivation layer 161 and the second passivation layer 162 can be used to protect the drain electrode 140 and the source electrode 130 respectively, so as to avoid leakage when the field effect transistor biosensor 100 works in a liquid environment.

[0103] like Figure 1 As shown, the inner side of the source 130 contacts the side of the dielectric layer 150 and the side of the channel layer 140, and the first passivation layer 161 covers the drain 140 and can cover the area near the contact side of the drain 140 and the dielectric layer 150 above the dielectric layer 150. The first passivation layer 161 can isolate the drain 140 from the liquid environment during testing, and open a window in the device channel for electrical detection.

[0104] like Figure 1 As shown, the inner side of the drain electrode 140 contacts the side of the dielectric layer 150 and the side of the channel layer 140, and the second passivation layer 162 covers the source electrode 130 and can cover the area near the contact side of the source electrode 130 and the dielectric layer 150 on the dielectric layer 150. The second passivation layer 162 can isolate the source electrode 130 from the liquid environment during the test, and expose the functionalized sensitive area above the channel (that is, the area covered by the functionalized molecular connection layer 170 on the dielectric layer 150, that is, the area between the first passivation layer 161 and the second passivation layer 162 on the dielectric layer 150) to facilitate the subsequent regulation of the channel by the liquid gate electrode and the charged biological molecules, and to perform electrical detection and sensing tests. The length of this exposed area (that is, the length of the functionalized sensitive area above the channel) should account for more than 90% of the entire channel length. For example, if the channel length is 20μm, the window length (that is, the length of the functionalized sensitive area above the channel) is preferably 18μm.

[0105] The functionalized molecular connection layer 170 is used to provide functional group sites for connecting the nucleic acid aptamer 180. In some embodiments, the functionalized molecular connection layer 170 can be, but is not limited to, one of the following: gold nanoparticles deposited on the dielectric layer; 3-aminopropyltriethoxysilane (APTES) coupled to the dielectric layer. In addition, the functionalized connection molecular layer 170 is not limited thereto, and can also be any other functional group molecular layer that can provide subsequent covalent reaction active sites with the biological probe molecule (i.e., the nucleic acid aptamer 180).

[0106] Different types of functionalized molecular connection layers 170 can use different fixing methods to fix the nucleic acid aptamer 180. In some embodiments, the nucleic acid aptamer 180 can be fixed on the functionalized molecular connection layer 170 in the form of a gold-thiol bond or an amide bond. DNA nucleic acid sequences can be easily thiolated, and the reaction of thiol groups with gold is very efficient and stable. Through this linking method, the modified probe molecule (i.e., the nucleic acid aptamer 180) can be firmly fixed to the gate sensitive area in a covalent bond manner.

[0107] In some embodiments, the thickness of the functionalized molecular connection layer 170 may be, but is not limited to, 1 nm to 8 nm. For example, the functionalized molecular connection layer 170 may be a gold nanoparticle functional layer formed by discretely distributed gold particles having a thickness not exceeding 5 nm. In specific applications, the thickness of the functionalized molecular connection layer 170 may be flexibly adjusted as required, for example, the thickness of the functionalized molecular connection layer 170 may be less than 1 nm, as long as the connection is stable and firm.

[0108] In some embodiments, the functionalized molecular connection layer 170 can be randomly formed on the surface of the dielectric layer 150 by, for example, evaporation.

[0109] In the present disclosure, the nucleic acid aptamer 180 corresponds to the serum protein marker of Alzheimer's disease. Therefore, in some embodiments, the nucleic acid aptamer 180 can be, but is not limited to, a nucleic acid aptamer of thiol-modified Aβ protein. Since the nucleic acid aptamer 180 is a small molecule aptamer, it has a good capture ability for the serum marker Aβ protein aggregates of AD.

[0110] Figure 2 1 shows an example of the sequence of the nucleic acid aptamer 180. Figure 2 As shown, the nucleic acid aptamer 180 may include:

[0111] Aβ 42 Aggregate probe nucleic acid sequence: 5′-AGTCTAGGATTCGGCGTGGGT TAATTTTTTGCTGCCTGTGGTGTTGGGGCGGGTGCG-3′;

[0112] Aβ 40 Aggregate probe nucleic acid sequence: 5'-GCTGCCTGTGGTGTTGGGGCGGGTGCG -3'.

[0113] In some embodiments, the blocking molecule 190 may include, but is not limited to, one or more of the following: bovine serum albumin (BSA), 6-mercapto-1-hexanol (MCH), and Tween 20. Preferably, the blocking molecule may be a mixed blocking molecule, which may include BSA, MCH, and Tween 20 at the same time. The blocking molecule 190 may be used to block the active sites on the medium layer 150 that are exposed and not bound to the nucleic acid aptamer, thereby preventing the active sites on the medium layer 150 from generating nonspecific adsorption, thereby effectively improving the selectivity of the field effect tube biosensor to the serum marker Aβ protein aggregates of AD and the anti-interference ability to non-target substances.

[0114] Figure 3 The three-dimensional decomposition structure of the carbon nanotube biosensor according to one embodiment of the present disclosure and an exemplary diagram of its principle of detecting Aβ protein aggregates, a serum marker of AD, are shown. Figure 3 In the example, Si and SiO2 are used as the substrate layer, carbon nanotubes (CNTs) are used as the channel layer 120, Y2O3 is used as the dielectric layer 150, and the gold nanoparticle (Au NPs) functional layer is used as the functional molecular connection layer 170 for fixing the nucleic acid aptamer. Figure 3 As shown, before capturing the target (i.e., Aβ protein aggregates, a serum marker for AD), the secondary structure of the aptamer is in a folded state. After capturing the target, the secondary structure of the aptamer changes from its folded state to an extended state.

[0115] Figure 4 The schematic diagram of the working mechanism principle of the nucleic acid aptamer in the field effect transistor biosensor 100 in the ionic liquid is shown, that is, an example diagram of the electrostatic gating effect (Electrostatic Gating Effect) of the field effect transistor biosensor 100.

[0116] like Figure 4 As shown, taking the P-type field effect transistor biosensor as an example, before capturing the target (i.e., Aβ peptide), the secondary structure of the negatively charged nucleic acid aptamer is in a folded state, close to the channel, attracting positive charges to accumulate on the upper surface of the channel, causing the current to rise and the voltage Vgs of the gate relative to the source to increase. After capturing the target, the secondary structure of the nucleic acid aptamer changes from a folded state to an extended state, which means that the effective capture area is away from the channel, the negative charge on the nucleic acid aptamer is away from the channel, and the positive charge is away from the upper surface of the channel, causing the current to drop and the voltage Vgs of the gate relative to the source to decrease. In this way, the nucleic acid aptamer field effect transistor biosensor 100 can accurately detect the concentration of the target. At the same time, from Figure 4It can be seen that the blocking effect of various blocking molecules effectively prevents the non-specific adsorption of active sites on the dielectric layer 150, and successfully hinders the adsorption of various non-target substances (i.e., Figure 4 The anti-interference ability of field effect transistor biosensors has been significantly improved.

[0117] Figure 5 A schematic flow chart of a method for preparing a field effect transistor biosensor 100 in some embodiments of the present disclosure is shown. Figure 6~Figure 10 A schematic diagram of the device structure formed by each preparation step is shown.

[0118] like Figure 5 As shown, the preparation process S50 of the field effect transistor biosensor 100 may include:

[0119] Step S52, providing a substrate;

[0120] Step S54, etching to form a channel layer on the substrate;

[0121] In some embodiments, step S54 may include: transferring the etching layer pattern to the substrate by ultraviolet lithography, electron beam exposure, etc., and then removing the excess carbon nanotubes in the pattern by reactive ion etching, etc., so that the remaining carbon nanotubes form a channel layer. Taking the preparation of a 4-inch wafer field effect tube biosensor 100 as an example, the power of the reactive ion etching can be 140w, the reaction time is 40s, the gas used is O2, and the length of the formed channel layer can be 20μm and the width is 40μm.

[0122] Figure 6 The schematic diagram of the device structure prepared in this step is shown.

[0123] Step S56, forming a source electrode and a drain electrode on the channel layer;

[0124] For example, the source and drain electrodes can be formed using electron beam evaporation.

[0125] In some embodiments, step S56 may include: first forming a source electrode pattern and a drain electrode pattern on the channel layer by photolithography, and then depositing a metal film on the source electrode pattern and the drain electrode pattern by electron beam evaporation (for example, a stacked metal film with a 0.3 nm thick Ti layer as the first metal layer, a 20 nm thick Pd layer as the ohmic contact layer, and a 40 nm thick Au layer as the second metal layer), and finally forming the source and drain by stripping.

[0126] Figure 7 The schematic diagram of the device structure prepared in this step is shown.

[0127] Step S58, depositing a dielectric layer in the channel between the source and the drain on the channel layer;

[0128] In some embodiments, step S58 may include: forming a metal having a first thickness in the channel between the source and the drain on the channel layer by electron beam evaporation, and oxidizing at a certain temperature to form a metal oxide film having a second thickness.

[0129] In some implementations, in step S58 , the oxide high-κ dielectric layer may be directly formed by atomic layer deposition (ALD) or magnetron sputtering.

[0130] Taking the preparation of a 4-inch wafer field effect transistor biosensor 100 as an example, yttrium (Y) with a thickness of 3 nm can be formed in the channel by electron beam evaporation, and oxidized at 270° for 30 minutes to form a 6 nm thick dense yttrium oxide (Y2O3) film as a high-k gate dielectric.

[0131] Step S510, preparing a functionalized molecular connection layer on the dielectric layer;

[0132] In some implementations, step S510 may include: preparing gold nanoparticles in the region corresponding to the channel on the dielectric layer by physical vapor deposition or electrochemical deposition. Physical vapor deposition may include but is not limited to electron beam evaporation, magnetron coating, and the like.

[0133] Taking the preparation of a 4-inch wafer field effect transistor biosensor 100 as an example, physical vapor deposition or electrochemical deposition can be used to prepare gold nanoparticles with a thickness of 0.3-0.6 nm, which are deposited in the center of the channel as a functional molecular connection layer.

[0134] Figure 8 The schematic diagram of the device structure prepared in this step is shown.

[0135] Step S512, spin coating a first passivation layer on the drain electrode, and spin coating a second passivation layer on the source electrode;

[0136] In some embodiments, step S512 may include: passivating the drain and source with photoresist, and spin coating the first passivation layer and the second passivation layer. In this way, the liquid environment during the test can be isolated, and a window is opened in the channel of the field effect transistor biosensor so that the biomolecules can be connected to the exposed gate area to regulate the electrical properties of the channel. It is also convenient to load the liquid gate voltage and detect the electrical characteristics of the sensor device.

[0137] Taking the preparation of the field effect transistor biosensor 100 on a 4-inch wafer as an example, S1813 photoresist may be used.

[0138] Fig. 9 The schematic diagram of the device structure prepared in this step is shown.

[0139] Step S514, fixing the nucleic acid aptamer on the functionalized molecular connection layer to achieve functional modification of the device;

[0140] In some embodiments, in step S514, the nucleic acid aptamer can be fixed on the gold nanoparticle functional layer on the dielectric layer in the form of gold-sulfhydryl bonds (Au-S).

[0141] In one implementation, fixing the nucleic acid aptamer on the gold nanoparticle functional layer on the dielectric layer in the form of gold-thiol bonds may include:

[0142] A nucleic acid aptamer solution of thiol-modified Aβ protein is prepared, wherein the solution environment is pure water, phosphate buffer solution (PBS) or 4-morpholineethanesulfonic acid buffer solution (MES), and the solution pH range is 7-7.8;

[0143] The nucleic acid aptamer solution is dripped onto the gold nanoparticle functional layer region above the dielectric layer for 12-24 hours to allow the gold nanoparticles to fully react with the thiol groups, thereby fixing the nucleic acid aptamer on the functionalized molecular connection layer above the dielectric layer via gold-sulfur bonds.

[0144] In some implementations, in step S514, the nucleic acid aptamer can be fixed on the APTES-modified medium layer by amide bond linkage.

[0145] In one implementation, the nucleic acid aptamer is fixed on the medium layer modified with APTES by amide bond linkage, which may include:

[0146] Prepare a nucleic acid aptamer solution of Aβ protein with a modified carboxyl group at the 5' end, the solution environment is pure water, PBS or MES, and the solution pH range is 5-7.4;

[0147] The nucleic acid aptamer solution is dripped onto the silane amino-functionalized dielectric layer, and 0.05 mol to 0.1 mol of carbodiimide and 1 mmol to 10 mmol of N-hydroxysuccinimide (EDC / NHS) are added to activate the carboxyl group for 2-4 hours, so that the amino functional group of the functionalized molecular connection layer fully reacts with the carboxyl group of the nucleic acid aptamer to form an amide bond, thereby fixing the nucleic acid aptamer on the functionalized molecular connection layer above the dielectric layer.

[0148] The silane amino functionalized dielectric layer is a dielectric layer modified by APTES, and the dielectric layer modified by APTES and the silane amino functionalized dielectric layer are formed by the reaction of 3-aminopropyltriethoxysilane (APTES) coupled on the dielectric layer and the dielectric layer.

[0149] In some embodiments, step S514 may include: preparing a nucleic acid aptamer solution of thiol-modified Aβ protein, using 10 μL of 10 mM tris(2-carboxyethyl)phosphine TCEP and 10 μM (10 μL) of thiol-modified aptamer to mix and activate for 30 minutes, incubating on the chip for 12-24 hours, repeatedly washing with pure water, and gently blowing dry with nitrogen gas to allow the functional groups of the functionalized molecular connection layer to fully react with the thiol groups, thereby fixing the nucleic acid aptamer on the medium layer.

[0150] In some embodiments, the nucleic acid aptamer is a nucleic acid aptamer of a thiol-modified Aβ protein.

[0151] Taking the preparation of a 4-inch wafer field effect transistor biosensor 100 as an example, a 1× phosphate buffer (PBS) can be used to prepare a nucleic acid aptamer solution of thiol-modified Aβ protein with a solution concentration of 10 μM. The solution is incubated on the chip gate window dielectric layer for 12 hours, and the nucleic acid aptamer is fixed in the area above the channel in the form of gold nanoparticles (Au NPs) and thiol (-HS) to achieve functional modification of the device.

[0152] In some embodiments, the thickness of the functionalized molecular connecting layer may be 1 nm to 8 nm.

[0153] Fig.10 The schematic diagram of the device structure prepared in this step is shown.

[0154] Step S516, sealing the redundant sites on the dielectric layer and the functionalized molecular connection layer with a sealing agent.

[0155] In some embodiments, in step S516, the blocking agent may be a mixed blocking agent comprising bovine serum albumin, 6-mercapto-1-hexanol and Tween 20.

[0156] In some embodiments, step S516 may include: performing multiple blocking operations on redundant sites on the dielectric layer in a predetermined order by using one or two of the bovine serum albumin, 6-mercapto-1-hexanol, and Tween 20 for each blocking operation. Through multi-step blocking, non-specific adsorption of complex non-target molecules in serum can be more effectively avoided, thereby improving the anti-interference ability of the device.

[0157] For example, in step S516, 6-mercapto-1-hexanol may be used as a blocking agent to block for 0.5-1 hour, and then Tween 20 and bovine serum albumin may be used to block for 1.5-2.5 hours.

[0158] For another example, in step S516, 6-mercapto-1-hexanol, bovine serum albumin, and Tween 20 can be used for blocking in a random order, respectively. The blocking time of 6-mercapto-1-hexanol is 0.5-1 hour, the blocking time of bovine serum albumin is 1.5-3 hours, and the blocking time of Tween 20 is more than 0.5 hour.

[0159] For another example, in step S516, 6-mercapto-1-hexanol may be used as a blocking agent to block for a second predetermined time, and then Tween 20 and bovine serum albumin may be used to block for a third predetermined time.

[0160] Taking the preparation of the FET biosensor 100 of a 4-inch wafer as an example, 6-mercapto-1-hexanol (MCH) with a concentration of 2 millimolar (mM) can be used as a blocking agent for 0.5 hours, and then blocked with Tween20 with a mass fraction of 0.5% and BSA with a concentration of 1 millimolar (mM) for 1.5 hours.

[0161] The device structure obtained in step S516 is as follows: Figure 1 shown.

[0162] The carbon nanotube biosensor disclosed in the present invention can be used for the detection of Alzheimer's disease. For example, the carbon nanotube biosensor disclosed in the present invention can be specifically used for the detection of early Alzheimer's disease.

[0163] The field effect tube biosensor disclosed in the present invention can achieve the following beneficial effects:

[0164] The field effect transistor biosensor disclosed in the present invention uses small molecule nucleic acid aptamers as probe molecules of functionalized carbon nanotube transistor sensors, and can utilize the configuration change of the aptamer charge skeleton to achieve target biomolecule detection in high ionic strength solution environments such as serum. In addition, the present invention can directly detect serum and plasma samples without complex sample processing.

[0165] The field effect transistor biosensor disclosed in the present invention realizes high-sensitivity, high-selectivity and anti-interference detection of Aβ protein aggregates and Tau protein, which are AD markers in serum. The detection is real-time, the detection time is less than 5 minutes, and the lower limit of detection can reach 1fM (Fermi), which can meet the sensitivity required by human serum-related indicators.

[0166] The following experimental data proves that the present disclosure can achieve the above technical effects:

[0167] Fig.11 and Fig.12The schematic diagram shows the test results of direct detection of serum plasma samples by three field effect tube biosensors at the same time. Serum plasma samples were added every 200 seconds, the concentrations of control group BSA and control group IgG were 100 pM, and the serum plasma samples ranged from 1 femtomolar (fM) to 10 pM. Fig.11 and Fig.12 In the figure, the horizontal axis is time (Times) and the vertical axis is response (△I / I (%)).

[0168] pass Fig.11 and Fig.12 It can be seen that the detection of the field effect transistor biosensor disclosed in the present invention is real-time, the detection time is less than 5 minutes, and the detection lower limit can reach 1fM, which can meet the sensitivity required by human serum-related indicators. That is, the field effect transistor biosensor 100 disclosed in the present invention can achieve high-sensitivity detection of AD markers Aβ protein aggregates and Tau protein in serum.

[0169] In addition, through Fig.11 and Fig.12 It can be seen that the normalized current in the tests of the control group BSA and the control group IgG has almost no change, while the current change in the test results of the field effect transistor biosensor disclosed in the present invention exceeds 40%, that is, the detection sensitivity of the field effect transistor biosensor disclosed in the present invention is significantly higher than that of the related art.

[0170] Fig.13 Shown are modified Aβ 40 , Aβ 42 Selectivity test results of the aptamer field effect tube biosensor array in 1×PBS, serum environment and control groups with three orders of magnitude higher concentrations (i.e., blank control group (Blank), control group IgG, control group BSA). Fig.14 and Fig.15 The effects of various interfering substances on Aβ in serum environment are shown respectively. 42 , Aβ 40 The test results.

[0171] pass Figure 13~Figure 15 It can be seen intuitively that the field effect transistor biosensor 100 disclosed in the present invention has a strong affinity for Aβ 40 Aβ 42The responses were as high as 20% and 24% respectively. The responses of the control group IgG, BSA and the blank control group were all within 5%. It can be seen that whether in a serum environment or in PBS, whether it is a single interferent or a complex mixture, although the concentration of the control group is three orders of magnitude higher than the concentration of the target, the field effect tube biosensor 100 disclosed in the present invention has good selectivity. That is, the field effect tube biosensor 100 disclosed in the present invention has a high anti-interference ability to both single interferents and complex mixtures.

[0172] Fig.16 The following Table 1 shows the detection results of the field effect transistor biosensor 100 disclosed in the present invention and other various detection platforms such as electrochemical, optical and colorimetric methods. Fig.16 As shown in Table 1, the AD serum marker Aβ detected by the field effect transistor biosensor 100 of the present disclosure in human serum 40 The detection limit was 55aM, Aβ 42 The detection limit is 45aM. This is the highest detection limit of AD serum marker Aβ in serum so far. 40 , Aβ 42 minimum concentration.

[0173] Table 1

[0174]

[0175] Fig.17 The results show that the field effect transistor biosensor 100 detects the AD serum marker Aβ in serum environment using different blocking agent combinations. 40 , Aβ 42 Schematic diagram of the selectivity of Fig.17 As shown in Table 1, the use of MCH, BSA and Tween 20 as blocking agents simultaneously has very good blocking performance, which greatly improves the selectivity of the field effect tube biosensor 100 for Aβ 40 The selectivity ratio can reach 730% for Aβ 42 The selectivity ratio can reach 800%, which is much higher than the reported values ​​of other detection platforms.

[0176] In the description of this specification, the description with reference to the terms "one embodiment / method", "some embodiments / methods", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment / method or example are included in at least one embodiment / method or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment / method or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments / methods or examples. In addition, those skilled in the art may combine and combine the different embodiments / methods or examples described in this specification and the features of the different embodiments / methods or examples, unless they are contradictory.

[0177] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0178] Those skilled in the art should understand that the above embodiments are only for the purpose of clearly illustrating the present disclosure, and are not intended to limit the scope of the present disclosure. For those skilled in the art, other changes or modifications may be made based on the above disclosure, and these changes or modifications are still within the scope of the present disclosure.

Claims

1. A field effect transistor biosensor for Alzheimer's disease, characterized in that: The field effect tube biosensor is used for detecting serum markers of Alzheimer's disease, and the field effect tube biosensor comprises: A substrate layer, comprising a first layer formed of a silicon material and a second layer located on the first layer, wherein the second layer is formed of a silicon dioxide material; A channel layer is formed on the substrate layer, wherein the material of the channel layer is carbon nanotubes; A source electrode and a drain electrode are formed on the channel layer, and the source electrode and the drain electrode respectively adopt a laminated metal structure, and the laminated metal structure includes a first metal layer as a bottom layer, an ohmic contact layer as a middle layer, and a second metal layer as a top layer; A dielectric layer formed in the channel, the dielectric layer is a high-κ dielectric layer, the κ value is greater than 10, and the channel is a region between the source and the drain on the channel layer; A first passivation layer is formed on the drain electrode and a second passivation layer is formed on the source electrode, the first passivation layer covers the drain electrode, and the drain electrode is isolated from the liquid environment during the test by the first passivation layer, and the second passivation layer covers the source electrode, and the source electrode is isolated from the liquid environment during the test by the second passivation layer; A functionalized molecular connection layer is formed on the dielectric layer, the functionalized molecular connection layer is used to provide functional group sites for connecting nucleic acid aptamers, the functionalized molecular connection layer is gold nanoparticles deposited on the dielectric layer or 3-aminopropyltriethoxysilane coupled to the dielectric layer; the thickness of the functionalized molecular connection layer is 1nm~8nm; A nucleic acid aptamer fixed on the functionalized molecular connection layer, wherein the nucleic acid aptamer corresponds to a serum protein marker of Alzheimer's disease; The blocking molecules are located above the medium layer and are used to block the redundant sites on the medium layer and the functionalized molecular connection layer. The blocking molecules are mixed blocking molecules, which include bovine serum albumin, 6-mercapto-1-hexanol and Tween 20.

2. The field effect transistor biosensor for Alzheimer's disease according to claim 1, characterized in that: The nucleic acid aptamer is fixed on the functionalized molecular connection layer in the form of gold-thiol bonds or amide bonds.

3. The field effect transistor biosensor for Alzheimer's disease according to claim 1, characterized in that: The nucleic acid aptamer is a nucleic acid aptamer of thiol-modified Aβ protein.

4. The field effect transistor biosensor for Alzheimer's disease according to claim 1, characterized in that: The sequence of the nucleic acid aptamer includes: Aβ 42 Aggregate probe nucleic acid sequence: 5′-AGTCTAGGATTCGGCGTGGGTTAATTTTTTGCTGCCTGTGGTGTTGGGGCGGGTGCG-3′; Aβ 40 Aggregate probe nucleic acid sequence: 5'-GCTGCCTGTGGTGTTGGGGCGGGTGCG-3'.

5. The field effect transistor biosensor for Alzheimer's disease according to claim 1, characterized in that: The field effect tube biosensor is used for detecting early stage Alzheimer's disease.

6. A method for preparing a field effect transistor biosensor for Alzheimer's disease, characterized in that: The field effect tube biosensor is used for detecting serum markers of Alzheimer's disease, and the method comprises: Providing a substrate, the substrate comprising a first layer formed of a silicon material and a second layer located on the first layer, the second layer being formed of a silicon dioxide material; Etching on the substrate to form a channel layer, wherein the material of the channel layer is carbon nanotubes; A source electrode and a drain electrode are formed on the channel layer, wherein the source electrode and the drain electrode respectively adopt a laminated metal structure, wherein the laminated metal structure includes a first metal layer as a bottom layer, an ohmic contact layer as a middle layer, and a second metal layer as a top layer; Depositing a dielectric layer in the channel between the source and the drain on the channel layer, wherein the dielectric layer is a high-κ dielectric layer, and the κ value is greater than 10; A functionalized molecular connection layer is prepared on the dielectric layer, wherein the functionalized molecular connection layer is gold nanoparticles deposited on the dielectric layer or 3-aminopropyltriethoxysilane coupled on the dielectric layer; the thickness of the functionalized molecular connection layer is 1 nm to 8 nm; Spin-coat a first passivation layer on the drain electrode, and spin-coat a second passivation layer on the source electrode, wherein the first passivation layer covers the drain electrode, and the drain electrode is isolated from the liquid environment during the test by the first passivation layer, and the second passivation layer covers the source electrode, and the source electrode is isolated from the liquid environment during the test by the second passivation layer; Immobilizing nucleic acid aptamers on the functionalized molecular connection layer, wherein the nucleic acid aptamers correspond to serum protein markers of Alzheimer's disease; The redundant sites on the medium layer and the functionalized molecular connection layer are blocked by a blocking agent, wherein the blocking agent is a mixed blocking agent including bovine serum albumin, 6-mercapto-1-hexanol and Tween 20.

7. The method for preparing the field effect transistor biosensor for Alzheimer's disease according to claim 6, characterized in that: The method of fixing the nucleic acid aptamer on the functionalized molecular connection layer includes: fixing the nucleic acid aptamer on the gold nanoparticle functional layer on the dielectric layer through a gold-thiol bond; or fixing the nucleic acid aptamer on the dielectric layer modified with 3-aminopropyltriethoxysilane through an amide bond.

8. The method for preparing the field effect transistor biosensor for Alzheimer's disease according to claim 7, characterized in that: The method of fixing the nucleic acid aptamer on the gold nanoparticle functional layer above the medium layer in the form of a gold-sulfhydryl bond comprises: preparing a nucleic acid aptamer solution of thiol-modified Aβ protein, wherein the solution environment is pure water, a phosphate buffer solution or a 4-morpholineethanesulfonic acid buffer solution, and the pH value of the solution is in the range of 7-7.8; dripping the nucleic acid aptamer solution on the gold nanoparticle functional layer region above the medium layer for 12-24 hours, so that the gold nanoparticles react fully with the thiol groups, thereby fixing the nucleic acid aptamer on the functionalized molecular connection layer above the medium layer in the form of a gold-sulfhydryl bond.

9. The method for preparing the field effect transistor biosensor for Alzheimer's disease according to claim 7, characterized in that: The nucleic acid aptamer is fixed on the medium layer modified by 3-aminopropyltriethoxysilane in the form of amide bond linkage, comprising: configuring a nucleic acid aptamer solution of Aβ protein with a modified carboxyl group at the 5' end, wherein the solution environment is pure water, phosphate buffer or 4-morpholineethanesulfonic acid buffer solution, and the pH value of the solution is in the range of 5-7.4; dropping the nucleic acid aptamer solution on the medium layer functionalized by silane amino groups, adding 0.05 mol to 0.1 mol of carbodiimide and 1 mmol to 10 mmol of N-hydroxysuccinimide to activate the carboxyl group for 2-4 hours, so that the amino functional group of the functionalized molecular connection layer fully reacts with the carboxyl group of the nucleic acid aptamer to form an amide bond, thereby fixing the nucleic acid aptamer on the functionalized molecular connection layer above the medium layer.

10. The method for preparing a field effect transistor biosensor for Alzheimer's disease according to claim 6, characterized in that: The method of blocking the redundant sites on the medium layer and the functionalized molecular connection layer by using a blocking agent includes: first using 6-mercapto-1-hexanol as a blocking agent to block for 0.5-1 hour, and then using Tween 20 and bovine serum albumin to block for 1.5-2.5 hours.

11. The method for preparing a field effect transistor biosensor for Alzheimer's disease according to claim 6, characterized in that: The method of sealing the redundant sites on the medium layer and the functionalized molecular connection layer by using a sealing agent comprises: using 6-mercapto-1-hexanol, bovine serum albumin, and Tween 20 to seal in a random order; wherein the sealing time of the 6-mercapto-1-hexanol is 0.5-1 hour, the sealing time of the bovine serum albumin is 1.5-3 hours, and the sealing time of the Tween 20 is more than 0.5 hour.

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