Method for detecting AD biomarker in serum based on carbon-based field effect transistor biosensor

Abstract

According to the method for detecting the AD biomarker in the serum based on the carbon-based field effect transistor biosensor, an AL-BSA nano-film is deposited on a channel region of a CNT-FET, and the Debye volume of a device is increased by utilizing a porous structure of the AL-BSA nano-film, so that the Debye length of the device under high ionic strength (0.1 * PBS buffer solution) is increased, and the detection sensitivity of the AD biomarker in the serum is improved. The biological probe can sense the AD biomarker in the serum, and a signal is acquired, so that the biological probe and the AD biomarker are combined within the Debye length, and the AD biomarker in the serum can be directly and sensitively detected under the condition of relatively low Debye length; the technical problem that an existing FET biosensor cannot detect AD biomarkers in serum under high ionic strength is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a method for detecting AD biomarkers in blood based on a carbon-based field effect transistor biosensor. Background Art

[0002] Alzheimer's disease (AD) is a degenerative neurological disease characterized by the aggregation of amyloid-β (Aβ) and neurofibrillary tangles formed by phosphorylated tau filaments. The National Institute on Aging and the Alzheimer's Institute (NIA-AA) proposed to use Aβ deposition, pathological Tau, and neurodegenerative biomarkers to define the biological characteristics of AD. Studies have shown that Tau pathology is closely related to the onset of neurodegeneration and the manifestation of clinical symptoms in space and time. Higher levels of P-tau181 are associated with an increased chance of autopsy-confirmed AD and distinguish between AD and non-AD pathology within 8 years before death. Compared with the invasive method of obtaining cerebrospinal fluid (CSF) through lumbar puncture, less invasive and cost-effective blood biomarkers are ideal for detecting AD. However, due to the blood-brain barrier, the concentration of biomarkers in blood is usually 1 to 2 orders of magnitude lower than that in CSF, which makes high-performance detection of P-tau181 biomarkers more difficult.

[0003] Current methods for detecting AD biomarkers in blood include enzyme-linked immunosorbent assay, electrochemistry, and electrochemiluminescence, but the low sensitivity, high cost, and complexity of these methods limit their application in blood testing. Field effect transistors (FETs) have become one of the most promising technologies for detecting low-abundance biomarkers due to their low cost, high sensitivity, simple operation, and no labeling required. Semiconductor carbon nanotubes have ultrahigh mobility and good biocompatibility, which makes them a good candidate for high-performance FET biosensors and has attracted widespread attention.

[0004] However, existing FET biosensors have a Debye length in the buffer, which causes a double electric layer to form at a specific distance above the sensor surface. Charged molecules cannot be detected beyond this distance, thus limiting the detection of biomolecules. Since the size of the bioprobe is usually about 10nm, which exceeds the Debye length, it is usually not detectable under bare FET. Strategies that have been explored include using shorter bioprobes, dilute electrolyte solutions, and modified polyethylene glycol polymers to address the Debye length limitation. However, these methods can usually only detect targets in 0.01× PBS buffer and cannot be detected at higher ionic strengths, limiting the application of FET biosensors. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor, aiming to achieve direct and sensitive detection of AD biomarkers in serum under low Debye length conditions by using a carbon-based field effect transistor biosensor with an amyloid protein membrane as a dielectric layer, thereby solving the technical problem that existing FET biosensors cannot detect AD biomarkers in serum under higher ionic strengths.

[0006] According to a first aspect of the present invention, a method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor is provided, comprising the following steps:

[0007] S1. Prepare an AL-FET biosensor, use a semiconductor single-walled carbon nanotube network film as a channel layer, and deposit a phase-change bovine serum albumin (AL-BSA) nanofilm layer on the surface of the channel region, and a biological probe for detecting target molecules is provided on the AL-BSA nanofilm layer; wherein the length of the biological probe is greater than the Debye length of the AL-FET biosensor in 0.1×PBS buffer;

[0008] S2, incubate and clean the sensing interface of the AL-FET biosensor with 0.1×PBS buffer, then use 0.1×PBS buffer as a liquid gate, measure at a set gate voltage and drain bias, record the current between the source and drain, and obtain the corresponding transfer curve as a baseline;

[0009] S3, cleaning and drying the AL-FET biosensor after step S1, then dropping the serum sample to be tested onto the sensing interface of the AL-FET sensor for incubation, cleaning with 0.1×PBS buffer after incubation, and after drying, using 0.1×PBS buffer as a liquid gate to measure under the set gate voltage and drain bias, recording the current between the source and drain, and obtaining the corresponding transfer curve;

[0010] S4, calculating the responsiveness of the serum sample to be tested according to the baseline obtained in step S2 and the transfer curve obtained in step S3, if the responsiveness of the serum sample to be tested is greater than the response threshold value, it indicates that AD biomarkers are present in the serum sample to be tested;

[0011] Among them, the Debye length of the AL-FET sensor in 0.1×PBS buffer is increased by the porous structure of the AL-BSA nanomembrane, so that the biological probe and the target molecule are combined within the Debye length, thereby achieving the purpose of detecting AD biomarkers in serum under the condition that the length of the biological probe is greater than the Debye length of the AL-FET sensor in 0.1×PBS buffer.

[0012] As an optional embodiment, the difference between the length of the biological probe and the Debye length of the AL-FET sensor in 0.1×PBS buffer is 1.6 nm to 2.6 nm.

[0013] As an optional implementation, the responsivity is calculated according to formula (1):

[0014] Responsivity = |II 0 ∣ / I 0 (1)

[0015] Where, I is the maximum current value between the source and the drain measured in step S3, I 0 is the maximum current value between the source and the drain measured in step S2.

[0016] As an optional implementation, the response threshold is 0.02.

[0017] As an optional implementation, in step S2, the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

[0018] As an optional implementation, in step S3, the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

[0019] As an optional embodiment, the AL-BSA nanofilm is formed by dropping a BSA amyloid protein solution onto the channel region between the source and drain electrodes.

[0020] As an optional embodiment, the BSA amyloid solution is formed by mixing a BSA solution and a TCEP solution.

[0021] As an optional embodiment, the AD biomarker is P-tau181.

[0022] As an optional embodiment, the preparation of the AL-FET biosensor comprises:

[0023] S11, in Si / SiO 2 A carbon nanotube network film is deposited on the substrate to form a CNT layer;

[0024] S12, forming a source-drain region on the CNT layer by ultraviolet lithography, and then depositing a Ti / Pd / Au metal layer on the formed source-drain region by electron beam vapor deposition to form a source-drain electrode, obtaining a CNT channel region connected to the source-drain electrode, and etching away excess carbon nanotube network film;

[0025] S13, passivating the device obtained in step S12 with a photoresist, and performing exposure, development and fixing of the channel region with a laser direct writing lithography machine, so that the carbon nanotube network film in the channel region is exposed, thereby obtaining a carbon nanotube field effect transistor CNT-FET;

[0026] S14, mixing the BSA solution and the TCEP solution to obtain a BSA amyloid protein solution, dropping the BSA amyloid protein solution onto the channel region of the CNT-FET to react and form an AL-BSA nanofilm, and then washing with ultrapure water and drying with nitrogen;

[0027] S15. A mixed solution of EDC and NHS is added dropwise onto the AL-BSA nanomembrane and incubated at room temperature to activate the carboxyl groups of the AL-BSA nanomembrane. Then, a biological probe is added dropwise onto the treated AL-BSA nanomembrane to form an amide bond between the amino group on the biological probe and the carboxyl group of the AL-BSA nanomembrane, thereby obtaining an AL-FET biosensor.

[0028] It can be seen from the above technical solutions of the present invention that the method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor proposed by the present invention deposits an AL-BSA nanofilm on the channel region of the CNT-FET, and utilizes the porous structure of the AL-BSA nanofilm to increase the Debye volume of the device, thereby increasing the Debye length of the device under high ionic strength (0.1×PBS buffer), so that the biological probe can sense the target molecules in the serum, obtain signals so that the biological probe and the target molecules are combined within the Debye length, and the interconnected porous structure on the AL-BSA nanofilm can enhance the diffusion of molecules by promoting the movement of fluids, molecules and ions, thereby enhancing signal detection, thereby achieving the purpose of detecting AD biomarkers in serum under the condition that the biological probe length is greater than the Debye length of the AL-FET sensor in 0.1×PBS buffer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the structure of the AL-FET biosensor in the example of the present invention.

[0030] Figure 2 It is a flow chart of the preparation of the AL-FET biosensor in an example of the present invention.

[0031] Figure 3 Schematic diagram of using AL-FET biosensor to detect AD biomarkers in serum in an example of the present invention.

[0032] Figure 4It is an AFM image of the AL-FET biosensor in the example of the present invention; wherein, 4a is an atomic force microscope characterization image of CNT, 4b is an atomic force microscope characterization image of AL-BSA nanofilm modified on the CNT surface, and 3c is an atomic force microscope characterization image of antibody modified on the surface of 4b (on AL-BSA).

[0033] Figure 5 is a box plot that distinguishes healthy individuals from AD patients in the example of the present invention.

[0034] Figure 6 a is the change of the transfer curve of the AL-FET biosensor in the present invention for detecting different concentrations of P-tau181, Figure 6 b is the responsiveness fitting curve of the AL-FET biosensor to different concentrations of P-tau181 in the example of the present invention, Figure 6 c is a graph showing the relationship between the responsiveness of the AL-FET biosensor and the concentration of P-tau181 in an example of the present invention.

[0035] Figure 7 a is the change of the transfer curve of the AL-FET biosensor in the present invention for detecting different concentrations of P-tau181, Figure 7 b is the change in the transfer curve of the F-FET biosensor in the example of the present invention detecting different concentrations of P-tau181.

[0036] Figure 8 This is a statistical chart comparing the responsiveness of AL-FET and F-FET to P-tau181 in the example of the present invention.

[0037] Fig. 9 It is a schematic diagram of AL-FET and F-FET detecting P-tau181 in the example of the present invention.

[0038] Fig.10 is the transfer curve of the spiked detection in the example of the present invention. DETAILED DESCRIPTION

[0039] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

[0040] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

[0041] Buffer solutions play an important role in helping to maintain the acid-base balance in the human body, protecting the stability of drugs, protecting tissues, and in drug development. In order to increase the Debye length, most FET biosensors currently use 0.01×PBS (Debye length 7.4nm) as the liquid gate for detection, but low-concentration PBS is not conducive to the binding of aptamers and targets, as well as the storage of antibodies, aptamers, etc., while increasing the PBS concentration (increasing the ionic strength) will cause the Debye length to be insufficient, making it impossible for aptamers and targets to be sensed.

[0042] Therefore, the present invention constructs a method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor. By using a carbon-based field effect transistor biosensor with an amyloid protein membrane as a dielectric layer, the Debye length of the carbon-based field effect transistor biosensor in 0.1×PBS is increased to ensure that the aptamer and the target can be sensed. On this basis, the concentration of PBS as a liquid gate is increased, which promotes the binding of the aptamer and the target. In this way, the sensitivity of detection is improved, and the AD biomarker in serum can be more effectively detected under the condition that the length of the biological probe is greater than the Debye length of the AL-FET sensor in 0.1×PBS buffer.

[0043] AL-FET biosensor

[0044] Combination Figure 1 As shown, the exemplary AL-FET sensor of the present invention includes, from bottom to top, SiO 2 Dielectric layer 2, forming Si / SiO 2 Substrate, located on Si / SiO 2 A semiconductor single-walled carbon nanotube network film channel layer 3 on a substrate, a metal source electrode 4 and a metal drain electrode 5 located on the semiconductor carbon nanotube network film, a passivation layer 6 located on the source and drain electrodes, a phase change bovine serum albumin (AL-BSA) nanofilm layer 7 located between the source and drain electrodes, and

[0045] The biological probe 8 for detecting the target is located on the AL-BSA nano-membrane layer, and the length of the biological probe 8 is greater than the Debye length of the AL-FET biosensor in 0.1×PBS buffer.

[0046] As an optional example, the length of the biological probe is 4 to 5 nm.

[0047] As an optional example, the silicon-based substrate is a P-type silicon substrate, Si / SiO 2 The substrate can be purchased directly.

[0048] As an optional example, the thickness of the semiconductor single-walled carbon nanotube network film is less than 2 nm, the channel length is designed to be 60 μm, and the width is 20 μm.

[0049] As an optional example, the thickness of the AL-BSA nanofilm is 10 nm to 15 nm, and particularly preferably 12 nm.

[0050] Preparation method of AL-FET biosensor

[0051] Combination Figure 2 As shown, the preparation process of the aforementioned AL-FET biosensor includes the following steps:

[0052] Step 1-1: On Si / SiO 2 A semiconductor single-walled carbon nanotube network film is deposited on the substrate to form a CNT layer.

[0053] Step 1-2: Form a source-drain region on the CNT layer by ultraviolet lithography, and then deposit a Ti / Pd / Au metal layer on the formed source-drain region by electron beam vapor deposition to form a source-drain electrode, obtain a CNT channel region connecting the source-drain electrode, and etch away excess carbon nanotube network film.

[0054] Step 1-3: passivate the obtained device with photoresist, and use a laser direct writing lithography machine to expose, develop and fix the channel area to expose the carbon nanotube network film in the channel area to obtain a carbon nanotube field effect transistor CNT-FET.

[0055] Step 1-4, mixing the BSA solution and the TCEP solution to obtain a BSA amyloid protein solution, dropping the BSA amyloid protein solution onto the channel region of the CNT-FET to react and form an AL-BSA nanofilm, and then rinsing with ultrapure water and drying with nitrogen.

[0056] Step 1-5: Add a mixed solution of EDC and NHS onto the AL-BSA nanomembrane and incubate at room temperature to activate the carboxyl group of the AL-BSA nanomembrane. Then, add the biological probe onto the treated AL-BSA nanomembrane to form an amide bond between the amino group on the biological probe and the carboxyl group of the AL-BSA nanomembrane to obtain an AL-FET biosensor.

[0057] As an optional example, the volume ratio of the BSA solution to the TCEP solution is 1:1, the concentration of the BSA solution is 5 mg / mL, the concentration of the TCEP solution is 50 mM, and the pH is adjusted to 5.0 using a NaOH solution.

[0058] As an optional example, the mixed solution of EDC and NHS contains 0.4M EDC and 0.1M NHS, and the pH value of the mixed solution is 5.0. In a preferred embodiment, maleic acid (MES) is used to adjust the pH value of the mixed solution.

[0059] The formation mechanism of the phase-change bovine serum albumin (AL-BSA) nanofilm in the AL-FET biosensor of the present invention is as follows:

[0060] The phase transition process is triggered by TCEP reducing the disulfide bonds of BSA, which converts native BSA into phase-change BSA oligomer nanoparticles and fibrils in the solution, and then the fibrils in the solution aggregate to form microparticles. The hydrophobic aggregation caused by TCEP reducing the disulfide bonds within the BSA molecule is mediated by the net charge of the protein colloid. When the pH is close to the isoelectric point of the protein, the electrostatic repulsion between the colloids is weakened and the aggregation of amyloid proteins is enhanced. BSA oligomer nanoparticles can form a stable bond with the substrate through hydrogen bonds, electrostatic interactions, and hydrophobic interactions, so that they can be assembled at the solid-liquid interface to form a nanofilm.

[0061] Method for detecting AD biomarkers in serum based on carbon-based field-effect transistor biosensor

[0062] Combination Figure 3 As shown, in an exemplary embodiment of the present invention, the method for detecting AD biomarkers in serum using the aforementioned AL-FET biosensor comprises the following steps:

[0063] (1) The sensing interface of the AL-FET biosensor was incubated and cleaned with 0.1×PBS buffer. Then, 0.1×PBS buffer was used as a liquid gate. Measurements were performed under set gate voltages and drain biases. The currents between the source and drain were recorded, and the corresponding transfer curves were obtained as baselines.

[0064] (2) After step (1), the AL-FET biosensor is cleaned and dried, and then the serum sample to be tested is added to the sensing interface of the AL-FET sensor for incubation. After the incubation is completed, it is cleaned with 0.1×PBS buffer. After drying, 0.1×PBS buffer is used as a liquid gate to measure under the set gate voltage and drain bias, and the current between the source and the drain is recorded to obtain the corresponding transfer curve.

[0065] (3) Calculating the responsiveness of the serum sample to be tested based on the baseline obtained in step (1) and the transfer curve obtained in step (2). If the responsiveness of the serum sample to be tested is greater than the response domain value, it indicates that AD biomarkers are present in the serum sample to be tested.

[0066] Among them, the Debye length of the AL-FET biosensor in 0.1×PBS buffer is increased by the porous structure of the AL-BSA nanomembrane, so that the biological probe and the target molecule are combined within the Debye length, thereby achieving the purpose of detecting AD biomarkers in serum under the condition that the length of the biological probe is greater than the Debye length of the AL-FET sensor in 0.1×PBS buffer.

[0067] The Debye length of the sensor in 0.1×PBS buffer solution is about 2.4nm. The antibody (bioprobe) used in the AL-FET biosensor of the present invention is about 4-5nm long, which exceeds the 2.4nm Debye length of 0.1×PBS. Therefore, the target molecules in the serum can hardly be detected under the existing sensor.

[0068] The present invention utilizes the porous structure of the AL-BSA nanomembrane in the AL-FET biosensor to increase the Debye volume, thereby increasing the Debye length, and the interconnected porous structure can enhance the diffusion of molecules by promoting the movement of fluids, molecules and ions, thereby enhancing signal detection, so that the AL-FET biosensor can detect target molecules in 0.1×PBS buffer, and increase the ionic strength from 0.01×PBS to 0.1×PBS, which is more conducive to the binding of aptamers and targets, as well as the storage of antibodies, aptamers, etc.

[0069] As an optional example, the difference between the length of the biological probe and the Debye length of the AL-FET sensor in 0.1×PBS buffer is 1.6 nm to 2.6 nm.

[0070] As an optional example, the responsivity is calculated according to formula (1):

[0071] Responsivity = |II 0 ∣ / I 0 (1)

[0072] Where I is the maximum current between the source and drain measured in step (2), I 0 is the maximum current value between the source and the drain measured in step (1).

[0073] As an optional example, the response threshold is 0.02.

[0074] As an optional example, in step (1), the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

[0075] As an optional example, in step (2), the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

[0076] As an alternative example, the AD biomarker is P-tau181.

[0077] As an optional example, the serum sample to be tested is diluted to 50% with 0.1×PBS buffer. There is a very high concentration of interfering proteins in the whole serum, which will cause undesirable effects on the sensor. Appropriate dilution of the sample can reduce the interference of the protein.

[0078] It can be understood that the dilution concentration includes but is not limited to 50%, as long as it is within the detection limit of the biosensor used for detection and makes the sensor relatively stable.

[0079] In other optional examples, the aforementioned AL-FET biosensor can be used to detect the AD biomarker P-tau181 in other solvent environments. For example, when PBS buffer is used as the solvent, the blank response of PBS is used as the threshold to compare whether P-tau181 exists in the sample to be tested.

[0080] In other examples, in some samples, the P-tau181 in the sample to be tested can be quantitatively tested by drawing a P-tau181 standard curve.

[0081] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0082] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0083] Example 1

[0084] [Preparation of AL-FET biosensor]

[0085] In Si / SiO 2 A semiconductor single-walled carbon nanotube network film is deposited on a substrate to obtain a CNT wafer, which is directly purchased from Suzhou Enjing Semiconductor Technology Co., Ltd.

[0086] 1. Form a source-drain region on the CNT layer by UV lithography, and then deposit a Ti / Pd / Au metal layer (Ti / Pd / Au thicknesses are 0.3 / 30 / 40 nm, respectively) on the formed source-drain region by electron beam vapor deposition to form source-drain electrodes, obtain a CNT channel region connecting the source-drain electrodes, and etch away excess carbon nanotube network film; the channel length is 60 μm and the width is 20 μm.

[0087] 2. Use photoresist to passivate the obtained device, and use a laser direct writing lithography machine to expose, develop and fix the channel area to expose the carbon nanotube network film in the channel area, obtain a carbon nanotube field effect transistor CNT-FET, and store it in a vacuum environment.

[0088] 3. Prepare the BSA amyloid solution by mixing BSA solution (5 mg / mL) and TCEP solution (50 mM, pH 5.0, pH adjusted with NaOH solution) in a volume ratio of 1:1, and drop the BSA amyloid solution onto the channel region of CNT-FET to react for 2 h to form an AL-BSA nanofilm, which is then rinsed with ultrapure water and blown dry with nitrogen.

[0089] 4. Add 10 μL of solution (containing 0.4 M EDC and 0.1 M NHS, pH 5.0) on the AL-BSA nanomembrane and incubate at room temperature for 30 minutes to activate the carboxyl groups of the AL-BSA nanomembrane. Then, add 10 μL of 100 μg / mL antibody on the treated AL-BSA nanomembrane at room temperature for 2.5 hours to form an amide bond between the amino group on the antibody and the carboxyl group of the AL-BSA nanomembrane to obtain the AL-FET biosensor.

[0090] like Figure 4 As shown, the AFM image illustrates the uniform deposition of carbon nanotubes on the substrate ( Figure 4 a); After AL-BSA modification, the protein nanofilm evenly covers the CNT surface ( Figure 4 b); The surface roughness (Ra) of AL-BSA nanofilm after antibody modification decreased from 2.08 nm to 1.96 nm ( Figure 4 c), indicating that the antibody filled the gaps in the AL-BSA nanomembrane, resulting in a decrease in roughness.

[0091] Example 2

[0092] [Serum sample testing]

[0093] There were 25 serum samples, including 20 patients and 5 normal subjects.

[0094] 1. Dilute the obtained serum sample to 50% with 0.1× PBS to prepare the test solution.

[0095] 2. 10 μL 0.1×PBS was added to the surface of the prepared AL-FET (prepared according to the method of Example 1) and incubated for 0.5 h, then washed with 0.1×PBS and then with ultrapure water, and finally dried with nitrogen gas, and 10 μL 0.1×PBS was added as a liquid gate, the gate voltage was set to -0.6V-0.6V, the drain bias was -0.1V, and a data point was collected every 0.02V. The current between the source and the drain was recorded using a semiconductor parameter characteristic analyzer, and the obtained transfer curve was recorded as the baseline, and the current I at -0.6V was recorded. 0 .

[0096] 3. After step 2, clean the AL-FET with ultrapure water and blow dry with nitrogen. Then, drop the test liquid onto the surface of AL-FET and incubate for 0.5h. Then, wash it with 0.1×PBS and then with ultrapure water. Finally, blow dry with nitrogen. Add 10μL 0.1×PBS as the liquid gate, set the gate voltage to -0.6V-0.6V, the drain bias to -0.1V, collect a data point every 0.02V, use a semiconductor parameter characteristic analyzer to record the current between the source and drain, obtain the transfer curve, and record the current I at -0.6V.

[0097] 4. Pass | II 0 ∣ / I 0 The response of each unknown sample was calculated, and the response of normal (HC) and patients (AD) was recorded by counting the responses of different samples. The significant difference between the responses of patients and normal subjects and the response threshold were also counted.

[0098] like Figure 5 As shown, according to the data, the AL-FET sensor can well distinguish normal people from patients (P<0.0001), and the response threshold of the sensor is 0.02. When the response exceeds 0.02, the AD biomarker P-tau181 can be detected and the patient is considered to be ill. When the response is less than or equal to 0.02, the AD biomarker P-tau181 cannot be detected and the patient is considered to be normal.

[0099] Example 3

[0100] [Detection limit]

[0101] 1. Add 10 μL 0.1×PBS to the surface of the prepared AL-FET (prepared according to the method of Example 1) and incubate for 0.5 h, then wash with 0.1×PBS and then with ultrapure water, finally blow dry with nitrogen, add 10 μL 0.1×PBS as the liquid gate, set the gate voltage to -0.6V-0.6V, the drain bias to -0.1V, collect a data point every 0.02V, use a semiconductor parameter characteristic analyzer to record the current between the source and the drain, and record the obtained transfer curve as the baseline.

[0102] 2. Prepare P-tau181 solutions of different concentrations (from 1fg / mL to 100ng / mL) (1fg / mL, 10fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, 10ng / mL, 100ng / mL). For different concentrations of P-tau181, add them to the AL-FET surface and incubate for 0.5h, then wash with 0.1×PBS and then with ultrapure water, finally blow dry with nitrogen, add 10μL 0.1×PBS as liquid gate, set the gate voltage to -0.6V-0.6V, the drain bias voltage to -0.1V, collect a data point every 0.02V, and use a semiconductor parameter characteristic analyzer to record the current between the source and the drain to obtain the transfer curve at each concentration, such as Figure 6 As shown in a.

[0103] 3. Record the source-drain current (-0.6V) obtained after adding different concentrations of P-tau181, and use ∣II 0 ∣ / I 0 (I is the source-drain current obtained by testing different concentrations of P-tau181, I 0 The response of the P-tau181 sensor at different concentrations is calculated by using the baseline current recorded. Figure 6 As shown in b.

[0104] 4. By fitting the curve obtained by AL-FET's response to different concentrations of P-tau181, the standard curve equation and detection limit were calculated, such as Figure 6 As shown in c.

[0105] The source-drain current of AL-FET increases with the increase of the concentration of target P-tau181. According to the equation of the fitting curve y=

[0106] 0.03323lg(x)+0.5381, three times the standard deviation, the detection limit is calculated to be 0.1fg / mL.

[0107] Example 4

[0108] [Sensitivity]

[0109] 1. Drop AL-BSA solution onto the CNT-FET device (prepared according to the method of Example 1) and incubate at room temperature for 2 h. Then, rinse the CNT-FET device with deionized water and dry it under nitrogen.

[0110] 2. Then, a lysozyme solution with a concentration of 5 mg / mL was added dropwise to the FET surface and incubated overnight at room temperature to ensure that the lysozyme was incorporated into the pores of AL-BSA.

[0111] 3. To activate the carboxyl groups of AL-BSA, 10 μL of a solution containing 0.4 M EDC and 0.1 M NHS (pH 5.0) was incubated on the device at room temperature for 0.5 h.

[0112] 4. Subsequently, 10 μL of antibody solution with a concentration of 100 μg / mL was added dropwise onto the FET surface and incubated at room temperature for 2.5 h to obtain a flat F-FET biosensor.

[0113] 5. Using the F-FET biosensor prepared in step 4 and the AL-FET (prepared according to the method of Example 1), P-Tau 181 was detected according to steps 1-3 in Example 3. The transfer curves were as follows: Figure 7 a and 7b.

[0114] like Figure 8 As shown in the results, it can be seen that the AL-FET biosensor shows a greater response to the detection of different concentrations of P-Tau 181, while the responsiveness of the F-FET biosensor is much smaller than that of the AL-FET biosensor.

[0115] Combination Fig. 9 As shown, this is because the F-FET biosensor uses Lys to seal the pores of the film, so the antibody can only be modified on the surface of the film, and the height of the antibody itself is 4-5nm. Therefore, when using the F-FET biosensor, the target binds to the antibody outside the Debye length of 0.1×PBS (about 2.4nm), resulting in the inability to sense the charge of the target, thereby having a low sensitivity; while in the AL-FET biosensor, the antibody is modified in the pores of the AL-BSA nanomembrane, and the porous structure increases the Debye volume, thereby increasing the Debye length, which can make the target bind to the antibody within the Debye length of 0.1×PBS, thereby having a higher sensitivity; this also proves that the method of the present invention can achieve the purpose of detecting AD biomarkers under the condition that the length of the biological probe is greater than the Debye length of the AL-FET sensor in 0.1×PBS buffer.

[0116] Example 5

[0117] [Spiked recovery]

[0118] 1. Add 10 μL 0.1×PBS to the surface of the prepared AL-FET (prepared according to the method of Example 1) and incubate for 0.5 h, then wash with 0.1×PBS and then with ultrapure water, finally blow dry with nitrogen, add 10 μL 0.1×PBS as a liquid gate, set the gate voltage to -0.6V-0.6V, the drain bias to -0.1V, collect a data point every 0.02V, use a semiconductor parameter characteristic analyzer to record the current between the source and the drain, record the obtained transfer curve as the baseline, and record the current I at -0.6V 0 .

[0119] 2. Prepare 10fg / mL of P-tau181 as the test sample, add the test sample to the surface of AL-FET and incubate for 0.5h, then wash with 0.1×PBS and then with ultrapure water, finally blow dry with nitrogen, add 10μL0.1×PBS as the liquid gate, set the gate voltage to -0.6V-0.6V, the drain bias to -0.1V, collect a data point every 0.02V, use a semiconductor parameter characteristic analyzer to record the current between the source and drain, obtain the transfer curve, and record the current I at -0.6V.

[0120] Combination Fig.10 As shown, through ∣II 0 ∣ / I 0 Calculate the response of the unknown sample and substitute it into the standard curve obtained in Example 3, |II 0 ∣ / I 0 =0.03323lg(x)+0.5381, the unknown sample concentration is calculated to be 9.77fg / mL, and the recovery rate is 97.7%. Taking 95%-105% as the judgment standard, it shows that it has good recovery efficiency and can be used to determine P-tau181 in buffer solution.

[0121] The method of the present invention can detect the low-abundance target P-tau181 under the condition that the length of the biological probe is greater than the Debye length of the AL-FET sensor in 0.1×PBS buffer.

[0122] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. A method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor, characterized in that: The following steps are involved: S1. Prepare an AL-FET biosensor, use a semiconductor single-walled carbon nanotube network film as a channel layer, and deposit a phase-change bovine serum albumin (AL-BSA) nanofilm layer on the surface of the channel region, and a biological probe for detecting target molecules is provided on the AL-BSA nanofilm layer; wherein the length of the biological probe is greater than the Debye length of the AL-FET biosensor in 0.1×PBS buffer; S2, incubate and clean the sensing interface of the AL-FET biosensor with 0.1×PBS buffer, then use 0.1×PBS buffer as a liquid gate, measure at a set gate voltage and drain bias, record the current between the source and drain, and obtain the corresponding transfer curve as a baseline; S3, cleaning and drying the AL-FET biosensor after step S1, then dropping the serum sample to be tested onto the sensing interface of the AL-FET sensor for incubation, cleaning with 0.1×PBS buffer after incubation, and after drying, using 0.1×PBS buffer as a liquid gate to measure under the set gate voltage and drain bias, recording the current between the source and drain, and obtaining the corresponding transfer curve; S4, calculating the responsiveness of the serum sample to be tested according to the baseline obtained in step S2 and the transfer curve obtained in step S3, if the responsiveness of the serum sample to be tested is greater than the response threshold value, it indicates that AD biomarkers are present in the serum sample to be tested; Among them, the Debye length of the AL-FET sensor in 0.1×PBS buffer is increased by the porous structure of the AL-BSA nanomembrane, so that the biological probe and the target molecule are combined within the Debye length, thereby achieving the purpose of detecting AD biomarkers in serum under the condition that the length of the biological probe is greater than the Debye length of the AL-FET sensor in 0.1×PBS buffer.

2. The method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor according to claim 1, characterized in that: The difference between the length of the bioprobe and the Debye length of the AL-FET sensor in 0.1×PBS buffer was 1.6 nm to 2.6 nm.

3. The method for detecting AD biomarkers in serum based on carbon-based field effect transistor biosensor according to claim 1, characterized in that: The responsivity is calculated according to formula (1): Responsivity = |I-I0| / I0(1) Wherein, I is the maximum current value between the source and the drain measured in step S3, and I0 is the maximum current value between the source and the drain measured in step S2.

4. The method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor according to claim 1, characterized in that: The response threshold was 0.

02.

5. The method for detecting AD biomarkers in serum based on carbon-based field effect transistor biosensor according to claim 1, characterized in that: In step S2, the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

6. The method for detecting AD biomarkers in serum based on carbon-based field effect transistor biosensor according to claim 1, characterized in that: In step S3, the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

7. The method for detecting AD biomarkers in serum based on carbon-based field effect transistor biosensor according to claim 1, characterized in that: The AL-BSA nanofilm is formed by dropping a BSA amyloid protein solution onto the channel region between the source and drain electrodes.

8. The method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor according to claim 7, characterized in that: The BSA amyloid solution is formed by mixing a BSA solution and a TCEP solution.

9. The method for detecting AD biomarkers in serum based on carbon-based field effect transistor biosensor according to claim 1, characterized in that: The AD biomarker is P-tau181.

10. The method for detecting AD biomarkers in serum based on carbon-based field effect transistor biosensor according to claim 1, characterized in that: The method for preparing the AL-FET biosensor comprises: S11, depositing a carbon nanotube network film on a Si / SiO2 substrate to form a CNT layer; S12, forming a source-drain region on the CNT layer by ultraviolet lithography, and then depositing a Ti / Pd / Au metal layer on the formed source-drain region by electron beam vapor deposition to form a source-drain electrode, obtaining a CNT channel region connected to the source-drain electrode, and etching away excess carbon nanotube network film; S13, passivating the device obtained in step S12 with a photoresist, and performing exposure, development and fixing of the channel region with a laser direct writing lithography machine, so that the carbon nanotube network film in the channel region is exposed, thereby obtaining a carbon nanotube field effect transistor CNT-FET; S14, mixing the BSA solution and the TCEP solution to obtain a BSA amyloid protein solution, dropping the BSA amyloid protein solution onto the channel region of the CNT-FET to react and form an AL-BSA nanofilm, and then washing with ultrapure water and drying with nitrogen; S15. A mixed solution of EDC and NHS is added dropwise onto the AL-BSA nanomembrane and incubated at room temperature to activate the carboxyl groups of the AL-BSA nanomembrane. Then, a biological probe is added dropwise onto the treated AL-BSA nanomembrane to form an amide bond between the amino group on the biological probe and the carboxyl group of the AL-BSA nanomembrane, thereby obtaining an AL-FET biosensor.

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