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

Abstract

The application provides a method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor, which comprises the following steps: depositing an AL-BSA nanofilm on a channel region of a CNT-FET, increasing the Debye volume of the device by using the porous structure of the AL-BSA nanofilm, thereby increasing the Debye length of the device under high ion strength (0.1*PBS buffer), enabling the biological probe to sense the AD biomarkers in the serum, obtaining a signal to make the biological probe combine with the AD biomarkers within the Debye length, and directly and sensitively detecting the AD biomarkers in the serum under the condition of a lower Debye length, so as to solve the technical problem that the existing FET biosensor cannot detect the AD biomarkers in the serum under a higher ion strength.

Description

TECHNICAL FIELD

[0001] The present application 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

[0002] Alzheimer's disease (AD) is a degenerative neurological disease characterized by the aggregation of amyloid-beta (Aβ) and neurofibrillary tangles of phosphorylated tau protein filaments. The National Institute on Aging and Alzheimer's Disease (NIA-AA) proposes 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 probability of AD confirmed by autopsy, and can distinguish AD from non-AD pathology within 8 years before death. Compared with the invasive method of obtaining cerebrospinal fluid (CSF) through lumbar puncture, blood biomarkers with less invasiveness and cost-effectiveness are an ideal choice 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 it more difficult to detect the P-tau181 biomarker with high performance.

[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 detection. Field effect transistor (FET) is one of the most promising technologies for detecting low-abundance biomarkers due to its low cost, high sensitivity, simple operation and no need for labeling. Semiconductor carbon nanotubes have ultra-high mobility and good biocompatibility, making them a good candidate for high-performance FET biosensors and attracting widespread attention.

[0004] However, existing FET biosensors have a Debye length in the buffer, which leads to the formation of a double layer at a certain distance above the sensor surface, beyond which charged molecules cannot be detected, thus limiting the detection of biomolecules. Since the size of biological probes is usually about 10 nm, which exceeds the Debye length, it is usually not possible to detect them under a bare FET. Strategies that have been explored include the use of shorter biological probes, dilution of electrolyte solutions and modification of polyethylene glycol polymers to address the Debye length limitation. However, these methods can only detect target materials in 0.01xPBS buffer and cannot detect them at higher ionic strengths, limiting the application of FET biosensors. SUMMARY

[0005] The purpose of this invention is to provide a method for detecting AD biomarkers in serum based on a carbon-based field-effect transistor biosensor. The aim is 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 membrane as the dielectric layer, thereby solving the technical problem that existing FET biosensors cannot detect AD biomarkers in serum under high ionic intensities.

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

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

[0008] S2. 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, and measurements were taken at the set gate voltage and drain bias voltage. The current between the source and drain was recorded, and the corresponding transfer curve was obtained as a baseline.

[0009] S3. After completing step S1, clean and dry the AL-FET biosensor. Then, add the serum sample to be tested to the sensing interface of the AL-FET sensor for incubation. After incubation, clean with 0.1×PBS buffer and dry. Then, use 0.1×PBS buffer as a liquid gate to measure the current between the source and drain at the set gate voltage and drain bias voltage. Record the current between the source and drain to obtain the corresponding transfer curve.

[0010] S4. Calculate the response of the serum sample to be tested based on the baseline obtained in step S2 and the transfer curve obtained in step S3. If the response of the serum sample is greater than the response threshold, it indicates that AD biomarkers are present in the serum sample to be tested.

[0011] Specifically, the porous structure of the AL-BSA nanomembrane increases the Debye length of the AL-FET sensor in 0.1×PBS buffer, enabling the biological probe to bind to the target molecule within the Debye length. This allows for the detection of AD biomarkers in serum when 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 0.1x PBS buffer is 1.6nm-2.6nm.

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

[0014] Responsivity = |I-I0| / I0 (1)

[0015] In the formula, 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.

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

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

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

[0019] As an optional embodiment, the AL-BSA nanofilm is formed by dropping the amyloid solution of BSA on the channel region between the source and the drain electrode.

[0020] As an optional embodiment, the amyloid solution of BSA is formed by mixing BSA solution and 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, depositing a carbon nanotube network film on a Si / SiO2 substrate to form a CNT layer;

[0024] S12, forming a source-drain region on the CNT layer by ultraviolet lithography, 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 the excess carbon nanotube network film;

[0025] S13, passivating the device obtained in step S12 using photoresist, and using a laser direct writing lithography machine to expose, develop and fix the channel region, so that the carbon nanotube network film of the channel region is exposed, to obtain a carbon nanotube field effect transistor CNT-FET;

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

[0027] S15, dropping a mixed solution of EDC and NHS onto the AL-BSA nanofilm and incubating at room temperature to activate the carboxyl groups of the AL-BSA nanofilm, then dropping a biological probe onto the treated AL-BSA nanofilm to form an amide bond between the amino groups on the biological probe and the carboxyl groups of the AL-BSA nanofilm, to obtain an AL-FET biosensor.

[0028] As can be seen from the technical solutions of the present application above, the method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor provided by the present application deposits an AL-BSA nanofilm on a channel region of a CNT-FET, uses 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 (0.1x PBS buffer), enabling the biological probe to sense target molecules in the serum, obtaining a signal to enable the biological probe to combine with the target molecules 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 length of the biological probe is greater than the Debye length of the AL-FET sensor in 0.1x PBS buffer. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a structural schematic diagram of the AL-FET biosensor in the example of the present application.

[0030] Figure 2 is a preparation flowchart of the AL-FET biosensor in the example of the present application.

[0031] Figure 3 is a schematic diagram of detecting AD biomarkers in serum using the AL-FET biosensor in the example of the present application.

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

[0033] Figure 5is a box plot of the present application in the example of distinguishing healthy individuals and AD patients.

[0034] Figure 6 a is the transfer curve change of the AL-FET biosensor in the example of the present application for detecting different concentrations of P-tau181, Figure 6 b is the fitting curve of the response degree of the AL-FET biosensor in the example of the present application to different concentrations of P-tau181, Figure 6 c is a graph of the response degree of the AL-FET biosensor in the example of the present application versus the concentration of P-tau181.

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

[0036] Figure 8 is a statistical chart of the response degree of AL-FET and F-FET to P-tau181 in the example of the present application.

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

[0038] Figure 10 is a transfer curve of the present application in the example of the standard addition detection. DETAILED DESCRIPTION

[0039] In order to better understand the technical content of the present application, specific embodiments are described below with reference to the accompanying drawings.

[0040] Aspects of the present disclosure are described in the detailed description with reference to the accompanying drawings, in which numerous examples are illustrated. Embodiments of the present disclosure are not necessarily intended to encompass all aspects of the present application. It should be understood that various concepts and embodiments introduced above and those described below can be implemented in any of numerous ways, as the skilled artisan will understand upon reading the present disclosure.

[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 research and development. In order to increase the Debye length, most FET biosensors currently select 0.01xPBS (Debye length 7.4nm) as the liquid grid for detection, but low concentration PBS is not conducive to the binding of aptamers and targets, and the storage of antibodies, aptamers, etc., and increasing the concentration of PBS (increasing the ionic strength) will cause the Debye length to be insufficient, so that the aptamer and the target cannot be sensed.

[0042] Therefore, the application constructs a method for detecting AD biomarkers in serum based on a carbon-based field effect transistor biosensor, uses a carbon-based field effect transistor biosensor with an amyloid membrane as a medium layer, increases the Debye length of the carbon-based field effect transistor biosensor in 0.1×PBS, ensures that the aptamer and the target can be sensed, and on this basis, promotes the binding of the aptamer and the target due to the increase in the PBS concentration as a liquid grid, thereby improving the detection sensitivity, and more effectively 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.

[0043] AL-FET biosensor

[0044] In combination Figure 1 As shown in the drawings, the exemplary AL-FET sensor of the application sequentially includes a SiO2 dielectric layer 2 above a silicon-based substrate 1, forming a Si / SiO2 substrate, a semiconductor single-walled carbon nanotube network film channel layer 3 on the Si / SiO2 substrate, a metal source electrode 4 and a metal drain electrode 5 on the semiconductor carbon nanotube network film, a passivation layer 6 on the source and drain electrodes, a phase-change bovine serum albumin (AL-BSA) nanomembrane layer 7 between the source and drain electrodes, and

[0045] A biological probe 8 for detecting target substances on the AL-BSA nanomembrane layer, the length of the biological probe 8 being 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 4-5 nm.

[0047] As an optional example, the silicon-based substrate is a P-type silicon substrate, and the Si / SiO2 substrate can be directly purchased.

[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 nanomembrane is 10-15 nm, and 12 nm is particularly preferred.

[0050] Method of making an AL-FET biosensor

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

[0052] Step 1-1, depositing a semiconductor single-walled carbon nanotube network film on the Si / SiO2 substrate to form a CNT layer.

[0053] Step 1-2, forming source-drain regions on the CNT layer by ultraviolet lithography, then depositing a Ti / Pd / Au metal layer on the formed source-drain regions by electron beam vapor deposition to form source-drain electrodes, obtaining a CNT channel region connected to the source-drain electrodes, and etching away the excess carbon nanotube network film.

[0054] Step 1-3, passivating the obtained device with photoresist, and using a laser direct writing lithography machine to expose, develop and fix the channel region, so that the carbon nanotube network film of the channel region is exposed, and a carbon nanotube field effect transistor (CNT-FET) is obtained.

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

[0056] Step 1-5, dropping a mixed solution of EDC and NHS onto the AL-BSA nanofilm and incubating at room temperature to activate the carboxyl groups of the AL-BSA nanofilm, then dropping a biological probe onto the treated AL-BSA nanofilm to form an amide bond between the amino groups on the biological probe and the carboxyl groups of the AL-BSA nanofilm, and obtaining an AL-FET biosensor.

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

[0058] As an optional example, the mixed solution of EDC and NHS contains 0.4M EDC and 0.1M NHS, and the pH of the mixed solution is 5.0. In a preferred example, MES is used to adjust the pH 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 application is as follows:

[0060] The phase transition process is triggered by TCEP reducing the disulfide bonds of BSA to convert native BSA in solution into phase transition BSA oligomer nanoparticles and fibrils, and then the fibrils in solution aggregate to form microparticles. The hydrophobic aggregation caused by TCEP reducing the intramolecular disulfide bonds of BSA 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 colloids is weakened, and the amyloid aggregation is enhanced. The BSA oligomer nanoparticles can form stable binding with the substrate through hydrogen bonding, electrostatic interaction and hydrophobic interaction, so as to assemble the nanofilm at the solid-liquid interface.

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

[0062] In combination Figure 3 As shown in the exemplary embodiments of the present application, the method for detecting AD biomarkers in serum by using the aforementioned AL-FET biosensor includes the following steps:

[0063] (1) The sensing interface of the AL-FET biosensor is incubated and cleaned with 0.1×PBS buffer, then 0.1×PBS buffer is used as a liquid gate to measure at a set gate voltage and drain bias, and the current between the source and the drain is recorded to obtain the corresponding transfer curve as a baseline.

[0064] (2) After the AL-FET biosensor in step (1) is completed, it is cleaned and dried, then the serum sample to be tested is added to the sensing interface of the AL-FET sensor for incubation, after incubation, it is cleaned with 0.1×PBS buffer, dried, and then measured with 0.1×PBS buffer as a liquid gate at a 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) The responsivity of the serum sample to be tested is calculated according to the baseline obtained in step (1) and the transfer curve obtained in step (2), if the responsivity of the serum sample to be tested is greater than the response domain value, it indicates that there is an AD biomarker in the serum sample to be tested.

[0066] Wherein, the porous structure of the AL-BSA nanofilm increases the Debye length of the AL-FET biosensor in 0.1×PBS buffer, 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 0.1x PBS buffer solution is about 2.4 nm, and the length of the antibody (biological probe) used in the AL-FET biosensor of the present application is about 4-5 nm, which exceeds the Debye length of 2.4 nm of 0.1x PBS, so it is almost impossible to detect the target molecules in the serum under the existing sensor.

[0068] The present application uses the porous structure of the AL-BSA nanofilm 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, enabling the AL-FET biosensor to detect target molecules in 0.1x PBS buffer, increasing the ionic strength from 0.01x PBS to 0.1x PBS, thereby more conducive to the binding of aptamers and targets, and 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 0.1x PBS buffer is 1.6-2.6 nm.

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

[0071] Responsivity = |I-I0| / I0 (1)

[0072] In the formula, I is the maximum current value between the source and the drain measured in step (2), and I0 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-0.6V, and the drain bias is -0.1V.

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

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

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

[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 AD biomarker P-tau181 in other solvent environments, for example, in PBS buffer as the solvent, and the blank response of PBS is taken as the threshold value to determine whether P-tau181 exists in the sample to be tested.

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

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

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

[0083] Example 1

[0084] [Preparation of AL-FET biosensor]

[0085] A semiconductor single-walled carbon nanotube network film is deposited on a Si / SiO2 substrate to obtain a CNT wafer, which is directly purchased from Suzhou Enchips Semiconductor Technology Co., Ltd.

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

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

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

[0089] 4. Add 10 μL of solution (containing 0.4 M EDC and 0.1 M NHS, pH 5.0) to 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 to the treated AL-BSA nanomembrane at room temperature for 2.5 h to form amide bonds between the amino groups on the antibody and the carboxyl groups of the AL-BSA nanomembrane, thus obtaining the AL-FET biosensor.

[0090] like Figure 4 As shown, the AFM images illustrate the uniform deposition of carbon nanotubes on the substrate. Figure 4 a); After AL-BSA modification, the protein nanofilm uniformly covered the CNT surface ( Figure 4 b); The surface roughness (Ra) of the antibody-modified AL-BSA nanofilm decreased from 2.08 nm to 1.96 nm. Figure 4 c) indicates that the antibody filled the voids in the AL-BSA nanofilm, resulting in a decrease in roughness.

[0091] Example 2

[0092] [Serum sample testing]

[0093] Of the 25 serum samples, 20 were patients and 5 were healthy individuals.

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

[0095] 2. 10 μL of 0.1×PBS was dropped onto the surface of the prepared AL-FET (prepared according to the method in Example 1) and incubated for 0.5 h. Then, it was washed with 0.1×PBS and then with ultrapure water. Finally, it was dried with nitrogen gas. 10 μL of 0.1×PBS was dropped on as a liquid gate. The gate voltage was set to -0.6V to 0.6V, and the drain bias was -0.1V. A data point was collected every 0.02V. The current between the source and drain was recorded using a semiconductor parameter characteristic analyzer. The obtained transfer curve was recorded as the baseline, and the current I0 at -0.6V was also recorded.

[0096] 3. After completing step 2, the AL-FET is cleaned with ultrapure water and dried with nitrogen. Then, the test solution is dropped onto the surface of the AL-FET and incubated for 0.5 h. After cleaning with 0.1×PBS, it is cleaned with ultrapure water and dried with nitrogen. 10 μL of 0.1×PBS is added as a liquid gate. The gate voltage is set to -0.6V to 0.6V, and the drain bias is -0.1V. A data point is collected every 0.02V. The current between the source and drain is recorded using a semiconductor parameter characteristic analyzer to obtain the transfer curve. The current I at -0.6V is also recorded.

[0097] 4. Calculate the response of each unknown sample by |I-I0| / I0, record the response of HC and AD, and calculate the significant difference between the response of HC and AD and the response threshold.

[0098] As shown in Figure 5 According to the data, the AL-FET sensor can well distinguish between normal people and patients (P<0.0001), and the response threshold of the sensor is 0.02. When the response is greater than 0.02, the AD biomarker P-tau181 can be detected, and it 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 it is considered to be normal.

[0099] Example 3

[0100] [Detection limit]

[0101] 1. Add 10 μL of 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 dry with nitrogen, add 10 μL of 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, and obtain the transfer curve as the baseline.

[0102] 2. Prepare P-tau181 solutions of different concentrations (from 1 fg / mL to 100 ng / mL) (1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL). For P-tau181 of different concentrations, add to the surface of the AL-FET and incubate for 0.5 h, then wash with 0.1×PBS and then with ultrapure water, finally dry with nitrogen, add 10 μL of 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, and obtain the transfer curve at each concentration, as shown in Figure 6 a.

[0103] 3. Record the source-leakage current (-0.6V) obtained after adding different concentrations of P-tau181. Calculate the response of the sensor at different concentrations of P-tau181 using |I-I0| / I0 (where I is the source-leakage current obtained at different concentrations of P-tau181, and I0 is the recorded baseline current). Figure 6 As shown in b.

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

[0105] The source-drain current of the AL-FET increases with increasing target P-tau181 concentration, according to the equation y = ...

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

[0107] Example 4

[0108] [Sensitivity]

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

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

[0111] 3. To activate the carboxyl group of AL-BSA, a 10 μL 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 to the FET surface and incubated at room temperature for 2.5 hours 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 in Example 1), P-Tau 181 was detected according to steps 1-3 in Example 3, and the transfer curves are shown below. Figure 7 As shown in 7a and 7b.

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

[0115] In combination Figure 9 As shown, this is because the F-FET biosensor uses Lys to seal the pores of the thin film, so that the antibody can only be modified on the surface of the thin film, and the height of the antibody itself is 4-5nm, so when using the F-FET biosensor, the target binds to the antibody outside the Debye length (about 2.4nm) of 0.1xPBS, resulting in the inability to sense the charge of the target, thereby having lower sensitivity; while in the AL-FET biosensor, the antibody is modified in the pores of the AL-BSA nanofilm, and the porous structure increases the Debye volume, thereby increasing the Debye length, so that the target can bind to the antibody within the Debye length of 0.1xPBS, thereby having higher sensitivity; this also proves that the method of the present application 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 0.1xPBS buffer.

[0116] Example 5

[0117] [Spiked recovery]

[0118] 1. Add 10 μL of 0.1xPBS 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 0.1xPBS and then with ultrapure water, finally dry with nitrogen, add 10 μL of 0.1xPBS 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, obtain the transfer curve as the baseline, and record the current I0 at -0.6V.

[0119] 2. Configure 10fg / mL of P-tau181 as the sample to be tested, add the sample to be tested to the surface of the AL-FET and incubate for 0.5 h, then wash with 0.1xPBS and then with ultrapure water, finally dry with nitrogen, add 10 μL of 0.1xPBS 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, obtain the transfer curve, and record the current I at -0.6V.

[0120] In combination Figure 10As shown, the response of the unknown sample is calculated by |I-I0| / I0, and the standard curve obtained in Example 3 is brought in, |I-I0| / I0=0.03323lg(x)+0.5381, and the concentration of the unknown sample is calculated as 9.77 fg / mL, and the recovery rate is 97.7%, which is within the range of 95%-105%, indicating that the recovery efficiency is good, and the P-tau181 in the buffer solution can be determined.

[0121] The method of the present application can detect 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 application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application. Those skilled in the art can make various modifications and improvements without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application shall be subject to the scope defined by the claims.

Claims

1. A method for detecting non-diagnostic and / or therapeutic targets of AD markers in serum based on carbon-based field effect transistor biosensors, characterized by, The method comprises the following steps: S1, preparing an AL-FET biosensor, using a semiconductor single-walled carbon nanotube network film as a channel layer, and depositing a phase transition albumin from bovine serum (AL-BSA) nanomembrane layer on the surface of the channel region, and a biological probe for detecting target molecules is arranged on the AL-BSA nanomembrane layer; wherein the AL-BSA nanomembrane is formed by dropping an amyloid protein solution of BSA on the channel region between the source electrode and the drain electrode, and the length of the biological probe is greater than the Debye length of the AL-FET biosensor in 0.1*PBS buffer solution; S2, incubating and cleaning the sensing interface of the AL-FET biosensor by using 0.1*PBS buffer solution, and then measuring under the set gate voltage and drain bias by using 0.1*PBS buffer solution as a liquid gate, recording the current between the source electrode and the drain electrode, and obtaining 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 on the sensing interface of the AL-FET biosensor for incubation, cleaning with 0.1*PBS buffer solution after incubation, and then measuring under the set gate voltage and drain bias by using 0.1*PBS buffer solution as a liquid gate after drying, recording the current between the source electrode and the drain electrode, and obtaining the corresponding transfer curve; S4, calculating the responsivity 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 responsivity of the serum sample to be tested is greater than the response domain value, it indicates that there is an AD biomarker in the serum sample to be tested; Wherein, the Debye length of the AL-FET sensor in the 0.1*PBS buffer solution 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 the AD biomarker in the serum under the condition that the length of the biological probe is greater than the Debye length of the AL-FET sensor in the 0.1*PBS buffer solution.

2. The method for detecting a non-diagnostic and / or therapeutic target of an AD marker 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 biological probe and the Debye length of the AL-FET sensor in the 0.1*PBS buffer solution is 1.6 nm to 2.6 nm.

3. The method of claim 1, wherein the method is a non-diagnostic and / or therapeutic target detection method for AD markers in serum based on carbon-based field effect transistor biosensors, characterized in that, The responsivity is calculated according to formula (1): Responsivity = |I-I0| / I0 (1) In the formula, I is the maximum current value between the source electrode and the drain electrode measured in step S3, and I0 is the maximum current value between the source electrode and the drain electrode measured in step S2.

4. The method of claim 1, wherein the method is a non-diagnostic and / or therapeutic target detection method for AD markers in serum based on carbon-based field effect transistor biosensors. The response threshold is 0.

02.

5. The method of claim 1, wherein the method is a non-diagnostic and / or therapeutic target detection method for AD markers in serum based on carbon-based field effect transistor biosensors, characterized in that, In the step S2, the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

6. The method of claim 1, wherein the method is a non-diagnostic and / or therapeutic target detection method for AD markers in serum based on carbon-based field effect transistor biosensors. In the step S3, the gate electrode is -0.6V to 0.6V, and the drain bias is -0.1V.

7. The method of claim 1, wherein the method is a non-diagnostic and / or therapeutic target detection method for AD markers in serum based on carbon-based field effect transistor biosensors. The amyloid protein solution of BSA is formed by mixing BSA solution and TCEP solution.

8. The method of claim 1, wherein the method is a non-diagnostic and / or therapeutic target detection method for AD markers in serum based on carbon-based field effect transistor biosensors, characterized in that, The AD biomarker is P-tau181.

9. The method of claim 1, wherein the method is a non-diagnostic and / or therapeutic target detection method for AD markers in serum based on carbon-based field effect transistor biosensors, characterized in that, The preparation of the AL-FET biosensor specifically comprises the following steps: 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, 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 the excess carbon nanotube network film; S13, passivating the device obtained in step S12 using photoresist, and using a laser direct writing photoetching machine to expose, develop and fix the channel region, so that the carbon nanotube network film of the channel region is exposed, to obtain a carbon nanotube field effect transistor CNT-FET; S14, mixing a BSA solution and a TCEP solution to obtain an amyloid solution of BSA, dropping the amyloid solution of BSA onto the channel region of the CNT-FET to react and form an AL-BSA nanofilm, then washing with ultrapure water and blowing dry with nitrogen; S15, dropping a mixed solution of EDC and NHS onto the AL-BSA nanofilm and incubating at room temperature to activate the carboxyl groups of the AL-BSA nanofilm, then dropping a biological probe onto the treated AL-BSA nanofilm to form an amide bond between the amino groups on the biological probe and the carboxyl groups of the AL-BSA nanofilm, to obtain an AL-FET biosensor.

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