Method for preparing biosensor for detecting Alzheimer's disease biomarker and biosensor prepared by method

Abstract

The present disclosure provides a method of preparing a biosensor for detecting Alzheimer's disease biomarkers, comprising depositing an alumina film on a Si substrate by an atomic layer deposition system to form an Al2O3 / Si substrate; depositing an electric contact part Cr / Au on the Al2O3 / Si substrate through a thermal evaporator, and forming a source electrode, a drain electrode and a planar grid electrode on the Al2O3 / Si substrate; providing double-layer graphene on the Al2O3 / Si substrate through thermal annealing in a vacuum environment; performing low-damage plasma treatment (LDPT) on the double-layer graphene with a mixture of oxygen and hydrogen to form a graphene oxide / graphene (GO / G) layered composite material on the Al2O3 / Si substrate; an antibody is immobilized on the surface of a GO / G layered composite material by a reaction between an amine group of the antibody and a carboxyl group of GO of the GO / G layered composite material, where the antibody is specific for p-tau217 protein.

Description

[0001] References to electronic sequence listings

[0002] The contents of the electronic sequence listing (sequencelisting.xml; size: 3.04 KB, creation date: January 12, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to a method for preparing a biosensor, particularly a biosensor for detecting Alzheimer's disease biomarkers and a method for preparing the same. Background Art

[0004] Dementia is characterized by a loss of cognitive function beyond what would be expected as a normal consequence of biological aging. According to a 2021 report from the World Health Organization, over 55 million people worldwide currently suffer from dementia; this number is projected to increase to over 78 million by 2030 and to 139 million by 2050 (Dementia, Newsroom, World Health Organization, 2022). It has become a serious threat to our society and healthcare systems. Alzheimer's disease (AD) is the most common form of dementia, accounting for over 60% of dementia cases. Clinicopathological evidence suggests that cognitive decline can occur more than 15 years before clinical detection (Amieya et al., 2014; Villemagne et al., 2013). While the progression of AD is irreversible, effective treatments exist when managed in its early clinical course. Therefore, early diagnosis and treatment are of great clinical importance for the clinical management of AD.

[0005] Biomarkers are biochemical indicators used to assess disease risk. Among these biomarkers, the accumulation of amyloid-β (Aβ) peptide in extracellular plaques and hyperphosphorylated tau (p-tau) protein in neurofibrillary tangles have been incorporated into the AD diagnostic framework (Huang et al., 2009; Varesi et al., 2022; Teunissen et al., 2022). Aβ and p-tau can be visualized using positron emission tomography (PET) or quantitatively measured in cerebrospinal fluid for AD diagnosis (Valotassiou et al., 2018; Brier et al., 2016; Palmqvist et al., 2015). However, these methods are invasive, cumbersome, expensive, and not widely available to all AD patients. Therefore, there is a strong need to develop blood-based biomarkers for AD diagnosis, as peripheral blood collection is simple, low-cost, and less invasive. Recent studies have shown that tau protein enters the blood after being injected into the brain, and high levels of p-tau in the blood indicate neuronal degeneration in the brain, which is a key factor that ultimately leads to AD (Fiandaca et al., 2015; Banks et al., 2017; Chiu et al., 2013). Several studies using different assays and technologies, including electrolyte-insulator-semiconductor devices (Bhalla et al., 2014; Bhalla et al., 2015), localized surface plasmon resonance (Bhalla et al., 2015), and electrochemical sensors (Formisano et al., 2015), have demonstrated that p-tau isoforms, including p-tau181, p-tau217, and p-tau231, are highly specific for the detection of PET-confirmed Aβ and tau pathology across the clinical AD continuum in terms of phosphorylation (Suárez-Calvet et al., 2020; Bayoumy et al., 2021; Karikari et al., 2022; Leuzy et al., 2021). However, due to the low concentration of p-tau protein in peripheral blood, which is beyond the detection range of traditional enzyme-linked immunosorbent assays, and the presence of nonspecific proteins and various interfering substances when detecting in blood samples, the sensitivity of p-tau protein detection remains very challenging (Derkus et al., 2016; Galasko et al., 2013; Lue et al., 2017; Yang et al., 2018; Hampel et al., 2018). Therefore, it is urgent to develop a point-of-care (POC) biosensor to accurately detect tau protein in blood-based samples.Nanomaterial-based immunosensors have been reported for detecting p-tau protein. MESchneider et al. fabricated a carbon screen-printed electrode modified with multi-walled carbon nanotubes decorated with platinum nanoparticles to develop an electrochemical-based immunosensor for detecting p-tau181 (Schneider et al., 2022). The electrode was functionalized with polyallylamine hydrochloride to interact with the carboxyl groups of the p-tau181 detection antibody. Square-wave voltammetry was performed to detect p-tau181 in phosphate-buffered saline (PBS). The biosensor demonstrated a limit of detection (LOD) of 0.24 pg / mL (~1.1 nM) and a linear range from 8.6 to 1100 pg / mL (~4.1 to 523.8 nM). After a 10-fold dilution in fetal bovine serum, the sensing performance, both in terms of sensitivity and LOD, was approximately halved by dilution in PBS. K. Kim et al. used the Langmuir-Blodgett technique to prepare densely packed carbon nanotubes to develop a chemiresistive sensor array for multiple core AD biomarkers (Kim et al., 2020). The device demonstrated an LOD of 2.72 fM for p-tau181 detection in plasma, with a linear range from 1 fM to 100 nM. HTN Le and S. Cho developed an electrochemical biosensor based on cross-shaped electrodes via activated self-assembled monolayers to retain specific antibodies for p-tau231 detection (Le et al., 2022). Electrochemical impedance spectroscopy was performed to detect p-tau231 in human serum. The biosensor showed an LOD of 140 pg / ml (~66.7 nM) with a linear range from 100 pg / ml to 10 ng / ml (~47.6 to 476.2 nM). Accurate detection of p-tau231 was also demonstrated by the low dissociation constant between the antibody and p-tau231. L.M. T. Phan and S. Cho developed a colorimetric gold nanoparticle-based aptablot immunoassay for the detection of p-tau231 in human serum albumin (Phan et al., 2022). The color intensity of the changes in p-tau231 concentration can be analyzed by the naked eye or with a digital camera using ImageJ software. The method resulted in an LOD of 4.71 pg / ml (~2.2 nM) and a linear range of 0.064 to 1000 ng / ml (~30.5 nM to 476.2 μM).S. Janelidze et al. reported that p-tau217 showed a stronger correlation with the tau PET tracer [18F]flortaucipir and showed that individuals with abnormally increased [18F]flortaucipir retention were more easily accurately identified, indicating that it is more useful than other biomarkers in diagnosing AD (Janelidze et al., 2020). Unfortunately, a POC biosensor for detecting p-tau217 has not yet been reported.

[0006] Geim and Novoselov demonstrated single-crystalline graphene films (Novoselov et al., 2004). Due to its many excellent properties, such as electrical conductivity, mechanical strength, biocompatibility, and high surface area, graphene (G) has been widely used to develop various types of biosensors (Kanagavalli et al., 2021; Justino et al., 2017; Jangir et al., 2022). Its high sensitivity to bioanalytes containing aromatic groups, such as DNA, RNA, and proteins, has made it a suitable transducer in biosensors. However, the affinity between G and bioanalytes is nonspecific, making it unsuitable for detecting them individually. Therefore, G surfaces must be functionalized to facilitate the binding of biorecognition elements for subsequent detection or capture of target analytes. Pyrene-based liners, such as pyrenebutyric acid succinimidyl ester and pyrenebutyric acid, are commonly used to functionalize G surfaces via π-π interactions (Nekrasov et al., 2022; Hinnemo et al., 2017). These wet chemical processes for G surface functionalization require hours or even days, and the resulting functionalization occurs via a non-covalent process that is less stable than its covalent counterparts.

[0007] In our previous study (Govindasamy et al., 2022), we developed atomic layer composites of graphene oxide / graphene (GO / G) using chemical vapor deposition (CVD)-grown bilayer graphene (BG) and our developed low-damage plasma treatment (LDPT), an atomic layer oxidation process for the development of chemi-impedance biosensors. CVD-grown BG was converted to GO / G by LDPT, which can be achieved using a mixture of hydrogen and oxygen to achieve atomic layer oxidation. Only the top layer of BG was functionalized with oxidizing groups, which served as active sites for forming covalent bonds with biorecognition elements (e.g., antibodies in the present disclosure). Furthermore, after LDPT, the conductivity of the bottom G layer remained almost unchanged, allowing it to act as a transducer in response to the attachment of target analytes to the top GO conjugated with the biorecognition element via π-π interactions between the GO and G layers.

[0008] The present disclosure provides a solution-gated field effect transistor (SGFET) featuring an atomic layer composite of graphene oxide / graphene (GO / G) for detecting the p-tau217 biomarker. Summary of the Invention

[0009] The present disclosure aims to provide a biosensor with high sensitivity for detecting biomarkers of Alzheimer's disease.

[0010] To achieve at least the foregoing objectives, a method for preparing a biosensor for detecting biomarkers of Alzheimer's disease comprises the following steps: depositing an aluminum oxide thin film on a Si substrate using an atomic layer deposition system to form an Al2O3 / Si substrate; depositing a plurality of electrical contacts, Cr / Au, on the Al2O3 / Si substrate using a thermal evaporator to form a source electrode, a drain electrode, and a planar gate electrode on the Al2O3 / Si substrate; forming a bilayer graphene on the Al2O3 / Si substrate between the source electrode and the drain electrode by thermal annealing under a vacuum environment; subjecting the bilayer graphene to low-damage plasma treatment (LDPT) with a mixture of oxygen and hydrogen to form a graphene oxide / graphene (GO / G) layered composite material on the Al2O3 / Si substrate; and immobilizing an antibody on the surface of the GO / G layered composite material by reacting amine groups of the antibody with carboxyl groups of GO in the GO / G layered composite material, wherein the antibody is specific for the p-tau217 protein.

[0011] In one embodiment, the method further comprises the step of supplying an epoxy type adhesive to define a sensing area.

[0012] In one embodiment, the size of the sensing area ranges from 5x5 to 10x10 mm. 2 .

[0013] In one embodiment, the step of immobilizing the antibody on the surface of the GO / G layered composite is performed by incubating the sensing region with 100 μg / ml of the antibody in a volume range of 20 to 100 μl aliquots at a temperature range of 4 to 37° C. for 1 to 24 hours.

[0014] Another object of the present disclosure is to provide a method for detecting biomarkers of Alzheimer's disease.

[0015] To achieve this goal, the method for detecting an Alzheimer's disease biomarker includes the steps of detecting the amount of p-tau217 protein in a sample using a biosensor, wherein the biosensor includes a Si substrate; depositing an aluminum oxide thin film on the Si substrate using an atomic layer deposition system to form an Al2O3 / Si substrate; depositing a plurality of electrical contact portions Cr / Au on the Al2O3 / Si substrate using a thermal evaporator; forming a graphene oxide / graphene (GO / G) layered composite material on the Al2O3 / Si substrate by low-damage plasma treatment (LDPT); and immobilizing an antibody on the surface of the GO / G layered composite material by reacting an amine group of the antibody with a carboxyl group of GO in the GO / G layered composite material, wherein the antibody is specific for the p-tau217 protein.

[0016] In one embodiment, an epoxy-type adhesive is supplied to define a sensing area of the biosensor.

[0017] In one embodiment, the size of the sensing area ranges from 5x5 to 10x10 mm. 2 .

[0018] In one embodiment, the antibody is immobilized on the surface of the GO / G layered composite by incubating the sensing region with 100 μg / ml of the antibody in a volume range of 20 to 100 μl aliquots at a temperature range of 4 to 37° C. for 1 to 24 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A process showing a method for preparing the biosensor of the present disclosure is shown.

[0020] Figure 2 Schematic diagram of a solution-gate field-effect crystal.

[0021] Figure 3 Shown are (a) a schematic diagram of a GFET-based biosensor featuring a GO / G layered composite and (b) p-tau217 protein bound to an antibody immobilized on GO / G, (c) the sensing mechanism based on Dirac point shift, and (d) a photograph of the measurement setup and solution-gated graphene transistor (SGGT).

[0022] Figure 4Shown are (a) transmittance of BG before and after LDPT, (b) Raman spectrum of BG before and after LDPT, (c) XPS spectrum of BG before and after LDPT, (d) contact angle of BG before and after LDPT, (e) resistance of SLG and BG before and after LDPT, and (f) TEM image and interlayer spacing of GO / G atomic layer composite.

[0023] Figure 5 (a) is a graph showing the transfer curves of SGFETs containing BG and GO / G atomic layer composite materials, Figure 5 (b) is a graph showing the output characteristics of the GO / G atomic layer composite material.

[0024] Figure 6 (a) is a graph showing the transfer curve of an antibody-modified SGFET based on GO / G. Figure 6 (b) is a graph showing VCNPs versus incubation time, Figure 6 (c) shows a fluorescence microscopy image (left) and corresponding optical image (right) of a GO / G sample that has been exposed to antibodies modified with amines and FAM.

[0025] Figure 7 (a) is a graph showing the transfer curve of an antibody-modified SGFET based on GO / G. Figure 7 (b) shows ΔV CNP Plot relative to p-tau217 protein concentration in PBS, Figure 7 (c) is a graph showing the concentration of vehicle relative to the concentration of p-tau217 protein, and Figure 7 (d) is a graph showing the mobility relative to the p-tau217 protein concentration obtained by Hall measurement.

[0026] Figure 8 (a) is a graph showing the transfer curves of antibody-modified GO / G-based SGFETs, and Figure 8 (b) shows ΔV CNP Plotted against p-tau217 protein concentration in HSA.

[0027] Figure 9 (a) is a graph showing the transfer curve of the antibody-modified GO / G-based SGFET, and Figure 9 (b) shows V CNP and plots of the changes recorded after different storage periods. DETAILED DESCRIPTION

[0028] In order to facilitate understanding of the purpose, features and effects of the present disclosure, specific embodiments are provided in conjunction with the accompanying drawings for detailed description of the present disclosure.

[0029] Materials and instruments

[0030] In the present disclosure, p-tau217 protein and its antibody (rich in amine groups at the C-terminus) were obtained from Chang Gung Memorial Hospital, Taiwan, China. The details of protein preparation, clone screening and quality control are described below.

[0031] Peptide Synthesis and Protein Preparation: The p-tau-217 peptide GSRSRTPSLPTPPTREPKKVAVVR (SEQ ID NO: 1) and the control peptide GSRSRTPSLPTPPTREPKKVAVVR (SEQ ID NO: 2) were both >95% pure, with the 11th amino acid of SEQ ID NO: 1 being phosphorylated threonine and the 11th amino acid of SEQ ID NO: 2 being unphosphorylated threonine, as guaranteed by the manufacturer (BIOTOOLS Co., Ltd., Taiwan, China). Peptides (20 μg / μl) were prepared and stored according to the manufacturer's recommendations. Tau (MAPT) protein was purchased from Origene (NM_005910, USA) and phosphorylated by glycogen synthase kinase 3β (GSK-3β, Sino Biological, China). Deactivated viral lysates, influenza A / B (Flu A / B), human parainfluenza virus, adenovirus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome (SARS) coronavirus (ZeptoMatrix, USA) were purchased and used according to the manufacturer's instructions.

[0032] Immunization: 6-8 week old female BALB / c mice were injected intraperitoneally with 100 μg of p-tau-217 emulsified in complete Freund's adjuvant (Sigma-Aldrich, USA). On days 14, 28, and 42, booster injections of 100 μg of peptide in incomplete Freund's adjuvant (Sigma-Aldrich, USA) were given. Before sacrifice, antibody responses were elicited by two injections of 50 μg of peptide emulsified in IFA at three-day intervals.

[0033] Hybridoma cell preparation and antibody purification: To generate monoclonal antibodies against the p-tau-217 peptide, the synthesized p-tau-217 peptide was inoculated into BALB / c mice three times every two weeks. Two booster injections were given three days apart before sacrifice. The spleens of the mice were immediately harvested and fused with myeloma cells to prepare hybridoma cells and subsequently subjected to semi-solid-state screening (ClonaCell Hybridoma Kit, STEMCELL Technology, USA). Hybridoma strains were propagated in 96-well microtiter plates (Corning, USA) until cell confluence reached saturation, and their supernatants were collected and tested by ELISA for antibody reactivity against the p-tau-217 peptide, full-length p-tau protein, or a control peptide. Antibodies with high affinity for the p-tau-217 peptide and / or p-Tau protein were selected and used on Protein G Sepharose resin (Cytiva, USA). The purified mAbs were dialyzed against PBS buffer to remove glycine and concentrated using Amicon Ultra-15 centrifugal filter units (10 kDa, Merck Millipore, USA). The mAbs were stored at -80°C for subsequent experiments.

[0034] Enzyme-linked immunosorbent assay (ELISA): 100 ng / well of p-tau protein, p-tau-217 peptide, or tau-217 in coating buffer (150 mM Na2CO3, 150 mM NaHCO3, pH 9.6) was coated onto a 96-well microtiter plate and incubated overnight at 4°C. For cross-reactivity testing, 1 μg / well of deactivated viral lysate was used. After blocking with 1% bovine serum albumin, 100 μL of cell supernatant or 1 μg of purified mAb was added and incubated at room temperature for 1 hour. After incubation, the microtiter plates were washed four times with TBST (TBS containing 0.05% Tween 20) and the bound antibodies were detected with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse IgG Fc region (Jackson ImmunoResearch Laboratories, USA) for 1 hour at room temperature. After washing, horseradish peroxidase (HRP) activity was measured at 450 nm using a microplate enzyme-linked immunosorbent assay (ELISA) reader (EZ Read 400) using 3,3',5,5'-tetramethylbenzidine (TMB, Sigma Aldrich, USA) as a substrate.

[0035] Modification of mAbs: Amine-reactive esters of carboxylic acid groups on mAbs were prepared using a reactive amination kit following the manufacturer's recommendations (G-BIOSCIENCES, USA). Briefly, 1 mg / mL of purified mAb was dissolved in 1X optimization buffer. EDC and Sulfo-NHS were added to give final concentrations of 2 mM and 5 mM, respectively, and incubated at room temperature for 15 minutes. β-Mercaptoethanol (Sigma Aldrich, USA) was then added to the antibody solution to a final concentration of 20 mM and incubated at room temperature for 10 minutes to deactivate EDC. Free EDC, NHS, and EDC byproducts were removed by desalting columns, and the antibody solution was adjusted with PBS (pH 7) and concentrated through a 10 kDa cutoff centrifugal filter. Amine-reactive sites were blocked with hydroxylamine (Sigma Aldrich, USA) at a final concentration of 10 mM and incubated at room temperature for 5 minutes. Buffer exchange was performed with PBS (pH 7) through a 10 kDa cutoff centrifugal filter. The antibody solution was aliquoted into 100 μL / vial (0.25-0.5 μg / μL) and stored at -80°C.

[0036] To prepare monoclonal antibodies (mAbs) against p-tau 217, BALB / c mice were immunized with p-tau 217 peptide to induce anti-p-tau 217 antibodies. At the end of immunization, mouse spleens were harvested and used to fuse hybridomas with bone marrow cells to generate hybridoma clones that produce anti-p-tau 217 mAbs. In a preliminary screening, more than 500 hybridoma clones were selected to examine antibody reactivity against p-tau 217 peptide and p-tau protein. Table 1 lists the top 20 clones with the highest reactivity against p-tau 217 and p-tau. Table 1 shows the affinity test results of the mAbs for p-tau protein and p-tau 217 peptide, where OD is the abbreviation for optical density.

[0037] Table 1

[0038]

[0039]

[0040] Ten mAb clones (No. 3, No. 49, No. 72, No. 83, No. 103, No. 118, No. 123, No. 127, No. 140, and No. 170) were selected for detection limit testing using ELISA (Table 2). Table 2 shows the detection limit of mAbs against p-tau 217 peptide.

[0041] Table 2

[0042]

[0043] To further exclude the possibility that the antibodies cross-react with other antigens, two mAb clones (No. 72 and No. 123) with high affinity for p-tau 217 were examined for cross-reactivity with other viral antigens, such as influenza A / B, human parainfluenza virus, adenovirus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome (SARS) coronavirus. In addition, p-tau 217 and tau 217 peptides were used as positive and negative controls (Table 3). Table 3 shows the results of the cross-reactivity test of the monoclonal antibodies with viral lysates, where Flu A / B represents influenza A / B, para-Flu represents human parainfluenza virus, RSV represents respiratory syncytial virus, SARS represents severe acute respiratory syndrome coronavirus, and the control group represents tau-217 peptide. Clone No. 72, which has high antibody affinity for p-tau-217 and p-tau and little cross-reactivity with other antigens, was used in this study.

[0044] Table 3

[0045]

[0046] 1X phosphate buffered saline (PBS; containing 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.47 mM KH2PO4) with a pH of 7.4 and HSA were purchased from Sigma-Aldrich, USA. All other chemicals were of analytical grade and used as received. Deionized water was obtained from a Millipore water purification system (18.2 MΩ resistor, Milli-Q Direct8). 25 μm copper foil for the preparation of bilayer graphene (BG) was purchased from Alfa Aesar (ThermoFisher Scientific). BG was grown on copper foil using CVD in a 3-inch diameter tubular quartz furnace. The details of the growth procedure and its subsequent transfer to the target substrate are described in the literature of Govindasamy et al. in 2022.

[0047] The optical and structural characterization of the G sample was carried out using an ultraviolet-visible (UV-Vis) spectrometer (V-650, JASCO, Japan) and a Raman spectrometer (HORIBA iHR-550, equipped with a 532 nm laser, Japan). Its chemical composition was studied by an X-ray photoelectron spectrometer (XPS; PHI 5000VersaProbe III, ULVAC, Japan) with a monochromatic Al Kα source. The layered composite material was observed by a transmission electron microscope (TEM, JEOL, JSM-2100, Japan). Water contact angle (WCA) measurements were performed using a PSC-100B instrument (Pentad Scientific, Taiwan, China) to examine its hydrophilicity. The electronic properties of the SGFET were measured using a semiconductor parameter analyzer (B1500A, Agilent Tech, USA) at a homemade probe station. The V g and a 0.02V interval and a 0.5V source-drain bias (V sd ) is measured under the condition of source-drain current (I sd ) versus gate voltage (V g ) transfer curves. A Hall-effect measurement system (AHM-800B, Agilent Tech, USA) was used to determine the carrier concentration and mobility of devices treated with p-tau217 solutions of varying concentrations. All properties were measured for at least five samples; the averages and standard deviations are reported.

[0048] Methods for preparing biosensors and operating instructions for p-tau217 detection

[0049] Figure 1 The preparation process of the biosensor in the present disclosure is shown. A 6 nm aluminum oxide (Al2O3) thin film is deposited on a Si substrate using an atomic layer deposition system to obtain a 3×2.5 cm 2 Al2O3 / Si substrate. Cr / Au (5 / 50nm) electrode contacts were deposited on the Al2O3 / Si substrate using shadow masking to create source, drain, and planar gate electrodes. Two sets of electrode contacts were prepared on the substrate, with detailed dimensions shown in Figure 2Then, the bilayer graphene (BG) formed by the chemical vapor deposition (CVD) process (Govindasamy et al., 2022) was transferred to the substrate between the source and drain electrodes, followed by thermal annealing under a vacuum environment to remove any other polymer residues and enhance the contact between the BG and the electrodes to obtain BG on the Al2O3 / Si substrate (BG / Al2O3 / Si). Thereafter, the bilayer graphene was subjected to LDPT treatment with a gas mixture of oxygen and hydrogen to form a GO / G layered composite material on the electrode-deposited Al2O3 / Si substrate. The details of the LDPT process have been reported previously (Govindasamy et al., 2022). Finally, an epoxy resin type adhesive was supplied to define the 4×9 mm of each device. 2 Sensing area. After preparing the GO / G layered composite on the electrodeposited Al2O3 / Si substrate, the antibody was immediately immobilized on the GO / G surface via a carbodiimide-mediated reaction between the amine group (-NH2) at the C-terminus of the aminated antibody and the carboxyl group (-COOH) of GO. A 20μl aliquot of a 100μg / ml antibody solution was dropped onto the sensing area and incubated at 4°C for 16 hours. Next, the device incubated with the antibody solution was gently rinsed three times with PBS to wash away excess unbound antibody. A total of 20μl of p-tau217 solution at different concentrations in PBS was added dropwise to the sensing area and allowed to bind to the antibody for 30 minutes at room temperature. The sample was then gently rinsed with PBS to remove excess unbound target p-tau217 protein. In parallel, different concentrations of p-tau217 solutions in HSA were prepared according to the operating instructions, as were different concentrations of p-tau217 solutions in PBS, to verify the practicality of the detection method. Figure 3 Figure 3. Schematic diagram of the SGFET-based biosensor featuring GO / G layered composite for p-tau217 protein.

[0050] Characterization of bilayer graphene (BG) and graphene oxide / graphene (GO / G) layered composites

[0051] A GO / G layered composite material is provided. The top layer of GO reacts with an antibody as a biorecognition element, and the bottom layer of G acts as a converter. The preparation of the GO / G layered composite material begins with the growth of BG, followed by LDPT. UV-Vis spectroscopy, Raman spectroscopy, XPS, CA measurement, and resistance measurement were performed to examine the formation of the GO / G layered composite material ( Figure 4 ). Figure 4(a) shows the UV-Vis spectra of BG before and after LDPT. The transmittance of BG before LDPT at 550 nm is 95.3%, which is almost consistent with the 2.3% transmittance reduction of single-layer graphene (SLG). Figure 4 The Raman spectrum shown in (b) shows that the 2D to G intensity ratio of BG before LDPT is about 0.83, the D band intensity is low, and the 2D band half maximum width is ∼59 cm -1 , indicating the formation of AB-stacked BG (Sheng et al., 2015; Liu et al., 2012). Considering these material analyses, high-quality BG was used in the present disclosure. The transmittance of BG has slightly increased after LDPT. It has been reported that the band gap of graphene oxide increases due to the incorporation of oxygen into graphene (Jin et al., 2020). Therefore, the absorption of light in the visible range is reduced, resulting in an increase in transmittance, such as Figure 4 (a) As shown in Figure 2. After the BG sample was subjected to LDPT, the D band intensity increased and the intensity ratio of the 2D- to G bands (I 2D / I G ) decreased significantly, which was attributed to the incorporation of oxidized functional groups into BG. Notably, a blue shift was observed after LDPT, which pointed out the p-doping effect on BG (Tang et al., 2010). Figure 4 (c) shows the XPS spectrum of the BG sample after LDPT. Five carbon atom bonding states can be decomposed: sp at 284.5 eV 2 C=C bond, sp at 285.5eV 3 C—C bonds, hydroxyl groups (C—OH) at 286.6 eV, carbonyl groups (C═O) at 288 eV, and carboxyl groups (COOH) at 288.9 eV (Krishnamoorthy et al., 2013; Huang et al., 2021). These oxygen functional groups cause defect structures in BG, which correspond to the increase of D band and I in Raman spectrum. 2D / I G Reduce, such as Figure 4 (b) COOH groups are the main functional groups required for the covalent bond formation between the amine groups of the antibody and the GO top layer, accounting for 6.5% of all carbon bonding states. The hydrophilicity of the BG samples was evaluated in terms of their WCAs. Figure 4 As shown in (d), the BG sample becomes hydrophilic by observing the change in CAs from 83° before LDPT to 18° after LDPT. This result confirms that the hydrophilic surface is caused by the formation of oxidized functional groups, which is consistent with the XPS spectrum. Figure 4(e) Shows the resistance of BG and single-layer graphene (SLG) samples before and after LDPT between the source and drain electrodes. The resistance of SLG increases significantly after LDPT, exceeding millions of ohms, indicating that it becomes almost an electrically insulating material.

[0052] In contrast, the resistance of BG after LDPT increased only slightly, and the conductivity remained at the same level. Based on these results, it was concluded that in the BG sample, the upper layer of G was converted to GO, while the lower layer of G was only slightly modified by LDPT. TEM measurements were performed to confirm the layered G composite of GO / G. Figure 4 As shown in (f), the double-layer structure of GO / G is clearly observed, and the distance between the two layers is about 0.361nm. Figure 4 As a result, atomic layer oxidation was achieved by LDPT to form a GO / G layered G composite material as a converter, while the upper layer of GO could react with antibodies for subsequent antigen binding.

[0053] Figure 5 (a) Plotting a representative transfer curve of an SGFET characterized by BG (taking into account the gate voltage (V g ) of the sink-source current (I d )). The typical characteristics of the V-shaped curve of BG are observed, indicating its bipolar characteristics. CNP Located in positive V g (~0.6 V), which indicates that p-type doping is caused by adsorbates during sample preparation, which is widely observed in CVD-grown G (Pirkle et al., 2011; Liao et al., 2022). With LDPT, the BG sample is transformed into a GO / G layered composite, and the V CNP Value to the corrected V g movement because the electronegative oxygen functional groups withdraw π-electrons from graphene, leading to its p-type behavior (Dey et al., 2016). Figure 5 (b) shows the electrical characteristics of GO / G based SGFET with I d -V sd Curve. With V g Slightly lower, I d The value is significantly reduced, which is speculated to be a sensitive regulation of electrical transport related to the gate voltage.

[0054] Sensing performance for p-tau217

[0055] In order to optimize the sensing performance of the SGFET-based biosensor, the incubation time of the antibody immobilized on the GO / G surface was studied. d -V gTransfer curve, such as Figure 6 (a) and extracted from each transfer curve Figure 6 V in (b) CNP When the incubation time is 4 hours, V CNP To negative V g The negative charge of the antibody in this disclosure is consistent with the literature. As the immobilization time increases to 16 hours, V CNP Continue to more negative V g This indicates that more antibodies were immobilized on the GO / G surface. It is worth noting that when the incubation time was extended to 24 h, V CNP The results were almost identical, indicating that the amount of antibody immobilized on the GO / G surface reached saturation. Therefore, a 16-hour incubation time was used for subsequent p-tau217 detection. At the same time, GO / G samples were exposed to amine- and FAM (carboxyfluorescein)-modified antibodies, and the immobilization of antibodies on the GO / G surface was then examined under a 16-hour incubation time. Figure 6 (c) Fluorescence microscopy images (left) and corresponding optical images (right) of GO / G samples exposed to amine- and FAM-modified antibodies. The fluorescence observed on GO / G, indicated by the dotted line, confirms the successful immobilization of the antibodies by the incubation process.

[0056] After optimizing the antibody incubation time, the sensing performance of the GO / G-based SGFET biosensor for different concentrations of the target p-tau217 protein was determined. The detection of target p-tau217 was performed in ten-fold increments, from 10 fg / ml to 100 pg / ml. Figure 7 As shown in (a), when the target p-tau217 binds, V CNP Positive V g It has been reported that p-tau217 has a high positive charge density around the aggregation-prone microtubule-binding region and has long-range N-terminal interactions with the middle and C-terminal regions (Limorenko et al., 2022). Therefore, it is believed that this rightward shift when the target p-tau217 binds to the GO / G-based SGFET biosensor is due to the p-type doping effect imposed by the charge from the positively charged p-tau217 to the GO / G active channel. Because the target p-tau217 protein is close to the surface of the graphene composite, it leads to Coulombic interactions between it and the hole-rich tau protein. As the concentration of the target p-tau217 increases, a larger rightward shift occurs due to the transfer of more hole charges from the target p-tau217 protein. Figure 7 (b) is a graph showing V after target binding relative to V before binding. CNP The offset of the value is expressed as ΔV CNP, and the shift is related to the logarithmic concentration.

[0057] The biosensor of the present disclosure showed a high sensitivity of 18.6 mV / decade with a linearity (R 2 ) and a limit of detection (LOD) of 10 fg / ml. Since this disclosure is the first to demonstrate the detection of p-tau217 using a nanomaterial-based POC biosensor, it cannot be compared with other methods. Other p-tau isomers, p-tau231, were previously reported with an LOD of 4.71 pg / ml using a gold nanoparticle-based colorimetric circle blot immunoenzyme assay (Phan et al., 2022) and an LOD of 60 pg / ml using an electrochemical biosensor based on a corrugated gold thin film electrode (Le et al., 2022). These LODs are much higher than the LODs in this disclosure. Hall measurements were performed to examine the p-type doping effect due to binding of the target p-tau217 protein, using the same immobilization and binding protocol as previously described in this disclosure. As Figure 7 As shown in (c), the carrier concentration (n), which is the main carrier of holes in GO / G, increases with the increase of the target p-tau217 protein concentration. This result confirms that the response to V CNP The hole transfer from the positively charged p-tau217 protein to the GO / G is shifted to the right. In the Hall measurement, the mobility (μ) of GO / G according to the target p-tau217 protein concentration is also obtained ( Figure 7 (d)). As the concentration of the target p-tau217 protein increases, the mobility decreases. G is known to be a typical 2D material, highly sensitive to the attachment of foreign atoms or molecules, leading to internal carrier scattering. Therefore, the attachment of more target p-tau217 protein leads to more scattering in the G bottom layer through the π-π interaction between GO and G, and thus lower carrier mobility. Notably, across the entire concentration range, the carrier concentration increases approximately threefold while the mobility decreases approximately twofold. According to the van der Pauw equation:

[0058] R∝1 / μn

[0059] Where R is the resistivity. R is proportional to the inverse of the product of μ and n. n reduces R by approximately three times, while μ increases R by approximately two times. Overall, R decreases slightly. This can explain the increase in I with increasing target p-tau217 protein amount. d Slightly increased.

[0060] Specificity and stability of biosensors

[0061] For clinical testing of any biosensor, specific sensing is a very high requirement. In this disclosure, the specificity of the GO / G-based SGFET biosensor for p-tau217 protein was tested in undiluted HSA over the same concentration range as tested in PBS, following the same assay protocol. Figure 8 (a) Shows representative transfer curves of GO / G-based SGFETs measured at different concentrations of target p-tau217 protein. Bipolar transport behavior was also observed in HSA solution. When the concentration of p-tau217 protein increased, V CNP The value is also positive V g The trend of Id changes according to p-tau217 protein concentration was almost the same as that measured in PBS. Figure 8 (b) shows the V values measured only in HSA solution. CNP ΔV CNP Based on a linear fit with a high R of 0.994 2 value, with a sensitivity of 16.7mV / decade. Notably, the sensitivity remained at approximately 90% of the value measured in PBS, indicating high specificity in a complex matrix. Stability is another key factor for analysis in clinical testing. In order to examine the stability of the GO / G-based SGFET biosensor, the transfer curve of the antibody-modified GO / G-based SGFET after antibody immobilization was measured, in preparation for the target p-tau217 protein detection. Before each measurement, the antibody-modified GO / G-based SGFET was stored in PBS solution. Figure 9 (a) shows representative transfer curves of antibody-modified GO / G-based SGFETs obtained after different storage days. Figure 9 As shown in (b), V CNP The values are extracted from each curve. Even after seven days of storage, V CNP The values remained almost the same, with a change of less than 2%. It is worth noting that with the storage days, I d The S and D electrodes decrease gradually and slightly as they are repeatedly contacted by the needle probe, leading to inevitable electrode degradation. d The decrease is due to repeated contact, but the intrinsic properties of the G layered composite as the sensing mechanism, namely V CNP The offset was very stable, indicating the good stability of the biosensor prepared in the present disclosure.

[0062] The present disclosure provides a biosensor based on SGFET, which features a GO / G layered composite material for detecting p-tau217 protein. This is the first study of the most effective biomarker for detecting AD. The top layer of GO covalently immobilizes an amine-functionalized antibody; while the bottom layer of G acts as a transducer to respond to the attachment of the target p-tau217 protein via π-π interactions between the GO and G layers. When the logarithmic concentration of the target p-tau217 protein increases (from 10 fg / ml to 100 pg / ml), the ΔV of the biosensor increases. CNP The values increased linearly, with a sensitivity of 18.6 mV / decade, a linearity of 0.991, and an LOD of 10 fg / ml in a PBS environment. Simultaneously, detection in HSA achieved approximately 90% of the sensitivity and similar linearity and LOD as in HSA. In addition to excellent specificity, the stability of the antibody-coated biosensor ready for sensing was confirmed by observing only approximately 2% change after 7 days of storage in PBS. Accordingly, it is believed that the SGFET-based biosensor prepared by the method of the present disclosure improves the accuracy of early diagnosis of AD.

[0063] Although the present disclosure has been described in terms of specific embodiments, numerous modifications and variations can be made by one skilled in the art without departing from the scope and spirit of the present disclosure as set forth in the claims.

Claims

1. A method for preparing a biosensor for detecting Alzheimer's disease biomarkers, characterized in that: It includes the following steps: An aluminum oxide film is deposited on a Si substrate using an atomic layer deposition system to form an Al2O3 / Si substrate; Depositing a plurality of electrical contact portions Cr / Au on the Al2O3 / Si substrate by a thermal evaporator to form a source electrode, a drain electrode and a planar gate on the Al2O3 / Si substrate; forming a double-layer graphene on the Al2O3 / Si substrate between the source electrode and the drain electrode by thermal annealing in a vacuum environment; The double-layer graphene is subjected to a low-damage plasma treatment (LDPT) using a mixture of oxygen and hydrogen to form a graphene oxide / graphene GO / G layered composite material on the Al2O3 / Si substrate; as well as The antibody is immobilized on the surface of the GO / G layered composite material through a reaction between the amine group of the antibody and the carboxyl group of GO of the GO / G layered composite material, wherein the antibody is specific for the p-tau217 protein.

2. The method according to claim 1, further comprising the step of supplying an epoxy type adhesive to define the sensing area.

3. The method according to claim 2, wherein the size of the sensing area ranges from 5x5 to 10x10 mm 2 .

4. The method according to claim 2, wherein the step of immobilizing the antibody on the surface of the GO / G layered composite material is performed by incubating the sensing area with 100 μg / ml of the antibody in a volume ranging from 20 to 100 μl aliquots at a temperature ranging from 4 to 37°C for 1 to 24 hours.

5. A biosensor for detecting an Alzheimer's disease biomarker, manufactured by a method comprising the following steps: An aluminum oxide film is deposited on a Si substrate using an atomic layer deposition system to form an Al2O3 / Si substrate; Depositing a plurality of electrical contact portions Cr / Au on the Al2O3 / Si substrate by a thermal evaporator to form a source electrode, a drain electrode and a planar gate on the Al2O3 / Si substrate; forming a double-layer graphene on the Al2O3 / Si substrate between the source and the drain by thermal annealing in a vacuum environment, subjecting the double-layer graphene to low-damage plasma treatment (LDPT) with a mixture of oxygen and hydrogen to form a graphene oxide / graphene GO / G layered composite material on the Al2O3 / Si substrate; and The antibody is immobilized on the surface of the GO / G layered composite material through a reaction between the amine group of the antibody and the carboxyl group of GO of the GO / G layered composite material, wherein the antibody is specific for the p-tau217 protein.

6. A biosensor for detecting a biomarker of Alzheimer's disease, comprising: Si substrate; an aluminum oxide film of forming an Al2O3 / Si substrate on the Si substrate by an atomic layer deposition system; A plurality of electrical contact portions Cr / Au deposited on the Al2O3 / Si substrate by a thermal evaporator; Graphene oxide / graphene GO / G layered composite material formed on the Al2O3 / Si substrate by low damage plasma treatment LDPT; and The antibody is immobilized on the surface of the GO / G layered composite material through a reaction between the amine group of the antibody and the carboxyl group of GO of the GO / G layered composite material, wherein the antibody is specific for the p-tau217 protein.

7. The biosensor according to claim 6, wherein an epoxy type adhesive is supplied to define the sensing area.

8. The biosensor according to claim 7, wherein the size of the sensing area ranges from 5x5 to 10x10 mm 2 .

9. The biosensor according to claim 7, wherein the antibody is immobilized on the surface of the GO / G layered composite material by incubating the sensing area with 100 μg / ml of the antibody in a volume ranging from 20 to 100 μl aliquots at a temperature ranging from 4 to 37°C for 1 to 24 hours.

10. A method for detecting a biomarker for Alzheimer's disease, comprising the following steps: The amount of p-tau217 protein in a sample is detected using a biosensor, wherein the biosensor is manufactured by the following steps: An aluminum oxide film is deposited on a Si substrate using an atomic layer deposition system to form an Al2O3 / Si substrate; Depositing a plurality of electrical contact portions Cr / Au on the Al2O3 / Si substrate by a thermal evaporator to form a source electrode, a drain electrode and a planar gate on the Al2O3 / Si substrate; forming a double-layer graphene on the Al2O3 / Si substrate between the source electrode and the drain electrode by thermal annealing in a vacuum environment; treating the double-layer graphene with a mixture of oxygen and hydrogen using a low-damage plasma (LDPT) to form a graphene oxide / graphene GO / G layered composite material on the Al2O3 / Si substrate; and The antibody is immobilized on the surface of the GO / G layered composite material through a reaction between the amine group of the antibody and the carboxyl group of GO of the GO / G layered composite material, wherein the antibody is specific for the p-tau217 protein.

11. The method of claim 10, wherein an epoxy-type adhesive is supplied to define the sensing area of the biosensor.

12. The method according to claim 11, wherein the size of the sensing area ranges from 20 to 80 mm 2 .

13. The method of claim 11, wherein the antibody is immobilized on the surface of the GO / G layered composite material by incubating the sensing region with 100 μg / ml of the antibody in a volume ranging from 20 to 100 μl aliquots at a temperature ranging from 4 to 37°C for 1 to 24 hours.

14. A method for detecting a biomarker for Alzheimer's disease, comprising the following steps: The amount of p-tau217 protein in a sample is detected using a biosensor, wherein the biosensor comprises: Si substrate; an aluminum oxide film of forming an Al2O3 / Si substrate on the Si substrate by an atomic layer deposition system; A plurality of electrical contact portions Cr / Au deposited on the Al2O3 / Si substrate by a thermal evaporator; Graphene oxide / graphene GO / G layered composite material formed on the Al2O3 / Si substrate by low damage plasma treatment LDPT; and The antibody is immobilized on the surface of the GO / G layered composite material through a reaction between the amine group of the antibody and the carboxyl group of GO of the GO / G layered composite material, wherein the antibody is specific for the p-tau217 protein.

15. The method of claim 14, wherein an epoxy type adhesive is supplied to define the sensing area of the biosensor.

16. The method of claim 15, wherein the size of the sensing area ranges from 5x5 to 10x10 mm 2 .

17. The method of claim 15, wherein the antibody is immobilized on the surface of the GO / G layered composite material by incubating the sensing region with 100 μg / ml of the antibody in a volume ranging from 20 to 100 μl aliquots at a temperature ranging from 4 to 37°C for 1 to 24 hours.

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