Method for manufacturing a biosensor for detecting biomarkers for Alzheimer's disease and biosensor manufactured thereby

Abstract

The present disclosure provides a method for fabricating a biosensor for detecting biomarkers of Alzheimer's disease, the method including the steps of depositing an aluminum oxide film on a Si substrate by an atomic layer deposition system to form an Al2O3 / Si substrate; depositing electrical contacts Cr / Au on the Al2O3 / Si substrate by a thermal evaporator to form source, drain, and planar gate on the Al2O3 / Si substrate; subjecting bilayer graphene to thermal annealing in a vacuum environment on the Al2O3 / Si substrate; subjecting the bilayer graphene to low-damage plasma treatment (LDPT) using a mixture of oxygen and hydrogen to form a graphene oxide / graphene (GO / G) layered composite on the Al2O3 / Si substrate; and immobilizing an antibody specific to p-tau217 protein on the surface of the GO / G layered composite via a reaction between the amine group of the antibody and the carboxyl group of GO in the GO / G layered composite.

Description

[Technical Field]

[0001] The present disclosure relates to a method for manufacturing a biosensor, and in particular to a method for manufacturing a biosensor for detecting biomarkers for Alzheimer's disease and the biosensor manufactured thereby. [Background technology]

[0002] Dementia is characterized by a loss of cognitive function beyond that expected from the normal outcome of biological aging. According to a 2021 World Health Organization report, more than 55 million people worldwide currently suffer from dementia, a number predicted to increase to more than 78 million by 2030 and 139 million by 2050 (Dementia, Newsroom, World Health Organization, 2022). Dementia poses a serious threat to our society and healthcare systems. Alzheimer's disease (AD) is the most common form of dementia, accounting for more than 60% of dementia cases. Clinicopathological evidence suggests that cognitive decline may manifest more than 15 years before clinical diagnosis (Amieya et al., 2014; Villemagne et al., 2013). Although AD progression is irreversible, effective treatments exist if AD is controlled early in its clinical course. Therefore, early diagnosis and treatment have important clinical implications for AD management.

[0003] Biomarkers are biochemical indicators used to assess disease risk. Among these biomarkers, the accumulation of amyloid beta (Aβ) peptide in extracellular plaques and the accumulation of hyperphosphorylated tau (p-tau) protein in neurofibrillary tangles have been incorporated into the diagnostic framework for AD (Huang et al., 2009; Varesi et al., 2022; Teunissen et al., 2022). For AD diagnosis, Aβ and p-tau can be visualized by positron emission tomography (PET) or quantitatively measured in cerebrospinal fluid (CSF) (Valotassiou et al., 2018; Brier et al., 2016; Palmqvist et al., 2015). However, these methods are invasive, cumbersome, and expensive, making them inaccessible to all AD patients. Therefore, there is a strong need for the development of blood-based biomarkers for AD diagnosis, as peripheral blood sampling is convenient, low-cost, and minimally invasive. Recent studies have shown that tau protein enters the bloodstream after being injected into the brain, and that high p-tau levels in the blood are a sign of neuronal degeneration in the brain, ultimately contributing to the development of AD (Fiandaca et al., 2015; Banks et al., 2017; Chiu et al., 2013). Several studies using different assays and techniques, 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 protein isoforms, including p-tau181, p-tau217, and p-tau231, are highly specific for the detection of PET-confirmed Aβ and tau pathology across the continuum of clinical AD progression (Suarez-Calvet et al., 2020; Bayoumy et al., 2021; Karikari et al., 2022; Leuzy et al., 2021).However, the detection sensitivity of p-tau protein remains a challenge due to its low concentration in peripheral blood, which is outside the detection range of conventional enzyme-linked immunosorbent assays (ELISAs), and the presence of nonspecific proteins and various interfering substances when detected from blood samples (Derkus et al., 2016; Galasko et al., 2013; Lue et al., 2017; Yang et al., 2018; Hampel et al., 2018). Therefore, the development of point-of-care (POC) biosensors for accurate detection of tau protein in blood-based samples is urgently needed. Nanomaterial-based immunosensors for detecting p-tau protein have been reported. Schneider et al. fabricated a carbon screen-printed electrode decorated with platinum nanoparticles decorated with multiwalled carbon nanotubes to develop an electrochemical-based immunosensor for p-tau detection (Schneider et al., 2022). The electrode was functionalized with amine groups using polyallylamine hydrochloride to interact with the carboxyl groups of the antibody for p-tau181 detection. Square-wave voltammetry was performed to detect p-tau181 in phosphate-buffered saline (PBS). The biosensor demonstrated a linear range of 8.6–1100 pg / mL (approximately 4.1–523.8 nM) and a limit of detection (LOD) of 0.24 pg / mL (approximately 1.1 nM). The sensitivity and LOD of a 10-fold dilution in fetal bovine serum were approximately half that of a dilution in PBS. K. Kim et al. developed a multicore chemiresistive sensor array as an AD biomarker by preparing densely aligned carbon nanotubes using the Langmuir-Blodgett technique (Kim et al., 2020). This device showed an LOD of 2.72 fM with a linear range from 1 fM to 100 nM for the detection of p-tau181 in plasma. H.T.N. Le and S. Cho developed an electrochemical biosensor based on interdigitated corrugated electrodes via activated self-assembled monolayers to preserve specific antibodies for p-tau231 detection (Le et al., 2022).Electrochemical impedance spectroscopy was used to detect p-tau231 in human serum. The biosensor demonstrated an LOD of 140 pg / ml (approximately 66.7 nM) with a linear range of 100 pg / ml to 10 ng / ml (approximately 47.6–476.2 nM). Precise detection of p-tau231, even with a low dissociation constant between the antibody and p-tau231, was also demonstrated. L.M.Phan and S.Cho developed a colorimetric aptablot based on gold nanoparticles to detect p-tau231 in human serum albumin (Phanet et al., 2022). The color saturation, which changes depending on the p-tau231 concentration, can be analyzed by naked eye or with a digital camera using ImageJ software. This approach yielded a linear range of 0.064–1000 ng / ml (approximately 30.5 nM to 476.2 μM) and an LOD of 4.71 pg / ml (approximately 2.2 nM). S. Janelidze et al. reported that p-tau217 showed a stronger correlation with the tauPET tracer [18F]flortaucipir and could more accurately identify individuals with abnormally elevated [18F]flortaucipir uptake, indicating its utility over other biomarkers in AD diagnosis (Janelidze et al., 2020). Unfortunately, no point-of-care biosensors for detecting p-tau217 have yet been reported.

[0004] Geim and Novoselov disclosed single-crystalline graphite films (Novoselov et al., 2004). Graphene (G) has been widely used in the development of various types of biosensors due to its many excellent properties, including electrical conductivity, mechanical strength, biocompatibility, and high surface area (Kanagavalli et al., 2021; Justino et al., 2017; Jangir et al., 2022). Its high sensitivity to the binding of aromatic base-containing bioanalytes, such as DNA, RNA, and proteins, makes it a promising transducer in biosensors. However, the nonspecific affinity between G and bioanalytes makes it unsuitable for their sole detection. Therefore, for subsequent detection or capture of target analytes, the surface of the G must be functionalized to facilitate the binding of biorecognition elements. Pyrene-based liners, such as pyrenebutanoic acid succinimidyl ester and pyrenebutyric acid, are often used to functionalize the surface of G via π-π interactions (Nekrasov et al., 2022; Hinnemo et al., 2017). The wet chemical processes for surface functionalization of G require hours or even days, and as a result, functionalization occurs via noncovalent processes that are less stable than those covalent processes.

[0005] In our previous work (Govindasamy et al., 2022), we developed an atomically layered composite of graphene oxide / graphene (GO / G) using bilayer graphene (BG) grown by chemical vapor deposition (CVD) and our proprietary low-damage plasma treatment (LDPT). LDPT is an atomic-layer oxidation process for developing chemiresistive biosensors. CVD-grown BG is transformed into GO / G through LDPT, which can achieve atomic-layer oxidation using a hydrogen and oxygen gas mixture. Only the top layer of BG is functionalized with oxidized groups, which serve as active sites for forming covalent bonds with biorecognition elements (e.g., antibodies of the present disclosure). Furthermore, the conductivity of the bottom G remains almost unchanged after LDPT, and it can function as a transducer responding to the attachant of a target analyte on the top GO, which is covalently bonded to the biorecognition element through π-π interactions between the GO and G layers.

[0006] The present disclosure provides a solution-gated field-effect transistor (SGFET) featuring an atomically layered composite of graphene oxide / graphene (GO / G) for detecting the p-tau217 biomarker. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present disclosure is to provide a highly sensitive biosensor for detecting biomarkers of Alzheimer's disease. [Means for solving the problem]

[0008] To achieve at least the above objectives, a method for fabricating a biosensor for detecting biomarkers of Alzheimer's disease includes the steps of depositing an aluminum oxide film on a Si substrate by an atomic layer deposition system to form an Al2O3 / Si substrate; depositing Cr / Au electrical contacts on the Al2O3 / Si substrate by a thermal evaporator to form a source, drain, and planar gate on the Al2O3 / Si substrate; subjecting bilayer graphene to thermal annealing in a vacuum environment across the source and drain on the Al2O3 / Si substrate; subjecting the bilayer graphene to low-damage plasma treatment (LDPT) using a mixture of oxygen and hydrogen to form a graphene oxide / graphene (GO / G) layered composite on the Al2O3 / Si substrate; and immobilizing an antibody specific to p-tau217 protein on the surface of the GO / G layered composite via a reaction between the amine group of the antibody and the carboxyl group of GO in the GO / G layered composite.

[0009] In one embodiment, the method further comprises dispensing an epoxy adhesive to define the sensing area.

[0010] In one embodiment, the sensing area is 5x5 to 10x10 mm 2 The sizes range from

[0011] In one embodiment, the step of immobilizing the antibody on the surface of the GO / G layered composite is carried out by incubating the sensing area with 100 μg / ml antibody in an aliquot ranging from 20 to 100 μl at a temperature ranging from 4 to 37°C for 1 to 24 hours.

[0012] Another object of the present disclosure is to provide methods for detecting biomarkers for Alzheimer's disease.

[0013] To this end, a method for detecting a biomarker for Alzheimer's disease includes detecting the amount of p-tau217 protein in a sample with a biosensor, the biosensor including a Si substrate, an aluminum oxide film disposed on the Si substrate by an atomic layer deposition system to form an Al2O3 / Si substrate, Cr / Au electrical contacts disposed on the Al2O3 / Si substrate by a thermal evaporator, a graphene oxide / graphene (GO / G) layered composite formed on the Al2O3 / Si substrate by low-damage plasma treatment (LDPT), and an antibody immobilized on the surface of the GO / G layered composite via a reaction between the amine group of the antibody specific for p-tau217 protein and the carboxyl group of GO in the GO / G layered composite.

[0014] In one embodiment, an epoxy adhesive is dispensed to define the sensing area of ​​the biosensor.

[0015] In one embodiment, the sensing area is 5x5 to 10x10 mm 2 The sizes range from

[0016] In one embodiment, the antibody is immobilized on the surface of the GO / G layered composite by incubating the sensing area with 100 μg / ml antibody in aliquots ranging from 20 to 100 μl at temperatures ranging from 4 to 37°C for 1 to 24 hours. [Brief explanation of the drawings]

[0017] [Figure 1] 1A-1C are diagrams illustrating a process for manufacturing a biosensor according to the present disclosure. [Figure 2] FIG. 1 is a schematic diagram illustrating a solution-gate field-effect transistor. [Figure 3](a) Schematic of the GFET-based biosensor featuring GO / G layered composite. (b) Schematic of p-tau217 protein bound to an antibody immobilized on GO / G. (c) Schematic of the sensing mechanism based on the shift of the tracking point. (d) Photograph of the setup and solution-gated graphene transistor (SGGT). [Figure 4] (a) shows the transmittance of BG before and after PT. (b) shows Raman spectra of BG before and after LDPT. (c) shows XPS spectra of BG before and after LDPT. (d) shows the contact angle of BG before and after LDPT. (e) shows the electrical resistance of SLG and BG before and after LDPT. (f) shows a TEM image of a GO / G atomic layered composite and the interlayer spacing. [Figure 5] (a) is a graph showing the transfer curves of SGFETs containing GO / G atomic layer composites, and (b) is a graph showing the output characteristics of the GO / G atomic layer composites. [Figure 6] (a) Graph showing the transfer curve of the SGFET based on the modified GO / G. (b) Graph showing VCNP versus incubation time. (c) Fluorescence microscopy image (left) and corresponding optical image (right) of the GO / G sample exposed to amine- and FAM-modified antibody. [Figure 7] (a) is a graph showing the transfer curve of the SGFET based on the modified GO / G. (b) is a graph showing ΔVCNP versus p-tau217 protein concentration in PBS. (c) is a graph showing carrier concentration versus p-tau217 protein concentration. (d) is a graph showing mobility versus p-tau217 protein concentration obtained by Hall measurement. [Figure 8] (a) is a graph showing the transfer curve of the SGFET based on the modified GO / G, and (b) is a graph showing ΔVCNP versus p-tau217 protein concentration in HSA. [Figure 9](a) Transfer curve of SGFET based on modified GO / G. (b) VCNP and its variation recorded after different storage periods. DETAILED DESCRIPTION OF THE INVENTION

[0018] In order to facilitate understanding of the objectives, features and effects of the present disclosure, embodiments are provided in conjunction with the accompanying drawings for detailed description of the present disclosure.

[0019] Materials and equipment

[0020] In this disclosure, p-tau217 protein and its various C-terminally amine-conjugated antibodies were obtained from Chang Gung Memorial Hospital, Taiwan. Details of protein preparation, colon screening, and qualification are described below.

[0021] Peptide synthesis and protein preparation: The manufacturer (BIOTOOLS, Taiwan) guaranteed that the purity of the p-tau217 peptide GSRSRTPSLPTPPTREPKKVAVVR (SEQ ID NO: 1) and the control peptide GSRSRTPSLPTPPTREPKKVAVVR (SEQ ID NO: 2) exceeded 95%. The 11th amino acid of SEQ ID NO: 1 is phosphorylated threonine, while the 11th amino acid of SEQ ID NO: 2 is non-phosphorylated threonine. The peptides were prepared and stored according to the manufacturer's recommendations (20 μg / μl). Tau (MAPT) protein was purchased from OriGene (NM_005910, USA) and phosphorylated with glycogen synthase kinase 3β (GSK-3β, Sino Biological Co., Ltd., China). Inactivated virus lysates, influenza A / B (FluA / B), human parainfluenza virus, adenovirus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus (SARS) were purchased (ZeptoMatrix, USA) and used according to the manufacturer's instructions.

[0022] Immunization: Each 6-8 week-old female BALB / c mouse received an intraperitoneal injection of 100 μg of p-tau217 emulsified in complete Freund's adjuvant (Sigma-Aldrich, USA). Booster injections of 100 μg of peptide in incomplete Freund's adjuvant (Sigma-Aldrich, USA) were administered on days 14, 28, and 42. Before sacrifice, the mice were injected twice with 50 μg of peptide emulsified in IFA, 3 days apart, to elicit antibody responses.

[0023] Hybridoma generation and antibody purification: To generate monoclonal antibodies against the p-tau217 peptide, BALB / c mice were inoculated with synthetic p-tau217 peptide three times every two weeks. Two boosts were administered at 3-day intervals before sacrifice. Mouse spleens were immediately harvested and fused with myeloma cells for hybridoma generation and subsequent semi-solid selection (ClonaCell Hybridoma Kit, STEMCELL Technology, USA). Hybridoma colonies were grown in 96-well microtiter plates (Corning, USA) until confluent, and the supernatants were harvested and tested for antibody reactivity against the p-tau217 peptide, full-length p-tau protein, or control peptides by ELISA. High-affinity antibodies against the p-tau217 peptide and / or p-tau protein were selected for application to protein G Sepharose resin (Cytiva, USA). These purified monoclonal antibodies were dialyzed against PBS buffer to remove glycine and concentrated with an Amicon Ultra 15 centrifugal filter unit (10 kDa, Merck Millipore, USA). The monoclonal antibodies were stored at −80°C for the following experiments.

[0024] Enzyme-linked immunosorbent assay (ELISA): 100 ng / well of p-tau protein, p-tau217 peptide, or tau-217 was coated onto a 96-well microtiter plate in coating buffer (150 mM Na2CO3, 150 mM NaHCO3, pH 9.6) overnight at 4°C. For cross-reactivity tests, 1 μg / well of inactivated virus lysate was used for testing. After blocking with 1% bovine serum albumin, 100 μL of cell supernatant or 1 μg of purified monoclonal antibody was added and incubated for 1 hour at room temperature (RT). At the end of the incubation period, the microtiter plate was washed four times with TBST (TBS containing 0.05% Tween 20) and the bound antibody was detected with horseradish peroxidase (HRP)-conjugated anti-mouse IgG Fc region (Jackson ImmunoResearch Laboratories, USA) at a dilution of 1:2000 for 1 hour at room temperature. After washing, the activity of horseradish peroxidase (HRP) was read at 450 nm using 3,3',5,5'-tetramethylbenzidine (TMB, Sigma-Aldrich, USA) as the substrate in a microELISA reader (EZ read 400).

[0025] Modification of monoclonal antibodies: Amine-reactive esters of carboxyl groups on monoclonal antibodies were prepared using a reactive amination kit according to the manufacturer's recommendations (G-BIOSCIENCES, USA). Briefly, 1 mg / mL of purified monoclonal antibody was dissolved in 1x Optimizer buffer. EDC and sulfo-NHS were added to final concentrations of 2 mM and 5 mM, respectively, and the mixture was incubated at room temperature for 15 minutes. Next, β-mercaptoethanol (Sigma-Aldrich, USA) was added to the antibody solution to a final concentration of 20 mM and incubated at room temperature for 10 minutes to quench the EDC. Free EDC, NHS, and EDC by-products were removed using a desalting column, and the antibody solution, adjusted to PBS (pH 7), was concentrated using a 10 kDa cutoff centrifugal filter unit. Hydroxylamine (Sigma-Aldrich, USA) was added to a final concentration of 10 mM to block the amine-reactive sites for 5 minutes at room temperature. Buffer exchange was performed with PBS (pH 7) using a 10 kDa cutoff centrifugal filter. The antibody solution was aliquoted at 100 μL / vial (0.25–0.5 μg / μL) and stored at -80°C.

[0026] To prepare monoclonal antibodies (mAbs) against p-tau217, BALB / c mice were immunized with p-tau217 peptide to induce antibodies against p-tau217. At the end of the immunization, the spleens of the mice were harvested for hybridoma fusion with bone marrow cells to generate hybridoma clones producing anti-p-tau217 mAbs. In preliminary selection, over 500 hybridoma clones were selected to verify the reactivity of the antibodies against p-tau217 peptide and p-tau protein. The top 20 clones that exhibited high reactivity against p-tau217 and p-tau are shown in Table 1. Table 1 also shows the affinity test results of the monoclonal antibodies against p-tau protein and p-tau217 peptide. OD stands for optical density.

[0027] [Table 1]

[0028] 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, and the detection limits were measured using an ELISA test (Table 2). Table 2 shows the detection limits of monoclonal antibodies against the p-tau217 peptide.

[0029] [Table 2]

[0030] To further exclude the possibility that the antibodies might cross-react with other antigens, we tested the cross-reactivity of two mAb clones (No. 72 and No. 123) with higher affinity for p-tau217 with other viral antigens, such as influenza A / B, human parainfluenza virus, adenovirus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus (SARS). p-tau217 was used as a positive control, and the tau-217 peptide was used as a negative control (Table 3). Table 3 shows the results of the cross-reactivity test between the monoclonal antibodies and viral lysates. Flu A / B represents influenza A / B, para-Flu represents human parainfluenza virus, RSV represents respiratory syncytial virus, and SARS represents severe acute respiratory syndrome coronavirus (SARS). The control represents the tau-217 peptide. In this study, we used clone No. 72, which has high antibody affinity for p-tau217 and p-tau and little cross-reactivity with other antigens.

[0031] [Table 3]

[0032] 1x phosphate-buffered saline (PBS containing 137 mM NaCl, 2.7 mM KCl, 8.1 mM NaHPO, and 1.47 mM KHPO) at pH 7.4 and HSA were purchased from Sigma-Aldrich, USA. All other chemicals were analytical grade and used as received. Deionized water was obtained from a Millipore water purification system (resistance 18.2 MΩ, Milli-QDirect 8). 25 μm copper foil for bilayer graphene (BG) preparation was purchased from Alfa-Acer (Thermo Fisher Scientific). BG was grown on the copper foil by chemical vapor deposition (CVD) using a 3-inch diameter tubular quartz furnace. Details of the growth procedure and subsequent transfer to the target substrate are as described in Govindasamy et al., 2022.

[0033] Optical and structural characterization of the G sample was performed using ultraviolet-visible (UV-Vis) spectroscopy (V-650, JASCO Corporation, Japan) and Raman spectroscopy (Horiba, Ltd., iHR-550 equipped with a 532 nm laser, Japan). Its chemical composition was analyzed using X-ray photoelectron spectroscopy (XPS, PHI 5000 VersaProbe III, ULVAC, Japan) with a monochromated AlKα source. The layered composite was observed by transmission electron microscopy (TEM, JEOL Ltd., JSM-2100, Japan). Water contact angle (WCA) measurements were performed using a PSC-100B instrument (Pentad Scientific, Taiwan) to confirm hydrophilicity. The electrical characteristics of the SGFET were measured using a semiconductor parameter analyzer (B1500A, Agilent Technologies, USA) in a home-built probe station. The source-drain current (I sd ) vs. gate voltage (V g ) transfer curve is V g is -0.4 to 1.2V, the interval is 0.02V, and the source-drain bias (V sd) was measured under the condition of 0.5 V. The carrier concentration and mobility of devices were determined for p-tau217 solutions with different concentrations using a Hall effect measurement system (AHM-800B, Agilent Technologies, USA). All characteristics were measured for at least five samples, and the average values ​​and standard deviations are reported herein.

[0034] Method for fabricating biosensor and p-tau217 detection protocol

[0035] Figure 1 shows the fabrication procedure of the biosensor of the present disclosure. Using an atomic layer deposition system, a 6 nm aluminum oxide (Al2O3) film was coated on a Si substrate, measuring 3 × 2.5 cm. 2 An Al2O3 / Si substrate was obtained. The source, drain, and planar gate were established by forming Cr / Au (5 / 50 nm) electrical contacts on the Al2O3 / Si substrate through a shadow mask using a thermal evaporator. Two sets of electrical contacts were fabricated on the substrate, and their detailed dimensions are shown in Figure 2. Next, bilayer graphene (BG) formed by a chemical vapor deposition (CVD) process (Govindasamy et al., 2022) was transferred to the substrate across the source and drain electrodes. All other polymer residues were then removed by thermal annealing in a vacuum environment to strengthen the contact between the BG and the electrodes, resulting in a BG on the Al2O3 / Si substrate (BG / Al2O3 / Si). The bilayer graphene was then subjected to low-density annealing (LDPT) using a mixture of oxygen and hydrogen gases to form a GO / G layered composite on the electrode-deposited Al2O3 / Si substrate. Details of the LDPT process have been previously reported (Govindasamy et al., 2022). Finally, epoxy resin adhesive was dispensed to create a 4 × 9 mm 2A sensing area of ​​10 μL was defined. Immediately after preparing the GO / G layered composite on the Al2O3 / Si substrate with deposited electrodes, an antibody was immobilized on the GO / G surface via a carbodiimide-mediated reaction between the C-terminal amine group (-NH2) of the aminated antibody and the carboxyl group (-COOH) of GO. A 20 μL aliquot of a 100 μg / mL antibody solution was dispensed onto the sensing area and incubated at 4 °C for 16 h. The device was then gently rinsed three times with PBS to wash away excess unbound antibody. A total of 20 μL of p-tau217 solutions with various concentrations in PBS were dropped onto the sensing area for 30 min at room temperature to allow binding to the antibody. The sample was then gently rinsed with PBS to remove excess unbound p-tau217 protein of interest. Additionally, p-tau217 solutions with different concentrations in HSA were prepared using the same protocol as in PBS to verify the usefulness of the detection method. Figure 3 shows a schematic diagram of the SGFET-based biosensor featuring the GO / G layered complex of p-tau217 protein.

[0036] Characterization of BG and GO / G layered composites

[0037] A GO / G layered composite was prepared. The top layer of GO reacts with antibodies as a biorecognition element, while the bottom layer of G acts as a transducer. The preparation of the GO / G layered composite began with the growth of BG, followed by LDPT. UV-Vis spectroscopy, Raman spectroscopy, XPS, CA measurements, and electrical resistance measurements were performed to confirm the structure of the GO / G layered composite (Figure 4). Figure 4(a) shows the UV-Vis spectra of BG before and after LDPT. The transmittance of BG at 550 nm before LDPT was 95.3%, which is roughly consistent with the 2.3% transmittance reduction of single-layer graphene (SLG). The Raman spectrum shown in Figure 4(b) reveals that the 2D to G intensity ratio of BG before LDPT was approximately 0.83, with low intensity in the D band and a full width at half maximum of the 2D band of approximately 59 cm. -1This indicates that the BG is a crystalline material, suggesting the formation of an AB-stacked BG (Sheng et al., 2015; Liu et al., 2012). Based on these material analyses, high-quality BG was used in this study. The transmittance of the BG after LDPT increased slightly. It has been reported that the band gap of graphene oxide increases with the incorporation of oxygen into graphene (Jin et al., 2020). Therefore, the optical absorption in the visible range decreases, resulting in an increase in transmittance, as shown in Figure 4(a). After LDPT was performed on the BG sample, the incorporation of oxidized functional groups into the BG increased the intensity of the D band, and the intensity ratio of the 2D band to the G band (I 2D / I G ) dramatically decreased. Note that a blue shift was observed after LDPT, indicating the p-type doping effect in BG (Tang et al., 2010). Figure 4(c) shows the XPS spectrum of the BG sample after LDPT. The bonding state of the carbon atoms was observed as sp 2 The bond states can be resolved into five types: C=C bond, sp3 C-C bond at 285.5 eV, hydroxyl group (C-OH) at 286.6 eV, carbonyl group (C=O) at 288 eV, and carboxyl group (COOH) at 288.9 eV (Krishnamoorthy et al., 2013; Huang et al., 2021). These oxygen functional groups are characterized by an increase in the D band and I band in the Raman spectrum, as shown in Figure 4(b). 2D / I GThis leads to the formation of defect structures within the BG, corresponding to a decrease in the valence energy. The COOH group, the main functional unit required for covalent bond formation between the amino groups of the antibody and the top layer of GO, contributes up to 6.5% to the total carbon bonding state. The hydrophilicity of the BG sample was evaluated based on its WCA. As shown in Figure 4(d), the WCA was observed to change from 83° before LDPT to 18° after LDPT, making the BG sample hydrophilic. This result confirmed that the formation of oxidized functional groups resulted in a hydrophilic surface, as indicated by the XPS spectra. Figure 4(e) shows the electrical resistance between the source and drain electrodes of the BG and single-layer graphene (SLG) samples before and after LDPT. After LDPT, the resistance of the SLG dramatically increased, exceeding several megaohms, indicating that the SLG became a nearly electrically insulating material.

[0038] Conversely, the resistance of the BG sample after LDPT increased slightly, while the conductivity remained at the same order of magnitude. From these results, we can conclude that the top layer of G in the BG sample was converted to GO, while the bottom layer of G remained largely unchanged by LDPT. TEM measurements were performed to confirm the formation of the GO / G layered G composite. As shown in Figure 4(f), the bilayer structure of GO / G was clearly observed, with a spacing of approximately 0.361 nm between the two layers. According to the results in Figure 4, atomic layer oxidation was achieved by LDPT, forming a GO / G layered G composite as a transducer. At the same time, the top layer of GO was also able to react with antibodies for subsequent antigen binding.

[0039] Figure 5(a) shows a typical transfer curve of an SGFET characterized by a gate voltage (V g ) versus drain-source current (I d )) were plotted. The typical characteristics of the V-shaped curve of BG were observed, suggesting bipolarity. V CNP is positive V g(approximately 0.6 V), indicating p-type doping caused by adsorbates during sample preparation, which is commonly observed in CVD-grown GO (Pirkle et al., 2011; Liao et al., 2022). LDPT transformed the BG sample into a GO / GO layered composite, while also increasing the V of the SGFET. CNP The value of V is more positive g This is due to the abstraction of π electrons from graphene by the presence of electronegative oxygen functional groups, resulting in its p-type behavior (Dey et al., 2016). Figure 5(b) shows that V g is changed from -0.1V to -0.9V. d -V sd The curves are used to show the electrical characteristics of SGFETs based on GO / G. V g The value of I d The value of σ decreased significantly, suggesting a high sensitivity of the modulation of electrical transport with respect to gate voltage.

[0040] Sensing performance for p-tau217

[0041] To optimize the sensing performance of the SGFET-based biosensor, we investigated the incubation time of antibody immobilization on the GO / G surface. As shown in Figure 6(a), after the antibody immobilization process, I d -V g Transfer curves were measured, and the V values ​​in Figure 6(b) were extracted from each transfer curve. For an incubation time of 4 hours, V CNP is negative V g The shift towards n-type doping indicates that the antibody in this disclosure is negatively charged, which is consistent with the reference. When the immobilization time is increased to 16 hours, V CNP is more negative V g The V value continued to shift toward V, indicating that more antibodies were immobilized on the GO / G surface. CNPThe almost identical fluorescence intensity (FAM) of the GO / G sample suggests that the amount of immobilized antibody on the GO / G surface was saturated. Therefore, a 16-hour incubation time was used for subsequent detection of p-tau217. We also confirmed the immobilization of the antibody on the GO / G surface after 16 hours of incubation by exposing the GO / G sample to amine- and FAM-modified antibodies. Figure 6(c) shows a fluorescence microscope image (left) and the corresponding optical image (right) of the GO / G sample after exposure to amine- and FAM-modified antibodies. The fluorescence observed on the GO / G sample, depicted by the dotted line, confirmed the successful immobilization of the antibody through the incubation process.

[0042] After optimizing the antibody incubation time, the sensing performance of the GO / G-based SGFET biosensor was measured for target p-tau217 protein at various concentrations. The detection of target p-tau217 was performed from 10 fg / ml to 100 pg / ml, increasing by 10-fold. As shown in Figure 7(a), upon binding of target p-tau217, V CNP is a positive V g p-tau217 has been reported to have a high positive charge density around its aggregation-prone microtubule-binding region and long-range N-terminal interactions with the intermediate and C-terminal domains (Limorenko et al., 2022). Therefore, the proximity of the target p-tau217 protein to the graphene composite surface allows for Coulombic interactions between the surface and the hole-rich tau protein. Therefore, when the target p-tau217 binds to the GO / G-based SGFET biosensor, the rightward shift is believed to be due to the p-type doping effect caused by charge transfer from the positively charged p-tau217 to the GO / G active channel. As the concentration of target p-tau217 increases, a larger rightward shift occurs due to increased hole charge transfer from the target p-tau217 protein. Figure 7(b) shows the V after target binding with respect to logarithmic concentration. CNP and V before bonding CNPThe relative shift in the value of (ΔV CNP Graph showing the relationship between the temperature and humidity.

[0043] The biosensor of the present disclosure has a high sensitivity of 18.6 mV / decade and a linearity (R 2 ), and a limit of detection (LOD) of 10 fg / ml. Because this disclosure provides the first demonstration of p-tau217 detection using a nanomaterial-based POC biosensor, comparisons with other methods are not provided. Previously reported LODs for another p-tau isoform, p-tau231, were 4.71 pg / ml using a gold nanoparticle-based colorimetric aptablot (Phan et al., 2022), while 60 pg / ml using an electrochemical biosensor based on a corrugated gold film electrode (Le et al., 2022). These LODs are much higher than those in this disclosure. To verify the p-type doping effect of binding with the target p-tau217 protein, hole measurements were performed using the immobilization and binding protocol described above. As shown in Figure 7(c), increasing the concentration of the target p-tau217 protein increases the carrier concentration (n) of the majority hole carriers in GO / G. We confirmed that the hole transfer from the positively charged p-tau217 protein to GO / G was V CNP This causes a rightward shift in the mobility. Hall measurements also revealed the mobility (μ) of GO / G as a function of the target p-tau217 protein concentration (Figure 7(d)). As the target p-tau217 protein concentration increased, the mobility decreased. G is well known to be a typical 2D material, highly sensitive to the attachment of foreign atoms or molecules, causing carrier scattering within its interior. Therefore, the attachment of more target p-tau217 protein leads to greater scattering due to π-π interactions between GO and G in the bottom layer of G, thereby decreasing carrier mobility. Note that the carrier concentration increased approximately threefold over the entire concentration range, while the mobility decreased approximately twofold. This is based on the van der Pauw equation.

[0044]

number

[0045] where R is the resistivity. R is proportional to the inverse of the product of μ and n. n decreases R by about 3-fold, while μ increases R by about 2-fold. Overall, R decreased slightly. This is because, when the amount of target p-tau217 protein increased, I d This may explain the slight increase in

[0046] Biosensor specificity and stability

[0047] Specific sensing is highly desirable in clinical trials of any biosensor. In this study, we verified the specificity of the GO / G-based SGFET biosensor for p-tau217 protein in undiluted HSA over the concentration range of the detection protocol tested in PBS. Figure 8(a) shows representative transfer curves of the GO / G-based SGFET measured at various concentrations of the target p-tau217 protein. Ambipolar transport behavior was also observed in HSA solution. As the concentration of p-tau217 protein increased, V CNP The value of V is also positive g The I depending on the concentration of p-tau217 protein shifted towards d The change in V measured only in the HSA solution was almost the same as that measured in the PBS solution. CNP Regarding ΔV CNP The plot of the values ​​of R is shown. 2Using a value of 0.994, the sensitivity obtained based on linear fitting is 16.7 mV / decade. Note that the sensitivity measured in PBS still maintained approximately 90%, suggesting high specificity in complex matrices. Stability is another factor for assays in clinical trials. To investigate the stability of the GO / G-based SGFET biosensor, we measured the transfer curve of the antibody-modified GO / G-based SGFET after antibody immobilization prepared for the detection of the target p-tau217 protein. Before each measurement, the antibody-modified GO / G-based SGFET was stored in PBS solution. Figure 9(a) shows representative transfer curves of the antibody-modified GO / G-based SGFET obtained after various storage days. As shown in Figure 9(b), V CNP The value of V was extracted from each curve. CNP The value of I remained almost the same even after 7 days of storage, with a variation of less than 2%. d The gradual slight decrease in is due to the repeated contact between the S and D electrodes by the needle probe, which inevitably causes electrode deterioration. d Although the V CNP The shifts are very stable, demonstrating the good stability of the biosensors fabricated according to the present disclosure.

[0048] This disclosure provides an SGFET-based biosensor featuring a GO / G layered composite for detecting p-tau217 protein. This was the first study to identify the most effective biomarker for AD. The top layer of GO covalently immobilized an amine-functionalized antibody, and the bottom layer of G acted as a transducer that responded to the attachment of the target p-tau217 protein through π-π interactions between the GO and G layers. As the logarithmic concentration of the target p-tau217 protein increased (from 10 fg / ml to 100 pg / ml), the ΔV of the biosensor decreased. CNPThe value of increased linearly, and in a PBS environment, the sensitivity was 18 / 6 mV / decade, the linearity was 0.991, and the LOD was 10 fg / ml. Detection in HSA was also performed to obtain a sensitivity of approximately 90% in HSA with similar linearity and LOD. In addition to excellent specificity, the stability of the antibody-coated biosensor prepared for sensing was confirmed by only approximately 2% variation observed even after 7 days of storage in PBS. Therefore, it is believed that the SGFET-based biosensor fabricated by the disclosed method will improve the accuracy of early diagnosis of AD.

[0049] While the present disclosure has been described with reference to specific embodiments, numerous modifications and variations may be made by those skilled in the art without departing from the scope and spirit of the present disclosure as set forth in the claims.

[0050] The entire electronic sequence listing (sequencelisting.xml, size: 3.04 kb, created date: January 12, 2024) is incorporated herein by reference.

Claims

1. 1. A method of manufacturing a biosensor for detecting a biomarker for Alzheimer's disease, comprising: An aluminum oxide film is deposited on a Si substrate by an atomic layer deposition system, and the surface of the Si substrate is covered with the aluminum oxide film. ２ O ３ / forming a Si substrate; The Al is evaporated by a thermal evaporator. ２ O ３ / Si substrate and depositing electrical contact Cr / Au on the Al ２ O ３ / forming a source, a drain, and a planar gate on a Si substrate; The bilayer graphene is then bonded to the Al layer across the source and drain by thermal annealing in a vacuum environment. ２ O ３ / Si substrate; The bilayer graphene was subjected to low damage plasma treatment (LDPT) using a mixture of oxygen and hydrogen to remove the Al ２ O ３ forming a graphene oxide / graphene (GO / G) layered composite on a Si substrate; and immobilizing the antibody specific to p-tau217 protein on the surface of the GO / G layered complex via a reaction between the amine group of the antibody and the carboxyl group of GO of the GO / G layered complex.

2. The method of claim 1 further comprising dispensing an epoxy adhesive to define the sensing area.

3. The sensing area is 5 x 5 to 10 x 10 mm ２ The method of claim 2 , wherein the size ranges from

4. 3. The method of claim 2, wherein the step of immobilizing the antibody on the surface of the GO / G layered complex is carried out by incubating the sensing area with 100 μg / ml of the antibody in an aliquot ranging from 20 to 100 μl at a temperature ranging from 4 to 37° C. for 1 to 24 hours.

5. 1. A biosensor for detecting a biomarker for Alzheimer's disease, comprising: An aluminum oxide film is deposited on a Si substrate by an atomic layer deposition system, and the surface of the Si substrate is covered with the aluminum oxide film. ２ O ３ / forming a Si substrate; The Al is evaporated by a thermal evaporator. ２ O ３ / Si substrate and depositing electrical contact Cr / Au on the Al ２ O ３ / forming a source, a drain, and a planar gate on a Si substrate; Thermal annealing in a vacuum environment bonds the bilayer graphene to the Al layer across the source and drain. ２ O ３ / Si substrate; The bilayer graphene was subjected to low damage plasma treatment (LDPT) using a mixture of oxygen and hydrogen to remove the Al ２ O ３ forming a graphene oxide / graphene (GO / G) layered composite on a Si substrate; and immobilizing an antibody on the surface of the GO / G layered complex via a reaction between the amine group of the antibody specific to p-tau217 protein and the carboxyl group of GO of the GO / G layered complex.

6. 1. A biosensor for detecting a biomarker for Alzheimer's disease, comprising: a Si substrate; The surface of the Si substrate is coated with an aluminum oxide film. ２ O ３ / An aluminum oxide film disposed on a Si substrate by an atomic layer deposition system to form a Si substrate; The Al is evaporated by a thermal evaporator. ２ O ３ / Si substrate and an electrical contact Cr / Au disposed on the substrate; Low damage plasma treatment (LDPT) ２ O ３ a graphene oxide / graphene (GO / G) layered composite formed on a Si substrate; and an antibody immobilized on the surface of the GO / G layered complex via a reaction between the amine group of the antibody specific to the p-tau217 protein and the carboxyl group of GO of the GO / G layered complex.

7. The biosensor of claim 6, wherein an epoxy resin adhesive is dispensed to define the sensing area.

8. The sensing area is 5 x 5 to 10 x 10 mm ２ 8. The biosensor of claim 7, having a size in the range of

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

10. 1. A method for detecting a biomarker for Alzheimer's disease, comprising:

1. A method comprising detecting the amount of p-tau217 protein in a sample using a biosensor, wherein the biosensor is manufactured by the steps of: An aluminum oxide film is deposited on a Si substrate by an atomic layer deposition system, and the surface of the Si substrate is covered with the aluminum oxide film. ２ O ３ / forming a Si substrate; The Al is evaporated by a thermal evaporator. ２ O ３ / Si substrate and depositing electrical contact Cr / Au on the Al ２ O ３ / forming a source, a drain, and a planar gate on a Si substrate; Thermal annealing in a vacuum environment causes the Al to be deposited across the source and drain. ２ O ３ / Providing bilayer graphene on a Si substrate; The bilayer graphene was subjected to low damage plasma treatment (LDPT) using a mixture of oxygen and hydrogen to remove the Al ２ O ３ forming a graphene oxide / graphene (GO / G) layered composite on a Si substrate; Immobilizing an antibody on the surface of the GO / G layered complex via a reaction between the amine group of the antibody specific for the p-tau217 protein and the carboxyl group of GO of the GO / G layered complex.

11. The method of claim 10, wherein an epoxy resin adhesive is dispensed to define the sensing area of ​​the biosensor.

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

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

14. 1. A method for detecting a biomarker for Alzheimer's disease, comprising: detecting the amount of p-tau217 protein in the sample using a biosensor, wherein the biosensor a Si substrate; The surface of the Si substrate is coated with an aluminum oxide film. ２ O ３ / An aluminum oxide film disposed on a Si substrate by an atomic layer deposition system to form a Si substrate; The Al is evaporated by a thermal evaporator. ２ O ３ / Si substrate and an electrical contact Cr / Au disposed on the substrate; Low damage plasma treatment (LDPT) ２ O ３ a graphene oxide / graphene (GO / G) layered composite formed on a Si substrate; and an antibody immobilized on the surface of the GO / G layered complex via a reaction between the amine group of the antibody specific to the p-tau217 protein and the carboxyl group of GO of the GO / G layered complex.

15. The method of claim 14, wherein an epoxy resin adhesive is dispensed to define the sensing area of ​​the biosensor.

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

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

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