A method of universal epitope molecularly imprinted membrane transfer to two-dimensional surface construction field effect transistor for detection of biomarkers

Abstract

The application discloses a method for transferring a general epitope molecular imprinting film to a two-dimensional surface to construct a field effect transistor for detecting biomarkers, and belongs to the technical field of biosensors. The method is based on preparing an epitope imprinting film on a copper foil surface, removing the copper foil by etching, and then transferring the epitope imprinting film to the surface of a two-dimensional material to prepare a field effect transistor for biological detection. First, a glycosylated amino acid sequence is used as an epitope molecule, and the epitope molecule is fixed to the surface of the copper foil by boron affinity. A film is formed by imprinting a plurality of functional monomers capable of interacting with the template. The copper foil substrate is removed by etching, and thus an independent epitope molecular imprinting film is obtained. The film can be transferred to the surface of any two-dimensional material. The application realizes the transfer of the epitope molecular imprinting film for the first time, fully utilizes the high selectivity of molecular imprinting and the super-sensitivity of two-dimensional materials, and can realize the high specificity, rapid and super-sensitive detection of biological samples.

Description

Technical Field

[0001] This invention belongs to the field of biosensors, specifically relating to a method for transferring a universal epitope molecular imprinted membrane to a two-dimensional surface to construct a field-effect transistor for detecting biomarkers. Background Technology

[0002] Two-dimensional materials such as graphene, molybdenum disulfide, and hexagonal boron nitride offer advantages such as large specific surface areas, extremely high density of active sites, and strong responses to surface adsorption events, making them ideal for sensing applications requiring high sensitivity and strong candidates for biosensors and other healthcare applications. However, traditional surface functionalization typically uses chemical reagents such as coupling agents and catalysts, as well as organic solvents such as dichloromethane and acetonitrile. These reagents can remain on the surface of two-dimensional materials, causing chemical contamination and altering the physicochemical properties of the material, such as conductivity, mechanical strength, or catalytic activity. Contaminants can block or alter the adsorption sites of target molecules, reducing detection sensitivity. Especially in trace analysis, surface contamination can mask weak signals, making it impossible to detect low concentrations of target substances. Inconsistent surface cleanliness leads to poor reproducibility of experimental results. For two-dimensional materials, even trace amounts of surface contamination can lead to different performance characteristics in different experiments, making it difficult to obtain consistent detection results. Therefore, the surface cleanliness of two-dimensional materials has a critical impact on the accuracy, sensitivity, stability, and reproducibility of subsequent detections. Most current research simply uses solutions for cleaning after each functionalization process, which is not effective in removing contaminants from two-dimensional surfaces.

[0003] Inspired by natural receptor-ligand interactions, molecular imprinting technology (MIT) creates highly selective recognition sites by embedding template molecules into polymer matrices, demonstrating significant potential in chemical and biosensor fields in recent years. Epitope molecularly imprinted membranes are based on specific epitopes (typically a short amino acid sequence on a target molecule) for molecular imprinting. By designing templates highly complementary to the epitopes, imprinted membranes can achieve highly selective recognition of target macromolecules. Compared to traditional whole-molecule imprinting, epitope molecularly imprinted membranes are simpler and avoid macromolecular denaturation. Epitope molecularly imprinted membranes (EMIMs) exhibit excellent specificity in biosensing and analytical detection. Furthermore, compared to biomacromolecules, epitope molecularly imprinted membranes also demonstrate superior stability. However, obtaining independent epitope molecularly imprinted membranes remains a significant challenge. Nevertheless, transferring molecularly imprinted membranes to the surface of two-dimensional materials is a versatile method that avoids surface contamination, offers high stability, and provides good selectivity. Summary of the Invention

[0004] To address the aforementioned problems, this invention discloses a method for transferring a universal epitope molecular imprinted membrane to a two-dimensional surface to construct a field-effect transistor for detecting biomarkers.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A method for transferring a general epitope molecularly imprinted film to a two-dimensional surface to construct a field-effect transistor for detecting biomarkers involves first growing a molecularly imprinted film on a copper foil, and then transferring it to the surface of any sensing material.

[0007] The transfer of the molecularly imprinted membrane includes the following steps:

[0008] Step (1): Preparation of molecularly imprinted membrane. The cleaned copper foil was placed in a solution of 95% ethanol and 5% 3-aminopropyltriethoxysilane and allowed to stand for 6 hours. Then, the solution residue on the surface of the copper foil was removed with ethanol and dried with nitrogen. The copper foil was placed in a solution of 5 mg / mL 2,4-difluoro-3-carboxyphenylboronic acid and sodium cyanoborohydride in ethanol at room temperature for 24 hours. The surface residue was washed with ethanol and dried with nitrogen. A 1 mM solution of functionalized peptide containing cis-dihydroxy compound was added to the surface of the copper foil and allowed to stand at room temperature for 1-3 hours. After washing again, an ethanol solution of APTES-UPTES-IBTES was added and allowed to stand at room temperature for 1-3 hours. After washing, an ethanol solution of TEOS was added and allowed to stand for 40-90 minutes. After washing, an acetonitrile-ethanol-acetic acid mixed solution was added and allowed to stand for 1-10 hours to complete the functionalization of the molecularly imprinted film on the surface of the copper foil.

[0009] Step (2): Spin-coat a layer of PMMA onto copper-based monolayer graphene, heat at 80°C for 10 min, then place it in a 1 m ammonium persulfate solution for etching and washing; ultrasonically clean the single-sided polished silicon dioxide with 500 nm oxide, then etch it with oxygen plasma, and then transfer the sensing material to the silicon wafer surface; transfer the etched epitope molecular imprint film to the silicon wafer with the sensing material, vacuum preserve it, and then heat it at 130°C for 90 min.

[0010] Furthermore, the compound containing cis-dihydroxy compounds mentioned in step (1) is either fructose or glucose.

[0011] Furthermore, the functionalized peptide in step (1) is any one of the 8-12 amino acid peptides at the N-terminus or C-terminus of a biological macromolecule.

[0012] Further, the volume ratio of the acetonitrile-ethanol-acetic acid mixed solution in step (1) is 50:49:1.

[0013] Furthermore, the molecularly imprinted thin film on the copper foil surface described in step (1) is functionalized.

[0014] Furthermore, the vacuum preservation time in step (2) is 24 hours.

[0015] Furthermore, the molecularly imprinted film obtained after etching away the copper foil can be transferred to any two-dimensional substrate and material surface, which is a universal method.

[0016] This invention relates to the preparation and transfer of epitope molecular imprinted membranes and their combination with field-effect transistors (FETs) for the detection of biomacromolecules. The steps are as follows: Prepare a reaction solution with a total volume of 20-300 μL, wherein the reaction system contains the target Aβ protein and 1xPBS buffer solution; then add it to the reaction cell of the FET, connect it to a lock-in or semiconductor analyzer, and react at 25°C for 15-30 min, observing the changes in electrical signals in real time.

[0017] The beneficial effects of this invention are as follows: This invention enables the transfer of molecularly imprinted membranes to the surface of any two-dimensional material for the detection of biomolecules. The molecularly imprinted membrane prepared on the copper foil surface in this invention has surface-specific cavities that can specifically identify the biomolecules to be tested. When the N-terminal or C-terminal short peptide of the protein being detected enters the molecularly imprinted cavity on the surface of the sensing material, it causes a change in the electrical signal, achieving ultrasensitive, rapid, and highly specific amplified detection of biomolecules. This avoids contamination from the solution during functionalization and allows transfer to the surface of any two-dimensional material. Attached Figure Description

[0018] Figure 1 This is a diagram of the glycosylated epitope structure of the present invention;

[0019] Figure 2 This is a schematic diagram illustrating the preparation and transfer of EMIM on copper foil according to the present invention;

[0020] Figure 3 This is a graph showing the transfer characteristics of the molecularly imprinted film of the present invention after it has been transferred to the graphene surface. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0022] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0023] Example 1: Process of preparing molecularly imprinted films on copper foil surfaces

[0024] Step (1): Pretreatment of copper foil. Cut the copper foil into small squares of 1cm×1cm, then soak it in acetone for 5 minutes, then soak it in isopropanol for 5 minutes, and finally dry it with nitrogen.

[0025] Step (2): Graft phenylboronic acid onto the surface of the copper foil. After cleaning, the copper foil is placed in a solution of 95% ethanol and 5% 3-aminopropyltriethoxysilane and left to stand for 6 hours. Then, the solution residue on the surface of the copper foil is removed with ethanol and dried with nitrogen. The copper foil is then placed in a solution of 5 mg / mL 2,4-difluoro-3-carboxyphenylboronic acid and sodium cyanoborohydride in ethanol at room temperature for 24 hours. The surface residue is washed with ethanol and dried with nitrogen.

[0026] Step (3): Imprinting process, 1 mM of glycosylated epitope molecules are dropped onto the surface of the copper foil, with the structure as shown in the figure. Figure 2 As shown, the functionalized peptide solution was allowed to stand at room temperature for 1-3 hours; after washing again, an ethanol solution of APTES-UPTES-IBTES was added and allowed to stand at room temperature for 1-3 hours; after washing, an ethanol solution of TEOS was added and allowed to stand for 40-90 minutes; after washing, an acetonitrile-ethanol-acetic acid mixed solution was added and allowed to stand for 1-10 hours to complete the functionalization of the molecularly imprinted film on the copper foil surface.

[0027] Step (4): Molecular imprinted membrane transfer process, such as Figure 2 As shown, a layer of PMMA was spin-coated onto copper-based monolayer graphene. After heating at 80°C for 10 min, it was etched and washed in a 1 mL ammonium persulfate solution. A single-sided polished silicon dioxide layer with 500 nm oxide was ultrasonically cleaned, etched with oxygen plasma, and then the sensing material was transferred onto the silicon wafer surface. The etched surface molecularly imprinted film was transferred onto the graphene-coated silicon wafer, vacuum-sealed, and then heated at 130°C for 90 min. The graphene transferred to the molecularly imprinted film was fabricated into a field-effect transistor device, and its transfer characteristic curve was tested. The test results are shown below. Figure 3 As shown.

[0028] Example 2: Using glucose-modified peptides and binding them to copper foil

[0029] Peptides were synthesized using solid-phase synthesis and their terminal groups were modified with glucose.

[0030] Functionalization process: Prepare a reaction solution with a total volume of 20-300 μL, which contains the target 50 mM ammonium bicarbonate, 500 mM sodium chloride and 1 mM glycopeptide solution. The epitope molecular imprinted membrane is transferred to the two-dimensional substrate and the material surface to complete the functionalization of glucose-modified peptides on graphene.

[0031] Example 3: Detection of β-amyloid (Aβ) in cerebrospinal fluid using GFET combined with EMIM

[0032] A 100 μL reaction solution was prepared, containing the target Aβ (50 aM, 500 aM, 5 fM, 50 fM, 500 fM, 5 pM, 50 pM) and 1 x PBS buffer solution. The solution was then added to the reaction cell of the FET and connected to the phase-locked loop using a constant current method. The reaction was carried out at 25 °C for 20 min, and the changes in the electrical signal were observed in real time. Each concentration (50 aM, 500 aM, 5 fM, 50 fM, 500 fM, 5 pM, 50 pM) was measured three times, and the working curve of the biosensor for Aβ detection was plotted.

[0033] A 100 μL Aβ sample was prepared using 1x PBS solution and then added to the reaction cell of a FET. The sample was connected to a phase-locked loop using a constant current method and reacted at 25°C for 20 min. The changes in the electrical signal were observed in real time. The content of Aβ in the cerebrospinal fluid of Alzheimer's patients could be determined based on the working curve.

[0034] Example 4: Detection of tau protein in blood by combining GFET with EMIM

[0035] A 100 μL reaction solution was prepared, containing the target tau protein at concentrations of 50 aM, 500 aM, 5 fM, 50 fM, 500 fM, 5 pM, and 50 pM, along with 1 x PBS buffer. This solution was then added to the reaction chamber of a FET and connected to a lock-in circuit using a constant current method. The reaction was carried out at 25 °C for 20 min, and the changes in the electrical signal were observed in real time. Each concentration (50 aM, 500 aM, 5 fM, 50 fM, 500 fM, 5 pM, and 50 pM) was measured three times, and a working curve for tau protein detection by the biosensor was plotted.

[0036] A 100 μL tau protein sample was prepared using 1x PBS solution and then added to the reaction cell of a GFET. The sample was connected to a phase-locked loop using a constant current method and reacted at 25°C for 20 min. The changes in the electrical signal were observed in real time, and the content of tau protein in the blood of Alzheimer's patients could be determined based on the working curve.

[0037] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A method for transferring epitope molecular imprinted films onto a two-dimensional surface to construct field-effect transistors for detecting biomarkers. The method of the object, characterized in that, First, a molecularly imprinted thin film is grown on a copper foil, and then the molecularly imprinted film is transferred to the surface of any sensing material. The transfer of the molecularly imprinted membrane includes the following steps: Step (1): Preparation of molecularly imprinted membrane. The cleaned copper foil was placed in a solution of 95% ethanol and 5% 3-aminopropyltriethoxysilane and allowed to stand for 6 hours. Then, the solution residue on the surface of the copper foil was removed with ethanol and dried with nitrogen. The copper foil was placed in a solution of 5 mg / mL 2,4-difluoro-3-carboxyphenylboronic acid and sodium cyanoborohydride in ethanol at room temperature for 24 hours. The surface residue was washed with ethanol and dried with nitrogen. A 1 mM solution of functionalized peptide containing cis-dihydroxy compound was added to the surface of the copper foil and allowed to stand at room temperature for 1-3 hours. After washing again, an ethanol solution of APTES-UPTES-IBTES was added and allowed to stand at room temperature for 1-3 hours. After washing, an ethanol solution of TEOS was added and allowed to stand for 40-90 minutes. After washing, an acetonitrile-ethanol-acetic acid mixed solution was added and allowed to stand for 1-10 hours to complete the functionalization of the molecularly imprinted film on the surface of the copper foil. Step (2): Spin-coat a layer of PMMA onto copper-based monolayer graphene, heat at 80°C for 10 min, then place it in a 1 m ammonium persulfate solution for etching and washing; ultrasonically clean the single-sided polished silicon dioxide with 500 nm oxide, then etch it with oxygen plasma, and then transfer the sensing material to the silicon wafer surface; transfer the etched epitope molecular imprint film to the silicon wafer with the sensing material, vacuum preserve it, and then heat it at 130°C for 90 min.

2. The preparation method as described in claim 1, characterized in that, The compound containing cis-dihydroxy compounds mentioned in step (1) is either fructose or glucose.

3. The preparation method as described in claim 1, characterized in that, The functionalized peptide in step (1) is any one of the 8-12 amino acid peptides at the N-terminus or C-terminus of a biological macromolecule.

4. The preparation method as described in claim 2, characterized in that, The volume ratio of the acetonitrile-ethanol-acetic acid mixed solution in step (1) is 50:49:

1.

5. The preparation method as described in claim 1, characterized in that, The copper foil surface molecular imprinted film functionalized in step (1).

6. The preparation method as described in claim 1, characterized in that, The vacuum preservation time mentioned in step (2) is 24 hours.

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