Method for detecting ultra-sensitive biological detection using a combination of epitope molecularly imprinted membrane library and field effect transistor

Abstract

The application discloses a method for detecting super-sensitive biological detection by combining an epitope molecular imprinting membrane library with a field effect transistor, and is used for detecting various biological molecules; meanwhile, the prepared epitope molecular imprinting membrane library is combined with the field effect transistor technology to develop a high-sensitivity biological detection method, and belongs to the technical field of biosensors; a molecular imprinting membrane library is established by preparing a molecular imprinting membrane on the surface of a copper foil and imprinting different peptide segment molecules; after the copper foil is etched and removed, the membrane is transferred to the surface of graphene to construct a field effect transistor, and the high sensitivity of graphene can realize the detection of different molecules; the peptide segment or protein can enter the specific cavity of the molecular imprinting, thereby causing the change of an electric signal, and realizing the amplified detection of biological molecules; the establishment of the epitope molecular imprinting membrane library is realized for the first time, the universality of the method is proved, and the high specificity, rapid and super-sensitive detection of various biological samples is realized.

Description

Technical Field

[0001] This invention belongs to the field of sensing, specifically relating to a method for constructing an epitope molecular imprinted membrane library and combining it with a field-effect transistor for ultrasensitive biological detection. This method enables the detection of a variety of biological macromolecules and is a general detection method. Background Technology

[0002] Epitope molecularly imprinted membranes (EMIMs), compared to traditional protein molecularly imprinted polymers, imprint a small, specific fragment of the protein, rather than the entire protein. Imprinting specific peptides avoids the harsh reagents or conditions required for imprinting whole proteins, which can lead to protein denaturation. Furthermore, this method can detect not only the imprinted specific peptides but also the entire protein. Compared to biomacromolecules, epitope molecularly imprinted membranes also exhibit superior stability. This method has shown great potential in chemical sensors and biosensors in recent years. Epitope imprinted membranes demonstrate excellent specificity in biosensing and analytical detection. However, current research focuses on the in-situ synthesis of EMIMs on the surface of sensing materials; obtaining independent epitope molecularly imprinted membranes remains a significant challenge. Summary of the Invention

[0003] To address the aforementioned problems, this invention discloses a method for constructing epitope molecularly imprinted membrane libraries and combining them with field-effect transistors for ultrasensitive biological detection. The purpose of this invention is to provide a universal epitope molecularly imprinted membrane method and establish a library of various biomarker peptides, combining this technology with field-effect transistors for the detection of biomolecules. In this invention, the prepared epitope molecularly imprinted membrane library enables the detection of multiple biomarkers. When the N-terminal or C-terminal short peptide of the target protein enters the molecularly imprinted cavity on the surface of the sensing material, it causes a change in electrical signal, achieving ultrasensitive detection of multiple proteins.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows:

[0005] This invention firstly describes a method for constructing an epitope molecularly imprinted membrane library combined with a field-effect transistor for ultrasensitive biological detection. It involves the preparation of various epitope molecularly imprinted membranes, growing molecularly imprinted membranes with different peptide imprints on the surface of copper foil, and constructing an epitope molecularly imprinted membrane library composed of various analytes.

[0006] The establishment of the molecularly imprinted membrane library includes the following steps:

[0007] Step (1): Cleaning the copper foil. Cut the copper foil into 1cm×1cm pieces and soak them in acetone and isopropanol in sequence. After drying with nitrogen, soak them in 1M hydrochloric acid and then soak them in water.

[0008] Step (2): The copper foil from step (1) was placed in a 5 mg / mL solution of 2,4-difluoro-3-carboxyphenylboronic acid and sodium cyanoborohydride in ethanol and allowed to stand at room temperature. The surface residue was washed with ethanol and then dried with nitrogen.

[0009] Step (3): Add 1 mM fructose-functionalized peptide (containing cis dihydroxy compound) solution to the copper foil surface treated in step (2) and let it stand at room temperature for 1-3 h; after washing again, add APTES-UPTES-IBTES ethanol solution and let it stand at room temperature for 1-3 h; after washing, add TEOS ethanol solution and let it stand for 40-90 min; after washing, add acetonitrile-ethanol-acetic acid mixed solution and let it stand for 1-10 h to complete the functionalization of the molecular imprinted film on the copper foil surface;

[0010] Step (4): Spin-coat a layer of poly(methylmethacrylate, PMMA) onto copper-based monolayer graphene, heat it, place it in a 1M ammonium persulfate solution for etching and washing; a molecularly imprinted film that can be transferred to any two-dimensional surface can be obtained.

[0011] Step (5): Using the same method, but replacing the glycosylated peptides in step (3) with the N-segment 9-peptide (fructose-modified) of Alzheimer's disease marker Aβ protein, prostate cancer marker PSA protein, inflammatory factor IL-6, and heart failure marker B-type natriuretic peptide (BNP) to construct an epitope molecular imprinted membrane on copper foil, thereby establishing an epitope molecular imprinted membrane library.

[0012] Furthermore, in step (1), the soaking time in acetone and isopropanol is 5 minutes, the soaking time in hydrochloric acid is 5 seconds, and the soaking time in water is 5 minutes.

[0013] Furthermore, the settling time in step (2) is 24 hours.

[0014] Furthermore, the volume ratio of the acetonitrile-water-acetic acid mixed solution in step (3) is 50:49:1.

[0015] Furthermore, the heating temperature in step (4) is 80°C and the heating time is 10 min.

[0016] Furthermore, using the same method, but replacing the glycosylated peptide in step (3) with any one of the following: Alzheimer's disease marker Aβ protein, prostate cancer marker PSA protein, inflammatory factor IL-6, and the N-segment 9-peptide (fructose-modified) of heart failure marker B-type natriuretic peptide (BNP), an epitope molecular imprinted membrane is constructed on copper foil, thereby establishing an epitope molecular imprinted membrane library.

[0017] Furthermore, the detection method also includes the preparation of an epitope molecular imprint membrane library to achieve the detection of various biomacromolecules, such as Aβ protein, BNP and PSA.

[0018] Further, the preparation steps of the epitope molecular imprinted membrane library are as follows: using the same method, but replacing the glycosylated peptide in step (3) with any one of the following: Alzheimer's disease marker Aβ protein, prostate cancer marker PSA protein, inflammatory factor IL-6, and the N-segment 9-peptide (fructose-modified) of heart failure marker B-type natriuretic peptide (BNP) to construct an epitope molecular imprinted membrane on copper foil, thereby establishing an epitope molecular imprinted membrane library.

[0019] The beneficial effects of this invention are as follows: This invention is a universal method for detecting biomarkers, which can rapidly and accurately determine Aβ protein, PSA protein, IL-6, and BNP. Attached Figure Description

[0020] Figure 1 A schematic diagram of a molecularly imprinted film library on the surface of a copper foil;

[0021] Figure 2 The graph shows the transfer characteristics before and after the transfer to the graphene surface. Detailed Implementation

[0022] 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.

[0023] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0024] The room temperature described in this invention is 25°C.

[0025] Example 1: Functionalization of copper foil using fructose-modified B-type natriuretic peptide (BNP) N-terminal 9-peptide (fructose-modified). The peptide was synthesized using a solid-phase synthesis method, and its terminal group was modified with fructose.

[0026] Functionalization process: Prepare a peptide solution with a total volume of 1 mL (using 50 mM ammonium bicarbonate / 500 mM sodium chloride as solvent), then drop it onto the copper foil surface and react at room temperature for 1-5 h to complete the functionalization of fructose-modified peptides on the copper foil surface.

[0027] Transfer process: A layer of PMMA is spin-coated onto a copper-based monolayer graphene, heated, and then etched and washed in a 1M ammonium persulfate solution. This allows for transfer to any two-dimensional surface.

[0028] Example 2: Copper foil modified with prostate-specific antigen (PSA) modified peptides

[0029] Peptides were synthesized using a solid-phase synthesis method, and fructose was used to modify the N-terminal 9-peptide terminal of the PSA protein.

[0030] Functionalization process: Prepare 1 mL of fructose-functionalized peptide solution (50 mM ammonium bicarbonate / 500 mM sodium chloride buffer solution as solvent). After washing, add the copper foil to an ethanol solution of APTES-UPTES-IBTES and let it stand at room temperature for 1-3 h. After washing, add TEOS ethanol solution and let it stand for 40-90 min. After washing, add an acetonitrile-ethanol-acetic acid mixed solution and let it stand for 1-10 h to complete the preparation of the PSA molecularly imprinted film.

[0031] like Figure 1 As shown, molecular imprinted libraries of different proteins were established on the surface of copper foil.

[0032] Example 3: Detection of β-amyloid (Aβ) protein in cerebrospinal fluid using a graphene field-effect transistor (GFET) combined with EEMI.

[0033] 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.

[0034] A 100 μL Aβ sample was prepared using 1xPBS solution and then added to the reaction cell of a FET. The FET was connected to a lock-in circuit using a constant current method and reacted at 25 °C for 20 min. Changes in the electrical signal were observed in real time. Figure 2 As shown, the content of Aβ in the cerebrospinal fluid of Alzheimer's patients can be obtained based on the working curve.

[0035] 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 constructing an epitope molecularly imprinted membrane library and combining it with a field-effect transistor for ultrasensitive biological detection, characterized in that, Molecularly imprinted membranes with different peptide imprints were grown on the surface of copper foil, and a library of epitope molecularly imprinted membranes composed of various analytes was constructed. The establishment of the molecularly imprinted membrane library includes the following steps: Step (1): Cleaning the copper foil. Cut the copper foil into 1cm×1cm pieces and soak them in acetone and isopropanol in sequence. After drying with nitrogen, soak them in 1M hydrochloric acid and then soak them in water. Step (2): The copper foil from step (1) was placed in a 5 mg / mL solution of 2,4-difluoro-3-carboxyphenylboronic acid and sodium cyanoborohydride in ethanol and allowed to stand at room temperature. The surface residue was washed with ethanol and then dried with nitrogen. Step (3): Add 1 mM fructose-functionalized peptide (containing cis dihydroxy compound) solution to the copper foil surface treated in step (2) and let it stand at room temperature for 1-3 h; after washing again, add APTES-UPTES-IBTES ethanol solution and let it stand at room temperature for 1-3 h; after washing, add TEOS ethanol solution and let it stand for 40-90 min; after washing, add acetonitrile-ethanol-acetic acid mixed solution and let it stand for 1-10 h to complete the functionalization of the molecular imprinted film on the copper foil surface; Step (4): Spin-coat a layer of polymethyl methacrylate onto copper-based monolayer graphene, heat it, place it in a 1M ammonium persulfate solution for etching and washing; This allows for the preparation of molecularly imprinted films that can be transferred to any two-dimensional surface; Step (5): Replace the glycosylated peptide in step (3) with any one of the following: Alzheimer's disease marker Aβ protein, prostate cancer marker PSA protein, inflammatory factor IL-6, and N-segment 9 peptide (fructose modified) of heart failure marker B-type natriuretic peptide (BNP), and construct an epitope molecular imprinted membrane on copper foil to establish an epitope molecular imprinted membrane library.

2. The detection method as described in claim 1, characterized in that, The soaking time in acetone and isopropanol in step (1) is 5 min, the soaking time in hydrochloric acid is 5 s, and the soaking time in water is 5 min.

3. The detection method as described in claim 1, characterized in that, The settling time mentioned in step (2) is 24 hours.

4. The detection method as described in claim 1, characterized in that, The volume ratio of the acetonitrile-water-acetic acid mixed solution in step (3) is 50:49:

1.

5. The detection method as described in claim 1, characterized in that, The heating temperature in step (4) is 80°C and the heating time is 10 min.

6. The detection method according to any one of claims 1-5, characterized in that, The detection method also includes the preparation of an epitope molecular imprinted membrane library to enable the detection of a variety of biomacromolecules.

7. The detection method as described in claim 6, characterized in that, The preparation steps of the epitope molecular imprinted membrane library are as follows: The same method as the establishment of the molecular imprinted membrane library is used, but the glycosylated peptide in step (3) is replaced with any one of the following: Alzheimer's disease marker Aβ protein, prostate cancer marker PSA protein, inflammatory factor IL-6, and N-segment 9 peptide (fructose modified) of heart failure marker B-type natriuretic peptide (BNP). An epitope molecular imprinted membrane is constructed on copper foil to establish the epitope molecular imprinted membrane library.

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