1. A three-dimensional hydrogel-graphene-based biosensor comprises a substrate, an electrode layer, a graphene film and a three-dimensional hydrogel material layer which are sequentially stacked; the three-dimensional hydrogel material layer is formed by a hydrogel material with a three-dimensional network structure, the hydrogel material is obtained by polymerization reaction of raw materials including acrylamide monomers and modified probe molecules, and the modified probe molecules are the probe molecules modified with acrylamide groups.

2. The three-dimensional hydrogel-graphene-based biosensor as claimed in claim 1, wherein the pore size of the hydrogel material is 1.8-2.2 μm.

3. The three-dimensional hydrogel-graphene-based biosensor according to claim 1 or 2, wherein the thickness of the three-dimensional hydrogel material layer is 0.63-2.35 mm.

4. The three-dimensional hydrogel-graphene-based biosensor as claimed in claim 1, wherein the acrylamide monomer comprises acrylamide and methylene bisacrylamide, and the mass ratio of the acrylamide to the methylene bisacrylamide is (45-55): 1.

5. the three-dimensional hydrogel-graphene-based biosensor according to claim 1 or 4, wherein the molar ratio of the mass of the acrylamide monomer to the modified probe molecules is (5-20) g: 1. mu. mol.

6. The three-dimensional hydrogel-graphene-based biosensor according to claim 1, wherein the probe molecule is an aptamer.

7. The three-dimensional hydrogel-graphene-based biosensor according to claim 1, wherein the graphene thin film is formed of single-layer graphene.

8. The three-dimensional hydrogel-graphene-based biosensor according to claim 1, wherein the electrode layer comprises two electrodes disposed in parallel, and the graphene thin film is disposed on the surfaces of the two electrodes and the surface of the exposed substrate.

9. The method for preparing the three-dimensional hydrogel-graphene-based biosensor according to any one of claims 1 to 8, comprising the steps of:

preparing an electrode layer on one side of a substrate to obtain a substrate-electrode layer device;

arranging a graphene film on the surface of an electrode layer in the substrate-electrode layer device to obtain a substrate-electrode layer-graphene film device;

carrying out in-situ polymerization reaction on the surface of a graphene film in the substrate-electrode layer-graphene film device by using the reaction solution, and forming a three-dimensional hydrogel material layer on the surface of the graphene film to obtain a three-dimensional hydrogel-graphene-based biosensor; the reaction solution contains an acrylamide monomer, a modified probe molecule, an initiator, sodium nitrate and a solvent.

10. The method for preparing the graphene film according to claim 9, wherein the method for providing the graphene film on the surface of the electrode layer in the substrate-electrode layer device comprises:

providing a graphene film, and transferring the graphene film to the surface of an electrode layer by adopting a wet transfer method.

Background

The graphene-based electrical sensor has the advantages of high response speed and high sensitivity, can be used in cooperation with probe molecules such as aptamers, antibodies and enzymes capable of recognizing target substances with high specificity to form the graphene-based nano biosensor, and has important application value in the field of disease marker detection.

Graphene-based nano biosensors reported in the existing research are all surface affinity type sensors (DOI:10.1021/acsami.7b07684), that is, probe molecules are anchored on the surface of graphene through linked molecules containing aromatic ring structures, marker molecules with weak electric quantity in a solution to be detected and captured by the probe molecules in an identification mode can affect the quantity of movable carriers in unit cross section area in the graphene under the action of 'electrostatic induction', and the change of the concentration of the marker molecules can be determined by measuring the change of the quantity of the movable carriers, that is, the change of the conductivity of the graphene (the change of current at two ends of the graphene). In the whole process of detection by adopting the surface type sensor, the graphene surface is always exposed in a solution to be detected, and actual biological solution samples, such as blood, sweat, saliva and the like, contain a plurality of non-target biological macromolecules or impurities, which are easy to form biological deposition on the graphene surface, influence the stability and sensitivity of the sensor, and even cause the sensor to fail.

Disclosure of Invention

The invention aims to provide a three-dimensional hydrogel-graphene-based biosensor and a preparation method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a three-dimensional hydrogel-graphene-based biosensor, which comprises a substrate, an electrode layer, a graphene film and a three-dimensional hydrogel material layer which are sequentially stacked; the three-dimensional hydrogel material layer is formed by a hydrogel material with a three-dimensional network structure, the hydrogel material is obtained by polymerization reaction of raw materials including acrylamide monomers and modified probe molecules, and the modified probe molecules are the probe molecules modified with acrylamide groups.

Preferably, the pore diameter of the hydrogel material is 1.8-2.2 μm.

Preferably, the thickness of the three-dimensional hydrogel material layer is 0.63-2.35 mm.

Preferably, the acrylamide monomer comprises acrylamide and methylene bisacrylamide, and the mass ratio of the acrylamide to the methylene bisacrylamide is (45-55): 1.

preferably, the molar ratio of the mass of the acrylamide monomer to the modified probe molecule is (5-20) g: 1. mu. mol.

Preferably, the probe molecule is an aptamer.

Preferably, the graphene thin film is formed of single-layer graphene.

Preferably, the electrode layer comprises two electrodes arranged in parallel, and the graphene film is arranged on the surfaces of the two electrodes and the surface of the exposed substrate.

The invention provides a preparation method of the three-dimensional hydrogel-graphene-based biosensor, which comprises the following steps:

preparing an electrode layer on one side of a substrate to obtain a substrate-electrode layer device;

arranging a graphene film on the surface of an electrode layer in the substrate-electrode layer device to obtain a substrate-electrode layer-graphene film device;

carrying out in-situ polymerization reaction on the surface of a graphene film in the substrate-electrode layer-graphene film device by using the reaction solution, and forming a three-dimensional hydrogel material layer on the surface of the graphene film to obtain a three-dimensional hydrogel-graphene-based biosensor; the reaction solution contains an acrylamide monomer, a modified probe molecule, an initiator, sodium nitrate and a solvent.

Preferably, the method for providing the graphene thin film on the surface of the electrode layer in the substrate-electrode layer device comprises the following steps:

providing a graphene film, and transferring the graphene film to the surface of an electrode layer by adopting a wet transfer method.

The invention provides a three-dimensional hydrogel-graphene-based biosensor, which comprises a substrate, an electrode layer, a graphene film and a three-dimensional hydrogel material layer which are sequentially stacked; the three-dimensional hydrogel material layer is formed by a hydrogel material with a three-dimensional network structure, the hydrogel material is obtained by polymerization reaction of raw materials including acrylamide monomers and modified probe molecules, and the modified probe molecules are the probe molecules modified with acrylamide groups.

In the three-dimensional hydrogel-graphene-based biosensor provided by the invention, the graphene film is a conductive channel, and the hydrogel material has a three-dimensional network structure, so that target molecules in a solution to be detected can be combined with probe molecules through meshes of the hydrogel material; meanwhile, when the three-dimensional hydrogel-graphene-based biosensor is used for measuring an actual biological solution sample, a double electric layer is formed on the surface of a graphene conductive channel by the whole hydrogel-probe structure, and after target molecules are captured by probe molecules, the capacitance of the whole double electric layer changes correspondingly along with the change of the concentration of the captured target molecules, so that the quantity of freely moving electrons in graphene changes, the conductivity of the graphene changes, namely the transfer characteristic curve and Dirac points of the graphene move horizontally along with the target molecules with different concentrations, and detection signals are generated. Because the hydrogel material has a nanoscale three-dimensional reticular structure, like a filter screen is formed on the surface of the graphene film, macromolecular substances or impurities in an actual biological solution sample can be effectively blocked outside the hydrogel material, the separation of non-target molecules in the solution to be detected and the graphene conductive channel is realized, the formation of biological deposition on the surface of the graphene film is prevented, and simultaneously target small molecules are close to the surface of the graphene film through mutually communicated meshes in the hydrogel material along with the solution, so that the detection is effectively realized, and the stability and the sensitivity of the sensor are favorably improved.

Furthermore, the distance between the equivalent charge center of the grabbed charged target molecule and the graphene film can be adjusted by adjusting the thickness of the three-dimensional hydrogel material layer, so that the electrostatic induction effect is influenced, the sensitivity of the sensor is adjusted, and the adjustment of the target molecule detection range is finally realized. The results of the examples show that when the target molecule is cardiac troponin I (cTn I), the detection range of the target molecule is 5 aM-50 nM for a sensor with a three-dimensional hydrogel material layer thickness of 0.63-2.35 mm.

The invention provides a preparation method of the three-dimensional hydrogel-graphene-based biosensor, which comprises the following steps: preparing an electrode layer on one side of a substrate to obtain a substrate-electrode layer device; arranging a graphene film on the surface of an electrode layer in the substrate-electrode layer device to obtain a substrate-electrode layer-graphene film device; carrying out in-situ polymerization reaction on the surface of a graphene film in the substrate-electrode layer-graphene film device by using the reaction solution, and forming a three-dimensional hydrogel material layer on the surface of the graphene film to obtain a three-dimensional hydrogel-graphene-based biosensor; the reaction solution contains an acrylamide monomer, a modified probe molecule, an initiator, sodium nitrate and a solvent. In the three-dimensional hydrogel-graphene-based biosensor prepared by the method, the hydrogel material layer and the graphene film are fixed together in a contact manner through intermolecular force, and different from a probe molecule modification method of a traditional surface type sensor, probe molecules in the sensor are modified and fixed in a hydrogel material, so that the hydrogel material can be manually torn off after the sensor is used (when the hydrogel material is torn off, the graphene film cannot be brought down, the adhesion of the graphene film on a substrate is mainly realized through the intermolecular force, the acting force is far greater than the binding force between the graphene film and the hydrogel material), no use trace is left on the surface of the graphene film, and the sensor can be conveniently reused by preparing the hydrogel material layer again; and after the secondary processing, sensitive material graphene films used for the two times are not replaced (due to the fact that the graphene is different in production batches and artificial interference exists in the processing process, electrical signals of different devices are different, and calibration is not convenient), so that the electrical signals of the sensor are not changed, and calibration of the sensor is convenient.

Drawings

Fig. 1 is a schematic structural diagram of a three-dimensional hydrogel-graphene-based biosensor provided by the present invention;

fig. 2 is a flowchart illustrating a process of transferring a graphene film to the surface of an electrode layer by a wet transfer method according to the present invention;

FIG. 3 is a photograph and an optical microscope photograph of the hydrogel material prepared in example 1;

FIG. 4 is a graph of detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 in undiluted serum;

FIG. 5 is a graph showing detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 in undiluted blood;

FIG. 6 is a Hill equation fit curve based on the detection signals of a three-dimensional hydrogel-graphene based biosensor in undiluted serum, undiluted blood and PBS buffer;

fig. 7 is a graph of detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 after being placed in a room temperature environment for different periods of time;

FIG. 8 is a graph of detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 after being placed for 1 hour at different temperatures;

FIG. 9 is a graph of detection signals of three-dimensional hydrogel-graphene based biosensors of different three-dimensional hydrogel material layer thicknesses.

Detailed Description

The invention provides a three-dimensional hydrogel-graphene-based biosensor, which comprises a substrate, an electrode layer, a graphene film and a three-dimensional hydrogel material layer which are sequentially stacked; the three-dimensional hydrogel material layer is formed by a hydrogel material with a three-dimensional network structure, the hydrogel material is obtained by polymerization reaction of raw materials including acrylamide monomers and modified probe molecules, and the modified probe molecules are the probe molecules modified with acrylamide groups.

The three-dimensional hydrogel-graphene-based biosensor provided by the invention comprises a substrate. The substrate is not particularly limited in the present invention, and any substrate known to those skilled in the art may be used, and specifically, the substrate may be a flexible substrate or a rigid substrate. In embodiments of the present invention, the substrate is preferably surface coated with SiO₂Silicon wafer of layer, said SiO₂The thickness of the layers is preferably 285nm and the total thickness of the substrate is preferably 500 μm.

The three-dimensional hydrogel-graphene-based biosensor provided by the invention comprises electrode layers which are arranged on one side of the substrate in a laminated mode. In the examples of the present invention, tables are usedCoated with SiO₂The silicon chip of the layer is taken as a substrate, and the electrode layer is specifically arranged on the SiO₂The surface of the layer. In the present invention, the electrode layer preferably includes two electrodes disposed in parallel, and in particular, the electrodes are disposed at opposite ends of the substrate. In the invention, the electrode is preferably a metal electrode, and more preferably a gold/chromium composite conductive electrode, the gold/chromium composite conductive electrode specifically includes a chromium layer and a gold layer which are arranged in a laminated manner, the thickness of the chromium layer is preferably 2nm, and the thickness of the gold layer is preferably 43 nm; specifically, the chromium layer of the gold/chromium composite conductive electrode is contacted with the substrate.

The three-dimensional hydrogel-graphene-based biosensor provided by the invention comprises a graphene film which is arranged on the surface of an electrode layer in a laminated manner. In the present invention, when the electrode layer includes two electrodes disposed in parallel, the graphene thin film is specifically disposed on the surfaces of the two electrodes and the surface of the exposed substrate. In the present invention, the graphene thin film is preferably formed of single-layer graphene.

The three-dimensional hydrogel-graphene-based biosensor provided by the invention comprises a three-dimensional hydrogel material layer which is arranged on the surface of a graphene film in a laminated manner, wherein the three-dimensional hydrogel material layer is formed by a hydrogel material with a three-dimensional network structure, the hydrogel material is obtained by carrying out polymerization reaction on raw materials comprising an acrylamide monomer and modified probe molecules, and the modified probe molecules are probe molecules modified with acrylamide groups. In the invention, the pore diameter of the hydrogel material is preferably 1.8-2.2 μm, and more preferably 2 μm. In the invention, the thickness of the three-dimensional hydrogel material layer is preferably 0.63-2.35 mm. In the invention, the acrylamide monomer preferably comprises acrylamide and methylene bisacrylamide, and the mass ratio of the acrylamide to the methylene bisacrylamide is preferably (45-55): 1, more preferably 49: 1. in the invention, the molar ratio of the mass of the acrylamide monomer to the modified probe molecule is preferably (5-20) g: 1. mu. mol, more preferably (8 to 12) g: 1. mu. mol. In the present invention, the probe molecule is preferably an aptamer, and more preferably a single-stranded DNA having an acrylamide group at the 5-terminal or an RNA having an acrylamide group at the 5-terminal.

The invention provides a preparation method of the three-dimensional hydrogel-graphene-based biosensor, which comprises the following steps:

preparing an electrode layer on one side of a substrate to obtain a substrate-electrode layer device;

arranging a graphene film on the surface of an electrode layer in the substrate-electrode layer device to obtain a substrate-electrode layer-graphene film device;

carrying out in-situ polymerization reaction on the surface of a graphene film in the substrate-electrode layer-graphene film device by using the reaction solution, and forming a three-dimensional hydrogel material layer on the surface of the graphene film to obtain a three-dimensional hydrogel-graphene-based biosensor; the reaction solution contains an acrylamide monomer, a probe molecule modified with an acrylamide group, an initiator, sodium nitrate and a solvent.

In the present invention, unless otherwise specified, all the starting materials for the preparation are commercially available products well known to those skilled in the art.

The electrode layer is prepared on the single surface of the substrate to obtain the substrate-electrode layer device. The preparation method of the electrode layer is not particularly limited, and the method known by the technicians in the field can be adopted; the invention preferably adopts an electron beam evaporation method to prepare the electrode layer, and the invention has no special limitation on the operating conditions of the electron beam evaporation method and ensures that the electrode layer meeting the thickness requirement can be obtained.

After the substrate-electrode layer device is obtained, the graphene film is arranged on the surface of the electrode layer in the substrate-electrode layer device, and the substrate-electrode layer-graphene film device is obtained. In the present invention, the method of providing a graphene thin film on the surface of an electrode layer in the substrate-electrode layer device preferably includes:

providing a graphene film, and transferring the graphene film to the surface of an electrode layer by adopting a wet transfer method.

In the invention, the graphene film is preferably a graphene film prepared by a chemical vapor deposition method, the graphene film is preferably deposited on the surface of a copper foil, namely the graphene film is deposited on the front surface and the back surface of the copper foil, and a polymethyl methacrylate (PMMA) protective layer is arranged on one surface (marked as the front surface) of the copper foil attached with the graphene film; in the examples of the present invention, the copper foil (hereinafter referred to as a composite material) including the graphene thin film and the PMMA protective layer is commercially available.

Fig. 2 is a flowchart illustrating a process of transferring a graphene film to a surface of an electrode layer by using a wet transfer method according to an embodiment of the present invention, and the wet transfer method is described in detail with reference to fig. 2. In the present invention, the specific operation steps of the wet transfer method preferably include: cutting the composite material into 3 x 3mm²The graphene film on the back of the copper foil is removed by using an oxygen ion etching technology, then the front of the copper foil is upward (namely the side with the PMMA protective layer is upward), the copper foil floats in APS100 copper etching liquid with the concentration of 4 wt%, standing is carried out for 3 hours, the copper foil is completely etched, and the graphene film-PMMA protective layer material floats on the surface of the APS100 copper etching liquid; fishing out the graphene film by using a cover glass, putting the graphene film into a culture dish filled with deionized water, enabling the graphene film of the graphene film-PMMA protective layer material to be in contact with the deionized water, standing for 15min, removing APS100 copper etching liquid remained on the graphene film, marking the washing step as 1 washing step, and repeating the washing step for 2-3 times to ensure that impurities remained on the surface of the graphene film are fully removed and avoid precipitated crystal particles from being mixed between the graphene film and an electrode after water evaporation; after washing, immersing the substrate-electrode layer device in a culture dish, aligning the graphene film floating in deionized water to the center of an electrode, taking out the graphene film-PMMA protective layer material, naturally airing, putting the graphene film-PMMA protective layer material in a vacuum box, carrying out vacuum drying for 5 hours at the temperature of 25 ℃, and then putting the graphene film-PMMA protective layer material on a hot plate at the temperature of 180 ℃ for heating for 1 hour to soften the PMMA protective layer, so that the contact between the graphene film and the substrate and between the graphene film and the electrode is firmer; and then soaking the obtained device in acetone for 1h to completely remove the PMMA protective layer on the surface of the graphene film, and then sequentially washing with Isopropanol (IPA) and deionized water and drying with nitrogen to obtain the substrate-electrode layer-graphene film device.

Obtaining a substrate-electrode layerAfter the graphene film device is processed, carrying out in-situ polymerization reaction on the surface of the graphene film in the substrate-electrode layer-graphene film device by using the reaction solution, and forming a three-dimensional hydrogel material layer on the surface of the graphene film to obtain the three-dimensional hydrogel-graphene-based biosensor. In the invention, the reaction solution contains an acrylamide monomer, a modified probe molecule, an initiator, sodium nitrate and a solvent. In the present invention, the initiator is preferably Ammonium Persulfate (APS) and Tetramethylethylenediamine (TEMED), and the ratio of the amount of the ammonium persulfate to the amount of the tetramethylethylenediamine is preferably 2 mg: (0.8 to 1.2) μ L, more preferably 2 mg: 1 μ L. In the present invention, the reaction solution is preferably obtained by mixing a raw gum solution, a modified probe molecule solution, and an initiator solution. In the invention, the mass fraction of the acrylamide monomer in the original gum solution is preferably 10-20 wt%, more preferably 14-17 wt%, and further preferably 15-16 wt%. In the invention, the original glue solution is preferably obtained by diluting an acrylamide monomer mother liquor with a sodium nitrate solution, the mass fraction of an acrylamide monomer in the acrylamide monomer mother liquor is preferably 35-45 wt%, more preferably 38-42 wt%, and further preferably 39-40 wt%, and the solvent in the acrylamide monomer mother liquor is preferably water; the concentration of sodium nitrate in the sodium nitrate solution is preferably 1.8-2.2 mol/L, more preferably 2mol/L, and the solvent in the sodium nitrate solution is preferably Tris-HNO₃Buffer, said Tris-HNO₃The concentration of the buffer is preferably 0.5mol/L, and the pH value is preferably 8. In the invention, the concentration of the modified probe molecule solution is preferably 8-12 μ M, and more preferably 10 μ M; the solvent of the modified probe molecule solution is preferably phosphate buffered saline (PBS buffer), and the pH value of the PBS buffer is preferably 7.4. In the invention, the concentration of ammonium persulfate in the initiator solution is preferably 0.08-0.12 mg/muL, and more preferably 0.10 mg/muL; the solvent of the initiator solution is preferably water. In the present invention, the volume ratio of the original gel solution, the modified probe molecule solution, and the initiator solution in the reaction solution is preferably 20: (25-35): (3-5), more preferably 20: 30: 4. the invention preferably dissolves the original glue solution and the modified probe moleculeMixing the solutions, and then mixing the obtained mixed solution with an initiator solution.

According to the invention, a bottomless reaction container is preferably placed on the surface of the graphene film in the substrate-electrode layer-graphene film device, and the substrate-electrode layer-graphene film device and the reaction container are used as a mold to prepare the hydrogel material, so that a three-dimensional hydrogel material layer is formed on the surface of the graphene film in the substrate-electrode layer-graphene film device. The shape and the size of the reaction vessel are not particularly limited, and the shape and the size of the reaction vessel can be selected according to the shape and the size of the three-dimensional hydrogel material layer; in the examples of the present invention, a reaction vessel having a diameter of 5mm is specifically used. In the present invention, the material of the reaction vessel is preferably Polydimethylsiloxane (PDMS).

According to the invention, preferably, a bottomless reaction container is placed on the surface of the graphene film in the substrate-electrode layer-graphene film device, and after a reaction solution is prepared, the reaction solution is placed in the reaction container for in-situ polymerization reaction, and a three-dimensional hydrogel material layer is formed on the surface of the graphene film, so that the three-dimensional hydrogel-graphene-based biosensor is obtained. The amount of the reaction solution used in the present invention is not particularly limited, and may be selected according to the size of the three-dimensional hydrogel material layer actually required. In the present invention, the in situ polymerization reaction is preferably carried out at room temperature, i.e., without additional heating or cooling; in the embodiment of the invention, the room temperature is specifically 25 ℃; the time of the in-situ polymerization reaction is preferably 10-20 min, and more preferably 15 min. In the present invention, the in situ polymerization reaction is preferably carried out under a static condition. In the invention, in the in-situ polymerization reaction process, under the action of an initiator, an acrylamide monomer and a modified probe molecule form a three-dimensional hydrogel material on the surface of a graphene film through a free radical polymerization reaction.

In the invention, after the in-situ polymerization reaction, the reaction container is removed, and the three-dimensional hydrogel-graphene-based biosensor can be obtained.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Fig. 1 is a schematic structural diagram of a three-dimensional hydrogel-graphene-based biosensor in this embodiment, where the three-dimensional hydrogel-graphene-based biosensor includes a substrate, an electrode layer, a graphene film, and a three-dimensional hydrogel material layer, which are sequentially stacked; the substrate is coated with SiO on the surface₂A silicon wafer of layers, the total thickness of the substrate being 500 μm, the SiO₂The thickness of the layer was 285 nm; the electrode layer comprises two electrodes arranged in parallel, the electrode is an Au/Cr composite conductive electrode, the Au/Cr composite conductive electrode comprises a chromium layer and a gold layer which are arranged in a laminated manner, the thickness of the chromium layer is 2nm, the thickness of the gold layer is 43nm, and specifically, the chromium layer of the Au/Cr composite conductive electrode is in contact with a substrate; the graphene film is formed from single-layer graphene; the three-dimensional hydrogel material layer is formed by a hydrogel material with a three-dimensional network structure, the hydrogel material is obtained by polymerization reaction of raw materials including acrylamide monomers and modified probe molecules, the modified probe molecules are the probe molecules modified with acrylamide groups, and the thickness of the three-dimensional hydrogel material layer is 1.4 mm; wherein SiO is on the substrate₂The two opposite ends of the layer are respectively provided with the electrodes, and the graphene film is arranged on the surfaces of the two electrodes and the surface of the exposed substrate.

The preparation method of the three-dimensional hydrogel-graphene-based biosensor comprises the following steps:

with SiO coated on the surface₂A silicon wafer (commercially available) as a substrate, and electron beam evaporation is carried out on the SiO₂Preparing gold/chromium composite conductive electrodes at two opposite ends of the layer to obtain a substrate-electrode layer device;

transferring the graphene film to the surface of the electrode layer by adopting a wet transfer method, wherein the graphene film is basedThe method is characterized in that the method is deposited on the front surface and the back surface of a copper foil by a Chemical Vapor Deposition (CVD) method, a polymethyl methacrylate (PMMA) protective layer is arranged on the front surface of the copper foil attached with a graphene film (the composite material containing the graphene film is a commercial product), and the wet transfer method comprises the following specific operation steps: cutting the composite material into 3 × 3mm²The graphene film on the back of the copper foil is removed by using an oxygen ion etching technology, then the front of the copper foil is upward (namely the side with the PMMA protective layer is upward), the copper foil floats in APS100 copper etching liquid with the concentration of 4 wt%, standing is carried out for 3 hours, the copper foil is completely etched, and the graphene film-PMMA protective layer material floats on the surface of the APS100 copper etching liquid; fishing out the graphene film by using a cover glass, putting the graphene film into a culture dish filled with deionized water, enabling the graphene film of the graphene film-PMMA protective layer material to be in contact with the deionized water, standing for 15min, removing APS100 copper etching liquid remained on the graphene film, marking the step as 1 washing step, and repeating the washing step for 3 times to ensure that impurities remained on the surface of the graphene film are fully removed and avoid precipitated crystal particles from being mixed between the graphene film and an electrode after water evaporation; after washing, immersing the substrate-electrode layer device in a culture dish, aligning the graphene film floating in deionized water to the center of an electrode, taking out the graphene film-PMMA protective layer material, naturally airing, putting the graphene film-PMMA protective layer material in a vacuum box, carrying out vacuum drying for 5 hours at the temperature of 25 ℃, and then putting the graphene film-PMMA protective layer material on a hot plate at the temperature of 180 ℃ for heating for 1 hour to soften the PMMA protective layer, so that the contact between the graphene film and the substrate and between the graphene film and the electrode is firmer; soaking the obtained device in acetone for 1h to completely remove the PMMA protective layer on the surface of the graphene film, and then sequentially washing with Isopropanol (IPA) and deionized water and drying with nitrogen to obtain a substrate-electrode layer-graphene film device;

mixing Tris (hydroxymethyl) aminomethane (Tris) with nitric acid (HNO)₃) Preparing Tris-HNO with concentration of 0.5M, pH value of 8 by using deionized water₃A buffer solution; dissolving Acrylamide (Acrylamide) and methylenebisacrylamide (Bis-Acrylamide) in water to obtain a mixed monomer solution, wherein the Acrylamide and the methylenebisacrylamide in the mixed monomer solutionThe mass ratio of amine is 49:1, and the total concentration of acrylamide and methylene bisacrylamide in the mixed monomer solution is 40 wt%; subjecting the Tris-HNO to₃Mixing a buffer solution with sodium nitrate to obtain a sodium nitrate solution with the concentration of 2M, and diluting the total concentration of acrylamide and methylene bisacrylamide in the mixed monomer solution to 16 wt% by adopting the sodium nitrate solution to obtain an original glue solution; dissolving 50mg Ammonium Persulfate (APS) and 25 μ L Tetramethylethylenediamine (TEMED) in 500 μ L deionized water to obtain an initiator solution; mixing 20 μ L of the original gel solution and 30 μ L of an aptamer solution (the solvent is PBS buffer solution, pH value is 7.4) with concentration of 10 μ M in a centrifuge tube, wherein the aptamer is single-stranded DNA (purchased from Shanghai chemical company) of which the 5 end contains an acrylamide group; adding 4 mu L of initiator solution into the centrifuge tube, and uniformly mixing the initiator solution, the original gel solution and the aptamer solution to obtain a reaction solution; fixedly placing a Polydimethylsiloxane (PDMS) well with the diameter of 5mm on the surface of a graphene film in a substrate-electrode layer-graphene film device to serve as a reaction container; and adding the reaction solution into the PDMS well, standing and reacting for 15min at room temperature (25 ℃), allowing acrylamide, methylene bisacrylamide and an aptamer to form a hydrogel material layer on the surface of the graphene film through a free radical polymerization reaction, and removing the PDMS well to obtain the three-dimensional hydrogel-graphene-based biosensor.

FIG. 3 is a photograph and an optical microscopic image of the hydrogel material prepared in example 1, wherein (a) in FIG. 3 is a photograph (scale: 200 μm) of the hydrogel material and (b) is an optical microscopic image (scale: 50 μm) of the hydrogel material. As can be seen from FIG. 3, the hydrogel material prepared by the present invention has a porous structure with a pore size of about 2 μm.

Examples 2 to 9

A three-dimensional hydrogel-graphene-based biosensor was manufactured according to the method of example 1, except that the thicknesses of the three-dimensional hydrogel material layers were 0.63mm, 0.78mm, 1.05mm, 1.27mm, 1.6mm, 1.73mm, 1.96mm, and 2.35mm, respectively.

Test example

The performance test of the three-dimensional hydrogel-graphene-based biosensor is specifically as follows:

1. undiluted sterilized rabbit blood was used to prepare a cTnI protein solution (because undiluted sterilized rabbit blood is highly viscous and coagulable, the protein was uniformly dispersed in the blood as quickly as possible when preparing a cTnI protein solution of each concentration, but an oscillator was strictly prohibited during the preparation process to avoid protein inactivation and blood cell disruption), the concentrations were 100a, 500aM, 1fM, 5fM, 20fM, 100fM, 500fM, 2pM, 10pM, 50pM, 250pM, and 1000pM, respectively, and stored in an environment at 4 ℃ for use. Dropwise adding 40 mu L of cTnI protein solution with each concentration to the surface of the hydrogel material layer in the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 by using a pipette gun every 8min, respectively providing drain-source voltage and grid voltage by using two Keithley2400 single-channel power supply workstations, and respectively measuring graphene transfer characteristic curves; preparing a cTnI protein solution by adopting undiluted sterilized rabbit blood serum according to the method, and then respectively measuring the graphene transfer characteristic curves according to the method; and PBS buffer containing cTnI protein was used as a control.

The result shows that as the concentration of the cTnI protein in the undiluted blood is increased, the Dirac point moves 61mV in the negative direction of the x axis, and the change rate is less than 5% compared with that of the Dirac point which is measured in PBS buffer and moves 64mV in the negative direction of the x axis, so that the three-dimensional hydrogel-graphene-based biosensor provided by the invention can accurately identify the target molecule and generate a response signal in the undiluted blood.

Fig. 4 is a graph of detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 in undiluted serum, fig. 5 is a graph of detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 in undiluted blood, and fig. 6 is a hill equation fitted curve based on the detection signals of the three-dimensional hydrogel-graphene-based biosensor in undiluted serum, undiluted blood, and PBS buffer. Calculating to obtain K between the three-dimensional hydrogel-graphene-based biosensor and cTnI protein in undiluted blood according to the fitted curve in FIG. 6_DHas a size of 426fM, and K_D437fM, measured in PBS buffer, and 398fM, measured in undiluted serum, changed by less than 3% and 7%, respectively; meanwhile, the minimum limit concentration of the three-dimensional hydrogel-graphene-based biosensor in undiluted blood is calculated to be 13.9aM, the data shows obvious improvement even compared with the previously reported sensor for detecting cTnI protein in PBS buffer solution, the detection limit of the sensor is reduced by several times to dozens of times, and specific comparative data are shown in Table 1.

Table 1 comparative data of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 and other sensing platforms on the detection of cTnI protein

Note: in Table 1 [1] Jo H, Gu H, Jeon W, et al, electrochemical adapter of Cardiac Troponin I for the Early Diagnosis [ J ]. Analytical Chemistry,2015,87(19):9869 + 9875.

[2]Zhang L,Xiong C,Wang H,et al.A sensitive electrochemiluminescence immunosensor for cardiac troponin I detection based on dual quenching of the self-enhanced Ru(II)complex by folic acid and in situ generated oxygen[J].Sensors andActuators B:Chemical,2017,241:765-772。

[3]Ko S,Kim B,Jo S-S,et al.Electrochemical detection ofcardiac troponin I using a microchip with the surface-functionalized poly(dimethylsiloxane)channel[J].Biosensors&Bioelectronics,2007,23(1):51-59。

[4]Kong T,Su R,Zhang B,et al.CMOS-compatible,label-free silicon-nanowire biosensors to detect cardiac troponin I for acute myocardial infarction diagnosis[J].Biosensors&Bioelectronics,2012,34(1):267-272。

[5]Singal S,Srivastava A K,Dhakate S,et al.Electroactive graphene-multi-walled carbon nanotube hybrid supported impedimetric immunosensor for the detection ofhuman cardiac troponin-I[J].RSC Advances,2015,5(92):74994-75003。

2. The three-dimensional hydrogel-graphene-based biosensor prepared in example 1 was left in a room temperature environment for various times, and then a signal (target molecule is cTn I) was detected. Fig. 7 is a graph of detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 after being left in a room temperature environment for different periods of time, and in fig. 7, (a) is a graph of detection signals after being left for 1 day, (b) is a graph of detection signals after being left for 4 days, (c) is a graph of detection signals after being left for 7 days, and (d) is a hill equation fitting curve based on detection signals of the three-dimensional hydrogel-graphene-based biosensor after being left in a room temperature environment for different periods of time. The results show that the transfer characteristic curve of the three-dimensional hydrogel-graphene-based biosensor after being stored for 1 day in a room temperature environment obviously moves to the negative direction of the X axis, and the DeltaV_DiracThe value of (A) is significantly reduced by 58 mV. The transfer characteristic curves of the three-dimensional hydrogel-graphene-based biosensor stored for 4 and 7 days in a room temperature environment show a consistent change trend, V_DiracFrom 0.055V to 0.004V and from 0.102V to 0.046V, respectively, with the response signal av of the three-dimensional hydrogel-graphene based biosensor used immediately after processing_DiracThe comparison was carried out at 64mV with 8% and 12.5% respectively. The three-dimensional hydrogel-graphene-based biosensor provided by the invention has good stability, and the performance of the three-dimensional hydrogel-graphene-based biosensor cannot be obviously attenuated even being placed in a room temperature environment for a long time.

3. The three-dimensional hydrogel-graphene-based biosensor prepared in example 1 was left at room temperature, 40 ℃, 60 ℃ and 80 ℃ for 1h, and then a signal (target molecule is cTn I) was detected. Fig. 8 is a graph of detection signals of the three-dimensional hydrogel-graphene-based biosensor prepared in example 1 after being placed for 1 hour under different temperature conditions. The result shows that the three-dimensional hydrogel-graphene-based biosensor provided by the invention has good high temperature resistance, and the detection signal is still stable at 80 ℃.

4. The signals (target molecules are cTn I) of the three-dimensional hydrogel-graphene-based biosensors (the thicknesses of the three-dimensional hydrogel material layers are respectively 0.63mm, 0.78mm, 1.05mm, 1.27mm, 1.4mm, 1.6mm, 1.73mm, 1.96mm and 2.35mm) prepared in examples 1-9 and with different thicknesses of the three-dimensional hydrogel material layers are detected. Fig. 9 is a detection signal diagram of a three-dimensional hydrogel-graphene based biosensor with different three-dimensional hydrogel material layer thicknesses, wherein (a) is a relationship diagram of a signal gain Dirac point variation and the three-dimensional hydrogel material layer thickness, and (b) is a relationship diagram of a detection limit and the three-dimensional hydrogel material layer thickness. The result shows that the distance between the equivalent charge center of the grabbed charged target molecule and the graphene film can be adjusted by adjusting the thickness of the three-dimensional hydrogel material layer, so that the electrostatic induction effect is influenced, the sensitivity of the sensor is adjusted, and the adjustment of the target molecule detection range is finally realized.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.