CN108593719B - Immunosensor based on flexible electrode - Google Patents

Abstract

The invention discloses an immunosensor based on a flexible electrode and application thereof, and belongs to the technical field of biosensors. The immunosensor comprises a flexible electrode, the flexible electrode comprises a flexible electrode substrate and a biomolecule probe fixed on the surface of the flexible electrode substrate, and the flexible electrode substrate is made of polylactic acid and graphene. After the biomolecule probe on the immunosensor is specifically combined with a target substance to be detected, impedance change occurs, and the solubility of the target substance to be detected in a solution is calculated by detecting the impedance change. The electrode substrate prepared from the polylactic acid and graphene composite material is a flexible material, and the polylactic acid molecules contain rich carboxyl groups which can be used for fixing a biomolecule probe and keeping the bioactivity of the biomolecule probe; the graphene is doped in the polylactic acid matrix, so that the conductivity and flexibility of the electrode material are improved, and the performance of the sensor is greatly improved.

Description

An immunosensor based on flexible electrodes

technical field

The invention relates to the technical field of biosensors, in particular to an immunosensor based on flexible electrodes.

Background technique

Immunosensors have the advantages of good selectivity, small size, easy portability, and low cost. They have been widely used in food safety testing, clinical analysis, environmental monitoring, family medicine and other fields, such as pathogenic bacteria, antibiotics, urine protein, tumor markers detection of things, etc. For the on-site detection of trace substances, it is of great significance to further improve the sensitivity, repeatability and stability of the sensor.

When immunosensors are used for continuous target detection, the reproducibility of biomolecular probes is a major problem that restricts the practical application of this type of sensor. Therefore, the development of mass-produced and low-cost immunosensors, which can be used as disposable sensors, will overcome the cumbersome, time-consuming and labor-intensive drawbacks of immunosensors that require regeneration of biomolecular probes for multiple measurements.

In the face of more and more special detection environments, the development of new sensor technologies based on new materials and new processes has become a development trend. The flexible sensor based on flexible matrix material has the characteristics of flexibility, ductility, free bending or even folding, easy to carry, wearable, etc., and the structure is flexible and diverse, and can be placed arbitrarily according to the requirements of measurement conditions, suitable for many special applications scene and environment.

Common flexible materials include polyvinyl alcohol (PVA), polyester (PET), polyimide (PI), polyethylene naphthalene (PEN), paper sheets, textile materials, and the like. For example, Xia Shanhong's research group from the Institute of Electronics, Chinese Academy of Sciences has developed a flexible immunosensor based on paper materials. By self-assembling a monolayer film on the paper base and introducing nano-gold particles, the immunosensor for glycosylated hemoglobin detection has been realized; Xu Panju, Zhejiang Sci-Tech University Graphene oxide (GO)/reduced graphene oxide (rGO) microarray-based sensors were fabricated on flexible substrates (ITO/PET) to provide cell growth substrates and sensitive detection of hydrogen peroxide; Rice University, USA James Tour's research group uses laser to burn cheap polyimide plastic to create a flexible sensor based on graphene foam, which can be used for wrist pulse signal detection; Saudi King Abdullah University of Science and Technology PranatiNayak uses laser direct writing technology to prepare on PI substrate A graphene-based flexible sensor for molecular detection such as ascorbic acid has been developed.

At present, the research on flexible biosensors is still in its infancy, and there are not many related studies at home and abroad, and there is no report on the preparation of flexible immunosensors using polylactic acid and graphene composite materials.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a flexible electrode immunosensor with simple processing, low cost, mass production and excellent performance, which can be used as a disposable sensor to solve the problem of the high cost of existing immunosensors and biomolecular probes. The regenerative treatment is cumbersome, the scope of use is narrow, and the accuracy is reduced due to repeated use.

To achieve the above object, the present invention adopts the following technical solutions:

An immunosensor based on a flexible electrode, comprising a flexible electrode, the flexible electrode includes a flexible electrode substrate and a biomolecule probe fixed on the surface of the flexible electrode substrate, and the constituent materials of the flexible electrode substrate include polylactic acid and graphene.

The present invention adopts polylactic acid and graphene composite material to prepare flexible electrode substrate, high molecular weight polylactic acid is used as matrix, excellent plasticity can be made into various shapes, and graphene is evenly dispersed therein, because graphene has large specific surface area, electrical conductivity, High Young's modulus of elasticity can effectively improve the electrical conductivity and ductility of PLA.

The polylactic acid molecule is rich in carboxyl groups and can be used to immobilize biomolecular probes. The biomolecular probes are a class of substances with free amino groups. The amino groups react with the carboxyl groups on the surface of the electrode substrate to form amide bonds, making the biomolecular probes. fixed on the surface of the electrode substrate. Polylactic acid has good biocompatibility and can effectively maintain the biological activity of biomolecular probes.

The flexible electrode substrate can be made into a wire shape or a film shape, and the polylactic acid and graphene composite material is prepared by an extruder or a film processing device to obtain a wire or film shape electrode substrate.

Preferably, the diameter of the wire electrode is 0.01-3 mm, and the length is not limited; the thickness of the thin-film electrode is 50-500 μm.

The mass ratio of graphene and polylactic acid in the flexible electrode substrate is 0.5-10:100, and the effective doping amount of graphene is used to ensure the conductivity and flexibility of the flexible electrode.

The biomolecular probe of the present invention is a kind of substance that specifically binds to the target substance. Preferably, the biomolecular probe is any one of antibodies, antigens, DNA, nucleic acid aptamers, etc. The molecular structure contains free amino groups. Preferably, the antigen is a protein.

The preparation method of the flexible electrode comprises the following steps:

(1) Mixing graphene powder and polylactic acid particles, heating and melting, and processing into a wire-shaped or film-shaped flexible electrode substrate;

(2) The flexible electrode substrate is activated in a phosphate buffer containing N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

(3) Immerse the activated flexible electrode substrate in a solution containing biomolecular probes; or drop or spray the solution containing biomolecular probes onto the surface of the activated flexible electrode substrate, and after the reaction, biomolecular probes The needle is fixed on the surface of the flexible electrode substrate to prepare the flexible electrode;

The biomolecular probe has a free amino group, which reacts with the carboxyl group of polylactic acid to form an amide bond.

Preferably, in step (1), the mass ratio of graphene powder and polylactic acid particles is 1:20-30.

The polylactic acid particles are L-polylactic acid, D-polylactic acid or a mixture of L-polylactic acid and D-polylactic acid.

The graphene powder can be graphene oxide, reduced graphene oxide or element-doped graphene, wherein the element-doped graphene is nitrogen, phosphorus or sulfur and other element-doped graphene, and the doping elements include But not limited to the above.

Preferably, in step (1), the heating temperature is 170-230°C. Composites are processed into wire or film shapes using extruders or film processing equipment.

In step (2), a large number of carboxyl groups on the surface of the electrode substrate can pass through the coupling agent N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloric acid. Salt (EDC) activation.

Preferably, in step (2), the molar ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.1-10:1.

More preferably, the molar concentration of NHS in the buffer solution is 0.08 mol/L, and the molar concentration ratio of EDC is 0.05 mol/L.

Preferably, in step (2), the activation time is 3-5h.

Preferably, before activation, the flexible electrode substrate is soaked in N,N-dimethylformamide (DMF) for 5-120 minutes to dissolve the molten polylactic acid attached to the electrode surface, which is beneficial to increase the polylactic acid. Roughness and permeability.

More preferably, the flexible electrode substrate is soaked in N,N-dimethylformamide for 10 min.

After the DMF treatment, the surface of the flexible electrode was washed with ethanol and clean water in turn, and the activation step was carried out after drying.

In step (3), after the carboxyl group on the surface of the electrode substrate is activated, it reacts with the amino group of the biomolecular probe to form an amide bond, so that the biomolecular probe is immobilized on the surface of the electrode substrate.

Preferably, in step (3), the reaction conditions are placed at room temperature for 5min-24h. More preferably, the placing time is 15h.

The working principle of the flexible electrode immunosensor provided by the present invention is as follows: after the biomolecular probe on the electrode surface is specifically combined with the target substance to be measured, the electrode impedance changes. The concentration has a linear relationship, and the concentration of the target substance to be measured can be indirectly calculated by measuring the change in impedance by the direct impedance measurement method.

Specifically, the flexible electrode is first placed in the solution to be tested, so that the target substance is bound to the biomolecular probe on the surface of the flexible electrode, and then the two ends of the flexible electrode bound with the target substance are respectively connected to the LCR measuring instrument to measure impedance, and calculate the concentration of the target substance to be measured according to the calibration curve.

The flexible electrode-based immunosensor provided by the present invention can also adopt a three-electrode system or a two-electrode system. The flexible electrode is used as a working electrode to form a three-electrode system with an auxiliary electrode and a reference electrode; it can also form a two-electrode system with the auxiliary electrode. , measured by electrochemical measurement methods. Since the impedance changes after the flexible electrode is combined with the target substance, the current will also change accordingly. The impedance change is measured by detecting the current change, and then the concentration of the target substance to be measured is calculated. The auxiliary electrode adopts but is not limited to platinum electrode or carbon electrode, and the reference electrode adopts but not limited to Ag/AgCl electrode or saturated calomel electrode.

Specifically, the flexible electrode is first placed in the solution to be measured, so that the target substance is bound to the biomolecular probe on the surface of the flexible electrode, and then a three-electrode system or a two-electrode system is formed, which is measured by an electrochemical measurement method.

The present invention provides an application of the immunosensor in detecting the concentration of a target substance specifically bound to a biomolecular probe on a flexible electrode in a liquid or a gas.

The immunosensor provided by the present invention can be used to detect the target substance that can be specifically bound by the biomolecular probe immobilized on the surface of the flexible electrode. For example, the chloramphenicol antibody immobilized on the flexible electrode can specifically bind to chloramphenicol. The sensor can be used to detect chloramphenicol; if the cortisol antibody is immobilized on the flexible electrode, which can specifically bind to cortisol, then the immunosensor can be applied to detect cortisol; such as the nucleic acid of Listeria monocytogenes immobilized on the flexible electrode The aptamer can specifically bind to the nucleic acid of Listeria monocytogenes, then the immunosensor can be applied to detect Listeria monocytogenes; the flexible electrode of the present invention, the immobilized biomolecular probes include but are not limited to the above-mentioned ones.

The beneficial effects that the present invention has:

(1) The present invention adopts the electrode substrate prepared by polylactic acid and graphene composite material as a flexible material, and immobilizes biomolecular probes on the surface of the flexible electrode substrate to prepare a high-performance immunosensor. Polylactic acid has good biocompatibility and biodegradability. At the same time, its molecules are rich in carboxyl groups, which can be used to immobilize biomolecular probes and maintain their biological activity; the incorporation of graphene into the polylactic acid matrix improves the conductivity and flexibility of the electrode material, greatly improving the performance of the sensor .

(2) The flexible electrode-based immunosensor of the present invention can be mass-produced, has low cost, can be used as a disposable immunosensor, is bendable, occupies a small space, and greatly broadens application occasions and application fields.

Description of drawings

FIG. 1 is a flow chart of the preparation of the flexible electrode of Example 1. FIG.

FIG. 2 is a schematic diagram of the structure and composition of the flexible electrode in Example 1. FIG.

FIG. 3 is the electrochemical impedance response curve of the chloramphenicol immunosensor prepared in Example 1 without DMF treatment to chloramphenicol solutions of different concentrations.

FIG. 4 is a flow chart of the preparation of the flexible electrode of Example 2. FIG.

FIG. 5 is the impedance linear fitting curve of the DMF-treated chloramphenicol immunosensor prepared in Example 2 to chloramphenicol solutions with different concentrations.

FIG. 6 is a flow chart of the preparation of the flexible electrode of Example 3. FIG.

FIG. 7 is a schematic diagram of the structure and composition of the flexible electrode in Example 3. FIG.

8 is a linear fitting curve of the electrochemical impedance of the cortisol immunosensor without DMF treatment prepared in Example 3 to cortisol solutions of different concentrations.

FIG. 9 is a flow chart of the preparation of the flexible electrode of Example 4. FIG.

FIG. 10 is a schematic diagram of the structure and composition of the flexible electrode in Example 4. FIG.

FIG. 11 is the impedance linear fitting curve of the L. monocytogenes immunosensor prepared in Example 4 without DMF treatment to different concentrations of L. monocytogenes solution.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings: the implementation is carried out on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are provided, but the protection scope of the present invention is not limited to the following embodiments. .

The immunosensor includes a flexible electrode. The flexible electrode is composed of a flexible electrode substrate and biomolecular probes fixed on the flexible electrode substrate. The flexible electrode substrate is composed of polylactic acid and graphene, and the shape can be a wire or a thin film.

The immunosensor is used for the specific detection of the corresponding target substance according to the immobilized biomolecular probe, and the concentration of the target substance to be measured can be indirectly calculated by measuring the change of impedance by electrochemical measurement method or direct impedance measurement method.

The graphene powder, polylactic acid particles, chloramphenicol antibody, cortisol antibody, and Listeria monocytogenes nucleic acid aptamer used in the following examples are all commercially available materials.

Example 1

An immunosensor based on a flexible electrode provided in this embodiment is used to detect the concentration of chloramphenicol.

In this embodiment, the preparation method of the flexible electrode includes the following steps (FIG. 1):

S1, using a twin-screw extruder to prepare polylactic acid and graphene flexible wire electrodes.

The graphene powder and L-polylactic acid particles were mixed at a mass ratio of 1:30, added to the feeding box of the twin-screw extruder, and the temperature of the twin-screw extruder was adjusted to 180 degrees Celsius. After 60 minutes, the flexible wire electrode was extruded. Set the traction speed to 10cm/min.

S2, using ethanol and pure water to clean the prepared flexible electrode substrate.

The polylactic acid-graphene flexible wire electrodes prepared by the twin-screw extruder extrusion method were sonicated in ethanol and pure water for 20 min and dried.

S3. The chloramphenicol antibody is immobilized on the surface of the flexible wire electrode by EDC/NHS activation.

A certain amount of NHS and EDC were dissolved in phosphate buffer to prepare a mixture containing 0.08M NHS and 0.05M EDC, and the cleaned flexible wire electrode was immersed in the EDC/NHS mixture for activation for 3h;

After that, the activated flexible wire electrode was immersed in a solution containing 50 mg/mL chloramphenicol antibody, and left for 15 hours to prepare the flexible electrode of this embodiment, as shown in FIG. 2 .

After the flexible electrode of this embodiment is fabricated, the fabricated sensor is calibrated by configuring a standard concentration of chloramphenicol solution. The specific calibration method includes the following steps:

F1. Mix Na ₂ HPO ₄ (0.2M) and NaH ₂ PO ₄ (0.2M) in a certain proportion, adjust the pH to 7.2, add chloramphenicol of different quality, and configure chloramphenicol solutions with 7 concentration gradients.

F2. Immerse the prepared flexible electrode in a chloramphenicol solution with a minimum concentration, and wait for 30 minutes, so that the chloramphenicol molecules are bound to the chloramphenicol antibody molecules immobilized on the surface of the flexible electrode.

F3. Place the above-mentioned flexible electrode combined with the chloramphenicol molecule into a phosphate buffer (pH 7.4) containing 5mM potassium ferricyanide and 0.1M potassium chloride, and adopt the three-electrode electrochemical measurement method. The flexible electrode of the example is the working electrode, the Pt sheet is used as the auxiliary electrode, and the Ag/AgCl electrode is used as the reference electrode, and then the response of the sensor to this concentration of chloramphenicol solution is measured by electrochemical impedance method.

F4. The specific parameters of the electrochemical impedance method in this embodiment are as follows: the scanning frequency range is 0.1-10 ⁵ Hz, and the set voltage is the open-circuit voltage.

F5. According to the above method, measure the electrochemical impedance response value of the flexible electrode immunosensor of this embodiment to other concentrations of chloramphenicol.

After calibration, the electrochemical impedance response curves of the immunosensor without DMF treatment in this example to different concentrations of chloramphenicol were obtained, as shown in Figure 3, and the result achieved a concentration range of 0.1-10ng/mL of chloramphenicol. Solution testing.

Example 2

A flexible electrode-based immunosensor of this embodiment is used for chloramphenicol concentration detection.

In this embodiment, the preparation method of the flexible electrode immunosensor includes the following steps (FIG. 4):

The flexible electrode substrate is made of graphene oxide and D-polylactic acid, and the mass ratio of graphene oxide and D-polylactic acid is 1:25, and the processing and preparation are the same as step S1 in Example 1.

Further preferably, in the next step, such as step S2 in Example 1, the prepared flexible electrode substrate is first soaked in DMF for 10 minutes, then ultrasonicated in ethanol and pure water for 10 minutes and dried, and then continue with Example 1. In step S3, the flexible electrode immunosensor of this embodiment is prepared.

By configuring a standard concentration of chloramphenicol solution, the fabricated sensor is calibrated. The specific calibration method includes the following steps:

F1. Mix Na ₂ HPO ₄ (0.2M) and NaH ₂ PO ₄ (0.2M) in a certain proportion, adjust the pH to 7.2, add chloramphenicol of different quality, and configure chloramphenicol solutions with 6 concentration gradients.

F2. Immerse the prepared flexible electrode immunosensor in a solution of a minimum concentration of chloramphenicol, and wait for 30 minutes, so that the chloramphenicol molecules are bound to the chloramphenicol antibody molecules immobilized on the surface of the flexible electrode.

F3. Connect both ends of the obtained flexible electrode immunosensor combined with chloramphenicol molecules to a handheld LCR instrument (such as Keysight U1733C), select the first gear of impedance measurement, and set the scanning frequency to 100kHz until the displayed impedance value When it is a stable number, record this value as the impedance response of the sensor to the corresponding chloramphenicol concentration.

According to the method of steps F2 and F3, measure the impedance response of the sensor to other concentrations of chloramphenicol solution.

F4. After calibration, the impedance response linear fitting curve of the immunosensor treated with DMF to different concentrations of chloramphenicol in this example is obtained, as shown in FIG. 5 , the abscissa is the Ln concentration of chloramphenicol, and the ordinate is The impedance value of the flexible electrode combined with chloramphenicol realizes the detection of chloramphenicol concentration range of 0.05-10ng/mL.

Example 3

A flexible electrode-based immunosensor of this embodiment is used to detect cortisol concentration.

In this embodiment, the preparation method of the flexible electrode includes the following steps (FIG. 6):

S1, using a twin-screw extruder to prepare polylactic acid and graphene flexible wire electrodes.

The graphene powder and L-polylactic acid particles were mixed at a mass ratio of 1:40, added to the feeding box of the twin-screw extruder, and the temperature of the twin-screw extruder was adjusted to 180 degrees Celsius. After 60 minutes, the flexible wire electrode was extruded. Set the traction speed to 10cm/min.

S2, using ethanol and pure water to clean the prepared flexible electrode substrate.

The polylactic acid-graphene flexible wire electrodes prepared by the twin-screw extruder extrusion method were sonicated in ethanol and pure water for 20 min and dried.

S3. The cortisol antibody is immobilized on the surface of the flexible wire electrode by EDC/NHS activation.

A certain amount of NHS and EDC were dissolved in phosphate buffer to prepare a mixture containing 0.08M NHS and 0.05M EDC, and the cleaned flexible wire electrode was immersed in the EDC/NHS mixture for activation for 3h;

After that, the activated flexible wire electrode was immersed in a cortisol antibody solution containing 50 mg/mL and left for 15 hours to prepare the flexible electrode of this embodiment, as shown in FIG. 7 .

By configuring a standard concentration of cortisol solution, the fabricated sensor is calibrated. The specific calibration method includes the following steps:

F1. Mix Na ₂ HPO ₄ (0.2M) and NaH ₂ PO ₄ (0.2M) in a certain proportion, adjust the pH to 7.2, add cortisol of different quality, and configure cortisol solutions with 5 concentration gradients.

F2. Immerse the prepared immunosensor in a minimum concentration of cortisol solution and wait for 40 minutes, so that the cortisol molecules are bound to the cortisol antibody molecules immobilized on the surface of the flexible electrode.

F3. The above-mentioned flexible electrode combined with cortisol molecules is placed in a phosphate buffer (pH 7.4) containing 5mM potassium ferricyanide and 0.1M potassium chloride, and then a three-electrode electrochemical measurement method is used, in which the present The flexible electrode of the embodiment is the working electrode, the Pt sheet is used as the auxiliary electrode, and the Ag/AgCl electrode is used as the reference electrode, and then the response of the sensor to this concentration of cortisol solution is measured by electrochemical impedance method.

F4. The specific parameters of the electrochemical impedance method in this embodiment are as follows: the scanning frequency range is 0.1-10 ⁵ Hz, and the set voltage is the open-circuit voltage.

F5. According to the above method, measure the electrochemical impedance response value of the flexible electrode immunosensor of this embodiment to other concentrations of cortisol.

After calibration, the linear fitting curves of the electrochemical impedance of the immunosensor without DMF treatment in this example to different concentrations of ^cortisol are obtained, as shown in FIG. Alcohol solution detection.

Example 4

A flexible electrode-based immunosensor of this embodiment is used to detect the concentration of Listeria monocytogenes.

In this embodiment, the preparation method of the flexible electrode includes the following steps (FIG. 9):

S1. Preparation of polylactic acid and graphene flexible wire electrodes by twin-screw extruder

Mix nitrogen-doped graphene powder and L-polylactic acid particles in a mass ratio of 1:20, add them into the feeding box of the twin-screw extruder, adjust the extrusion temperature of the twin-screw extruder to 180 degrees Celsius, extrude and granulate, Then, the film is blown into a film by a single-screw extrusion film blowing machine. The film blowing temperature is 180 ° C. The blown film bubble is inflated and cooled by introducing air from the extrusion head. , and finally rolled and rolled by a winding device to obtain a flexible electrode substrate with a film shape of the present invention, and the film thickness is 200 μm.

S2, using DMF to treat the flexible electrode substrate, and washing the prepared flexible electrode substrate with ethanol and pure water respectively.

The polylactic acid-graphene flexible film-shaped electrode prepared above was cut into strips with a length of 5 cm and a width of 3 cm, and one of the strip flexible electrodes was first soaked in DMF for 10 min, and then sonicated in ethanol and pure water for 10 min respectively. and dried,

S3. The nucleic acid aptamer of Listeria monocytogenes with amino groups is immobilized on the surface of the flexible wire electrode by EDC/NHS activation.

Dissolve a certain amount of NHS and EDC in phosphate buffer to prepare a mixed solution containing 0.08M NHS and 0.05M EDC, immerse the cleaned flexible film-shaped electrode in the EDC/NHS mixture, and store in a sealed container for 3h ;

After that, the activated flexible wire electrode was immersed in a solution containing the nucleic acid aptamer of Listeria monocytogenes, and left for 15 hours to prepare the flexible electrode of this embodiment, as shown in FIG. 10 .

By configuring a standard concentration of Listeria monocytogenes solution, the fabricated sensor is calibrated. The specific calibration method includes the following steps:

F1. Mix Na ₂ HPO ₄ (0.2M) and NaH ₂ PO ₄ (0.2M) in a certain proportion, adjust the pH to 7.4, add different amounts of Listeria monocytogenes, and configure it into 6 concentration gradients of Listeria monocytogenes Special bacteria solution.

F2. Immerse the prepared flexible electrode in a solution of Listeria monocytogenes at a minimum concentration and wait for 40 minutes, so that Listeria monocytogenes binds to the nucleic acid aptamer molecule of Listeria monocytogenes immobilized on the surface of the immunosensor.

F3. The above-mentioned flexible electrode combined with Listeria monocytogenes is placed in a phosphate buffer solution of 2.5mM potassium ferricyanide (pH 7.4), and then a three-electrode electrochemical measurement method is used, in which the flexible electrode of this embodiment is used. The electrode was used as the working electrode, the Pt wire was used as the auxiliary electrode, and the Ag/AgCl electrode was used as the reference electrode, and then the response of the sensor to this concentration of Listeria monocytogenes solution was measured by electrochemical impedance method.

F4. The specific parameters of the electrochemical impedance method in this embodiment are as follows: the scanning frequency range is 0.1-10 ⁵ Hz, and the setting voltage is 0.24V.

F5. According to the above method, measure the electrochemical impedance response value of the flexible electrode immunosensor of this embodiment to other concentrations of Listeria monocytogenes.

After calibration, the linear fitting curve of the electrochemical impedance of the immunosensor without DMF treatment in this example to different concentrations of Listeria monocytogenes was obtained, as shown in Figure 11, and the result achieved a concentration range of 1-1×10 ⁵ CFU/mL of Listeria monocytogenes solution detection, the abscissa is the Lg concentration of Listeria monocytogenes, and the ordinate is the impedance value of flexible electrodes combined with different concentrations of Listeria monocytogenes.

Claims (1)

1. A flexible electrode-based immunosensor comprises a flexible electrode, and is characterized in that the flexible electrode comprises a flexible electrode substrate and a biomolecule probe Listeria monocytogenes nucleic acid aptamer fixed on the surface of the flexible electrode substrate,

the preparation method of the flexible electrode comprises the following steps:

s1, mixing nitrogen-doped graphene powder and L-polylactic acid particles according to a mass ratio of 1:20, adding the mixture into a feeding box of a double-screw extruder, adjusting the extrusion temperature of the double-screw extruder to 180 ℃, extruding and granulating, then performing blow molding through a single-screw extrusion film blowing machine to form a film, wherein the film blowing temperature is 180 ℃, air is introduced from an extrusion head to blow and cool the film bubble, then the film is shaped through a herringbone plate, is pulled by a traction roller, and finally is rolled and coiled by a coiling device to obtain a film-shaped flexible electrode substrate, wherein the thickness of the film is 200 mu m;

s2, cutting the prepared polylactic acid-graphene flexible film-shaped electrode substrate into strips with the length of 5cm and the width of 3cm, soaking one strip in DMF for 10min, performing ultrasonic treatment in ethanol and pure water for 10min respectively, drying,

s3, dissolving NHS and EDC in phosphate buffer solution to prepare mixed solution containing 0.08M NHS and 0.05M EDC, immersing the cleaned strip in the mixed solution of EDC and NHS, and sealing and storing for 3 h;

s4, soaking the activated strip into a solution containing the Listeria monocytogenes aptamer, and standing for 15h to obtain the flexible electrode.

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