CN104977343B - A kind of high performance biosensors based on graphene/mesoporous carbon nano-composite material and preparation method thereof - Google Patents

Abstract

The invention provides a biosensor based on graphene/mesoporous carbon nanocomposite material and a preparation method thereof. The invention includes the preparation of graphene/mesoporous carbon nanocomposite material by hydrothermal synthesis method, which is used as the immobilized material for adsorbing enzymes; the biosensing and electrochemical principles are used to produce the graphene/mesoporous carbon nanocomposite material by combining screen and inkjet printing Detection test paper, screen printing is used to print conductive lines, and the sensitive biological components are spray-printed on the electrode support by non-contact spraying method, and the spraying amount and spraying area of the spraying material can be controlled. The nanocomposite carrier material is to grow mesoporous carbon on both sides of the graphene sheet to make a graphene/mesoporous carbon composite material, which is used as a carrier to carry enzymes, physically mixed with biological enzyme solutions, and printed by inkjet printers Modified on the glassy carbon electrode for rapid and efficient detection of blood glucose.

Description

A high-efficiency biosensor based on graphene/mesoporous carbon nanocomposites and its preparation method

technical field

The invention belongs to the interdisciplinary fields of printed electronics technology, nanomaterial technology, biotechnology, etc., and specifically relates to a high-efficiency biosensor based on graphene/mesoporous carbon nanocomposite material and a preparation method thereof.

Background technique

Diabetes has become a social health problem. The global incidence of diabetes is increasing rapidly. Diabetes has become the third chronic disease that seriously threatens human health after tumors and cardiovascular diseases. At present, there are more than 120 million diabetic patients in the world, and the number of patients in my country ranks second in the world, exceeding 20 million. According to the World Health Organization, by 2025, the number of adult diabetic patients in the world will increase to 300 million, while the number of diabetic patients in China will reach 40 million. Diabetes will remain a serious public health problem in China in the next few decades.

In the field of modern medical testing, point-of-care testing is developing rapidly, and the development of this technology allows patients to perform self-examination at home. Glucose biosensor can directly detect whole blood without pretreatment of blood. Because of its convenient portability, high sensitivity, good selectivity, fast detection speed, simple operation and intelligent system, it can be used in ordinary households to monitor blood at any time. And long-term monitoring, has a very good prospect.

Contents of the invention

The technical problem to be solved by the present invention is to provide a high-efficiency biosensor based on graphene/mesoporous carbon nanocomposite material and its preparation method. Graphene/mesoporous carbon nanocomposites can overcome the disadvantages of general nanomaterials, efficiently maintain enzyme activity and enable rapid electron transfer, combined with inkjet technology, the prepared biosensor combined with supporting instruments can quickly and conveniently obtain test results , and the manufacturing method is simple, convenient for post-processing and packaging, and conducive to large-scale production.

The technical problems solved by the present invention can be realized by adopting the following technical solutions.

A biosensor for detecting glucose, consisting of a flexible substrate, a working electrode printed on the flexible substrate and a counter electrode, the working electrode is a double-layer conductive film, composed of a water-based conductive ink layer and a bio-enzyme conductive ink on it Composition, the bio-enzyme conductive ink is composed of graphene/mesoporous carbon nanocomposite material, glucose oxidase and Nafion film-forming material.

The flexible base adopts a polymeric material with good toughness and high insulation, such as polyvinyl chloride, polyester or polycarbonate.

In the above biosensor, the working electrode is sprayed with bio-enzyme conductive ink. It can not only catalyze the glucose reaction, but also adsorb enzymes and carry out rapid electron transfer between the enzyme-catalyzed reaction center and the electrodes, thus forming a circuit loop. During the reaction, according to the level of glucose concentration in the blood, a corresponding current signal is generated, and It is detected and read by the corresponding tester.

A kind of biosensor preparation method based on graphene/mesoporous carbon nanocomposite material, it comprises the following steps:

1) Fully mix conductive carbon black, graphite, water-based acrylic resin, deionized water, and 25~28wt% ammonia water in a mass ratio of 10:5:15:15:3 to prepare water-based conductive ink, and set aside;

2) Fix the 200-400 mesh metal or nylon polyester wire mesh on the square lightweight metal frame, apply photosensitive glue in the dark room, and dry it for later use;

3) Put the computer-drawn working electrode and the counter electrode negative on the photosensitive glue, expose it to 20-40 watts of ultraviolet light for 15-30 minutes, rinse off the unhardened photosensitive glue covered by the electrode negative with a high-pressure water gun, and form silk after drying net template;

4) Install the screen template with the working electrode and counter electrode pattern on the screen printing machine, and print the working electrode and counter electrode with water-based conductive ink;

5) Dissolve glucose oxidase with a specific activity ≥ 150u/mg in phosphate buffer at pH 7.0 to prepare an enzyme solution with a concentration of 10mg/ml, fully dissolve and store in a 0°C refrigerator for later use;

6) Add the graphene/mesoporous carbon nanocomposite material as a carrier material into the enzyme solution, mix and stir for 10-36 hours, and use it to adsorb glucose oxidase, and set aside;

7) Prepare a Nafion film-forming material solution with a mass fraction of 0.5%, add it to the mixed solution prepared in step 6), mix and stir for 30 minutes to prepare a bio-enzyme conductive ink, and then print it on the surface of the working electrode by spraying. After drying for 24 hours, the immobilization of the enzyme and the modification of the electrode can be completed to make a biosensor.

The specific steps for the preparation of the graphene/mesoporous carbon nanocomposite material in step 6) of the above scheme are:

Dissolve 1.5-2g of molecular directing agent in 10-30ml of deionized water, mix and stir for 10-30min, add 1-2ml of 1M HCl and continue stirring; add 30-50mg of graphene and 1.5-2g of β -Cyclodextrin, stirred for 0.5-1h, then transferred the mixed solution to the reaction kettle and heat-treated the reaction at 120-150°C for 24-36h, and finally, under the protection of nitrogen in the tube furnace, the heating rate was 10°C/min to React at 700-800°C for 3-5 hours to obtain the nanocomposite material.

The preparation of described graphene, utilizes cheap conductive graphite raw material, adopts electrophoresis method, by adjusting the composition of electrophoretic liquid, pH value, applied voltage value etc., separates few layer (<10 layers) graphene of high purity and high conductivity . It is characterized in that the electrophoresis voltage is set at 60-100V; the graphene refers to any one of single-layer graphene, few-layer graphene (<10) or mixtures thereof.

The present invention uses silk screen printing to print conductive lines for conduction, thereby reducing the requirements for process manufacturing. Screen printing materials and screen printing equipment do not need special processing, and can be obtained through commercially available channels at a very low cost; The extraction and preparation of few-layer graphite with good conductivity is simple and easy, the raw materials are easy to obtain, and the cost is low; the graphene/mesoporous carbon nanocomposite with large specific surface area, good biocompatibility and good conductivity is prepared by hydrothermal synthesis The material can not only immobilize glucose oxidase, but also modify it on the electrode to speed up the transfer of electrons between the enzyme active center and the electrode, increasing the sensitivity of the sensor; using spray printing technology to control the spraying amount and area of biologically sensitive materials, so that the final The accuracy and repeatability of the product are fully guaranteed.

The beneficial effects of the present invention are:

1) The combination of screen printing and spray printing can not only simplify the screen printing process and reduce costs, but also eliminate the adverse effects of the composite screen printing method on the biologically sensitive material-enzyme, and the spray printing method can effectively control Bio-sensitive material-enzyme spraying amount and spraying area, the whole process is simple and easy to operate, low cost, and can be mass-produced;

2) Electrophoresis of few-layer graphene from cheap and easy-to-obtain graphite is simpler than the existing method of preparing graphene, the cost is low, and the prepared graphene has good electrical conductivity;

3) The graphene/mesoporous carbon nanocomposite material prepared by hydrothermal method has good biocompatibility, porosity and large specific surface area, which is conducive to the immobilization of enzymes, and the interface resistance of the composite material is small and conductive Good performance, which is conducive to promoting the transfer of electrons;

4) The use of graphene composite materials with good conductivity to modify electrodes is beneficial to increase the sensitivity, response time and detection range of biosensors. This invention can greatly facilitate the detection of home portable blood glucose concentration.

Description of drawings

Fig. 1 is the biosensor detection test paper structural diagram prepared by the present invention;

Fig. 2 is the schematic diagram of the biosensor electrode prepared by the present invention;

Fig. 3 is the preparation flowchart of the biosensor electrode prepared by the present invention;

Figure 4 is a schematic diagram of the working principle of the graphene/mesoporous carbon composite.

detailed description

Below in conjunction with embodiment further illustrate.

Example 1

1) Weigh 10g of graphite, 5g of conductive carbon black, 15g of water-based acrylic resin, 15g of deionized water, and 3g of ammonia water, mix and grind for 1 hour to prepare water-based conductive ink and set aside.

2) Fix 200-mesh metal or nylon polyester material on a square lightweight metal frame, apply Kodak photosensitive adhesive in a dark room, and dry it for later use;

3) Put the computer-drawn working electrode and the counter electrode negative on the photosensitive glue, expose it to a 25-watt UV lamp for 15 minutes, rinse off the unhardened photosensitive glue covered by the electrode negative with a high-pressure water gun, and form a silk screen template after drying;

4) Install the screen template with the working electrode and counter electrode pattern on the (SYP) type screen printing machine, and use water-based conductive ink to print the working electrode and counter electrode;

5) Dissolve glucose oxidase with a specific activity ≥ 150u/mg in phosphate buffer solution with pH 7.0 to prepare an enzyme solution with a concentration of 10mg/ml. After fully dissolving, put it in a 0°C refrigerator for later use;

6) Preparation of few-layer graphene: Surfactant, ammonia conductive liquid and water are prepared in a certain proportion as electrolyte, and two copper sheets are connected to the positive and negative electrodes of the electrophoresis instrument through wires, and the distance between the upper and lower electrodes is about 10-15cm, the electrophoresis voltage is set to 60-100V for ionization;

7) Preparation of graphene/mesoporous carbon composite material: Take 1.5g of molecular directing agent-triblock copolymer F127 dissolved in 20ml of deionized water and stir for 10min, add 1ml of HCl with a concentration of 1M and continue stirring; add in sequence 30mg of graphene prepared in step 2) of right 2 and 1.5g of β-cyclodextrin, stirred for 0.5h. Then the mixture was transferred to the reactor and heat treated at 130°C for 24 hours. Finally, under the protection of nitrogen in a tube furnace, the heating rate was 10°C/min to 750°C for 3 hours to prepare nanocomposites for use.

8) Add the graphene/mesoporous carbon nanocomposite material prepared in step 7) as a carrier material to the enzyme solution in step 5), mix and stir for adsorption for 10 hours; add the Nafion film-forming material solution with a mass fraction of 0.5% to the preparation In the obtained mixed solution, mixed and stirred for 30 minutes, the obtained mixed solution was printed on the working electrode by spraying, and dried at room temperature for 24 hours to complete the immobilization of the enzyme and the modification of the electrode, and the graphene/mesoporous carbon-based Efficient biosensor.

Example 2

This example is roughly the same as Example 1, except that the applied DC voltage is increased to 100V when preparing few-layer graphene by electrophoresis in step 6), and other conditions remain unchanged.

Example 3

This example is roughly the same as Example 1, except that in step 7), the calcination temperature in the tube furnace is raised to 800°C, and other conditions remain unchanged.

Example 4

This example is roughly the same as Example 1, except that the adsorption time of the nanocomposite to the enzyme in step 8) is increased to 24 hours, and other conditions remain unchanged.

The invention adopts the screen printing method to prepare the circuit and the bare carbon electrode, which is low in production cost, convenient and easy; the biological enzyme is printed by the inkjet printing method under the condition that the amount of the sprayed material and the size of the covered area can be effectively controlled. ; At the same time, two carbon nanomaterials with high bio-enzyme adsorption capacity and high conductivity are combined, which is conducive to the rapid transfer of electrons and realizes high-sensitivity detection of biosensors. The modification of graphene by direct growth on the surface not only retains the two-dimensional sheet structure and high conductivity of graphene, overcomes the difficulty of high interface resistance of nanocomposites, but also solves the problem of easy interfacial contact between graphene sheets. The problem of agglomeration and the porosity of the growing mesoporous carbon provide a better biological microenvironment for the enzyme, which is conducive to the immobilization of the enzyme. In addition, by adjusting the pore size of mesoporous carbon, immobilization substrates suitable for biomolecules of different scales can be prepared, which can promote heterogeneous electron transfer and improve the bioelectrocatalytic performance of immobilized proteins. It is extremely valuable and broadens the development path of carbon-based biosensors.

Claims (3)

1. it is a kind of detect glucose biology sensor preparation method, described biology sensor by flexible substrates, be imprinted on it is soft Property substrate on working electrode and to electrode form, the working electrode is a kind of bilayer conductive film, by water-soluble conducting ink Layer and thereon biology enzyme electrically conductive ink composition, described biology enzyme electrically conductive ink by graphene/mesoporous carbon nano-composite material, Glucose oxidase and Nafion filmogens composition；

It is characterized in that comprise the following steps：

1）By conductive black, graphite, water-based acrylic resin, deionized water, 25 ~ 28wt% ammoniacal liquor with 10:5:15:15:3 matter Amount ratio, which is sufficiently mixed, uniformly prepares water-soluble conducting ink, standby；

2）The metal of 200-400 mesh numbers or nylon polyesters material web are fixed on square light-weight metal framework, in darkroom Coat photoresists, drying for standby；

3）It is placed in by the working electrode of computer drawing and to electrode negative film on photoresists, exposes 15- under 20-40 watts of ultraviolet 30 minutes, the unhardened photoresists covered by electrode negative film are washed with giant, screen template is formed after drying；

4）It will be installed on working electrode and to the screen template of electrode pattern on screen process press, using water-soluble conducting ink Print working electrode, to electrode；

5）Rate activity is dissolved in for >=150u/mg glucose oxidase and is configured to concentration in pH7.0 phosphate buffer and is It is standby that 0 DEG C of refrigerator is shelved after 10mg/ml enzyme solutions, fully dissolving；

6）Using graphene/mesoporous carbon nano-composite material as carrier material, it is added in enzyme solutions, mixes 10~36h, It is standby for adsorbing glucose oxidase；

7）The Nafion filmogen solution that mass fraction is 0.5% is prepared, adds step 6）In obtained mixed solution, mixing 30min is stirred, biology enzyme electrically conductive ink is made, then working electrode surface is printed onto by the way of spraying, dries at room temperature 24h can complete immobilized and electrode the modification of enzyme, and biology sensor is made.

2. preparation method according to claim 1, it is characterised in that step 6）The middle nano combined material of graphene/mesoporous carbon The preparation process of material is：

Take 1.5~2g molecular guide agent to be dissolved in 10~30ml deionized water 10~30min of mixing, add 1~2ml's Concentration is that 1M HCl continues to stir；Sequentially add 30~50mg graphene and 1.5~2g beta-schardinger dextrin, stirring 0.5~ 1h, mixed liquor is then transferred in reactor 24~36h of heat treatment reaction at 120~150 DEG C, finally the nitrogen in tube furnace Under the protection of gas, 10 DEG C/min programming rate is to reacting 3~5h at 700~800 DEG C, you can nano composite material.

3. preparation method according to claim 2, it is characterised in that the graphene uses electrically conductive graphite raw material, adopts Prepared with electrophoresis method.