CN103616357A - Visual biosensor device and preparation method thereof - Google Patents

Abstract

The invention discloses a visual biosensor device and a preparation method thereof, combining the excellent photoelectric properties of graphene quantum dot multicolor fluorescence and its biological advantages, and preparing graphene with different sizes and different luminescent colors by using a wet chemical method For quantum dot samples, fully consider the chemical characteristics of the surface and edge of graphene quantum dots and the assembly of subsequent biomolecules, select appropriate chemical groups to functionalize the surface and edges of GQD, and assemble different biomolecules on different fluorescent graphene quantum dots. Molecules build biosensors. Biomolecules have different effects on GQD fluorescence, and there are quantitative correlations between characteristic parameters such as spectral intensity and peak position and the concentration and type of biomolecules. On this basis, direct visual molecular detection can be applied to biomedical detection and analysis. Provide more accurate reference for disease diagnosis and treatment; and facilitate the detection of various biomolecules.

Description

A visual biosensor device and its preparation method

technical field

The present invention belongs to the field of biosensing technology research, and specifically refers to a visual biosensor constructed with graphene quantum dots.

Background technique

Fluorescence detection technology has always been one of the important means in biomedical detection. In recent years, biomedical researchers have paid more and more attention to the excellent characteristics of semiconductor quantum dots, which are adjustable in size and spectrum, and have been discussed in molecular labeling, biological detection, medical imaging, etc., and many important progress have been made. Most of the work reported so far focuses on semiconductor quantum dots of CdSe, CdS and other systems. When these materials are applied in biomedical environments, they will encounter problems such as poor stability, biological toxicity, poor water solubility, and low cell membrane penetration. It is a very meaningful research work to design a luminescent system with stable luminescence, excellent biocompatibility, and linear response, and to develop corresponding optical or spectroscopic detection methods.

On the other hand, the excellent optical and electronic properties of graphene and its derivatives have been highly valued. Among them, graphene oxide (GO) has a mixed electronic configuration of sp2 and sp3, and the ratio of sp2 and sp3 is adjustable. This unique atomic and electronic structure is the function regulation of this two-dimensional network material, especially the regulation of photoelectric properties. And exploit provides a very rich and flexible physical basis. Based on this, reports on the fluorescence of graphene oxide and graphene quantum dots have been emerging in the past one or two years. At present, people have realized graphene quantum dots and graphene oxide with different luminous colors such as blue and green. It will be a very meaningful research topic to study the quantum mechanical mechanism of multicolor fluorescence of graphene quantum dots and the process technology of luminescence regulation, and to further expand its functions and applications in the fields of optoelectronics and bioelectronics. At the same time, graphene quantum dots also have a high specific surface area, abundant surface states, and excellent biocompatibility, which provides an excellent material science basis for the assembly and detection of biomolecules, making it a biosensor important candidate materials.

The enzyme biosensors in the prior art can not only realize the high selectivity and high sensitivity of the enzyme to the determination of the substrate, but also combine the characteristics of fast response and easy operation of the electrochemical biosensor, and can quickly determine a certain amount in the sample. Given compound concentration. However, there are many difficulties in the development of enzyme biosensors, such as the need to find a suitable immobilization material to immobilize the enzyme and achieve a short response time to a given compound and a long service life. The key technology of biosensor research is how to immobilize biological components on the substrate efficiently and stably. At present, quantitative biological analysis and visual sensor construction based on graphene quantum dot multicolor fluorescence are still rare in the world, especially two-photon fluorescence and time-resolved detection will become the unique perspective of the present invention, and it is believed that a series of innovations can be carried out In order to further expand the application of graphene in biomedical detection and imaging, and develop new biosensing materials and technologies.

Invention content

The purpose of the present invention is to provide a visual biosensor and its preparation method, to find a suitable immobilization material to immobilize the enzyme and achieve the purpose of short response time and long service life to a given compound, so as to further expand the graphene Applications in biomedical detection, imaging, etc.

In order to solve the above technical problems, the present invention integrates the excellent photoelectric properties of graphene quantum dot multicolor fluorescence and its biological advantages, analyzes the regulation method and physical mechanism of the scale, shape and luminescence characteristics of graphene quantum dots, and explores the biomolecular The assembly method, and based on fluorescence spectroscopy, time-resolved spectroscopy, two-photon fluorescence technology and other characterization techniques, build a visualized biosensor. The specific technical scheme is as follows:

A method for preparing a visualized biosensor device, characterized in that the fluorescent properties of graphene quantum dots are used to construct a visualized biosensor, so that the visualized biosensor device can directly obtain the concentration of a substance to be detected by using visible fluorescence changes, specifically comprising the following steps :

Step 1, preparing graphene quantum dots of different sizes and different fluorescence, the specific process is as follows

Process 1, using wet chemical method, using flake graphite as raw material, adopting Hummers method to prepare graphite oxide: assemble a reaction bottle in an ice-water bath, add 46ml of 98% concentrated sulfuric acid, add 2g flake graphite and 1g nitric acid under magnetic stirring Slowly add 6g of potassium permanganate to the sodium solid mixture, control the temperature at 10-15°C, and stir for 1.5h; then place the beaker in a constant temperature water bath at 35°C, add deionized water during stirring, and continue stirring for 30min; then control React at 95°C for 30 minutes to obtain reaction solution 1; dilute reaction solution 1 to 200ml with deionized water, add an appropriate amount of hydrogen peroxide, stir to speed up the reaction, and centrifuge at 4000rpm for 10 minutes after the reaction to obtain reaction solution 2; The reaction solution 2 was repeatedly washed with hydrogen peroxide, dilute hydrochloric acid and deionized water to make the pH = 7, then dried at 80°C for 48 hours, ground and sieved to obtain graphite oxide, and stored in a dry box;

Process 2: Weighing graphite oxide and dispersing it in water of equal mass, ultrasonically forming a yellow transparent colloidal solution, then adding dilute hydrochloric acid, centrifuging and drying into powder to obtain graphene oxide powder;

Process 3: thermally reduce the graphene oxide powder to obtain graphene sheets, oxidize and ultrasonically decompose the graphene sheets with 30ml of concentrated nitric acid and 10ml of concentrated sulfuric acid for 15 hours to obtain solution 1; remove the acid by centrifuging solution 1, and then dissolve in water again to obtain a solution 2. Use NaOH to adjust the pH value of solution 2 to be greater than or equal to 10, perform hydrothermal reduction at 200°C for 10 hours in a reaction kettle, and filter with a microporous membrane to obtain liquid graphene quantum dots;

Step 2, select chemical groups to functionalize the graphene quantum dot surface and edge, and analyze the impact of the process of functionalization on the fluorescence of graphene quantum dots, the specific process is as follows:

Process 1, take 20mL of the graphene quantum dot solution and adjust the pH value to 5-6 with HAc;

In process 2, add 50mg of alkylamine and 20mL of toluene, mix well and put it in a 50mL reactor for 6 hours at 150°C to obtain solution 3;

Process 3, absorb the upper toluene solution of solution 3, and vacuum dry at 80°C to remove excess alkylamine. The -COO-functional group on the surface of the water-soluble graphene quantum dots makes it have good water solubility and is easy to carry out Surface functionalization;

Step 3, build a visualized biosensor, the process is as follows

Assembling fluorescent graphene quantum dots with biomolecules to construct biosensors: After the functionalization of graphene quantum dots, the functional groups will have very good interactions with biomolecules, including hydrogen bonds and π-π conjugation, thus combining Together, the fluorescent graphene quantum dots and biomolecules are assembled on the electrode to form a recognition element, and a biosensor is formed with a physical and chemical transducer and a signal amplification device.

The biomolecule is any one of glucose oxidase, CEA, CA19-9, DNA fragment and protein.

The chemical group is alkylamine or dodecylamine.

The concentration of the hydrogen peroxide is 4-6%.

A visualized biosensor device, characterized in that: the biosensor device is prepared by using the method for preparing a visualized biosensor device described in claims 1-4.

The working principle of the present invention is: the biomolecules in the biosensor can be combined with the measured target to form a complex through the recognition process, such as the combination of antibodies and antigens, and the combination of enzymes and substrates. These biomolecules have an impact on the fluorescence of graphene quantum dots . According to the change of fluorescence, the information of the measured target can be obtained directly.

The invention has beneficial effects . The invention directly uses the fluorescence information of graphene quantum dots to detect molecules, and can quickly and conveniently obtain the information of target molecules; the use of graphene quantum dots to construct visualized molecular detection and biosensing technology helps to deeply understand some life activities. To understand the pathogenesis of various diseases, promote the development of diagnosis and treatment technology, and then promote and maintain human health. The invention will promote the development of photoelectric properties of graphene and the expansion of biomedical application functions, and develop a new visual biological detection method, thereby providing an important reference for disease diagnosis and treatment.

Description of drawings

Fig. 1 is the TEM figure of graphene quantum dot of the present invention.

Fig. 2 is a schematic structural diagram of the substance recognition element of the biosensor of the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto, and the process parameters and methods not specifically indicated can be carried out according to conventional techniques.

Embodiment 1 : the preparation of graphene quantum dot

The specific preparation process of graphene quantum dots in this embodiment is as follows: take flake graphite as raw material, prepare graphite oxide according to Hummer's method, then disperse 25 mg graphite oxide in 25 mL water, ultrasonically disperse into a uniform yellow transparent colloidal solution, and The solution was centrifuged with dilute hydrochloric acid and dried into powder. The graphene oxide powder is subjected to thermal reduction of graphene oxide sheets in a tube furnace at 200°C for 2 hours, and 0.05 g of thermally reduced graphene sheets is oxidized and ultrasonically decomposed with 30 ml of concentrated nitric acid and 10 ml of concentrated sulfuric acid for 15 hours, and the pH of the solution is adjusted to 8 , the graphene quantum dots were prepared by hydrothermal reduction at 200°C for 10h in a reactor, and the TEM image of the obtained graphene quantum dots is shown in Figure 1.

Embodiment 2 : Taking glucose oxidase as an example to construct a biosensor

Spin-coat 10 μL of the functionalized graphene quantum dot solution onto the surface of the glassy carbon electrode, dry overnight at room temperature, then activate the electrode surface, and finally spin-coat 10 μL of the glucose oxidase suspension onto the electrode surface to obtain the enzymatic The substance recognition element of the sensor has a structure as shown in Figure 2, and the enzyme biosensor can be constructed.

Claims (5)

1. the preparation method of a visual bio-sensing device, it is characterized in that utilizing the fluorescent characteristic of graphene quantum dot to build visual biology sensor, thereby make visual bio-sensing device utilize visible change in fluorescence directly to obtain the concentration of material to be detected, specifically comprise the following steps:

Step 1, prepares the graphene quantum dot of different size, different fluorescence, and detailed process is as follows

Process one, adopt wet chemical method, take crystalline flake graphite as raw material, adopt Hummers legal system for graphite oxide: in ice-water bath, to assemble reaction bulb, add in the concentrated sulphuric acid of 46ml 98%, under magnetic agitation, add 2g crystalline flake graphite and 1g sodium nitrate solid mixture, slowly add 6g potassium permanganate, control temperature 10-15 ℃, stirring reaction 1.5h; Again beaker is placed in to 35 ℃ of waters bath with thermostatic control, in whipping process, adds deionized water, continue stirring reaction 30min; Control temperature at 95 ℃, reaction 30min obtains reactant liquor one again; With deionized water, reactant liquor one is diluted to 200ml, adds appropriate hydrogen peroxide, stir and to add fast response, with the rotating speed centrifugal treating 10min of 4000rpm, obtain reactant liquor two after waiting reaction to finish; Use hydrogen peroxide, watery hydrochloric acid and deionized water cyclic washing reactant liquor two, make PH=7 again, then be dried 48h under 80 ℃ of conditions, and grinding is sieved, and obtains graphite oxide, is placed in drying box and preserves;

Process two, takes in the water of the quality such as graphite oxide is scattered in, ultrasonic yellowly transparent colloidal solution, then add watery hydrochloric acid, after centrifugal treating, be dried to powder and obtain graphene oxide powder;

Process three, carries out thermal reduction by graphene oxide powder and obtains graphene film, and graphene film is obtained to solution one with 30ml red fuming nitric acid (RFNA) and 10ml concentrated sulphuric acid oxidation ultrasonic decomposition 15h; By solution one centrifugal filtration except disacidify, solution two soluble in water again, by the pH value of NaOH regulator solution two, to being more than or equal to 10, in reactor, 200 ℃ are carried out 10h hydrothermal reduction, with filtering with microporous membrane, obtain liquid graphene quantum dot;

Step 2, selects chemical group to process graphene quantum dot surface and limbic functionization, and the impact of the process of analytic functionization processing on graphene quantum dot fluorescence, and detailed process is as follows:

Process one, gets graphene quantum dot solution described in 20mL, and with HAc, being adjusted to pH value is 5-6;

Process two, then add 50mg alkyl amine and 20mL toluene, after mixing, put into 150 ℃ of reaction 6h of 50mL reactor and obtain solution three;

Process three, the upper toluene solution of draw solution three, under 80 ℃ of conditions, vacuum drying is removed unnecessary alkyl amine, water-soluble graphene quantum dots surface-COO-functional group makes it have good water-solublely, and easily to it, carries out surface-functionalized;

Step 3, builds visual biology sensor, and process is as follows

The assembling of fluorescence graphene quantum dot and biomolecule is built to biology sensor: after graphene quantum dot functionalization with functional group's meeting and biomolecule between produce extraordinary interaction and comprise hydrogen bond, π-πconjugation, thereby combine, fluorescence graphene quantum dot and biomolecule are assembled on electrode, form recognition component, form biology sensor with physics and chemistry transducer, signal amplifying apparatus.

2. the preparation method of a kind of visual bio-sensing device as claimed in claim 1, is characterized in that: described biomolecule is glucose oxidase, CEA, CA19-9, any in DNA fragmentation and albumen.

3. the preparation method of a kind of visual bio-sensing device as claimed in claim 1, is characterized in that: described chemical group is alkyl amine or lauryl amine.

4. the preparation method of a kind of visual bio-sensing device as claimed in claim 1, is characterized in that: the concentration of described hydrogen peroxide is 4-6%.

5. a visual bio-sensing device, is characterized in that: described bio-sensing device is utilize preparation method's preparation of a kind of visual bio-sensing device described in claim 1-4 and obtain.

Images