CN106770031A - A kind of preparation method of the graphene biosensor for specific proteins detection - Google Patents

Abstract

The invention relates to a preparation method of a graphene biosensor for specific protein detection. The graphene in the specific protein biosensor is prepared by a high-temperature thermal annealing method under an inert atmosphere. The invention designs and prepares a functional graphene film, and makes a graphene-specific protein biosensor. Tests have shown that this specific protein biosensor can detect specific proteins in real time without labeling, and can clearly see the impact of changes in the concentration of the target protein. This graphene biochip has great application prospects in biomolecular sensors and biomolecular detection.

Description

A preparation method of graphene biosensor for specific protein detection

technical field

The invention relates to the technical field of preparation of graphene sensing and biosensors, in particular to a graphene film-specific protein biosensor and a preparation method thereof.

Background technique

Proteins are the executors of life functions and contain important biological information. Protein-protein interactions play an important role in the process of biomolecular interactions. The interaction process between drugs and proteins is the main process of disease treatment, and the biological dynamic information at the molecular level is very important for the research of drug screening. Specific protein biosensors use various changes (binding, dissociation, elution) caused by specific reactions between proteins to record and analyze the reactions between the probe protein and the probe protein, and This reaction is converted into detectable light, electricity, force and other signals to complete the detection and monitoring of the probe protein.

Existing methods for detecting protein interactions are mainly divided into two categories: labeling and label-free methods. The labeling methods include fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assay (ELISA), mass spectrometry, yeast two-hybrid, tandem affinity analysis, and phage display technology. However, labeling proteins may change the structural characteristics of the protein and reduce the activity of the protein, and the difference in the labeling efficiency of different proteins will increase the difficulty of quantitative detection. Moreover, the protein labeling process is time-consuming and laborious. In addition, methods such as yeast two-hybrid, ELISA, and fluorescently-labeled protein chips are all end-point signal methods, which can only detect the equilibrium state of biomolecular interactions, but cannot detect the dynamic change process of biomolecular interactions. Therefore, label-free detection has attracted more attention in the process of studying protein interactions. Label-free methods mainly include microcantilever method, quartz crystal microbalance, atomic force microscopy, isothermal titration microcalorimetry and surface plasmon resonance (SPR) technology. Due to the real-time, label-free and highly sensitive detection of biomolecular interactions, SPR technology is widely used to study biomolecular (including but not limited to protein-protein) interactions.

SPR technology is an optical sensing technology, which not only has the advantages of no labeling and sensitivity, but also can detect the reversible dynamic process of binding and dissociation of biomolecular interactions in real time, and obtain the specificity, affinity and kinetics of intermolecular binding. It is an ideal technique for studying biomolecular interactions. However, the SPR instrument is expensive, and because of the expensive chip that needs to be replaced frequently, the subsequent use cost becomes more expensive, which brings a serious burden to users and enterprises.

Since graphene was first stripped out, the application of graphene and its derivatives (graphene oxide, reduced graphene oxide, etc.) in the field of life sciences, especially in the detection of proteins, has gradually increased. Graphene has excellent physical, chemical and structural properties, especially graphene oxide contains a large number of oxygen-containing functional groups, and has good biocompatibility. These properties of graphene have aroused great interest of researchers. Currently, graphene-based biosensors have been used to detect various biomolecules (DNA, RNA, and proteins, etc.) and cells.

Under the condition of total internal reflection, graphene has polarization-dependent absorption characteristics, so that the absorption of graphene to S light is much greater than that of P light. More importantly, this characteristic of graphene is sensitive to the medium close to graphene. of. When the medium close to graphene changes, the sensor prepared by using this characteristic of graphene can detect this change in real time. After the protein to be tested is introduced, the specific binding between the probe protein and the probe protein can be detected in real time. This detection method consumes less sample, does not require protein labeling, and has high sensitivity.

Contents of the invention

The object of the present invention is to propose a method for detecting specific proteins (including but not limited to) with a graphene-based sensor, which has the characteristics of real-time, label-free, and high sensitivity.

For this reason, the present invention adopts following technical scheme:

A preparation method for a graphene biosensor for specific protein detection, comprising the following steps:

1) Preparation of graphene:

First, clean and process the quartz sheet; secondly, 4 mg/ml graphene oxide aqueous solution is spin-coated onto the surface of the cleaned and treated quartz substrate, and spin-coated for 3 times; Under the atmosphere of a mixed gas of hydrogen (95% argon, 5% hydrogen), thermal annealing at 800 degrees for 1 hour to obtain a graphene film with a uniform thickness of ~8nm;

2) Surface treatment of graphene:

Oxygen plasma (or graphene oxide solution) is used to treat the graphene film on the quartz substrate, so that the surface of the graphene film contains abundant oxygen-containing functional groups, 1-ethyl-3-(3-trimethylaminopropyl) carbon dioxide A mixed solution of imine hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was used to activate the oxygen-containing functional groups on the surface of the graphene film;

3) Immobilization of probe protein:

Add the solution containing the probe protein to the graphene surface, react for a period of time at room temperature, couple a layer of probe protein on the graphene surface, and then rinse with PBS (phosphate buffer saline);

4) closed

Immerse the chip in BSA (bovine serum albumin) solution, place it at room temperature for a period of time, seal the residual carboxyl groups on the surface of the chip, and then wash it with PBS, then it can be used for the refractive index sensor based on the polarized absorption effect of graphene under the condition of total internal reflection detection.

Preferably, in the step 1), the cleaning of the quartz substrate specifically includes: ultrasonically cleaning the quartz substrate with acetone, isopropanol, and ultrapure water, respectively, and blowing it dry with nitrogen. The treatment of the quartz substrate is as follows: the quartz substrate is cleaned with oxygen plasma, the power of the machine is: 150W; the flow rate of oxygen used is: 300-400ml/min; the processing time: 1 minute.

Preferably, the size of the quartz substrate is 20mm×20mm×1mm.

Preferably, the preparation of the graphene oxide solution in the step 1) graphene preparation is specifically: dissolving 20 mg of graphene oxide powder in 5 ml of deionized aqueous solution, and then ultrasonically dispersing for 1 hour. After ultrasonic dispersion, centrifuge at 3500 rpm for 1 hour.

Preferably, the oxygen plasma treatment time in step 2) is 15 seconds.

Preferably, the concentration of EDC in step 2) is 0.4 mol/l, and the concentration of NHS is 0.1 mol/l.

Preferably, the probe protein in the immobilization of the probe protein in step 3) is rabbit immunoglobulin (including but not limited to).

Preferably, the concentration of PBS in the fixation of the probe protein in step 3) is 0.01mol/L, and the pH value is 7.4.

Preferably, the concentration of BSA used for blocking in step 4) is 10 mg/ml.

On the other hand, the present invention also discloses the detection method of the optical sensor based on the graphene polarization absorption effect under the condition of total internal reflection, comprising the following steps:

1) Attach the fluid cell to the side containing the probe protein graphene, then attach the graphene substrate with the fluid cell to one side of the equilateral prism with the aid of refractive index matching oil, and finally attach the It is installed in the measuring light path;

2) The measurement system uses a laser with a wavelength of 632.8nm as the light source. After passing through the polarizer, the lens is used to focus on the graphene containing the probe protein. Under the condition of total internal reflection, the reflected light is divided into P The light and the S light are respectively incident on the two probes of the balance detector, and the real-time signal acquisition and comparison are completed by a computer;

3) Baseline: inject buffer into the fluid cell;

4) Binding: the syringe pump is used to inject the solution containing the protein to be tested into the fluid pool, and the protein to be tested and the probe protein react specifically;

5) Dissociation: After the specific binding is completed, the buffer is injected into the fluid pool;

6) Regeneration: After the measurement is completed, the eluent and buffer are sequentially injected into the fluid cell.

The flow rate was maintained at 30 μL/min throughout the experiment. When detecting specific protein interactions, use the baseline as the zero point to measure protein association and dissociation. The function of the eluent is to completely release the specific protein from the surface of the chip, and the chip returns to its original state.

The present invention adopts the above technical scheme, the specific combination of the protein to be tested and the probe protein will change the refractive index of the graphene and the solution in contact with it, thereby causing the polarization-dependent absorption of the graphene to change, and finally leading to the balanced detector received The signal changes and the protein of interest is detected.

Description of drawings

Fig. 1 is the schematic diagram of the graphene biosensor adopted in the present invention.

Fig. 2 is the detection optical path system used in the present invention.

Fig. 3 is the immobilization process of the probe protein in the present invention.

Fig. 4 is the real-time kinetic process when the present invention detects protein to be tested;

Fig. 5 is the response curve of the present invention to detect different concentrations of the protein to be tested.

Fig. 6 is the specificity result of detecting different proteins in the present invention.

Fig. 7 is the repeatability result of detecting the minimum concentration of the present invention.

Fig. 8 is a comparison chart of the detection results of different concentrations of the protein to be tested according to the present invention and the detection results of the commercial BIA core X100 SPR sensor.

detailed description

In order to further illustrate the present invention, the method for specific protein detection provided by the present invention will be described in detail below in the form of drawings and examples, but it should not be construed as limiting the protection scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually in accordance with conventional conditions or conditions suggested by the manufacturer. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention.

The following will be further explained in conjunction with the examples of implementation.

Example 1

The preparation of graphene, its specific technical scheme is as follows:

1) Cleaning and processing of quartz slices:

The cleaning of the quartz substrate is specifically as follows: the quartz substrate is ultrasonically cleaned with acetone, isopropanol, and ultrapure water respectively, and dried with nitrogen. The treatment of the quartz substrate is as follows: the quartz substrate is cleaned with oxygen plasma, the power of the machine is: 150W; the flow rate of oxygen used is: 300-400ml/min; the processing time: 1 minute.

2) Preparation of graphene:

The graphene oxide aqueous solution of 4mg/ml is spin-coated to the quartz substrate surface that cleans and handles, spin-coats 3 times, then it is put into tube furnace, in the mixed gas of argon and hydrogen (95% argon gas, 5% hydrogen) atmosphere, 800 degrees of thermal annealing for 1 hour, to obtain a graphene film with a uniform thickness of ~ 8nm; Fig. 1 is the result of the thickness of our graphene film prepared by atomic force detection.

Example 2

Graphene polarization-dependent absorption sensor, its specific embodiment is as follows:

As shown in Figure 2, the graphene polarization-dependent absorption biosensor mainly consists of an optical system and a sensing system composed of a sandwich structure of prism-graphene/quartz sheet-fluid pool. Index matching oil was used to bond the graphene/quartz sheet to the prism. The red light emitted by the 633nm laser is adjusted to strictly linearly polarized light by the polarizer, and then focused onto the surface of the prism/graphene through the lens, where total reflection occurs at the graphene interface. A polarization beam splitter divides the reflected light into S light and P light, and a balanced detector is used to detect the separated P light and S light. When the protein to be tested in the solution reacts specifically with the probe protein on the graphene surface, the graphene’s absorption of P light and S light changes due to the polarization-dependent absorption characteristics of graphene, and the real-time signal changes are detected by the computer. Acquisition, the kinetic process (binding and dissociation) of the specific reaction of the test protein and the probe protein is presented by the computer in real time.

Example 3

The fixation of probe protein, its specific technical scheme is as follows:

First, oxygen plasma is used to treat the graphene surface prepared in Example 1, and the treatment time is 15 seconds, so that the graphene surface layer contains more oxygen-containing functional groups, and secondly, the mixed solution of EDC/NHS is used to activate graphene Oxygen-containing functional groups on the surface, then, as shown in Figure 3, PBS was used to wash the graphene surface, and then the protein (goat anti-rabbit IgG) used as a probe was injected into the graphene surface, and the probe protein was covalently The binding method was immobilized onto the graphene surface, and then, PBS was used to flush the fluidic cell.

Example 4

For the real-time detection of the kinetic process of the protein to be tested, the specific technical scheme is as follows:

As shown in Figure 4, first, PBS is injected into the fluid cell as the baseline (zero point) for detection; secondly, the solution containing the protein to be tested is injected into the fluid cell, and the protein to be tested is continuously combined with the probe protein on the graphene surface , the signal detected by the equilibrium probe changes accordingly, and the specific binding process between proteins is recorded in real time; again, PBS is injected into the fluid pool, and part of the protein to be tested is dissociated from the surface of the probe protein; with the glycine After introduction, the protein to be tested bound to the probe protein is eluted; finally, PBS is injected into the fluid cell again to complete the regeneration of the chip and the kinetic test of the protein to be tested.

Example 5

Sensitivity analysis of graphene biosensor to detect specific protein, its specific technical scheme is as follows:

Using a syringe pump, inject different concentrations of the protein solution to be tested (0.0625, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg/ml) into the fluid pool in sequence, the protein to be tested in the solution and the probe protein on the chip A specific reaction occurs, which causes the graphene to change the absorption of P-polarized light and S-polarized light, and finally causes the light intensity signal received by the balance detector to change. It can be seen from Figure 5 that as the concentration of the protein to be tested increases, the response of the detector increases (positive correlation), and the response sensitivity of the sensor to the protein to be tested is 0.0625 μg/ml. Figure 7. In order to prove the authenticity of the detection of the minimum concentration, we repeated the detection results of the minimum concentration. In Fig. 8, we compared the detection results of the graphene biosensor and the commercial BIAcore 100X SPR sensor. The detection sensitivity of the commercial BIAcore 100X SPR sensor was 0.625 μg/ml, which was lower than that of the graphene refractive index sensor of the present invention.

Example 6

The specificity detection of graphene biosensor, its specific scheme is as follows:

Rabbit IgG and bovine IGM at a concentration of 100 μg/ml were injected into the fluid pool using a syringe pump to react with the probe protein (goat anti-rabbit IgG), repeated 5 times. The specificity results are shown in Figure 6. Bovine IGM cannot specifically react with goat anti-rabbit IgG molecules, so the signal is very weak, while rabbit IgG specifically binds goat anti-rabbit IgG molecules, and a strong sensing signal is detected , indicating that the graphene glass chip in the present invention has the function of specific detection.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (8)

1. it is a kind of for specific proteins detection graphene biosensor preparation method, it is characterised in that：Including following Step：

1) preparation of Graphene：

Graphene oxide water solution is spun onto the quartz substrate surface cleaned and handle well；Then in putting it into tube furnace, Under the atmosphere of argon gas and the gaseous mixture of hydrogen, 800 degree of thermal annealings obtain the graphene film of in uniform thickness~8nm.

2) surface treatment of Graphene：

The graphene film in quartz substrate is processed using oxygen plasma (or graphene oxide solution), makes Graphene thin Film surface includes abundant oxygen-containing functional group, 1- ethyls -3- (3- front threes aminopropyl) carbodiimide hydrochlorides (EDC) and N- hydroxyls The mixed solution of succinimide (NHS) is utilized to activate the oxygen-containing functional group on graphene film surface；

3) fixation of probe proteins：

Solution containing probe proteins is added to graphenic surface, a period of time is reacted under room temperature condition, in graphenic surface coupling One layer of probe proteins of connection, are then rinsed with PBS (phosphate buffer)；

4) close：

Chip is immersed in BSA (bovine serum albumin) solution, is placed at room temperature a period of time, the carboxylic of closing chip surface residual Base, after then being rinsed with PBS, you can for the optical pickocff based on the Graphene polarization absorption effect under total internal reflection condition Detection.

2. the preparation method of a kind of graphene biosensor for specific proteins detection as claimed in claim 1, its Be characterised by, the step 1) in quartz substrate size be 20mm × 20mm × 1mm.

3. the preparation method of a kind of graphene biosensor for specific proteins detection as claimed in claim 1, its Be characterised by, the step 1) in prepared by Graphene the preparation of graphene oxide solution be specially：By the graphene oxide of 20mg Powder is dissolved in the deionized water solution of 5ml, then carries out ultrasonic disperse 1 hour.After ultrasonic disperse, 3500rpm is centrifuged 1 hour.

4. the preparation method of a kind of graphene biosensor for specific proteins detection as claimed in claim 1, its Be characterised by, the step 2) in oxygen plasma treatment time be 15 seconds.

5. the preparation method of a kind of graphene biosensor for specific proteins detection as claimed in claim 1, its Be characterised by, the step 2) in EDC concentration for 0.4mol/l, the concentration of NHS is 0.1mol/l.

6. the preparation method of a kind of graphene biosensor for specific proteins detection as claimed in claim 1, its It is characterised by, the step 3) probe proteins in the fixation of middle probe albumen are for rabbit immunoglobulin (including but limits to In).

7. the preparation method of a kind of graphene biosensor for specific proteins detection as claimed in claim 1, its It is characterised by, the step 3) concentration of PBS is 0.01mol/L in the fixation of middle probe albumen, pH value is 7.4.

8. the preparation method of a kind of graphene biosensor for specific proteins detection as claimed in claim 1, its Be characterised by, the step 4) in the concentration of BSA of closing be 10mg/ml.