CN109752347B

CN109752347B - Method for analyzing interaction between single carbon nanotube and protein

Info

Publication number: CN109752347B
Application number: CN201910155812.6A
Authority: CN
Inventors: 刘贤伟; 朱丽萍
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2021-04-23
Anticipated expiration: 2039-03-01
Also published as: CN109752347A

Abstract

The invention belongs to the field of analysis of biomolecular interaction, and discloses a method for analyzing interaction between a single carbon nanotube and protein. The analysis method provided by the invention is based on the surface plasmon resonance imaging (SPRM) technology, so that the complexity of fluorescence labeling and sample preparation can be avoided, and the real-time label-free analysis imaging of the in-situ action of different single carbon nanotubes and protein can be realized.

Description

Method for analyzing interaction between single carbon nanotube and protein

Technical Field

The invention belongs to the field of research on interaction of biomolecules, particularly relates to a method for analyzing interaction of a single carbon nanotube and protein, and particularly relates to a method for analyzing interaction of a single carbon nanotube and protein based on a surface plasmon resonance imaging (SPRM) technology.

Background

The carbon nanotube is also called buckytubes, and is a one-dimensional nano material with a special structure (the radial dimension is nano-scale, the axial dimension is micron-scale, and two ends of the tube are basically sealed). Carbon nanotubes are coaxial circular tubes consisting of several to tens of layers of carbon atoms arranged in a hexagonal pattern. The carbon nanotube can be regarded as a graphene sheet layer which is curled, and thus the number of layers of the graphene sheet can be divided into: in the single-walled carbon nanotube and the multi-walled carbon nanotube, when the multi-walled tube is formed, the layers are easy to become trap centers to capture various defects, so that the tube wall of the multi-walled tube is usually full of small-hole-like defects. The unique structure of the carbon nano tube enables the carbon nano tube to have a plurality of excellent functions, such as large specific surface area, high strength, good toughness, small density, excellent heat conductivity and electric conductivity and the like. Due to its excellent properties, carbon nanotubes are widely used in the fields of mechanics, energy, nano devices, electronic devices, sensors, catalysis, etc., and among them, industrialization has been achieved in the field of supercapacitors. The carbon nano tube has a unique hollow structure, a nano tube diameter and excellent cell penetrating performance, and with the rise of nano drugs, the carbon nano tube is often used as a carrier of the drugs to carry bioactive molecules into cells to be carried into a human body without generating toxicity.

The stable dispersion of the carbon nano tube in the aqueous solution and the action of the carbon nano tube with protein molecules are important research contents for realizing the biomedical application of the carbon nano tube, and the basic problems of specific action and the like of nano materials and proteins are often related, and the biological safety problem of the nano materials is also radiated, so that the research on the interaction of the carbon nano tube and blood proteins in human bodies is very important.

At present, methods for detecting the interaction between protein and carbon nanotube mainly include Scanning Electron Microscope (SEM) imaging technology, Atomic Force Microscope (AFM) imaging technology, and total internal reflection fluorescence microscope (TIRF) imaging technology. However, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) can only image but cannot obtain the binding kinetics curve, and finally cannot obtain the information related to the binding rate constant, the dissociation rate constant, and the like. However, the total internal reflection fluorescence microscope (TIRF) requires a fluorescence labeling method, which often results in a complicated sample preparation process, and the fluorescence labeling interferes with the normal sample condition, which is not favorable for in situ observation. In addition, the fluorescent label often has a phenomenon of quenching and bleaching, and cannot be observed for a long time.

Disclosure of Invention

In view of the above, the present invention provides a method for detecting interaction between a single carbon nanotube and a protein based on a surface plasmon resonance imaging (SPRM) technique, which can avoid the complexity of fluorescence labeling and sample preparation, and implement real-time label-free detection imaging of in-situ interaction between different single carbon nanotubes and proteins.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

a method for detecting interaction between a single carbon nanotube and protein comprises the steps of pretreating the carbon nanotube, spin-coating the pretreated carbon nanotube on a monolayer modified plasma resonance sensing chip, dispersing a protein solution around the carbon nanotube, and determining a binding kinetics curve of the protein solution and the single carbon nanotube on the plasma resonance sensing chip by using the change of surface plasma resonance light intensity.

Preferably, the method for detecting the interaction of the single carbon nanotube and the protein comprises the following steps:

1) the carbon nano tube is pretreated and then is spin-coated on the monolayer modified plasma resonance sensing chip, and a polydimethylsiloxane reaction tank is covered;

2) adding PBS into a polydimethylsiloxane reaction tank on the plasma resonance sensing chip under a 60X lens, collecting an SPR image, and establishing a standard baseline;

3) adding a protein solution into a polydimethylsiloxane reaction tank on the plasma resonance sensing chip after the base line is stable, and continuously collecting images to obtain corresponding combined images;

4) when the combination reaches the vicinity of the maximum platform, adding PBS into the polydimethylsiloxane reaction tank on the plasma resonance sensing chip again, and collecting an SPR image to obtain a corresponding dissociation image;

5) and obtaining an association rate constant and a dissociation rate constant by calculating an association dissociation curve according to the association image and the dissociation image.

Preferably, the pretreatment in the step 1) is ultrasonic treatment of carboxyl multi-walled carbon nanotubes obtained by carboxyl modification in an ethanol solution, filtration with a water-phase filter membrane, and taking the filtered supernatant. More preferably, the pore size d of the aqueous phase filter membrane is 0.22 μm.

Preferably, the plasmon resonance sensing chip in the step 1) is composed of a base material, a chromium layer and a gold layer which are sequentially arranged. In some embodiments, the substrate material is ordinary glass; the thickness of the chromium layer is 2-3nm, and the thickness of the gold layer is 40-50 nm. In a specific embodiment, the preparation method of the plasma resonance sensing chip comprises the steps of plating a cadmium layer with the thickness of 2-3nm on a glass substrate, and plating a gold film with the thickness of 40-50 nm.

Preferably, the monomolecular layer modified in the step 1) is obtained by modifying the surface of the plasma resonance sensing chip with SH-carrying PEG-OH, so that the interference of nonspecific binding shielding protein on the surface of the gold film on the gold film can be inhibited. In some embodiments, the monolayer on the plasmon resonance sensing chip is prepared by immersing the plasmon resonance sensing chip in a 1mM PEG-OH solution at 0 ℃ for 24h, so that a uniform monolayer is formed on the surface of the plasmon resonance sensing chip.

The method for detecting the interaction between a single carbon nanotube and a protein comprises the following steps of 2) adding PBS into a polydimethylsiloxane reaction pool on a plasma resonance sensing chip, and establishing a standard base line.

Wherein the PBS has a pH of 7.

Preferably, the Intensity of the light Intensity of the SPR at the time of acquiring the SPR image of step 2) is at 1/3Intensity MAX.

The method for detecting the interaction between a single carbon nanotube and protein comprises the step 3) of adding a protein solution into a polydimethylsiloxane reaction pool on a plasma resonance sensing chip to enable the carbon nanotube and the protein to be subjected to non-specific combination so as to obtain a combined image.

Preferably, the protein solution in step 3) is a PBS solution containing bovine serum albumin or wheat germ agglutinin.

In some embodiments, the protein solution has a concentration of bovine serum albumin or wheat germ agglutinin of 0.01mg/mL, 0.1mg/mL, or 1 mg/mL.

Preferably, the speed of acquiring the image in the step 3) is 106 fps.

The method for detecting the interaction between a single carbon nanotube and the protein comprises the step 4) of adding PBS into a polydimethylsiloxane reaction tank on the plasma resonance sensing chip, so that the protein adsorbed on the carbon nanotube is partially dissociated to obtain a corresponding dissociation image.

Furthermore, as a preferable mode, the method adopts an automatic drug delivery system to add PBS or protein solution into a polydimethylsiloxane reaction pool on the plasma resonance sensing chip, and rapid drug delivery can be realized.

The structure of the automatic drug delivery system is shown in figure 1. And (3) putting PBS into a container 1, opening a valve 1, and adding a PBS solution into a polydimethylsiloxane reaction tank on the plasma resonance sensing chip to obtain the stage I. And then quickly closing the valve 1, simultaneously opening the valve 2, and adding a protein solution into a polydimethylsiloxane reaction tank on the plasma resonance sensing chip to obtain the stage II. And finally, quickly closing the valve 2, quickly opening the valve 1, and adding the PBS solution into the polydimethylsiloxane reaction tank on the plasma resonance sensing chip again to obtain the stage III.

The automated drug delivery system can be tuned to a position 2 μm above the carbon nanotubes (MWNTs) with a response switching time of around 1 s.

Preferably, the flow rate of the PBS or protein solution is 1 s/drop.

Preferably, the calculation method in step 5) is to read the SPR Intensity on the carbon nanotubes by using Image J, and the SPR Intensity is recorded as SPR Intensity_MWNT-COOH(ii) a Surface strength of PEG-OH modified gold film, recorded as SPR Intensity_PEG-OH(ii) a Then SPR Intensity is used_MWNT-COOHMinus SPR Intensity_PEG-OHObtaining SPR Intensity with background interference being deducted_MWNT-COOHThen, using Matlab to remove noise and smooth to obtain the binding dissociation curve of the carbon nano tube and protein, substituting the binding part of the binding dissociation curve into formula (1), substituting the dissociation part into formula (2), and combining formula (1) and formula (2) to obtain K of the carbon nano tube_a、K_dThen, K of the carbon nanotube can be obtained from the formula (3)_D。

K_D＝K_a/K_d(3)

Wherein R is_tRepresenting the time-varying carbon nanotube SPR Intensity_MWNT-COOH，R_maxSPR Intensity representing the maximum of carbon nanotubes_MWNT-COOH，[A]Represents the concentration of protein BSA, t represents time, K_aDenotes the binding rate constant, K_dDenotes the dissociation rate constant, K_DIndicating the affinity of the carbon nanotubes to the protein.

Compared with the prior art, the invention has at least one of the following beneficial effects:

(1) the detection method is simple, convenient, real-time and efficient, and can carry out real-time in-situ monitoring only by injecting the protein around the carbon nano tube through a drug delivery system;

(2) the detection method can image the whole combination dissociation process of the protein and the carbon nano tube in real time and obtain a combination dissociation curve corresponding to each carbon nano tube;

(3) the preparation method of the plasma resonance sensing chip is simple;

(4) the detection protein in the detection method of the invention does not need fluorescent labeling, and the sample preparation is convenient and simple.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a diagram of a drug delivery system device;

FIG. 2 shows a diagram of a device for surface plasmon resonance imaging microscopy (SPRM) in combination with a drug delivery system;

FIG. 3 shows the Atomic Force Microscopy (AFM) characterization of the carboxyl multi-walled carbon nanotubes (MWNT-COOH) all in the diameter range of 15-30nm, which can be confirmed as single dispersed on the SPR sensing chip;

FIG. 4 shows the binding kinetics curves of single carbon tubes (No.. c.) (MWNT-COOH) obtained from three different carboxyl multi-walled carbon nanotubes (MWNT-COOH) in example 1 under the bright field at concentrations of 0.01mg/mL, 0.1mg/mL, and 1mg/mL, respectively;

FIG. 5 shows kinetic binding curves of single numbered (i) carboxy multi-walled carbon nanotubes (MWNT-COOH) selected from example 1 at three different concentrations at 0.01mg/mL, 0.1mg/mL, 1mg/mL and Bovine Serum Albumin (BSA);

FIG. 6 shows the binding kinetics curves of three different (No. (-) (carboxy multi-walled carbon nanotubes) (MWNT-COOH) in the open field of example 2 and a single (No. (-) (C.)) carbon tube obtained from phytohemagglutinin (WGA) with an optimal concentration of 0.1 mg/mL;

FIG. 7 shows a curve fit to a single (code. phi.) selected carboxyl multi-walled carbon nanotube (MWNT-COOH) from example 2.

Detailed Description

The invention discloses a method for analyzing interaction of a single carbon nanotube and protein based on a surface plasmon resonance imaging (SPRM) technology. . Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The method for detecting the interaction between the single carbon nanotube and the protein specifically comprises the following steps:

A. the gold film surface was modified with PEG-OH (m.w. ═ 340) monolayer (SAM). Firstly plating a chromium layer with the thickness of 2-3nm on the upper surface of a glass substrate, and then plating a gold film with the thickness of 40-50nm on the upper surface of the chromium layer; the gold film was immersed in a 1mM SH-PEG-OH (m.w. ═ 340) ethanol solution at 0 ℃ for 24 hours to form a uniform SAM layer of PEG-OH (m.w. ═ 340) on the surface of the gold film, which served as an SPR sensor chip.

B. And dispersing MWNT-COOH on the surface of the SPR sensing chip. And (2) ultrasonically dispersing 10mg of carboxyl multi-walled carbon nanotube MWNT-COOH in an ethanol solution for 5min, filtering the solution through a water-phase filter membrane with d being 0.22 mu m, and taking 10 mu L of supernatant to spin-coat on an SPR sensing chip. The carbon nanotubes were confirmed to be uniformly dispersed as single strands by Atomic Force Microscope (AFM) characterization.

C. Turn on the instrument and the drug delivery system, detect noise and stability. 0.01mg/mL, 0.1mg/mL, 1mg/mL BSA solution and WGA solution were prepared in 1 XPBS solution and injected into the administration system, respectively. The SPR chip was covered with a PDMS cuvette and 500. mu.L of 1 XPBS solution was added to the cuvette. Install the water pump by the cuvette, ensure that it is equal with the pumping rate to advance the medicine rate, advance the medicine and can not influence the liquid level in the cuvette and rise or descend to can not disturb SPR signal stability. The needle of the administration system is adjusted to be over the area of the carbon nano tube under the lens of 60 x, the administration system is introduced into the 1 x PBS solution to the SPR chip at the flow rate of 1s/drop due to gravity administration, the light Intensity of SPR is adjusted to be 1/3Intensity MAX, an SPR microscope, a water pump and an administration system valve are opened to continuously sample for 1min at 106fps, and signal noise and instrument stability are analyzed.

D. And after the instrument is determined to be stable, collecting sample information, and performing data processing by using ImageG and Matlab. WGA/BSA at 0.01mg/mL was first added to the system, and the sample was taken at a sampling rate of 106fps for 2min to reach the adsorption maximum plateau, then switched to 1 XPBS to dissociate the protein fraction on the carbon tubes, and the sample was continued at the same rate of 106fps for 1min, after which the sample was stopped, but the sample was continued to elute with 1 XPBS for 30 min. Then 0.1mg/mL WGA/BSA and 1mg/mL WGA/BSA are respectively added to repeat the steps, and the binding dissociation information of the WGA/BSA and single MWNT-COOH in the same area under different concentration gradients is collected. And reading the SPR intensity on the PEG-OH modified gold film surface and MWNT-COOH by using ImageG, then denoising and smoothing by using Matlab, subtracting to obtain a combined dissociation curve of a plurality of single-walled carbon tubes MWNT-COOH and WGA/BSA, and analyzing related information such as a combined rate constant, a dissociation rate constant and the like.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. Wherein the carbon nanotubes in each example are multi-walled carbon nanotubes. The carboxyl modified carbon nanotube, namely the carboxyl multi-walled carbon nanotube can be purchased from commercial sources.

Example 1: interaction of bovine serum albumin with carbon nanotubes

Detecting the interaction of 0.01, 0.1 and 1.0mg/mL BSA and three MWNT-COOH

(1) Firstly, plating a chromium layer with the thickness of 2-3nm on the upper surface of a glass substrate, and then plating a gold film with the thickness of 40-50nm on the upper surface of the chromium layer; soaking the gold film in 1mM SH-PEG-OH (M.W. ═ 340) ethanol solution at 0 deg.C for 24h to form a uniform PEG-OH (M.W. ═ 340) SAM layer on the surface of the gold film as an SPR sensing chip;

(2) taking 10mg of carboxyl modified multi-walled carbon nanotubes (MWNT-COOH) to ultrasonically disperse in an ethanol solution for 5min, passing the solution through a water-phase filter membrane with d being 0.22 mu m, taking 10 mu L of filtered supernatant to spin-coat on an SPR sensing chip, and verifying that the carbon nanotubes are uniformly dispersed as single fibers by Atomic Force Microscope (AFM) characterization;

(3) covering a PDMS small pool on an SPR sensing chip, adding 500 mu L of 1 XPBS solution, and installing a water pump beside the small pool to ensure that the medicine feeding rate is equal to the pumping rate of the water pump, so that the medicine feeding does not influence the rise or fall of the liquid level in the small pool, and the stability of an SPR signal is not interfered;

(4) preparing BSA solutions of 0.01, 0.1 and 1.0mg/mL in a 1 XPBS solution, and injecting the BSA solutions into three tubes of a drug delivery system respectively;

(5) opening an SPR instrument, adjusting a needle head of a drug delivery system to be above a carbon nano tube region under a 60 × lens in a bright field, ensuring that at least 3-4 dispersed carbon nano tubes are arranged in the searched region, opening a valve 1, introducing 1 × PBS solution to an SPR sensing chip at a flow rate of 1s/drop, adjusting the light Intensity of SPR to be at 1/3Intensity MAX, and collecting an SPR image for establishing a standard baseline, wherein the SPR response is the most sensitive at the moment;

(6) after the base line is stable, closing the valve 1, rapidly switching, opening the valve 2, adding 0.01mg/mL BSA solution on the carbon nano tube area by using a drug delivery system, and continuously collecting SPR images at the speed of 106fps by using a camera to obtain corresponding SPR microscope combined images;

(7) when the binding reaches the vicinity of the maximum platform, the drug delivery system is quickly switched back to 1 × PBS (the valve 2 is closed, the valve 1 is opened), so that BSA on the carbon tube is partially dissociated to obtain a corresponding SPR microscope dissociation image, the sampling is continuously carried out for 1min at the same speed of 106fps, then the sampling is stopped, but the sample is continuously eluted by 1 × PBS for 30 min;

(8): repeating the steps (6) and (7), but switching the concentration of the BSA solution to 0.1mg/mL to obtain corresponding SPR microscopic images of binding and dissociation;

(9): repeating the steps (6) and (7), but switching the concentration of the BSA solution to 1.0mg/mL to obtain corresponding SPR microscopic images of binding and dissociation;

(10): the SPR intensities on MWNT-COOH were measured using Image G and recorded as SPR Intensity_MWNT-COOH(ii) a Surface strength of PEG-OH modified gold film, recorded as SPR Intensity_PEG-OH(ii) a Then SPR Intensity is used_MWNT-COOHMinus SPR Intensity_PEG-OHObtaining SPR Intensity with background interference being deducted_MWNT-COOHThen, using Matlab to remove noise and smooth, obtaining the combination dissociation curve of four carbon tubes MWNT-COOH and 0.01, 0.1, 1.0mg/mL BSA

(11): substituting the binding part of the binding dissociation curve into formula (1), substituting the dissociation part into formula (2),

wherein R is_tRepresenting the time-varying carbon nanotube SPR Intensity_MWNT-COOH，R_maxSPR Intensity representing the maximum of carbon nanotubes_MWNT-COOH，[A]Represents the concentration of protein BSA, and t represents time.

The K of three MWNT-COOH can be obtained by combining the formulas (1) and (2)_a、K_dThen, K of four MWNT-COOH can be obtained from the formula (3)_D. The results are shown in Table 1.

K_D＝K_a/K_d (3)

Wherein the obtained K_aThe expression represents the size of the affinity of the carbon nano tube to the protein, and the larger the value is, the stronger the affinity is; k_dRepresents 1/Ka, the smaller the value, the greater the affinity; k_DShows the combined affinity effect of Ka and Kd, and the larger the value, the stronger the affinity.

TABLE 1K of three MWNT-COOH on bovine serum albumin_a、K_d、K_D

0.1mg/mL BSA	K_a(M^-1s^-1)	K_d(s^-1)	K_D(μM)
				MWNT-COOH_1	14.80×10⁴	0.0490	0.30
MWNT-COOH_2	12.32×10⁴	0.0537	0.23
				MWNT-COOH_3	15.14×10⁴	0.0662	0.23

The results show that the affinity of three carbon tubes for bovine serum albumin is as follows: MWNT _ COOH _1> MWNT _ COOH _2 ═ MWNT _ COOH _ 3.

Example 2: interaction of Wheat Germ Agglutinin (WGA) with carbon nanotubes

Detection of the interaction of 0.1mg/mL WGA with three MWNT-COOH

(4) preparing 0.1mg/mL WGA solution in 1 XPBS solution, and injecting the WGA solution into a drug delivery system;

(5) opening an SPR instrument, adjusting a needle head of a drug delivery system to be above a carbon nano tube region under a 60 Xlens in a bright field, ensuring that at least 3-4 dispersed carbon nano tubes are in the searched region, introducing 1 XPBS solution to an SPR sensing chip at a flow rate of 1s/drop, adjusting the light Intensity of SPR to be at 1/3Intensity MAX, collecting an SPR image and establishing a standard baseline, wherein the response of the SPR is most sensitive at the moment;

(6) after the baseline is stable, adding 0.1mg/mL WGA solution on the carbon nano tube area by using a drug delivery system, and continuously collecting SPR images at 106fps to obtain a corresponding binding curve;

(7) when the binding reaches the vicinity of the maximum platform, switching the drug delivery system back to 1 XPBS to partially dissociate WGA on the carbon tube to obtain a corresponding dissociation curve, continuously sampling at the same rate of 106fps for 1min, and then stopping sampling, but continuously eluting the sample with 1 XPBS for 30 min;

(8): read out on three MWNT-COOH by Image GThe SPR Intensity of (1) is recorded as SPR Intensity_MWNT-COOH(ii) a Surface strength of PEG-OH modified gold film, recorded as SPR Intensity_PEG-OH(ii) a Then SPR Intensity is used_MWNT-COOHMinus SPR Intensity_PEG-OHObtaining SPR Intensity with background interference being deducted_MWNT-COOHAnd then, utilizing Matlab to denoise and smooth to obtain a binding dissociation curve of the three carbon tubes MWNT-COOH and 0.1mg/mL WGA, and solving corresponding kinetic parameters Ka, Kd and KD to obtain related binding rate constants and dissociation rate constants by substituting a formula and performing curve fitting according to the scheme of the embodiment 1. The results are shown in Table 2.

TABLE 2K of three MWNT-COOH on wheat germ agglutinin_a、K_d、K_D

0.1mg/mL WGA	K_a(M^-1s^-1)	K_d(s^-1)	K_D(uM)
				MWNT-COOH_1	11.70×10⁴	0.0123	0.10
MWNT-COOH_2	14.22×10⁴	0.0092	0.15
				MWNT-COOH_3	9.68×10⁴	0.0138	0.07

The results show that the affinity of three carbon tubes for wheat germ agglutinin is: MWNT _ COOH _2> MWNT _ COOH _1> MWNT _ COOH _ 3.

Claims

1. A method for analyzing interaction between a single carbon nanotube and a protein, comprising the steps of:

5) obtaining an association dissociation curve according to the association image and the dissociation image, and calculating to obtain an association rate constant and a dissociation rate constant;

the pretreatment of step 1) is that the carbon nano tube is treated by ultrasonic treatment in ethanol solution after being modified by carboxyl, a water phase filter membrane is used for filtration, and the filtered supernatant is taken;

the plasma resonance sensing chip comprises a substrate material, a chromium layer and a gold layer which are sequentially arranged; the monolayer is modified by modifying the surface of the plasma resonance sensing chip with PEG-OH with SH to obtain a PEG-OH surface;

and step 2) acquiring the SPR image, wherein the light Intensity of the SPR is 1/3Intensity MAX.

2. The method of claim 1, wherein the protein solution of step 3) is a PBS solution containing bovine serum albumin or wheat germ agglutinin.

3. The method of claim 2, wherein the protein solution has a concentration of bovine serum albumin or wheat germ agglutinin of 0.01mg/mL, 0.1mg/mL or 1 mg/mL.

4. The method of claim 1, wherein the speed of step 3) said acquiring images is 106 fps.

5. The method of claim 1, wherein the PBS or the protein solution is added to the PDMS reaction pool on the SPR chip by an automatic drug delivery system, and the flow rate of the PBS or the protein solution is 1 s/drop.

6. The method of claim 1, wherein the step 5) of obtaining the association rate constant by calculating the association dissociation curve according to the association Image and the dissociation Image comprises reading out the SPR Intensity on the carbon nanotube by using Image G and recording the SPR Intensity as SPR Intensity_MWNT-COOH(ii) a Surface strength of PEG-OH modified gold film, recorded as SPR Intensity_PEG-OH(ii) a Then SPR Intensity is used_MWNT-COOHMinus SPR Intensity_PEG-OHObtaining SPR Intensity with background interference being deducted_MWNT-COOHThen, using Matlab to remove noise and smooth to obtain the binding dissociation curve of the carbon nano tube and protein, substituting the binding part of the binding dissociation curve into formula (1), substituting the dissociation part into formula (2), and combining formula (1) and formula (2) to obtain K of the carbon nano tube_a、K_dThen, K of the carbon nanotube can be obtained from the formula (3)_D，

K_D＝K_d/K_a (3)