CN111167409A - Preparation method and application of Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly - Google Patents
Preparation method and application of Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly, belonging to the field of materials. The preparation method comprises the following steps: solvothermal method for preparing Fe3O4preparation of Fe from nano-cluster by St baby sol-gel method3O4@SiO2Magnetic nanoparticles; adding silane coupling agent, and chemically finishing Fe3O4@SiO2Surface carboxylation (Fe)3O4@SiO2@ COOH); introduction of activating agents at the carboxyl groupsDissolving Fe3O4@SiO2NTA modification (Fe) is carried out on the surface of the magnetic nano-particles3O4@SiO2@ COOH @ NTA); adding Ni2+And preparing the assembly. The method has clear steps, and the prepared magnetic nano functional assembly has rich surface groups of Ni2+The binding capacity is large, the binding force with His label protein is strong, and the separation efficiency is high.
Description
Technical Field
The invention belongs to the field of materials, and particularly relates to a preparation method of a magnetic functional assembly for separating and extracting His-tagged protein.
Background
With the development of biosciences and biotechnology, methods for separating proteins and peptides have become diverse, including precipitation, ultrafiltration, chromatography (exclusion chromatography, ion exchange chromatography, affinity chromatography, etc.), two-dimensional electrophoresis, and the like. A good separation method should have the following three advantages: firstly, the coupling technology has high selectivity, good stability and reproducibility; secondly, the protein load is high and controllable, the reproducibility is good, and the detection is easy; thirdly, the protein has the ability of nonspecific protein adsorption resistance, and the activity of the protein is not affected.
With the research and development of magnetic nanoparticles, the magnetic nanoparticles are widely applied to the fields of nuclear magnetic resonance, protein purification, drug delivery, bacterial detection, enzyme immobilization, catalysis and the like based on the characteristics of strong magnetism, large specific surface area, controllable size, easiness in separation and the like. The method (magnetic affinity separation) for separating and purifying the protein by the magnetic nano particles after surface functionalization is economical and practical, the separation condition is mild, the requirement on a sample is low, and the separation process is simple, convenient, rapid and efficient.
The composite magnetic nano particle prepared by the existing method can control the thickness of a coating layer in a nano scale, has the capability of separating and purifying histidine-tagged protein, and can be repeatedly used. However, in the synthesis process, the process is complex, and acrylic acid is difficult to branch to the surface of the magnetic nanoparticles, so that the carboxyl density is too low, the absolute value of Zeta potential is small, and the NTA reaction efficiency is low (nitrilotriacetic acid structure has no amino group, and is difficult to react with the carboxyl group on the surface of the magnetic nanoparticles).
Disclosure of Invention
The invention aims to provide a preparation method of a magnetic nano functional assembly, which aims to solve the problems of complex preparation process, insufficient surface group density, low separation efficiency and the like in the existing method.
Another object of the present invention is to provide a magnetic nano-functional assembly obtained by the preparation method.
The invention also aims to provide an application of the magnetic nano functional assembly in His-tagged protein separation.
The technical scheme of the invention is as follows: a preparation method of a Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly comprises the following steps:
(1) solvothermal method for preparing Fe3O4Nanoclusters;
(2) preparation of Fe by utilizing St barber sol-gel method3O4@SiO2Magnetic nanoparticles;
(3) chemical method for completing Fe3O4@SiO2Surface carboxylation (Fe)3O4@SiO2@ COOH) by mixing the above Fe3O4@SiO2Dispersing magnetic nano particles in an organic solvent, adding a silane coupling agent N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate under the conditions of nitrogen protection and condensation reflux, heating, stirring for reaction, and then cleaning with the organic solvent and deionized water to obtain Fe3O4@SiO2@ COOH magnetic nanoparticles;
(4) subjecting said Fe to3O4@SiO2Dispersing the @ COOH magnetic nano-particles in deionized water, adding EDC and NHS for activation, adding NA, NA-bis (carboxymethyl) -L-Performing incubation reaction on lysine hydrate, and cleaning with deionized water after the reaction is finished to obtain Fe3O4@SiO2@ COOH @ NTA magnetic nanoparticle dispersion;
(5) in said Fe3O4@SiO2Ni is introduced into @ COOH @ NTA magnetic nano-particle dispersion liquid2+Obtaining Ni-NTA modified Fe after the reaction is finished3O4@SiO2A magnetic nano functional assembly.
In step (4) { Fe3O4@SiO2@ COOH @ NTA magnetic nanoparticle at 1624cm-1In the form of-COO-The peak intensity of C = O expansion/contraction vibration was higher than that of Fe obtained as described above3O4@SiO2The @ COOH magnetic nanoparticles were 3-fold enhanced.
Ni-NTA modified Fe in step (5)3O4@SiO2Ni in magnetic nano functional assembly2+The content is 1 × 10- 5mol/g-9×10-5mol/g。
Further, the adding amount of the sodium N- (trimethoxysilylpropyl) ethylenediamine triacetate in the step (3) is 0.5-3 ml; the heating temperature is 60-90 ℃; the reaction time is 5-10 h; produced Fe3O4@SiO2The density of the carboxyl of the @ COOH magnetic nano-particles is 0.1-0.5 mu mol/mg, and the Zeta potential is-30 mv-38 mv.
Further, EDC and NHS are respectively added in the step (4) in an amount of 10-40 mg; adding NA, the amount of NA-bis (carboxymethyl) -L-lysine hydrate is 1-4 mg; the incubation reaction time is 18-48 h.
Further, Ni is added in the step (5)2+The concentration is 0.1-1 mol/L; said addition of Ni2+The reaction time is 1.5-3 h. Prepared Ni of Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly2+The content of (A) is 2.19-8.69 x 10-5mol/g。
Ni-NTA modified Fe obtained by the above method3O4@SiO2The application of the magnetic nano functional assembly in the separation of His tag protein comprises the following steps: taking a certain amount of theNi-NTA modified Fe3O4@SiO2Respectively adding fluorescence-labeled His-tagged protein and non-His-tagged protein into the magnetic nano functional assembly, finally adding buffer solution to enable the concentration of a reaction system to be 2 mu M, and incubating for 1 h; after external magnetic separation, adding buffer solution for washing for 3 times, adding eluent, incubating for 10min, performing external magnetic separation, collecting supernatant, measuring fluorescence intensity by fluorescence spectrum, and detecting separation efficiency. The separation efficiency is 25-66.7%.
The mechanism of the invention is as follows: the functionalized modification of the surface of the magnetic nano-particles is the key importance of magnetic affinity separation. In view of the above, the invention adopts a carboxylated silane coupling agent to carry out carboxylation modification on the magnetic nano-particles coated by silicon dioxide, which is beneficial to overcoming the problems of low surface carboxyl density, small absolute value of Zeta potential and poor binding force, and meanwhile, the NA, NA-di (carboxymethyl) -L-lysine hydrate is used for replacing nitrilotriacetic acid, NA, NA-di (carboxymethyl) -L-lysine hydrate to form a ligand on the surface of the magnetic particles, so that the ligand is used for chelating metal Ni ions and is used for separation, extraction and detection of biomolecules. By directly combining amino in NA, NA-di (carboxymethyl) -L-lysine hydrate with carboxyl on the surface of magnetic beads and optimizing process conditions, the problems of insufficient NTA grafting and Ni are hopeful to be solved2+Low binding amount.
The invention is characterized in that: 1. the carboxylated magnetic beads are obtained by adding N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate and heating for reaction. 2. The structure is obtained by adding NA, NA-bis (carboxymethyl) -L-lysine hydrate for reaction, and compared with nitrilotriacetic acid, the compound has an amino group and has strong binding capacity with activated carboxyl, so that the obtained NTA structure is more stable, and linked groups are more. 3. The assembly and the intermediate are characterized and explained by means of infrared, Zeta potential, conductance titration, ICP test and the like; 4. by utilizing fluorescence detection, the detection method is simpler and more visual.
Compared with the prior art: the invention has the following advantages and beneficial effects:
1. the invention takes the magnetic nanocluster as the core and the surfaceProceed to SiO2The stability is increased by the inorganic coating; meanwhile, in the carboxylation process, a carboxyl silanization reagent is selected, so that SiO is improved2Surface carboxyl group bonding force and carboxyl density; the stability and reliability of the subsequent NTA group linkage are ensured.
2. In the NTA modification process, NA-di (carboxymethyl) -L-lysine hydrate is selected, amino in the structure of the hydrate and carboxyl on the surface of particles are utilized to carry out dehydration condensation, so that the combination amount of NTA is greatly improved, and 1624cm can be seen from figure 3-1About a 3-fold increase in peak intensity; thereby increasing Ni in the next step2+The amount of chelant.
3. The invention improves the Fe modified by Ni-NTA through refining the experimental steps, changing reactants and improving the process3O4@SiO2The magnetic nano functional assembly has the advantages that the overall performance of the magnetic nano functional assembly has stronger binding force with His tag protein, the protein of the His tag can be separated and extracted more efficiently, quickly and massively, the magnetic nano functional assembly has great application prospect in the separation of the His tag protein, and meanwhile, the magnetic nano functional assembly has potential value in the fields of biological detection, drug screening and the like.
Drawings
FIG. 1 shows Ni-NTA modified Fe3O4@SiO2A flow chart of a preparation method of the magnetic nano functional assembly;
FIG. 2 shows Fe prepared in the example of the present invention3O4Nanoclusters; fe3O4@SiO2；Fe3O4@SiO2-
@ COOH； Fe3O4@SiO2@COOH@NTA； Fe3O4@SiO2Zeta potential profile of @ COOH @ NTA-Ni;
FIG. 3 shows Fe prepared according to an example of the present invention3O4；Fe3O4@SiO2；Fe3O4@SiO2@COOH ；Fe3O4@SiO2Infrared spectrum of @ COOH @ NTA;
FIG. 4 shows Fe prepared in the examples of the present invention3O4@SiO2Conductance titration plot of @ COOH;
FIG. 5 shows Fe prepared in the example of the present invention3O4Nanoclusters; fe3O4@SiO2(ii) a Ni-NTA modified Fe3O4@SiO2TEM picture of magnetic nanometer functional assembly body interlinkage protein;
FIG. 6 shows Ni-NTA modified Fe prepared according to the present invention3O4@SiO2The magnetic nano functional assembly extracts fluorescence spectrograms of His-tagged protein and non-His-tagged protein.
FIG. 7 shows 2. mu.M pure His-tag protein, 2. mu.M pure His-tag protein and Ni-NTA modified Fe prepared according to the present invention3O4@SiO2Fluorescence spectrograms of the supernatant and the eluent after the reaction of the magnetic nanometer functional assembly.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention in any way.
In the examples, EDC: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and NHS: N-hydroxysuccinimide.
Example 1
(1)Fe3O4Preparing nanoclusters:
weighing 0.34g FeCl3·6H2O，0.9gNaAc·3H2Dissolving O in 60mL of glycol, stirring at room temperature to completely dissolve the solid to obtain a mixed solution; adding the mixed solution into a stainless steel high-temperature reaction kettle embedded with 100mL of polytetrafluoroethylene, reacting for 8h at 180 ℃, cooling, washing for 3 times respectively by using ethanol and deionized water, separating by using an external magnetic field, and freeze-drying to obtain Fe3O4Nanoclusters;
in fig. 5: (a) fe3O4；(b)Fe3O4@SiO2(ii) a (c) Ni-NTA modified Fe3O4@SiO2TEM picture of magnetic nanometer functional assembly body interlinkage protein;
fe can be seen from the transmission electron microscope of FIG. 5(a)3O4Nanocluster ofVery small amount of Fe3O4The nano particles are assembled, and the cluster particle size is about 200 nm.
Fe can be seen from the Zeta potential distribution diagram in FIG. 2(1)3O4The nanocluster surface is positively charged at 38 mv.
FIG. 3 shows Fe prepared according to an example of the present invention3O4；Fe3O4@SiO2；Fe3O4@SiO2@COOH ；Fe3O4@SiO2Infrared spectrum of @ COOH @ NTA; in the figure: (a) fe3O4；(b)Fe3O4@SiO2；(c)Fe3O4@SiO2@COOH ；(d)Fe3O4@SiO2Infrared spectrum of @ COOH @ NTA.
590cm can be seen from the Fourier transform infrared spectrogram of FIG. 3(a)-1The peak appeared is the stretching vibration peak of Fe-O bond, and is Fe3O4Is in the range of 3378cm-1The stretching vibration peak of O-H bond appears nearby, which is due to Fe3O4The nanocluster surface contains hydroxyl groups.
342The preparation of (1):
weighing 0.025g of Fe prepared in step (1)3O4Adding 12mL of deionized water and 20mL of ethanol into the nanoclusters, performing ultrasonic treatment for 10min, adding 2mL of concentrated ammonia water and 0.05mL of ethyl orthosilicate under the condition of ice-water bath, and performing ultrasonic treatment for 40 min; magnetic separating outside, washing with deionized water for 3 times, and freeze drying to obtain Fe3O4@SiO2。
From FIG. 5(b), it can be seen that Fe is present in the TEM3O4The surface of the nanocluster is uniformly coated with a layer of SiO about 20nm2And the surface is smooth.
Fe can be seen from the Zeta potential distribution diagram in FIG. 2(2)3O4@SiO2The surface is negatively charged and is-31 mv.
1084 cm can be seen from the Fourier transform infrared spectrogram of FIG. 3(b)-1The peak of reverse stretching vibration of Si-O appears at 795 cm-1Symmetric stretching vibration of Si-OPeak 955cm-1A bending vibration peak of Si-OH appears; at the same time at 3378cm-1The increased strength is due to SiO2The surface contains a large number of silicon hydroxyl groups.
(3)Fe3O4@SiO2Preparation of @ COOH magnetic nanoparticles:
0.05g of Fe prepared in step (2) was weighed3O4@SiO2Placing the mixture into a 100mL three-necked bottle, adding 40mL of ethanol, mechanically stirring at room temperature, adding 1mL of N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate, and reacting at 80 ℃ for 8 hours under the condition of introducing nitrogen; magnetic separation outside, washing three times with deionized water to obtain Fe3O4@SiO2A dispersion of @ COOH magnetic nanoparticles.
From the Zeta potential distribution chart in FIG. 2(3), Fe can be seen3O4@SiO2The surface of the @ COOH magnetic nano-particles is negatively charged and is-38 mv.
1624cm can be seen from the Fourier transform infrared spectrogram of FIG. 3(c)-1In the form of-COO-Medium C = O stretching vibration peak.
From the conductance titration chart of FIG. 4, Fe can be seen3O4@SiO2The density of carboxyl groups of the @ COOH magnetic nanoparticles was 0.5. mu. mol/mg.
(4)Fe3O4@SiO2Preparation of @ COOH @ NTA magnetic nanoparticles:
taking the Fe prepared in the step (3)3O4@SiO2@ COOH magnetic nanoparticle dispersion containing Fe3O4@SiO20.03g of @ COOH magnetic nanoparticles, 4mL of deionized water, 40mgEDC and 40mgNHS, uniformly mixing and stirring for 2h, adding 4mgNA, NA-bis (carboxymethyl) -L-lysine hydrate, and reacting for 36h at room temperature; performing external magnetic separation, adding 4mL of deionized water, and cleaning for 3 times to obtain Fe3O4@SiO2@ COOH @ NTA magnetic nanoparticle dispersion.
Fe can be seen from the Zeta potential distribution diagram in FIG. 2(4)3O4@SiO2The surface of the @ COOH @ NTA magnetic nano-particle is negatively charged and is-28 mv.
From FIG. 3(d), the Fourier transform infrared spectrogram can be 1385cm-1Peaks corresponding to C-N bonds, 1624cm-1In the form of-COO-Middle C = O stretching vibration peak and intensity is Fe3O4@SiO2@ COOH 3 times higher than the magnetic nanoparticles. This is due to the fact that the NTA structure contains 3 carboxyl groups.
(5) Ni-NTA modified Fe3O4@SiO2Preparing a magnetic nano functional assembly:
taking the Fe prepared in the step (4)3O4@SiO2@ COOH @ NTA magnetic nanoparticle dispersion, after external magnetic separation, 4mL of 1MNiCl was added2Mixing the aqueous solution and reacting for 2 hours; performing external magnetic separation, adding deionized water, cleaning for 3 times, and finally dispersing in 4mL of deionized water to obtain Ni-NTA modified Fe3O4@SiO2A dispersion of a magnetic nano-functional assembly.
From the Zeta potential distribution chart in FIG. 2(5), Fe can be seen3O4@SiO2The surface of the @ COOH @ NTA-Ni magnetic nano-particle is negatively charged and is-24 mv.
Ni-NTA modified Fe prepared by the embodiment of the invention3O4@SiO2Ni of magnetic nano functional assembly2+The contents are shown in Table 1, and Ni-NTA modified Fe obtained by Inductively Coupled Plasma (ICP) test can be seen from Table 13O4@SiO2Ni in magnetic nano functional assembly2+Content, 1g of the magnetic nano functional assembly Ni2+The content can reach 8.693 multiplied by 10-5mol。
Example 2
As described in example 1, except that:
step (1) Fe3O4The preparation of nanoclusters is unchanged;
step (2) Fe3O4@SiO2The preparation of (A) is not changed;
adding 0.5mL of N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate in the step (3), heating at the temperature of 60 ℃ for 10 hours;
adding 10mgEDC and 10mgNHS and adding 1mg NTA into the step (4), wherein the reaction time is 48 h;
adding 0.1MNiCl into the step (5)2Mixing the aqueous solution and reacting for 3 hours.
Example 3
As described in example 1, except that:
step (1) Fe3O4The preparation of nanoclusters is unchanged;
step (2) Fe3O4@SiO2The preparation of (A) is not changed;
adding 2mL of N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate in the step (3), heating the mixture at 70 ℃ for 9 h;
adding 20mgEDC and 20mgNHS and adding 2mg NA, NA-bis (carboxymethyl) -L-lysine hydrate in the step (4), wherein the reaction time is 36 h;
adding 0.5M TiCl in the step (5)2Mixing the aqueous solution and reacting for 2.5 h.
Example 4
As described in example 1, except that:
step (1) Fe3O4The preparation of nanoclusters is unchanged;
step (2) Fe3O4@SiO2The preparation of (A) is not changed;
adding 3mL of N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate in the step (3), heating at 90 ℃ for 5 h;
adding 40mgEDC and 40mgNHS and adding 4mg NA, NA-bis (carboxymethyl) -L-lysine hydrate in the step (4), wherein the reaction time is 18 h;
adding 1.5M TiCl in the step (5)2Mixing the aqueous solution and reacting for 1.5 h.
His tag protein extraction test
Ni-NTA modified Fe prepared in the above examples3O4@SiO2The magnetic nano functional assembly is used for separating, extracting and testing His label protein, and comprises the following specific stepsThe method comprises the following steps:
taking Ni-NTA modified Fe prepared in the above example3O4@SiO28.65. mu.L of the dispersion of the magnetic nano functional assembly. Adding 240 mu L of protein with His labels and fluorescence labeling at 5 mu M, adding a certain amount of PBS buffer solution to make the total volume be 4mL, wherein the concentration of the reaction system is 2 mu M, and reacting for 1 h;
while another set was added with 240. mu.L, 5. mu.M of fluorescently tagged His-tag-free protein under the same conditions. After the reaction is finished, washing the reaction product for three times by PBS buffer solution, adding 600 mu L of eluent, after external magnetic separation, measuring the fluorescence intensity of the supernatant, and measuring the Fe modified by Ni-NTA3O4@SiO2The magnetic nano functional assembly has the capability of extracting His label protein.
FIG. 5(c) projection electron microscopy shows Ni-NTA modified Fe3O4@SiO2Compared with fig. 5(b), the magnetic nano functional assembly is linked with His label protein, and the surface of the magnetic nano functional assembly is obviously raised.
The fluorescence spectrum of FIG. 6(a) shows that the supernatant after extraction has a strong fluorescence intensity, while that of FIG. 6(b) shows no fluorescence intensity. The protein of His label has good specific binding capacity with the magnetic nano functional assembly, and the protein without His label has no binding capacity with the magnetic nano functional assembly.
The fluorescence intensity of 2. mu.M-pure His-tagged protein was observed in the fluorescence spectrum of FIG. 7(a), and the extraction efficiency was calculated to be 66.7% in FIG. 7(b) based on the fluorescence intensity of the supernatant obtained after reaction of 2. mu.M-pure His-tagged protein with the assembly. Meanwhile, fig. 7(c) can see that the eluate has no fluorescence intensity, indicating no effect on extraction.
Performance and extraction efficiency of the functional assemblies of examples 1-4 are shown in Table 2, which shows Fe prepared in examples of the present invention3O4@SiO2Ni of @ COOH @ NTA-Ni2+Table 2 shows that the extraction effect of example 1 is the best, and can reach 66.7%, because: 1. in the preparation process of the assembly, the surface carboxyl density of the intermediate can change along with the change of process parameters, and the intermediate prepared by the inventionThe assembly has higher carboxyl density, which is beneficial to the next reaction; 2. ni on surface of assembly2+High content, and can chelate more protein.
Claims (5)
1. A preparation method of a Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly is characterized by comprising the following steps:
(1) solvothermal method for preparing Fe3O4Nanoclusters;
(2) preparation of Fe by utilizing St barber sol-gel method3O4@SiO2Magnetic nanoparticles;
(3) chemical method for completing Fe3O4@SiO2Surface carboxylation (Fe)3O4@SiO2@ COOH) by mixing the above Fe3O4@SiO2Dispersing magnetic nano particles in an organic solvent, adding a silane coupling agent N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate under the conditions of nitrogen protection and condensation reflux, heating, stirring for reaction, and then cleaning with the organic solvent and deionized water to obtain Fe3O4@SiO2@ COOH magnetic nanoparticles;
(4) subjecting said Fe to3O4@SiO2Dispersing the @ COOH magnetic nanoparticles in deionized water, adding EDC and NHS for activation, adding NA, NA-bis (carboxymethyl) -L-lysine hydrate for incubation reaction, and after the reaction is finished, washing with deionized water to obtain Fe3O4@SiO2@ COOH @ NTA magnetic nanoparticle dispersion;
(5) in said Fe3O4@SiO2Ni is introduced into @ COOH @ NTA magnetic nano-particle dispersion liquid2+Obtaining Ni-NTA modified Fe after the reaction is finished3O4@SiO2A magnetic nano functional assembly.
2. The preparation method of the Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly according to claim 1, characterized in that: the adding amount of the N- (trimethoxysilylpropyl) ethylenediamine sodium triacetate in the step (3) is 0.5-3 ml; the heating temperature is 60-90 ℃; the reaction time is 5-10 h.
3. The preparation method of the Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly according to claim 1, characterized in that: EDC and NHS are respectively added in the step (4) in an amount of 10-40 mg; adding NA, the amount of NA-bis (carboxymethyl) -L-lysine hydrate is 1-4 mg; the incubation reaction time is 18-48 h.
4. The preparation method of the Ni-NTA modified silicon dioxide coated ferroferric oxide magnetic nano functional assembly according to claim 1, characterized in that: adding Ni in the step (5)2+The concentration is 0.1-1 mol/L; said addition of Ni2+The reaction time is 1.5-3 h.
5. Ni-NTA modified Fe obtained according to the process of any one of claims 1 to 43O4@SiO2The application of the magnetic nano functional assembly in the separation of His tag protein comprises the following steps: taking a certain amount of the Ni-NTA modified Fe3O4@SiO2Respectively adding fluorescence-labeled His-tagged protein and non-His-tagged protein into the magnetic nano functional assembly, finally adding buffer solution to enable the concentration of a reaction system to be 2 mu M, and incubating for 1 h; after external magnetic separation, adding buffer solution for washing for 3 times, adding eluent, incubating for 10min, performing external magnetic separation, collecting supernatant, measuring fluorescence intensity by fluorescence spectrum, and detecting separation efficiency.
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