Membrane protein imaging technology

Abstract

The invention belongs to the technical field of biology, and particularly discloses a membrane protein imaging method and application thereof.The method comprises the steps that different DNA molecules are used as response molecules to mark cell membranes and target membrane proteins, then the response molecules marked on the target membrane proteins can trigger molecular dynamic recruitment based on entropy driven circulation through a strand displacement reaction, and the dynamic recruitment is obtained. Causing a large number of probes in the solution to be recruited to the cell membrane; signals are expanded to the whole cell membrane from the membrane interface protein, so that ectopic amplification imaging of the membrane protein based on molecular dynamic collection is realized; in addition, UCNPs are selected to replace a traditional fluorescent part to further enhance the signal, and the method can effectively enhance the imaging signal of the low-abundance target membrane protein; meanwhile, the biological significance and the dynamic regulation and control process of the cell membrane protein can be known more deeply, and the method has great application value in the field of biological medicine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, discloses a membrane protein imaging technology, and particularly relates to an imaging method for effectively amplifying membrane protein signals. Background Art

[0002] Cell membrane proteins are important components of the cell membrane and play an indispensable role in signal transduction, material transport and energy conversion. They are also important biomarkers and drug targets in clinical applications. At present, the imaging analysis methods of cell membrane proteins usually involve traditional immunofluorescence techniques and their derivatives. However, the signal amplification rate of this detection method is low, and there are defects in the analysis of low-abundance target proteins. Current methods mainly focus on trying to modify fluorescent secondary antibodies using nanotechnology. However, these technologies mainly rely on signal recognition and amplification processes based on "target-primary antibody-secondary antibody" and "target-aptamer". The properties of proteins fixed on the membrane are quite different from those in the solution, which greatly limits the efficiency of signal amplification and output. Summary of the invention

[0003] To solve the above problems, we disclosed a membrane protein imaging technology that utilizes DNA self-assembly technology to overcome the limitations of in situ imaging based on target protein imaging, improve signal amplification efficiency and stability, and realize ectopic analysis of target proteins.

[0004] To achieve this goal, we further designed an ectopic amplification imaging method based on molecular dynamic recruitment based on the traditional immunofluorescence analysis method. Specifically, different DNA molecules are used as response molecules to label the cell membrane and target membrane proteins. Subsequently, the response molecules labeled on the target membrane protein can trigger the molecular dynamic recruitment based on the entropy-driven cycle through a chain displacement reaction, resulting in a large number of probes in the solution being recruited to the cell membrane. The signal is expanded from the membrane interface protein to the entire cell membrane, realizing the ectopic amplification imaging of membrane proteins based on molecular dynamic recruitment. In addition, we selected upconversion nanoparticles (UCNPs) to replace the traditional fluorescent part to further enhance the signal. Our method can effectively enhance the imaging signal of low-abundance target membrane proteins. At the same time, it helps to have a deeper understanding of the biological significance and dynamic regulation process of cell membrane proteins.

[0005] The present invention includes the following specific technical solutions:

[0006] A membrane protein imaging method, characterized in that it comprises the following steps:

[0007] S1. Cell membrane protein labeling: through the process of target protein-primary antibody-biotin-labeled secondary antibody-streptavidin-biotin-labeled EM-a, the membrane target protein EpCAM is labeled with EM-a;

[0008] S2. Cell membrane engineering: Add cholesterol-modified DNA complex EM-ci to the cells to be imaged and incubate at room temperature to allow EM-ci to embed into the cell membrane;

[0009] S3, Synthesis of UCNP-EM-d: EM-d-NH2 was covalently coupled to citric acid functionalized upconversion nanoparticles CA-UCNP via EDC / NHS;

[0010] S4, Molecular dynamic recruitment: After cell membrane protein labeling and cell membrane engineering, EM-bi and EM-d-FAM or UCNP-EM-d were added to the cells, and after incubation, the cell nuclei were stained with DAPI and imaged with a confocal laser scanning microscope;

[0011] The nucleic acid sequence of the EM-a is shown in SEQ ID NO: 1;

[0012] The EM-bi includes EM-b1 and EM-b2, and the nucleic acid sequences thereof are shown in SEQ ID NOs: 2 and 3.

[0013] The EM-ci includes EM-c1, EM-c2 and EM-c3, and the nucleic acid sequences thereof are shown in SEQ ID NOs: 4-6 respectively;

[0014] The nucleic acid sequence of the EM-d is shown in SEQ ID NO:7.

[0015] Furthermore, in the above membrane protein imaging method, the CA-UCNP is synthesized by the following steps:

[0016] 1) YCl3, YbCl3, TmCl3 solution, oleic acid and 1-octadecene are mixed, and after the reaction, methanol solution of ammonium fluoride and sodium hydroxide is added to obtain NaYF4:Yb,Tm crystal nuclei;

[0017] 2) reacting with YCl3, oleic acid, 1-octadecene mixture, ammonium fluoride and sodium hydroxide to obtain core-shell UCNPs;

[0018] 3) After hydrophilic treatment with ethanol, water and hydrochloric acid, sodium citrate solution was added for incubation to obtain CA-UCNP.

[0019] Preferably, the CA-UCNP is synthesized by the following steps:

[0020] A. Mix 135.5 mg YCl3, 83.7 mg YbCl3, 50 μL TmCl3, 8 mL oleic acid and 15 mL 1-octadecene, react at 90°C for 20 minutes under stirring, and then react at 150°C for 1 hour. Add 5 mL of anhydrous methanol containing 148 mg ammonium fluoride and 100 mg sodium hydroxide, and heat at 90°C for 1 hour to remove methanol. Add 4 mL cyclohexane and 4 mL ethanol to the collected product, and wash 3 times to obtain the crystal core NaYF4:Yb,Tm.

[0021] B. Take 135.5 mg YCl3, 8 mL oleic acid and 15 mL 1-octadecene, mix them, react for 20 minutes under stirring at 90 ° C, and then react at 150 ° C for 1 hour. Add 5 mL of anhydrous methanol containing 148 mg ammonium fluoride and 100 mg sodium hydroxide, and the above-mentioned cyclohexane solution containing NaYF4:Yb, Tm. Heat at 90 ° C for 1 hour to remove methanol and cyclohexane. The resulting mixture is heated to 300 ° C under nitrogen protection for 1.5 hours. After natural cooling, 20 mL of ethanol is added and the product is collected by centrifugation at 13000 rpm for 5 minutes. The collected product is added with 4 mL cyclohexane and 4 mL ethanol to obtain core-shell UCNPs; the core-shell UCNPs are dispersed in 4 mL ethanol, 2 mL water and 2 mL hydrochloric acid for hydrophilic treatment. After stirring and ultrasonic treatment at room temperature for 2 hours, the mixture is washed twice with 50% ethanol, and the UCNPs are collected by centrifugation at 13000 rpm for 20 minutes.

[0022] C. 800 mg of sodium citrate was dissolved in 80 mL of water and slowly added to the core-shell UCNPs, followed by gentle stirring and incubation at room temperature overnight. CA-UCNPs were collected by centrifugation at 13,000 rpm for 20 minutes and dispersed in 4 mL of water.

[0023] Furthermore, in the above membrane protein imaging method, step S1 comprises the following steps:

[0024] 1) When the cells reach an appropriate density, remove the cell culture medium and wash the cells three times with 1× PBS;

[0025] 2) Add 4% paraformaldehyde to fix for 15 minutes at room temperature, and then wash three times with 1× PBST;

[0026] 3) Add primary antibody and incubate for 1 hour, then wash three times with 0.05% PBST;

[0027] 4) Add biotin-labeled secondary antibody and incubate for 1 hour, then wash three times with 0.05% PBST;

[0028] 5) Add streptavidin and incubate for 15 minutes, then wash three times with 0.05% PBST;

[0029] 6) Add 500 nM biotin-labeled EM-a and incubate for 30 minutes, then wash three times with 1×PBS. At this time, EpCAM on the cell membrane is labeled with EM-a.

[0030] Furthermore, in the above membrane protein imaging method, step S2 comprises the following steps:

[0031] 500 nM of cholesterol-modified DNA complex EM-ci was added and incubated at room temperature for 30 minutes; after washing with 1× PBS to remove free and unstable EM-ci, the obtained samples were used for subsequent experiments.

[0032] Furthermore, in the above membrane protein imaging method, step S3 comprises the following steps:

[0033] 1) 20 μL of CA-UCNP, 2 mg of EDC and 3 mg of Sulfo NHS were mixed in 1 mL of 1× PBS, stirred at 600 rpm for 30 min, and then centrifuged at 13,000 rpm for 20 min to collect the activated CA-UCNP;

[0034] 2) After activation, 1 mL of 200 nM EM-d-NH2 conjugate was added and stirred at 600 rpm overnight at room temperature; finally, the final product UCNP-EM-d was collected by centrifugation at 13000 rpm for 20 min, washed three times with 1× PBS, then resuspended in 1× PBS containing 10 mM MgCl2, and stored in a refrigerator at 4°C for subsequent experiments.

[0035] Furthermore, in the above membrane protein imaging method, step S4 comprises the following steps:

[0036] 1) After cell membrane protein labeling and cell membrane engineering, 200 μL 1× PBS consisting of 10 mM MgCl2, 25 nM EM-bi, and 100 nM EM-d-FAM or 0.1 mg / mL UCNP-EM-d was added to the cells, incubated for 30 min, and washed three times with 1× PBSs;

[0037] 2) Then, DAPI was added to stain the cell nucleus, incubated at room temperature in the dark for 10 minutes, washed three times with 1× PBS, and the cells were imaged using an LSM 900 confocal laser scanning microscope.

[0038] Furthermore, in the above-mentioned membrane protein imaging method, the cells are MCF7 or HepG2.

[0039] The invention also discloses the application of the membrane protein imaging method in the field of biopharmaceuticals.

[0040] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0041] The present invention discloses a membrane protein imaging technology. In the present invention, we simulate the recruitment process on the cell membrane surface based on DNA self-assembly technology, construct a cell membrane protein amplification imaging technology based on molecular dynamic recruitment, and successfully realize the amplification imaging of cell membrane proteins of different abundances on different cells. It overcomes the limitations of imaging based solely on the pivot of the target protein, so that the signal is diffused to the entire cell membrane at the fulcrum, greatly improving the signal amplification ability. In addition, this technology also has advantages in imaging and analyzing low-abundance membrane proteins. Compared with the previous imaging and analysis methods of cell membrane proteins, the present invention not only improves the signal amplification efficiency and stability, but also performs well in target detection specificity. The present invention also replaces the chemical fluorescence signal on EM-d with UCNP, proving that different signal molecules can be used as objects of molecular dynamic recruitment, and other signal amplification methods can be developed in the future. By further adjusting the imaging technology according to the specific situation to adapt to the detection requirements, it can be said that the promotion of the present invention provides a new perspective for the application of cell diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of in situ magnified imaging of membrane proteins through dynamic molecular recruitment;

[0043] Figure 2 (A) Schematic diagram of the signal quenching and signal generation process of molecular dynamic recruitment; (B) Fluorescence kinetic curves of each assembly during the molecular dynamic recruitment process, with the lines from bottom to top representing: c1+c2+c3, c1+c2+c3+d, c1+c2+c3+b(L), c1+c2+c3+b(M), c1+c2+c3+b(H);

[0044] Figure 3 (A) Laser scanning confocal imaging results of "target protein-primary antibody-biotin-labeled secondary antibody-streptavidin-biotin-labeled EM-a-FAM", PA represents primary antibody and SA represents secondary antibody; (B) Signal statistics of laser scanning confocal imaging results of cholesterol-modified EM-ci fixed on the cell membrane, the lines from bottom to top represent respectively;

[0045] Figure 4 (A) Imaging results and signal statistics of dynamic recruitment of MCF7 membrane protein molecules by UCNP-EM-d; (B) Imaging results and signal statistics of dynamic recruitment of HepG2 membrane protein molecules by UCNP-EM-d. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Table 1 Sequence Listing

[0048]

[0049] The primary antibody in the embodiment is: primary antibody (rabbit monoclonal antibody, 1:500 dilution)

[0050] Secondary antibody: goat anti-rabbit secondary antibody (1:1000 dilution)

[0051] Example 1

[0052] Step 1: Cell membrane protein labeling

[0053] The membrane target protein EpCAM is labeled with EM-a through the process of "target protein-primary antibody-biotin-labeled secondary antibody-streptavidin-biotin-labeled EM-a". The steps are as follows:

[0054] 1) When the cells (MCF7 or HepG2 in this example) reach an appropriate density, remove the culture medium and wash the cells three times with 1×PBS (NaCl 136.89 mM; KCl 2.67 mM; Na2HPO4; 8.1 mM; KH2PO4 1.76 mM).

[0055] 2) Add 4% paraformaldehyde to fix at room temperature for 15 minutes, and then wash three times with 1×PBST.

[0056] 3) Add primary antibody and incubate for 1 hour, then wash three times with 0.05% PBST for 30 minutes, and then wash three times with 1×PBS. At this time, EpCAM is labeled with EM-a.

[0057] Step 2: Cell membrane engineering

[0058] 500 nM cholesterol-modified DNA complexes EM-ci (including EM-c1, EM-c2, and EM-c3) were added and incubated at room temperature for 30 minutes. Cholesterol can be embedded in the cell membrane through hydrophobic interactions with the phospholipid bilayer. After washing with 1× PBS to remove free and unstable EM ci, the obtained samples were used for subsequent experiments. Cells were imaged using a LSM 900 confocal laser scanning microscope (Zeiss, Germany).

[0059] Step 3: Synthesis of citric acid functionalized upconversion nanoparticles (CA-UCNPs)

[0060] Take 135.5mg YCl3, 83.7mg YbCl3, 50μLTmCl3, 8mL oleic acid and 15mL 1-octadecene, mix them, react at 90℃ for 20 minutes under stirring, and then react at 150℃ for 1 hour. Add 5mL anhydrous methanol containing 148mg ammonium fluoride and 100mg sodium hydroxide. Heat at 90℃ for 1 hour to remove methanol. Add 4mL cyclohexane and 4mL ethanol to the collected product, wash 3 times to obtain the crystal core NaYF4:Yb,Tm.

[0061] Take 135.5mg YCl3, 8mL oleic acid and 15mL 1-octadecene, mix them, react at 90℃ for 20 minutes under stirring, and then react at 150℃ for 1 hour. Add 5mL anhydrous methanol containing 148mg ammonium fluoride and 100mg sodium hydroxide, and the above cyclohexane solution containing NaYF4:Yb,Tm. Heat at 90℃ for 1 hour to remove methanol and cyclohexane. The resulting mixture is heated to 300℃ under nitrogen protection for 1.5 hours. After natural cooling, 20mL ethanol is added and the product is collected by centrifugation at 13000rpm for 5 minutes. Add 4mL cyclohexane and 4mL ethanol to the collected product to obtain core-shell UCNP.

[0062] The core-shell UCNPs were dispersed in 4 mL of ethanol, 2 mL of water and 2 mL of hydrochloric acid for hydrophilic treatment. After ultrasonic treatment for 2 h at room temperature, the mixture was washed twice with 50% ethanol and the UCNPs were collected by centrifugation at 13,000 rpm for 20 min.

[0063] 800 mg of sodium citrate was dissolved in 80 mL of water and slowly added to the core-shell UCNPs, which were then gently stirred and incubated overnight at room temperature. CA-UCNPs were collected by centrifugation at 13000 rpm for 20 min and dispersed in 4 mL of water.

[0064] Step 4: Synthesis of UCNP-EM-d

[0065] EM-d-NH2 was covalently coupled to CA-UCNPs via EDC / NHS. The steps were as follows: 20 μL of CA-UCNPs, 2 mg of EDC, and 3 mg of Sulfo NHS were mixed in 1 mL of 1×PBS. After stirring at 600 rpm for 30 min, the activated CA-UCNPs were collected by centrifugation at 13,000 rpm for 20 min. After activation, 1 mL of 200 nM EM-d-NH2 conjugate was added and stirred at 600 rpm overnight at room temperature. Finally, the final product UCNP-EM-d was collected by centrifugation at 13,000 rpm for 20 min, washed 3 times with 1×PBS, then resuspended in 1×PBS containing 10 mM MgCl2 and stored in a refrigerator at 4°C for subsequent experiments.

[0066] Step 5: Dynamic recruitment of molecules

[0067] After cell membrane protein labeling and cell membrane engineering, 200 μL 1×PBS, consisting of 10 mM MgCl2, 25 nM EM-bi and 100 nM EM-d-FAM (or 0.1 mg / mL UCNP-EM-d), was added to the cells, incubated for 30 minutes, and washed three times with 1×PBSs. Then DAPI was added to stain the cell nucleus, incubated at room temperature in the dark for 10 minutes, and washed three times with 1×PBS. The cells were imaged using a LSM 900 confocal laser scanning microscope (Zeiss, Germany).

[0068] Example 2

[0069] 1. Principle of in situ amplified imaging of membrane proteins through dynamic molecular recruitment

[0070] exist Figure 1 -A, the biotin-labeled EM-a is first anchored to the target membrane protein. At the same time, the cholesterol-modified EM-ci is embedded in the cell membrane through hydrophobic interactions. Subsequently, EM-a dynamically recruits the signaling molecule EM-d ( Figure 1-B). EM-a undergoes a chain displacement reaction with the inactive free EM-bi dispersed in the solution, and is activated to EM-b. EM-b actively recognizes EM-ci embedded in the lipid bilayer of the cell membrane, and releases EM-c2 through a chain displacement reaction, activating EM-ci to EM-c. EM-d labeled with fluorescent molecules or fluorescent nanoprobes binds to EM-c with specific single-stranded binding sites through a chain displacement reaction, releasing EM-c3 and EM-b. The released EM-b enters the next cycle and activates other EM-ci anchored on the cell membrane until all EM-ci are labeled by EM-d and recruitment is terminated. Therefore, through this process, EM-a can continuously recruit the signal molecule EM-d to the cell membrane, causing a large amount of fluorescent signals to accumulate on the cell membrane, thereby realizing the amplified imaging of membrane proteins. Among them, EM-ci is a double-stranded structure formed by the partial complementarity of EM-c1, EM-c2, and EM-c3, and EM-bi is an unactivated nucleic acid structure, a double-stranded structure formed by the partial complementarity of EM-b1 and EM-b2. EM-c and EM-b are activated states. Figure 1 EM-d is a signal probe labeled with FAM or UCNP.

[0071] 2. Dynamic recruitment of molecules in solution

[0072] In order to observe the dynamic recruitment process of molecules, C1 and C3 in EM-CI were labeled with quencher BHQ1 and fluorophore FAM, respectively. Figure 2 A). In the initial state, the fluorescence signal of c3 is extinguished by BHQ1, showing a low background signal. Once molecular dynamics recruitment occurs, FAM gradually dissociates from the BHQ1 quencher, resulting in an increase in the fluorescence signal. The results show that in the absence of the b-chain, the fluorescence of EM-ci is quenched, while after the addition of the b-chain, the FAM signal released by c3 can be observed at the corresponding position. These results indicate that molecular dynamics recruitment is in a stable state when inactive, with a low background fluorescence signal, and exhibits high specificity and sensitivity when activated. In addition, the fluorescence kinetic curves of various molecular dynamics recruitment assemblies were determined ( Figure 2 -B). The results showed that only a low signal could be observed without the addition of b.

[0073] 3. Molecular recruitment of membrane proteins

[0074] Since EpCAM has different expression levels in MCF-7 and HepG2 cells, EpCAM was used as a model membrane protein to enhance the ability to compare dynamic molecular recruitment imaging. Effective labeling of target proteins and cell membranes using EM-a and EM-ci is a basic requirement for achieving dynamic molecular recruitment.

[0075] 3.1 Labeling of target membrane proteins by EM-a-FAM combination

[0076] EM-a-FAM labels the target membrane protein through the combination of "target protein-primary antibody-biotin-labeled secondary antibody-streptavidin-biotin-labeled EM-a-FAM". Three control groups were set up: no addition of any substance; addition of primary antibody and EM-a-FAM; addition of biotin-labeled secondary antibody, streptavidin and EM-a-FAM. Laser scanning confocal microscopy (CLSM) was used to image the membrane protein labeled with EM-a and found that ( Figure 3 -A), it can be seen that only when the target protein, primary antibody, biotin-labeled secondary antibody, streptavidin and EM-a-FAM are present at the same time, the FAM fluorescence signal can be detected on the cell membrane of MCF-7 cells, indicating that EM-a successfully and specifically labels the target protein.

[0077] 3.2 Cholesterol-modified EM-ci inserts into cell membrane

[0078] During cell culture, cells were co-cultured with cholesterol-modified EM-ci, where EM-c3 was labeled with FAM and then imaged using CLSM ( Figure 3 -B). Strong FAM fluorescence signals were detected on the cell membranes of both cell types, indicating that cholesterol-modified EM-ci can be inserted into the cell membrane and complementarily bind to EM-c3. Statistical analysis of the fluorescence signals showed that both signals were strong and of similar intensity, indicating that the signal peak depends on the number of EM-ci anchored on the cell membrane. In addition, cholesterol-mediated DNA insertion is different from EM-a labeling of target proteins, and it shows consistent labeling effects in both types of cells, providing basic conditions for the dynamic recruitment of molecules on the cell membrane.

[0079] 3.4 Dynamic recruitment imaging of UCNP-EM-d

[0080] After successfully verifying the dynamic recruitment of EM-d-FAM on the cell membrane, we used UCNP to replace FAM fluorescence as the signal output. This not only enhances the signal of molecular dynamic recruitment, but also further verifies the universality of molecular dynamic recruitment. For cells labeled with both Em-a and Em-ci, UCNP-EM-d is recruited to the cell membrane through EDC and produces upconversion luminescence (UCL) under the excitation of infrared light ( Figure 4A and 4B). Similarly, EpCAM dynamic recruitment imaging was performed on MCF-7 and HepG2 cells. The results showed that strong UCL signals appeared in both MCF-7 cells with high EpCAM expression and HepG2 cells with low EpCAM expression. When Em-d-FAM was recruited, the signal differences in MCF7 and HepG2 cells with high or low EpCAM expression levels still existed. However, when EM-d-UCNP was recruited, the difference in UCL signals between the two cell types was not obvious, indicating that the signal amplification intensity had reached a high point. Recruiting EM-d-UCNP demonstrated stronger imaging capabilities for proteins with low expression levels. Therefore, the molecular dynamic recruitment system can further amplify the imaging signal through new materials such as UCNP, and achieve excellent imaging effects even for targets with extremely low abundance.

[0081] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A membrane protein imaging method, characterized in that: The following steps are involved: S1. Cell membrane protein labeling: through the process of target protein-primary antibody-biotin-labeled secondary antibody-streptavidin-biotin-labeled EM-a, the membrane target protein EpCAM is labeled with EM-a; S2. Cell membrane engineering: Add cholesterol-modified DNA complex EM-ci to the cells to be imaged and incubate at room temperature to allow EM-ci to embed into the cell membrane; S3, Synthesis of UCNP-EM-d: EM-d-NH2 was covalently coupled to citric acid functionalized upconversion nanoparticles CA-UCNP via EDC / NHS; S4, Molecular dynamic recruitment: After cell membrane protein labeling and cell membrane engineering, EM-bi and EM-d-FAM or UCNP-EM-d were added to the cells, and after incubation, the cell nuclei were stained with DAPI and imaged with a confocal laser scanning microscope; The nucleic acid sequence of the EM-a is shown in SEQ ID NO: 1; The EM-bi includes EM-b1 and EM-b2, and the nucleic acid sequences thereof are shown in SEQ ID NOs: 2 and 3; The EM-ci includes EM-c1, EM-c2 and EM-c3, and the nucleic acid sequences thereof are shown in SEQ ID NOs: 4-6 respectively; The nucleic acid sequence of the EM-d is shown in SEQ ID NO:

7.

2. A membrane protein imaging method according to claim 1, characterized in that: The CA-UCNPs were synthesized by the following steps: 1) Mix YCl3, YbCl3, TmCl3 solution, oleic acid and 1-octadecene, add ammonium fluoride and methanol solution of sodium hydroxide after reaction, and obtain NaYF4:Yb,Tm crystal nucleus; 2) Then react with YCl3, oleic acid, 1-octadecene mixture, ammonium fluoride and sodium hydroxide to obtain core-shell UCNP; 3) After hydrophilic treatment with ethanol, water and hydrochloric acid, sodium citrate solution was added for incubation to obtain CA-UCNP.

3. A membrane protein imaging method according to claim 2, characterized in that: The CA-UCNP is synthesized by the following specific steps: A. Take 135.5 mg YCl3, 83.7 mg YbCl3, 50 μL TmCl3, 8 mL oleic acid and 15 mL 1-octadecene, mix them, react at 90°C for 20 minutes under stirring, then react at 150°C for 1 hour, add 5 mL anhydrous methanol containing 148 mg ammonium fluoride and 100 mg sodium hydroxide, heat at 90°C for 1 hour to remove methanol, add 4 mL cyclohexane and 4 mL ethanol to the collected product, wash 3 times to obtain the crystal core NaYF4:Yb,Tm; B. Take 135.5 mg YCl3, 8 mL oleic acid and 15 mL 1-octadecene, mix them, react at 90 ° C for 20 minutes under stirring, then react at 150 ° C for 1 hour, add 5 mL of anhydrous methanol containing 148 mg ammonium fluoride and 100 mg sodium hydroxide, and the above cyclohexane solution containing NaYF4:Yb, Tm, heat at 90 ° C for 1 hour to remove methanol and cyclohexane, and the resulting mixture is heated to 300 ° C under nitrogen protection for 1.5 hours. After natural cooling, 20 mL ethanol is added, and the product is collected by centrifugation at 13000 rpm for 5 minutes. The collected product is added with 4 mL cyclohexane and 4 mL ethanol to obtain core-shell UCNPs; the core-shell UCNPs are dispersed in 4 mL ethanol, 2 mL water and 2 mL hydrochloric acid for hydrophilic treatment. After stirring and ultrasonic treatment at room temperature for 2 hours, the mixture is washed twice with 50% ethanol, and the UCNPs are collected by centrifugation at 13000 rpm for 20 minutes; C. 800 mg of sodium citrate was dissolved in 80 mL of water and slowly added to the core-shell UCNPs, which were then gently stirred and incubated overnight at room temperature. CA-UCNPs were collected by centrifugation at 13,000 rpm for 20 min and dispersed in 4 mL of water.

4. A membrane protein imaging method according to claim 1, characterized in that: The step S1 includes the following steps: 1) When the cells reach an appropriate density, remove the cell culture medium and wash the cells three times with 1× PBS; 2) Add 4% paraformaldehyde to fix for 15 minutes at room temperature, and then wash three times with 1× PBST; 3) Add primary antibody and incubate for 1 hour, then wash three times with 0.05% PBST; 4) Add biotin-labeled secondary antibody and incubate for 1 hour, then wash three times with 0.05% PBST; 5) Add streptavidin and incubate for 15 min, then wash three times with 0.05% PBST; 6) Add 500 nM biotin-labeled EM-a and incubate for 30 minutes, then wash three times with 1× PBS. At this time, EpCAM on the cell membrane is labeled with EM-a.

5. A membrane protein imaging method according to claim 1, characterized in that: The step S2 includes the following steps: 500 nM of cholesterol-modified DNA complex EM-ci was added and incubated at room temperature for 30 minutes; after washing with 1× PBS to remove free and unstable EM-ci, the obtained samples were used for subsequent experiments.

6. A membrane protein imaging method according to claim 1, characterized in that: The step S3 includes the following steps: 1) 20 μL of CA-UCNP, 2 mg of EDC, and 3 mg of Sulfo NHS were mixed in 1 mL of 1× PBS, stirred at 600 rpm for 30 min, and then centrifuged at 13,000 rpm for 20 min to collect the activated CA-UCNP; 2) After activation, 1 mL of 200 nM EM-d-NH2 conjugate was added and stirred at 600 rpm overnight at room temperature; finally, the final product UCNP-EM-d was collected by centrifugation at 13000 rpm for 20 min, washed three times with 1× PBS, then resuspended in 1× PBS containing 10 mM MgCl2 and stored in a refrigerator at 4°C for subsequent experiments.

7. A membrane protein imaging method according to claim 1, characterized in that: The step S4 includes the following steps: 1) After cell membrane protein labeling and cell membrane engineering, 200 μL 1× PBS consisting of 10 mM MgCl2, 25 nM EM-bi, and 100 nM EM-d-FAM or 0.1 mg / mL UCNP-EM-d was added to the cells, incubated for 30 min, and washed three times with 1× PBSs; 2) Then, DAPI was added to stain the cell nuclei, incubated at room temperature in the dark for 10 min, washed three times with 1× PBS, and the cells were imaged using an LSM 900 confocal laser scanning microscope.

8. A membrane protein imaging method according to claim 1, characterized in that: The cells are MCF7 or HepG2.

9. Application of the membrane protein imaging method according to any one of claims 1 to 8 in the field of biopharmaceuticals.

Images