Key type DNA assembly for circulating tumor cell marker protein multiplex analysis

Abstract

The invention discloses a key type DNA assembly for circulating tumor cell marker protein multiplex analysis, and belongs to the technical field of biomedical molecular diagnosis. The lock-key type DNA assembly comprises a lock type DNA assembly and key type DNA, the lock type DNA assembly comprises a 16nt double-strand displacement region, a 10nt single-strand fulcrum region, a 7nt single-strand closed region and a covalent double-strand signal region containing a plurality of fluorophores, and the key type DNA is a single-strand sequence modified on an antibody. According to the present invention, based on the key type DNA assembly design, the signal in-situ amplification and the multiple analysis of the CTC marker protein on the single cell level are achieved, the theoretical basis is provided for the multiple detection analysis of the cancer circulating tumor cells, and the new method is provided for the further realization of the accurate diagnosis and the personalized treatment of the cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical molecular diagnosis, in particular to a lock-and-key type DNA assembly for multiple analysis of circulating tumor cell marker proteins. BACKGROUND

[0002] Circulating tumor cells (CTCs) are tumor cells released into the blood from primary or metastatic tumors, which interact with various cell components such as platelets, and through blood flow to other parts of the body, and even to other tissues or organs, and are markers of tumor metastasis. CTCs are extremely rare, with only 1-10 CTCs per 10 mL of cancer patient blood, and are affected by epithelial-mesenchymal transition, CTCs express a variety of different phenotypes, and have high heterogeneity. CTCs have been shown to be closely related to metastasis of breast cancer, colorectal cancer and other cancers, and CTC detection plays a major role in early diagnosis, targeted therapy and disease prognosis of cancer. As a non-invasive liquid biopsy technology, CTC detection overcomes the limitations of traditional tissue biopsy methods, and can achieve continuous sample collection and long-term dynamic monitoring. However, current CTC analysis methods can usually only detect 2 to 3 molecular phenotypes, and this narrow detection range cannot fully capture the heterogeneity between different CTCs, thereby limiting its application in precision diagnosis and personalized treatment.

[0003] CODEX technology (CO-Detection by Indexing) is a relatively advanced super-multiplex immunofluorescence technology on the market, which can realize high-throughput detection of more than 50 biomarkers by combining barcoded oligonucleotides with antibodies and using fluorescently labeled complementary oligonucleotides for target detection. However, this technology does not involve signal amplification, which makes it difficult to detect low-expression markers. Therefore, improving detection sensitivity is still the key to further development of this technology. SUMMARY

[0004] The purpose of the present application is to provide a lock-and-key type DNA assembly for multiple analysis of circulating tumor cell marker proteins to solve the problems existing in the prior art. By combining the lock-and-key type DNA assembly with immunofluorescence technology, the conversion of protein signal to DNA signal is realized, and based on the DNA orthogonal reaction, the signal is amplified and output in situ, which improves the detection throughput and provides a theoretical basis for multiple detection and analysis of circulating tumor cells in cancer, and provides a new method for further realizing precise diagnosis and personalized treatment of cancer.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The application provides a lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins, which comprises a lock-type DNA assembly and a key-type DNA, wherein the lock-type DNA assembly comprises a 16 nt double-stranded displacement region, a 10 nt single-stranded branch point region, a 7 nt single-stranded closed region, and a covalent double-stranded signal region containing a plurality of fluorescent groups with an excitation wavelength of 488 nm and pseudo-color labeled with green; and the lock-and-key DNA assembly has an observation signal amplification and output effect.

[0007] The key-type DNA is a single-stranded sequence modified on an antibody and complementary to the lock-type DNA assembly.

[0008] Preferably, the fluorescent group comprises any one of AF488, AF546, AF594, AF647 and AF750.

[0009] Preferably, the key-type DNA comprises a single-stranded sequence complementary to the single-stranded branch point region and the double-stranded displacement region in the lock-type DNA assembly.

[0010] Preferably, the lock-and-key DNA assembly is assembled in a manner that, based on DNA orthogonal reaction, realizes in-situ amplification and output of signals, and improves detection sensitivity. When the key-type DNA exists, the single-stranded branch point region is combined with the lock-type DNA assembly, and the double-stranded displacement region is displaced, and the displaced sequence is complementary to the 7 nt single-stranded closed region to form a stable lock-and-key DNA assembly.

[0011] Preferably, a plurality of lock-type DNA assemblies and a plurality of key-type DNAs correspond to each other and do not interfere with each other.

[0012] The application also provides a lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins of breast cancer, which comprises a lock-type DNA assembly and a key-type DNA, and the lock-type DNA assembly comprises sequences self-assembled in any one of the following:

[0013] (1) nucleotide sequences self-assembled as shown in SEQ ID NO. 1, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 12, wherein the nucleotide sequence shown in SEQ ID NO. 12 is modified with a fluorescent group at one end;

[0014] (2) nucleotide sequences self-assembled as shown in SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 12, wherein the nucleotide sequence shown in SEQ ID NO. 12 is modified with a fluorescent group at one end;

[0015] (3) a self-assembly construction of nucleotide sequences as shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9 and SEQ ID NO. 12, wherein the nucleotide sequence as shown in SEQ ID NO. 12 is modified with a fluorescent group at one end;

[0016] (4) a self-assembly construction of nucleotide sequences as shown in SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 10 and SEQ ID NO. 12, wherein the nucleotide sequence as shown in SEQ ID NO. 12 is modified with a fluorescent group at one end;

[0017] (5) a self-assembly construction of nucleotide sequences as shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 11 and SEQ ID NO. 12, wherein the nucleotide sequence as shown in SEQ ID NO. 12 is modified with a fluorescent group at one end;

[0018] The key-type DNA comprises any one of the nucleotide sequences as shown in SEQ ID NO. 13-17, and each of the nucleotide sequences is modified with a maleimide group or biotin at the end.

[0019] Preferably, the fluorescent group comprises any one of AF488, AF546, AF594, AF647 and AF750.

[0020] Preferably, the lock-type DNA assembly of the sequence construction as shown in (1) forms a lock-key type DNA assembly with the nucleotide sequence as shown in SEQ ID NO. 13 through DNA orthogonal reaction;

[0021] and / or the lock-type DNA assembly of the sequence construction as shown in (2) forms a lock-key type DNA assembly with the nucleotide sequence as shown in SEQ ID NO. 14 through DNA orthogonal reaction;

[0022] and / or the lock-type DNA assembly of the sequence construction as shown in (3) forms a lock-key type DNA assembly with the nucleotide sequence as shown in SEQ ID NO. 15 through DNA orthogonal reaction;

[0023] and / or the lock-type DNA assembly of the sequence construction as shown in (4) forms a lock-key type DNA assembly with the nucleotide sequence as shown in SEQ ID NO. 16 through DNA orthogonal reaction;

[0024] and / or the lock-type DNA assembly of the sequence construction as shown in (5) forms a lock-key type DNA assembly with the nucleotide sequence as shown in SEQ ID NO. 17 through DNA orthogonal reaction.

[0025] The application also provides the use of the lock-and-key DNA assembly for the multiplex analysis of circulating tumor cell marker proteins or the lock-and-key DNA assembly for the multiplex analysis of circulating tumor cell marker proteins of breast cancer in the preparation of a product for the multiplex analysis of circulating tumor cell marker proteins at the single-cell level. Further, the product includes a kit, reagents, etc.

[0026] The application also provides the use of the lock-and-key DNA assembly for the multiplex analysis of circulating tumor cell marker proteins or the lock-and-key DNA assembly for the multiplex analysis of circulating tumor cell marker proteins of breast cancer in the preparation of a product for the diagnosis of tumors, including breast cancer. Further, the product includes a kit, reagents, etc.

[0027] The application discloses the following technical effects:

[0028] The application constructs a lock-and-key DNA assembly structure for the multiplex analysis of CTC marker proteins, and realizes the conversion of protein signals to DNA signals in combination with an immune imaging technology, breaks through the limitation of antibody species, and improves the detection throughput. Meanwhile, in combination with the signal amplification effect of the DNA assembly structure, the signal output and the sensitivity of detection can be significantly improved, and the heterogeneity analysis of breast cancer CTCs is realized. The application realizes the in-situ amplification of signals and the multiplex analysis of CTC marker proteins at the single-cell level based on the lock-and-key DNA assembly, and provides a new direction for the precise diagnosis and personalized treatment of CTCs in cancer. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0030] Figure 1 It is a schematic diagram of the universal lock-and-key DNA assembly structure in the embodiments;

[0031] Figure 2 It is a result diagram of the orthogonality verification of the lock-and-key DNA assembly in the embodiments;

[0032] Figure 3 It is a result diagram of the cell detection orthogonality of the lock-and-key DNA assembly in the embodiments;

[0033] Figure 4 It is a result diagram of the actual sample detection of the lock-and-key DNA assembly in the embodiments;

[0034] Figure 5 It is a schematic diagram of the sequence structure shown in Table 1 as 12-16. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present application will now be described in detail, with reference to the drawings, which are not to be construed as limiting the application. The detailed description is made with reference to certain aspects, features and embodiments of the application, but it is to be understood that the application is not limited to the aspects, features and embodiments specifically described.

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each and every value, intermediate value and sub-range within the range of values. The upper and lower limits of these smaller ranges and intermediate values can independently be included or excluded from the range of values.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0038] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0039] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0040] Example 1: Characterization of lock-and-key DNA assembly, comprising the following steps.

[0041] 1. A lock-and-key DNA assembly structure for multiplex analysis of circulating tumor cell marker proteins, comprising:

[0042] A. "Lock" type DNA is a DNA assembly containing multiple fluorescent molecules, the whole is a stable covalent binding structure, characterized by containing a 16 nt double-stranded displacement region, a 10 nt single-stranded branch point region, a 7 nt single-stranded closed region, and a covalent double-stranded signal region containing multiple fluorophores;

[0043] B. "Key" type DNA modified on the antibody, characterized by 10+16 nt single-stranded sequence, can be assembled with the branch point region and the displacement region of "lock" type DNA;

[0044] C. When "key" type DNA exists, it can be combined with "lock" type DNA assembly through the branch point region, and the displacement region is displaced, and the sequence displaced is complementary to the 7 nt closed region to form a stable structure, so as to realize the stable combination of lock and key;

[0045] D. Multiple "lock" type DNA assemblies and multiple "key" type DNA correspond to each other and do not interfere with each other;

[0046] 2. Based on the structure of lock and key type DNA assembly, a plurality of analyses of circulating tumor cell marker proteins are carried out, and the fluorescent groups include one or more of AF488, AF546, AF594, AF647, AF750, or other fluorescent groups, quantum dots.

[0047] 1) The design concept of lock and key type DNA assembly is as shown in Figure 1 , and the specific composition and combination mode are as follows:

[0048] A. "Lock" type DNA is a DNA assembly containing multiple fluorescent molecules, and the whole is a stable covalent combination structure, characterized by containing a 16 nt double-stranded displacement region, a 10 nt single-stranded branch point region, a 7 nt single-stranded closed region, and a covalent double-stranded signal region containing multiple fluorescent groups;

[0049] B. "Key" type DNA modified on the antibody, characterized by 10 nt+16 nt single-stranded sequence, can be assembled with the single-stranded branch point region and the double-stranded displacement region of "lock" type DNA;

[0050] C. When "key" type DNA exists, it can be combined with "lock" type DNA assembly through the branch point region, and the displacement region is displaced, and the sequence displaced is complementary to the 7 nt closed region to form a stable structure, so as to realize the stable combination of lock and key;

[0051] D. Multiple "lock" type DNA assemblies and multiple "key" type DNA correspond to each other and do not interfere with each other.

[0052] 2) Agarose gel electrophoresis verifies the feasibility and orthogonality of lock and key type DNA assembly

[0053] The DNA sequences in Table 1 are evaluated for thermodynamic similarity and dimerization degree by NUPACK nucleic acid sequence analysis website, so as to comprehensively evaluate the characteristics of the designed sequences and the similarities and differences between them. The feasibility and orthogonality of the lock and key type DNA assembly are verified by the magnetic bead model.

[0054] Table 1

[0055]

[0056]

[0057] Note: Key1', Key2', Key3', Key4', Key5' respectively correspond to the sequences shown in Key1, Key2, Key3, Key4 and Key5, and are modified by replacing the maleimide group (Maleimide) with biotin (Biotin). The side chain modification CNVK in the sequence above is to form a covalent bond and increase structural stability.

[0058] According to the above lock-and-key type DNA assembly design concept, the combination mode of the lock-and-key type DNA assembly formed by the markers Her2, PanCK, Vim, PDL1 and Ki67 in Table 1 is (taking Her2 as an example, the complementary corresponding relationship between the "lock" type DNA assembly and the "key" type DNA assembly sequence is distinguished by different annotation methods, and the corresponding relationship also exists for other markers):

[0059] The first combination mode (for magnetic bead model experiment):

[0060] Her2-AF488: 1+6+7+12&Her2-bT@Streptavidin magnetic beads (Lock1-AF488&Key1');

[0061] PanCK-AF488: 2+6+8+12&PanCK-bT@Streptavidin magnetic beads (Lock2-AF488&Key2');

[0062] Vim-AF488: 3+6+9+12&Vim-bT@Streptavidin magnetic beads (Lock3-AF488&Key3');

[0063] PDL1-AF488: 4+6+10+12&PDL1-bT@Streptavidin magnetic beads (Lock4-AF488&Key4');

[0064] Ki67-AF488: 5+6+11+12&Ki67-bT@Streptavidin magnetic beads (Lock5-AF488&Key5').

[0065] The second combination mode (for CTC simulation sample orthogonal detection experiment):

[0066] Her2-AF488: 1+6+7+12&Her2-T@Her2 (Lock1-AF488&Key1);

[0067] PanCK-AF488: 2+6+8+12&PanCK-T@PanCK (Lock2-AF488&Key2);

[0068] Vim-AF488: 3+6+9+12 & Vim-T@Vim (Lock 3-AF488 & Key 3);

[0069] PDL1-AF488: 4+6+10+12 & PDL1-T@PDL1 (Lock 4-AF488 & Key 4);

[0070] Ki67-AF488: 5+6+11+12 & Ki67-T@Ki67 (Lock 5-AF488 & Key 5).

[0071] The third combination mode (for actual clinical breast cancer sample detection):

[0072] Her2-AF488: 1+6+7+12 & Her2-T@Her2 (Lock 1-AF488 & Key 1);

[0073] PanCK-AF546: 2+6+8+13 & PanCK-T@PanCK (Lock 2-AF546 & Key 2);

[0074] Vim-AF594: 3+6+9+14 & Vim-T@Vim (Lock 3-AF594 & Key 3);

[0075] PDL1-AF647: 4+6+10+15 & PDL1-T@PDL1 (Lock 4-AF647 & Key 4);

[0076] Ki67-AF750: 5+6+11+16 & Ki67-T@Ki67 (Lock 5-AF750 & Key 5).

[0077] The above 1+6+7+12 represents the sequence combination corresponding to the names 1, 6, 7, and 12 in Table 1, and the other 2+6+8+12, 3+6+9+12, 4+6+10+12, 5+6+11+12, 2+6+8+13, 3+6+9+14, 4+6+10+15, 5+6+11+16 are the same.

[0078] As shown in Figure 2 , only when the same group of "key" type DNA and "lock" type DNA assembly exists, the reaction can occur, and clear color will be produced on the magnetic beads. This phenomenon shows that the lock-key type DNA assembly structure has good orthogonality.

[0079] Example 2: Application of lock-key type DNA assembly to breast cancer CTC heterogeneity analysis

[0080] Take breast cancer CTC marker Her2, Panck, Vim, PDL1, Ki67 as an example, use 5 groups of lock and key type DNA assemblies to output signals, realize multi-index inspection and heterogeneity analysis of CTCs. Including the following steps:

[0081] Step a: separate breast cancer CTCs by cell filter, and coat CTC suspension on polylysine (pLL) coated glass slide for adhesion. The specific operation is as follows:

[0082] Take 3mL of breast cancer patient blood, add 4% paraformaldehyde at a ratio of 1:2, mix evenly, and fix at room temperature for 10min. Add 3mL PBS to rinse the filter membrane. Add the treated blood sample, and when the sample is about to be filtered, add 3mL of 4% paraformaldehyde again, pause and stand for 10min. After fixation, click continue to run, and when the cell fixation solution is about to be filtered, wash the cells with 1mL PBS, repeat the washing for 12 times. After the experiment, take out the cell filter, blow down the cells on the filter membrane and move to the EP tube. Finally, dilute the CTCs to the appropriate ratio, load the adhesion glass slide and sample chamber on the sample clamp in turn, and place it in the centrifugal chamber of the smear machine. Add 350μL of cell suspension to each sample chamber, set the speed of the smear machine to 1200RPM, and the time to 5min to make CTC sample.

[0083] Step b: incubate double-stranded displacement region, single-stranded branch point region, single-stranded closed region and covalent double-stranded signal region containing multiple fluorophores in PCR instrument and perform ultraviolet irradiation to promote the formation of lock and key type DNA assembly. The specific operation is as follows:

[0084] After dissolving the DNA sequence with 1xPBS buffer, first denature at 95℃ for 5min in PCR instrument, and slowly reduce to room temperature. Then mix the DNA sequence, incubate at 37℃ for 30min in PCR instrument to form lock and key type DNA assembly. Finally, irradiate with ultraviolet light for 1min to complete photo covalent connection and form covalent double-stranded signal region containing multiple fluorophores.

[0085] Step c: couple Her2, Panck, Vim, PDL1, Ki67 antibody with "key" type DNA by covalent coupling method. The specific operation is as follows:

[0086] First, use NHS-PEG4-N3 or other coupling agent as activated antibody, and the activation molar ratio is 1:50 to obtain N3 modified antibody. After washing, the obtained antibody is coupled with DBCO modified "key" type DNA, and the coupling molar ratio is 1:10 (v:v). After coupling, add antibody stabilizer, and store the antibody at a concentration of 1:3 in 4℃ refrigerator for standby.

[0087] Step d: incubate the Her2, Panck, Vim, PDL1, Ki67 antibody-DNA conjugates obtained in step c with the sample obtained in step a, after the completion of the immune reaction, incubate the corresponding "lock" type DNA assembly, after washing, use fluorescence microscope for imaging, and analyze the imaging results for heterogeneity. The specific steps are as follows:

[0088] Dilute the antibody 50 times with blocking solution (2% BSA) to prepare a mixture of primary antibodies, and add 70 μL of the mixture of primary antibodies to the CTC sample, incubate overnight in a 4°C refrigerator. After incubation, wash twice with blocking solution, 2 min each time; fix with PFA at room temperature for 10 min, wash 3 times with 1x PBS; incubate with ice methanol for 5 min, wash 3 times with 1x PBS, and incubate with blocking buffer for 30 min. Then, add 200 nM of "lock" type DNA assembly, incubate in a 37°C oven for 30 min, and then wash twice with blocking solution, 2 min each time. Finally, add 50 μL of DAPI, incubate at room temperature for 5 min, wash 3 times with PBS, and perform nuclear staining. The final result is imaged and analyzed under a confocal microscope.

[0089] Based on the above method, the orthogonal detection of CTC simulation samples using lock-key type DNA assembly: cultured MCF7 cells were used as CTC simulation samples, and 5 "key" type DNA labeled PanCK primary antibodies were incubated respectively, and combined with 5 "lock" type DNA probes (the combination of key and lock refers to the second combination method of Example 1). Results Figure 3 As shown, 5 probes only produce signals in the orthogonal case, fully verifying that the technology has cell detection capability.

[0090] Based on the above method, the actual sample detection using lock-key type DNA assembly: prepare the CTC sample as described above, and incubate the "key" type DNA labeled primary antibody (the combination of key and lock is shown in the third combination method of Example 1). Results Figure 4 As shown, the application of lock-key type DNA assembly structure to the detection of 5 tumor targets on the actual sample shows the potential of lock-key type DNA assembly structure in the heterogeneity analysis of CTCs in actual clinical applications.

[0091] The above-described embodiments are only descriptions of the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins, characterized in that: The lock-key DNA assembly includes a lock DNA assembly and a key DNA, wherein the lock DNA assembly includes a 16nt double-stranded displacement region, a 10nt single-stranded branch region, a 7nt single-stranded blocking region, and a covalent double-stranded signal region containing multiple fluorescent groups, and the key DNA is a single-stranded sequence modified on the antibody and complementary to the lock DNA assembly.

2. The lock-key DNA assembly according to claim 1, wherein The fluorescent group includes any one of AF488, AF546, AF594, AF647 and AF750.

3. The lock-key DNA assembly according to claim 1, wherein The key-type DNA comprises a single-stranded sequence complementary to the single-stranded branch region and the double-stranded displacement region in the lock-type DNA assembly.

4. The lock-key DNA assembly according to claim 1, wherein The assembly method of the lock-key DNA assembly is: based on DNA orthogonal reaction, when the key DNA is present, it combines with the lock DNA assembly through the single-stranded branch region, and displaces the double-stranded displacement region, and the displaced sequence is complementary to the 7nt single-stranded closed region to form a stable lock-key DNA assembly.

5. The lock-key DNA assembly according to claim 1, wherein Multiple lock-type DNA assemblies and multiple key-type DNAs correspond to each other and do not interfere with each other.

6. A lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer, characterized in that: The lock-key DNA assembly comprises a lock-type DNA assembly and a key-type DNA, and the lock-type DNA assembly comprises a sequence self-assembly structure shown in any one of the following: (1) Self-assembly of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.12, wherein one end of the nucleotide sequence shown in SEQ ID NO.12 is modified with a fluorescent group; (2) self-assembly of the nucleotide sequences shown in SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 12, wherein one end of the nucleotide sequence shown in SEQ ID NO. 12 is modified with a fluorescent group; (3) Self-assembly of the nucleotide sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.12, wherein one end of the nucleotide sequence shown in SEQ ID NO.12 is modified with a fluorescent group; (4) self-assembly of the nucleotide sequences shown in SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 10, and SEQ ID NO. 12, wherein one end of the nucleotide sequence shown in SEQ ID NO. 12 is modified with a fluorescent group; (5) Self-assembly of the nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.11 and SEQ ID NO.12, wherein one end of the nucleotide sequence shown in SEQ ID NO.12 is modified with a fluorescent group; The key-type DNA includes any one of the nucleotide sequences shown in SEQ ID NO. 13-17, and the end of each nucleotide sequence is modified with a maleimide group or biotin.

7. The lock-key DNA assembly according to claim 6, wherein The fluorescent group includes any one of AF488, AF546, AF594, AF647 and AF750.

8. The lock-key DNA assembly according to claim 6, wherein The lock-type DNA assembly constructed by the sequence shown in (1) and the nucleotide sequence shown in SEQ ID NO.13 form a lock-key DNA assembly through a DNA orthogonal reaction; and / or the lock-type DNA assembly constructed by the sequence shown in (2) and the nucleotide sequence shown in SEQ ID NO.14 are reacted by DNA orthogonal reaction to form a lock-key DNA assembly; and / or the lock-type DNA assembly constructed by the sequence shown in (3) and the nucleotide sequence shown in SEQ ID NO.15 are reacted by DNA orthogonal reaction to form a lock-key type DNA assembly; and / or the lock-type DNA assembly constructed by the sequence shown in (4) and the nucleotide sequence shown in SEQ ID NO.16 are reacted by DNA orthogonal reaction to form a lock-key DNA assembly; And / or the lock-type DNA assembly constructed by the sequence shown in (5) and the nucleotide sequence shown in SEQ ID NO.17 form a lock-key DNA assembly through DNA orthogonal reaction.

9. Use of the lock-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins according to any one of claims 1 to 5 or the lock-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer according to any one of claims 6 to 8 in preparing a product for multiplex analysis of circulating tumor cell marker proteins at the single-cell level.

10. Use of the lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins according to any one of claims 1 to 5 or the lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer according to any one of claims 6 to 8 in the preparation of a product for diagnosing tumors, characterized in that: The tumor includes breast cancer.

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