A lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins

Abstract

The application discloses a lock-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 a key type DNA. The lock type DNA assembly comprises a 16nt double-stranded displacement region, a 10nt single-stranded branch point region, a 7nt single-stranded closed region and a covalent double-stranded signal region containing a plurality of fluorescent groups. The key type DNA is a single-stranded sequence modified on an antibody. The application realizes in-situ signal amplification and CTC marker protein multiplex analysis on a single cell level based on the lock-key type DNA assembly design, provides a theoretical basis for circulating tumor cell multiplex detection analysis of cancer, and provides a new method for further realizing precise diagnosis and individualized treatment of cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical molecular diagnostics, and in particular to a key-type DNA assembly for multiplex analysis of circulating tumor cell marker proteins. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells released from primary or metastatic lesions into the bloodstream. These cells interact with various cellular components, including platelets, and travel through the bloodstream to other parts of the body, even metastasizing to other tissues or organs. They are a marker of tumor metastasis. CTCs are extremely rare, with only 1-10 CTCs per 10 mL of blood from cancer patients. Influenced by epithelial-mesenchymal transition, CTCs express a variety of phenotypes, exhibiting high heterogeneity. CTCs have been proven to be closely associated with metastasis in various cancers, including breast cancer and colorectal cancer. CTC detection plays a significant role in early cancer diagnosis, targeted therapy, and disease prognosis. As a non-invasive liquid biopsy technique, CTC detection overcomes the limitations of traditional tissue biopsy methods, enabling continuous sample collection and long-term dynamic monitoring. However, current CTC analysis methods typically only detect 2 to 3 molecular phenotypes. This narrow detection range cannot fully capture the heterogeneity among different CTC cells, thus limiting its application in precision diagnosis and personalized treatment.

[0003] CODEX (CO-Detection by Indexing) technology is currently one of the most advanced ultra-multiplex immunoassay techniques on the market. It combines barcode oligonucleotides with antibodies and uses fluorescently labeled complementary oligonucleotides for target detection, enabling high-throughput detection of over 50 biomarkers. However, this technology does not involve signal amplification, making the detection of low-expression biomarkers difficult. Therefore, improving detection sensitivity remains crucial for the further development of this technology. Summary of the Invention

[0004] The purpose of this invention is to provide a key-type DNA assembly for multiplex analysis of circulating tumor cell marker proteins, thereby addressing the problems existing in the prior art. By combining the key-type DNA assembly with immunoimaging technology, the conversion of protein signals to DNA signals is realized. Based on DNA orthogonal reactions, in-situ signal amplification and output are achieved, improving detection throughput. This provides a theoretical basis for multiplex detection and analysis of circulating tumor cells in cancer and offers a new method for further achieving precise diagnosis and personalized treatment of cancer.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a lock-and-key DNA assembly for multiplex analysis of circulating tumor cell marker proteins. The lock-and-key DNA assembly comprises a lock-type DNA assembly and a key-type DNA assembly. The lock-type DNA assembly includes a 16nt double-stranded substitution region, a 10nt single-stranded fulcrum region, a 7nt single-stranded blocking region, and a covalently double-stranded signal region containing multiple fluorescent groups. The excitation wavelength of the fluorescent groups is 488nm, and pseudo-color labeling is performed using green. The signal amplification and output effect are observed.

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

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

[0009] Preferably, the key-type DNA contains single-stranded sequences complementary to the single-stranded pivot region and double-stranded substitution region in the lock-type DNA assembly.

[0010] Preferably, the key-lock DNA assembly is assembled in a manner based on DNA orthogonal reactions to achieve in-situ signal amplification and output, thereby improving detection sensitivity. When the key-lock DNA is present, it binds to the key-lock DNA assembly through the single-stranded pivot region and replaces the double-stranded substitution region, forming a stable key-lock DNA assembly that is complementary to the 7nt single-stranded blocking region.

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

[0012] The present invention also provides a key-lock DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer, the key-lock DNA assembly comprising a lock-type DNA assembly and a key-type DNA, wherein the lock-type DNA assembly comprises a sequence self-assembly of any of the following:

[0013] (1) Self-assembly of nucleotide sequences as 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;

[0014] (2) Self-assembly of nucleotide sequences as 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;

[0015] (3) Self-assembly of nucleotide sequences as 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;

[0016] (4) Self-assembly of nucleotide sequences as 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;

[0017] (5) Self-assembly of nucleotide sequences as 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;

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

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

[0020] Preferably, the lock-type DNA assembly constructed from the sequence shown in (1) and the nucleotide sequence shown in SEQ ID NO.13 are combined via a DNA orthogonal reaction to form a lock-and-key DNA assembly;

[0021] And / or the lock-type DNA assembly constructed from the sequence shown in (2) and the nucleotide sequence shown in SEQ ID NO.14 are used to form a lock-key DNA assembly through a DNA orthogonal reaction;

[0022] And / or the lock-type DNA assembly constructed from the sequence shown in (3) and the nucleotide sequence shown in SEQ ID NO.15 are used to form a lock-and-key DNA assembly through a DNA orthogonal reaction;

[0023] And / or the lock-type DNA assembly constructed from the sequence shown in (4) and the nucleotide sequence shown in SEQ ID NO.16 are used to form a lock-key DNA assembly through a DNA orthogonal reaction;

[0024] And / or the lock-type DNA assembly constructed from the sequence shown in (5) and the nucleotide sequence shown in SEQ ID NO.17 are used to form a lock-key DNA assembly through a DNA orthogonal reaction.

[0025] This invention also provides the application of the key-locked DNA assembly for multiplex analysis of circulating tumor cell marker proteins or the key-locked DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer in the preparation of products for multiplex analysis of circulating tumor cell marker proteins at the single-cell level. Further, the products include kits, reagents, etc.

[0026] This invention also provides the application of the keyed DNA assembly for multiplex analysis of circulating tumor cell marker proteins or the keyed DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer in the preparation of products for diagnosing tumors, wherein the tumor includes breast cancer. Further, the products include kits, reagents, etc.

[0027] The present invention discloses the following technical effects:

[0028] This invention constructs a key-lock DNA assembly structure for multiplex analysis of CTC marker proteins and combines it with immunoimaging technology to achieve the conversion of protein signals to DNA signals, overcoming the limitations of antibody species and improving detection throughput. Simultaneously, the signal amplification effect of the DNA assembly structure significantly improves signal output and detection sensitivity, enabling heterogeneity analysis of breast cancer CTCs. This invention, based on a key-lock DNA assembly, achieves in-situ signal amplification and multiplex analysis of CTC marker proteins at the single-cell level, providing a new direction for the precise diagnosis and personalized treatment of CTCs in cancer. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of a general key-locked DNA assembly structure in the embodiments;

[0031] Figure 2 This is a diagram showing the orthogonality verification results of the keyed DNA assemblies in the examples;

[0032] Figure 3 This is a diagram showing the orthogonality results of cell detection of keyed DNA assemblies in the examples;

[0033] Figure 4 The results of testing actual samples using the key-type DNA assembly in the examples;

[0034] Figure 5 The diagram shows the sequence structure labeled 12-16 in Table 1. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example 1: Characterization of key-locked DNA assemblies, specifically including the following steps.

[0041] 1. A key-lock 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. It has a stable covalently bound structure and is characterized by a 16nt double-stranded substitution region, a 10nt single-stranded fulcrum region, a 7nt single-stranded blocking region, and a covalently double-stranded signal region containing multiple fluorophores.

[0043] B. "Key" type DNA modification on the antibody is characterized by a 10+16nt single-stranded sequence that can complement the fulcrum region and substitution region of the "lock" type DNA assembly;

[0044] C. When the "key" type DNA is present, it can bind to the "lock" type DNA assembly through the fulcrum region and replace the substitution region. The substituted sequence complements the 7nt blocking region to form a stable structure, thereby achieving stable binding of the key and the lock.

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

[0046] 2. Based on the key-lock DNA assembly structure, it is used for multiplex analysis of circulating tumor cell marker proteins. The fluorescent groups include one or more of AF488, AF546, AF594, AF647, and AF750, or other fluorescent groups and quantum dots.

[0047] 1) Key-lock DNA assembly design concept, such as Figure 1 As shown, the specific structure and combination are as follows:

[0048] A. "Lock" type DNA is a DNA assembly containing multiple fluorescent molecules. It has a stable covalently bound structure and is characterized by a 16nt double-stranded substitution region, a 10nt single-stranded fulcrum region, a 7nt single-stranded blocking region, and a covalently double-stranded signal region containing multiple fluorophores.

[0049] B. "Key" type DNA modification on the antibody is characterized by a 10nt+16nt single-stranded sequence that can complement the single-stranded pivot region and double-stranded substitution region of the "lock" type DNA assembly.

[0050] C. When the "key" type DNA is present, it can bind to the "lock" type DNA assembly through the fulcrum region and replace the substitution region. The substituted sequence complements the 7nt blocking region to form a stable structure, thereby achieving stable binding of the key and the lock.

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

[0052] 2) Agarose gel electrophoresis was used to verify the feasibility and orthogonality of key-locked DNA assemblies.

[0053] Thermodynamic similarity and dimerization levels of the DNA sequences in Table 1 were assessed using the NUPACK nucleic acid sequence analysis website to comprehensively evaluate the characteristics and differences between the designed sequences. The feasibility and orthogonality of the key-locked DNA assemblies were verified using a magnetic bead model.

[0054] Table 1

[0055]

[0056]

[0057] Note: Key 1', Key 2', Key 3', Key 4', and Key 5' correspond to the sequences shown in Key 1, Key 2, Key 3, Key 4, and Key 5, respectively, and are obtained by replacing the maleimide group with biotin. The CNVK side chain modification in the sequences above is to form covalent bonds and increase structural stability.

[0058] Based on the above-mentioned lock-and-key DNA assembly design concept, the combination of lock-and-key DNA assemblies formed by the markers Her2, PanCK, Vim, PDL1, and Ki67 in Table 1 is as follows (taking Her2 as an example, the complementary correspondence between the "lock" type DNA assembly and the "key" type DNA assembly sequence is distinguished by different annotation methods; other markers also have corresponding relationships):

[0059] The first combination method (used for magnetic bead model experiments):

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

[0061] PanCK-AF488: 2+6+8+12 & PanCK-bT@Strepavidin magnetic beads (lock 2-AF488 & key 2');

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

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

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

[0065] The second combination method (used for orthogonal detection experiments of CTC simulated samples):

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

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

[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 method (used for actual clinical breast cancer sample testing):

[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. The same applies to the other combinations 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, and 5+6+11+16.

[0078] like Figure 2 As shown, a reaction occurs only when "key" and "lock" DNA assemblies of the same group are present, and only then will clear colors appear on the magnetic beads. This phenomenon indicates that the key-lock DNA assembly structure has good orthogonality.

[0079] Example 2: Application of key-locked DNA assemblies in heterogeneity analysis of breast cancer CTCs

[0080] Taking breast cancer CTC markers Her2, Panck, Vim, PDL1, and Ki67 as examples, five sets of keyed DNA assemblies were used for signal output to achieve multi-indicator detection and heterogeneity analysis of CTCs. The steps included are as follows:

[0081] Step a: Breast cancer CTCs were isolated using a cell filter. The CTC suspension was then coated onto a poly-L-lysine (pLL) coated glass slide for adhesion. The specific procedure was as follows:

[0082] Take 3 mL of blood from a breast cancer patient and add 4% paraformaldehyde at a 1:2 ratio. Mix well and fix at room temperature for 10 min. Add 3 mL of PBS to rinse the filter membrane. Add the treated blood sample, and when the sample is almost completely filtered, add another 3 mL of 4% paraformaldehyde. Pause the process and let it stand for 10 min. After fixation, click to continue the process. When the cell fixative is almost completely filtered, add 1 mL of PBS to the cell filter to wash the cells. Repeat the washing 12 times. After the experiment, remove the cell filter, pipette the cells off the filter membrane, and transfer them to an EP tube. Finally, dilute CTCs to an appropriate ratio, attach the adhesive slide and loading chamber to the sample holder, and place it in the centrifuge chamber of the smear machine. Add 350 μL of cell suspension to each loading chamber, set the smear machine speed to 1200 RPM, and the time to 5 min to prepare CTC samples.

[0083] Step b: Incubate the double-stranded substitution region, single-stranded fulcrum region, single-stranded blocking region, and covalently double-stranded signal region containing multiple fluorophores in a PCR instrument and irradiate with ultraviolet light to promote the formation of key-lock DNA assemblies. The specific operation is as follows:

[0084] After dissolving the DNA sequence in 1×PBS buffer, it was first denatured at 95°C for 5 min in a PCR instrument, and then slowly cooled to room temperature. The DNA sequences were then mixed and incubated at 37°C for 30 min in a PCR instrument to form a key-locked DNA assembly. Finally, it was irradiated with UV light for 1 min to complete photocovalent ligation, forming a covalent double-stranded signal region with multiple fluorophores.

[0085] Step c: Covalently conjugate Her2, Panck, Vim, PDL1, and Ki67 antibodies to key-type DNA. The specific procedure is as follows:

[0086] First, NHS-PEG4-N3 or other coupling agents were used as the activating antibody at a molar ratio of 1:50 to obtain N3-modified antibodies. After washing, the resulting antibodies were conjugated with DBCO-modified "key" DNA at a molar ratio of 1:10 (v:v). After conjugation, an antibody stabilizer was added, and the antibodies were stored at a concentration of 1:3 at 4°C for later use.

[0087] Step d: Incubate the Her2, Panck, Vim, PDL1, and Ki67 antibody-DNA conjugates obtained in step c with the sample obtained in step a. After the immune reaction is complete, incubate the corresponding "lock" type DNA assemblies. After washing, image the samples using a fluorescence microscope and perform heterogeneity analysis on the imaging results. The specific steps are as follows:

[0088] Antibodies were diluted 50-fold with blocking buffer (2% BSA) to prepare primary antibody mixtures. 70 μL of this mixture was added to CTC samples and incubated overnight at 4°C. After incubation, the samples were washed twice with blocking buffer for 2 min each time; fixed with PFA at room temperature for 10 min; washed three times with 1×PBS; incubated with ice-cold methanol for 5 min; washed three times with 1×PBS; and incubated with blocking buffer for 30 min. Subsequently, 200 nM of lock-type DNA assemblies were added, and the samples were incubated at 37°C for 30 min, followed by washing twice with blocking buffer for 2 min each time. Finally, 50 μL of DAPI was added, and the samples were incubated at room temperature for 5 min, washed three times with PBS, and stained for nuclear staining. The final results were analyzed using a confocal microscope.

[0089] Based on the above method, orthogonal detection of CTCs simulated samples was performed using key-lock DNA assemblies: cultured MCF7 cells were used as CTCs simulated samples, and five types of PanCK primary antibodies labeled with key-lock DNA were incubated, and binding attempts were made with five types of lock-lock DNA probes (the key-lock binding method refers to the second combination method in Example 1). Results Figure 3 As shown, all five probes only generate signals when orthogonal, fully verifying that the technology has cell detection capabilities.

[0090] Based on the above method, a key-lock DNA assembly was used to detect actual samples: CTCs samples were prepared as described above, and a primary antibody labeled with a key-lock DNA was incubated (the key-lock binding method is described in the third combination method of Example 1). Results Figure 4 As shown, the key-locked DNA assembly structure was used to detect five tumor targets on a real sample. The different distributions of target signals and the changes in fluorescence intensity demonstrate the potential of the key-locked DNA assembly structure for heterogeneity analysis of CTCs in actual clinical applications.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A key-type DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer, characterized in that, The lock-and-key DNA assembly comprises a lock-type DNA assembly and a key-type DNA assembly, specifically including: (1) The lock-type DNA assembly is constructed by self-assembly of nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.12, and the lock-type DNA assembly and the nucleotide sequence shown in SEQ ID NO.13 form a lock-key DNA assembly through DNA orthogonal reaction; And / or (2) the lock-type DNA assembly is constructed by self-assembly of nucleotide sequences as shown in SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.12, and the lock-type DNA assembly is formed by DNA orthogonal reaction with the nucleotide sequence shown in SEQ ID NO.14 to form a lock-key DNA assembly; And / or (3) the lock-type DNA assembly is constructed by self-assembly of nucleotide sequences as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.12, and the lock-type DNA assembly is formed by DNA orthogonal reaction with the nucleotide sequence shown in SEQ ID NO.15 to form a lock-key DNA assembly; And / or (4) the lock-type DNA assembly is constructed by self-assembly of nucleotide sequences as shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.10 and SEQ ID NO.12, and the lock-type DNA assembly is formed by DNA orthogonal reaction with the nucleotide sequence shown in SEQ ID NO.16 to form a lock-key DNA assembly; And / or (5) the lock-type DNA assembly is constructed by self-assembly of nucleotide sequences as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.11 and SEQ ID NO.12, and the lock-type DNA assembly is formed by DNA orthogonal reaction with the nucleotide sequence shown in SEQ ID NO.17 to form a lock-key DNA assembly; In each of the different lock-type DNA assemblies, one end of the nucleotide sequence shown in SEQ ID NO.12 is modified with a different fluorescent group; Any nucleotide sequence as shown in SEQ ID NO.13-17, wherein the end of each nucleotide sequence is modified with a maleimide group or biotin.

2. The key-locked DNA assembly as described in claim 1, characterized in that, The fluorescent group includes any one of AF488, AF546, AF594, AF647 and AF750.

3. The use of the key-type DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer as described in claim 1 or 2 in the preparation of products for multiplex analysis of circulating tumor cell marker proteins at the single-cell level.

4. The application of the key-type DNA assembly for multiplex analysis of circulating tumor cell marker proteins in breast cancer as described in claim 1 or 2 in the preparation of products for diagnosing tumors, characterized in that, The tumors include breast cancer.

Images