Kit for simultaneous detection of specific membrane protein marker and membrane protein dimer and its application

Abstract

The present invention belongs to the technical fields of functional nanoprobes and biological detection, and particularly relates to a kit for simultaneously detecting specific membrane protein markers and membrane protein dimers and its application. The detection kit includes a membrane proteome anchoring chain, recognition probes, SERS probes, and auxiliary signal amplification reagents. By combining DNA logic sensing based on aptamer recognition and DNA signal amplification based on the expression of EpCAM protein and dimerization of Met protein, a logic-responsive assembly is achieved, realizing the networked assembly of recognition probes and SERS probes, thereby achieving specific and precise recognition of tumor cells and highly sensitive SERS detection and imaging of membrane protein dimers. It is applicable to monitoring the HGF / Met signaling pathway mediated by Met protein dimerization between stromal cells and cancer cells in a complex tumor microenvironment and can evaluate the inhibitory effect of targeted drugs on receptor dimerization in real time.

Description

Technical Field

[0001] The present invention belongs to the fields of functional nanoprobe technology and biological detection, and particularly relates to a kit for simultaneously detecting specific membrane protein markers and membrane protein dimers and its application. Background Art

[0002] There are many specific membrane protein markers on the surface of tumor cells, and these markers play important roles in the occurrence, development, invasion, metastasis, and immune escape of tumors. For example, epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is abnormally highly expressed in various epithelial tumors, and its expression level is closely related to the malignancy, invasiveness, and metastatic ability of tumors. MUC1 mucin is a highly glycosylated transmembrane mucin that is abnormally highly expressed in tumors such as breast cancer and pancreatic cancer. It can promote tumor progression and enhance tumor drug resistance by regulating tumor cell metabolism and inhibiting tumor cell apoptosis. Similar membrane protein markers widely exist in different types of tumor cells, and their abnormal expression not only reflects the biological characteristics of tumors but also provides potential targets for tumor diagnosis and treatment. By detecting specific membrane protein markers, specific recognition of tumor cells can be achieved, providing an important basis for early diagnosis and personalized treatment of tumors.

[0003] The tumor microenvironment TME is a complex ecosystem composed of cancer cells, stromal cells, and acellular components (including extracellular matrix, growth factors, etc.). In this dynamic system, various stromal cells such as cancer-associated fibroblasts, immune cells, and stem cells interact with cancer cells by secreting signaling factors, activating transmembrane receptors and triggering downstream signaling pathways, thereby driving tumor proliferation, invasion, and drug resistance development. Among them, membrane protein dimerization, as a key step in receptor activation, is not only the initial event of intracellular signal transduction but also an important mechanism for regulating epithelial-mesenchymal transition EMT and tumor invasion. For example, hepatocyte growth factor HGF induces dimerization of its receptor mesenchymal epithelial transition factor Met, thereby activating the HGF / Met signaling pathway and triggering a pro-cancer signaling cascade reaction, inducing tumor cell proliferation, angiogenesis, and cell invasion. Epidermal growth factor EGF induces dimerization of epidermal growth factor receptor EGFR, thereby activating the EGF / EGFR signaling pathway, and its abnormal activation is closely related to the occurrence, proliferation, invasion, and metastasis of various tumors. Similar ligand-receptor interaction patterns widely exist in the dynamic regulation of the tumor microenvironment. However, how to accurately identify specific tumor cells in the complex tumor microenvironment, and simultaneously monitor weak membrane protein dimer events in real time, and analyze the transcellular signal transduction network mediated by them, remains a major challenge in current research.

[0004] Although traditional protein detection techniques can quantify the expression level of receptor proteins, they cannot capture their dynamic dimerization states and spatial distribution characteristics. Although fluorescence imaging strategies can detect the status of membrane protein receptors in situ, problems such as their low sensitivity and poor photostability limit their application in complex biological systems. These limitations make it difficult to reveal naturally occurring low-abundance signal events in the tumor microenvironment (such as receptor activation induced by trace factors secreted by stromal cells), and it is also difficult to achieve real-time monitoring of signal pathways or long-term tracking processes. To address these problems, surface-enhanced Raman scattering (SERS) is expected to provide a practical solution. It has single-molecule sensitivity, "fingerprint"-type characteristic Raman peaks, excellent photostability, and non-destructiveness, and is considered a reliable, easy-to-operate, and ultrasensitive analytical tool. In previous work, the patent application document with the application number 202210528479.0 has confirmed that the SERS technology has excellent imaging capabilities and application prospects in the detection of membrane protein dimers, and the patent application document with the application number 202311793420.5 has achieved the simultaneous detection of homologous and heterologous dimers of membrane proteins by the SERS technology. Although the existing SERS detection methods have greatly improved the detection sensitivity of membrane protein dimerization, there is still a challenge: cancer cells cannot be completely distinguished from normal cells, which may lead to difficult diagnosis and limited treatment effects. Therefore, there is an urgent need to develop a new SERS strategy to accurately identify specific tumor cells in a complex tumor microenvironment through specific membrane protein markers existing in tumor cells themselves, such as EpCAM, etc., and further improve the detection sensitivity of membrane protein dimerization in specific cells. Summary of the Invention

[0005] Based on the surface-enhanced Raman scattering technology, the present invention provides a kit for simultaneously detecting specific membrane protein markers and membrane protein dimers and its application, achieving highly sensitive SERS detection of membrane protein dimers in specific cancer cells. Through DNA logic sensing based on aptamer recognition and DNA signal amplification-based logic response assembly of EpCAM protein expression and Met protein dimerization, networked assembly of recognition probes and SERS probes is achieved, thereby realizing specific and accurate recognition of tumor cells and highly sensitive imaging of membrane protein dimers. It is possible to monitor the HGF / Met signal pathway mediated by Met protein dimerization between stromal cells and cancer cells in a complex tumor microenvironment, and can evaluate the inhibitory effect of targeted drugs on receptor dimerization in real time.

[0006] Technical Solution:

[0007] In the first aspect, the present invention provides a kit for simultaneously detecting specific membrane protein markers and membrane protein dimers, and the kit includes: membrane protein group anchor chains, recognition probes, SERS probes, and auxiliary signal amplification reagents;

[0008] The membrane proteome anchoring chain contains two membrane protein anchoring chains, T-Met-1 and T-Met-2, which are used to anchor two receptor protein monomers on the first membrane protein Met, namely Met-1 and Met-2. T-Met-1 and T-Met-2 can perform proximity hybridization when the two receptor protein monomers Met-1 and Met-2 dimerize. As Figure 1 shown, both T-Met-1 and T-Met-2 sequentially include: an aptamer sequence, a free sequence, a hybridization sequence, and a trigger sequence. It should be noted that, for the convenience of illustration, Figure 1 the illustration of the free sequence is omitted in

[0009] As Figure 2A shown, the recognition probe RP is prepared by simultaneously anchoring a first probe and a second probe on an AuNP with a diameter in the range of 20 - 50 nm. The first probe is a structure composed of a thiol-modified DNA single strand SYL3C anchored and connected to a DNA single strand c-aptamer through base complementary pairing. The second probe is a structure composed of a thiol-modified DNA single strand Linker anchored to a first RP hairpin H1 and a second RP hairpin H2 through base complementary pairing. The DNA single strand SYL3C can specifically recognize and bind to the second membrane protein and release the DNA single strand c-aptamer. Both the first RP hairpin H1 and the second RP hairpin H2 are assembled from DNA sequences, can initiate a CHA reaction after the release of the DNA single strand c-aptamer, form a stable double-stranded structure through toehold-mediated strand displacement reaction, and further release the DNA single strand c-aptamer.

[0010] As Figure 2BAs shown, the SERS probe (SERS tag) is prepared by anchoring a SERS hairpin and a Raman molecule on AuNPs with a diameter in the range of 20 - 50 nm. The SERS hairpin (H*) is assembled from a DNA sequence, and the Raman molecule is selected from any one of 4-MBA, DTNB, or 2-MBT;

[0011] The auxiliary signal amplification reagent is used to assist in signal amplification during dimerization and is an essential reagent for the detection kit described in the first aspect of the present invention. The auxiliary signal amplification reagent is a single-stranded DNAzyme with a hairpin structure; the single-stranded DNAzyme can specifically recognize and bind to the trigger sequence of the membrane protein anchoring strand, thereby forming a complex with the double-stranded T structure, and cleaving the SERS hairpin (H*) under the action of Mg 2+ ; due to the low level of Mg 2+ in the tumor microenvironment, the addition of Mg 2+ can enhance the auxiliary signal amplification. Therefore, preferably, the auxiliary signal amplification reagent further includes Mg 2+ .

[0012] As Figure 2C shown, the detection principle of the detection kit described in the first aspect of the present invention is specifically as follows: during the dimerization of the first membrane protein for proximity hybridization, the unhybridized trigger sequence parts of the two membrane protein anchoring strands can serve as footholds to open the hairpin structure of the single-stranded DNAzyme, and cleave the SERS hairpin (H*) of the SERS probe under the assistance of Mg 2+ ; at the same time, the single-stranded DNA SYL3C on the recognition probe can specifically recognize and bind to the second membrane protein, and the released single-stranded DNA c-aptamer can jointly trigger the CHA reaction with the recognition probe to form a more stable H1-H2 double strand and further release c-aptamer, enabling it to re-capture the recognition probe and trigger the next assembly cycle; the unhybridized part of the H2 sequence can hybridize with the cleaved SERS hairpin (H*), thereby enabling the recognition probe and the SERS probe to assemble; therefore, through the triggering of the first membrane protein dimerization and the second membrane protein, a network nanostructure composed of AuNPs with a diameter in the range of 20 - 50 nm is self-assembled around the cell membrane; the enhanced Raman scattering of the Raman molecule can be detected through the strong coupling surface plasmon resonance of the inter-particle nano-gaps of the network nanostructure.

[0013] As a more preferred embodiment of the detection kit described in the present invention, the molar ratio of the recognition probe (RP) to the SERS probe is 1:1;

[0014] As a more preferred embodiment of the detection kit of the present invention, the recognition probe RP is prepared by connecting H1 and H2 to the Linker, connecting c-aptamer to SYL3C, and co-labeling the Linker and SYL3C with 30 nm AuNP to obtain RP; the SERS tag is prepared by co-labeling 30 nm AuNP with the Raman molecule DTNB and H*; specifically, the preparation steps of the recognition probe RP include: incubating the solutions containing single-stranded DNA SYL3C, single-stranded DNA c-aptamer, the solution containing single-stranded DNA Linker, the first RP hairpin H1, and the second RP hairpin H2 at room temperature for 4 h each, incubating the two parts of the solution with 30 nm AuNP at room temperature for 5 h, and then performing probe aging treatment, that is, adding different gradient volumes of 2 M NaCl to the above solution at intervals of 30 min each time, so that the final concentration of NaCl is 0.2 M, gently shaking the mixture at room temperature for 6-12 h, centrifugally washing with PBS at 6000 rpm for 20 min, and after centrifugally washing three times, resuspending the precipitate in PBS to obtain the recognition probe RP; further preferably, the molar ratio of the c-aptamer to the SYL3C is (1:1)-(1.2:1); the molar ratio of H1, H2 to the Linker is (1:1:1)-(1.2:1.2:1); the molar ratio of the Linker, SYL3C to 30 nm AuNP is (5000:1000:1)-(10000:2000:1);

[0015] As a more preferred embodiment of the detection kit of the present invention, the preparation steps of the SERS probe SERS tag include: incubating the SERS hairpin H* with 30 nm AuNP at room temperature for 6-12 h, performing the same aging treatment as above, and then incubating the Raman molecule DTNB with the AuNP modified with the SERS hairpin H* at room temperature for 3 h, centrifugally washing with PBS at 6000 rpm for 20 min, and after centrifugally washing three times, resuspending the precipitate in PBS to obtain the SERS probe SERS tag; preferably, the molar ratio of H* to 30 nm AuNP is (1000:1)-(2000:1); the molar ratio of DTNB to the 30 nm AuNP modified with H* is (2000:1)-(3000:1).

[0016] In a second aspect, the present invention provides the use of the detection kit in the simultaneous detection of EpCAM protein and Met protein dimers. The second membrane protein is EpCAM protein, and T-Met-1 and T-Met-2 contain only 7 complementary bases; further preferably, the use includes the steps of: First, co-incubate the cells to be detected with the anchored strands T-Met-1 and T-Met-2 in a culture medium for 20 min to allow the anchored strands to bind to the Met protein on the cell membrane; after washing three times with PBS, add the DNAzyme and Mg 2+ solution, and incubate at 37 °C for 20 min; after washing three times with PBS, incubate the cells with the recognition probe RP and the SERS probe for 3 h; rinse the cells 3 times with PBS to remove free and non-specifically deposited probes, and then perform membrane protein dimer SERS imaging on live cells.

[0017] In the presence of EpCAM protein, an enhanced DTNB signal can be obtained when Met protein dimerizes, and this application method has been proven to be able to accurately identify specific tumor cells and perform highly sensitive detection of Met protein dimerization, thereby realizing the monitoring of the signal pathway between stromal cells and cancer cells in the tumor microenvironment and the real-time evaluation of the inhibitory effect of targeted drugs on Met protein dimerization;

[0018] The specially designed T-Met-1 and T-Met-2 contain only 7 complementary bases and it is difficult to form a stable T-Met structure in solution at room temperature. When the aptamer sequences of T-Met-1 and T-Met-2 are incubated with target cells, the aptamer sequences can specifically recognize and bind to the receptor protein Met on the live cell membrane. Once the dimerization inducer HGF is added to form Met protein dimers, the sequences of T-Met-1 and T-Met-2 can hybridize through 7 complementary bases to form a DNA double strand (i.e., proximity hybridization), leaving two single-stranded base sequences of T-Met-1 and T-Met-2 as footholds, which can hybridize with the Mg 2+ responsive DNAzyme, in Mg 2+Under the action of [DNAzyme], the hairpin H* of the SERS tag is cleaved. Meanwhile, SYL3C on the RP can specifically recognize and bind to the receptor protein EpCAM on the live cell membrane. At the same time, the released c-aptamer can jointly trigger the CHA reaction with the RP, that is, the c-aptamer can hybridize with H1 labeled on the Linker to form an H1-c structure. Then, with the help of H2 anchored on the Linker, the H1-c structure can dissociate because a more stable H1-H2 double strand is formed through toehold-mediated strand displacement reaction, and the c-aptamer is further released, enabling it to recapture the RP and trigger the next assembly cycle. At this time, the unhybridized part of H2 on the RP can hybridize with H* on the cleaved SERS tag, resulting in the successful assembly of the RP and the SERS tag. Therefore, through the dimerization of the Met protein and the triggering of the EpCAM protein, a network aggregate of 30 nm AuNPs (i.e., 30Au-30Au network nanostructure) is self-assembled on the EpCAM protein. At the same time, due to the strong coupled surface plasmon resonance at the interparticle nanogaps (i.e., hot spots) of the 30Au-30Au network nanostructure, the Raman scattering of the extremely strong Raman molecule DTNB can be detected.

[0019] As a preferred embodiment of the application described in the second aspect, the T-Met-1 sequence is as shown in SEQ ID NO: 1, the T-Met-2 sequence is as shown in SEQ ID NO: 2; the sequence of the first RP hairpin is as shown in SEQ ID NO: 4; the sequence of the second RP hairpin is as shown in SEQ ID NO: 5; the sequence of the SERS hairpin is as shown in SEQ ID NO: 6; the sequence of the SYL3C strand is as shown in SEQ ID NO: 7; the sequence of the c-aptamer strand is as shown in SEQ ID NO: 9; the sequence of the Linker strand is as shown in SEQ ID NO: 10; the sequence of the DNAzyme strand is as shown in SEQ ID NO: 11.

[0020] Beneficial effects: It is of practical significance to accurately identify specific tumor cells in a complex tumor microenvironment and monitor the transmembrane signaling pathway triggered by membrane protein dimerization. However, due to the often low levels of dimers on the cell membrane surface, the sensitivity of traditional detection methods is difficult to meet the application requirements. Although previous patent application documents with application numbers 202210528479.0 and 202311793420.5 have demonstrated the application potential of SERS technology in the detection of membrane protein dimers, it cannot fully distinguish cancer cells from normal cells, which may lead to difficulties in diagnosis and limited treatment effects. In contrast, the present invention can identify specific tumor cells in a complex tumor microenvironment through the specific markers existing in tumor cells themselves, namely the specific membrane protein markers described in the present invention, and then highly sensitively detect the membrane protein dimerization of specific cells;

[0021] The detection kit provided by the present invention realizes the networked assembly of the recognition probe RP and the SERS probe SERStag through DNA logic sensing based on aptamer recognition and DNA signal amplification-based logic response assembly based on EpCAM protein expression and Met protein dimerization, thereby achieving the specific and accurate recognition of tumor cells and the highly sensitive imaging of membrane protein dimers. It can monitor the HGF / Met signaling pathway mediated by Met protein dimerization between stromal cells and cancer cells in a complex tumor microenvironment and can evaluate the inhibitory effect of targeted drugs on receptor dimerization in real time. Brief Description of the Drawings

[0022] Figure 1 Schematic diagram of the structures of the two membrane protein anchoring chains T-Met-1 and T-Met-2 of the present invention and their dimerization under the action of HGF;

[0023] Figure 2A Composition and preparation process of the recognition probe RP;

[0024] Figure 2B Composition and preparation process of the SERS probe, i.e., SERS tag;

[0025] Figure 2C Schematic diagram of the detection principle of the detection kit described in the present invention;

[0026] Figure 3 Gel electrophoresis characterization diagram of the simulated working mechanism in Example 1;

[0027] Figure 4A Normalized absorption spectrum of the RP preparation process in Example 2 of the present invention;

[0028] Figure 4B Hydrodynamic diameter of the RP preparation process in Example 2 of the present invention;

[0029] Figure 4C Normalized absorption spectrum of the SERS tag preparation process in Example 2 of the present invention;

[0030] Figure 4D Hydrodynamic diameter of the SERS tag preparation process in Example 2 of the present invention;

[0031] Figure 5A Normalized absorption spectrum characterization of the network nanostructure of the probe in Example 3 of the present invention;

[0032] Figure 5B Hydrodynamic diameter characterization of the network nanostructure of the probe in Example 3 of the present invention;

[0033] Figure 5C SEM image of the network nanostructure of the probe in Example 3 of the present invention;

[0034] Figure 5D SERS spectrum of the network nanostructure of the probe in Example 3 of the present invention;

[0035] Figure 6 Magnetic field distribution before and after the probe assembly by FDTD simulation in Example 3 of the present invention, where Figure A is the electromagnetic field distribution of 30 nm AuNP; Figure B is the electromagnetic field distribution of the AuNP network nanostructure;

[0036] Figure 7 CCK-8 cytotoxicity test of MCF-7 cells after incubation under different conditions in Example 4 of the present invention;

[0037] Figure 8 SERS imaging of the dimerization of Met protein in MCF-7 cells under different treatment conditions over time in Example 5 of the present invention; among them, Figure A is the SERS imaging after treatment with HGF; Figure B is the average value of the SERS intensity calculated from Figure A; Figure C is the SERS image after no treatment; Figure D is the average value of the SERS intensity obtained from Figure C; Figure E is the SERS image after co-treatment with HGF and HGF inhibitor; Figure F is the average value of the SERS intensity obtained from Figure E;

[0038] Figure 9 SERS imaging of the dimerization of Met protein in MCF-7 cells in Example 6 of the present invention; among them, Figure A is the SERS images of MCF-7 cells before and after treatment with HGF, and after co-treatment with HGF and HGF inhibitor; Figure B is the analysis of the expression of phosphorylated Met (P-Met), Met and EpCAM in MCF-7 cells by Western blotting corresponding to Figure A;

[0039] Figure 10SEM images of 30 nm AuNP on MCF-7 cells in Example 7 of the present invention;

[0040] Figure 11 SERS imaging of different cells in Example 8 of the present invention; Figure A shows the expression of Met and EpCAM in four types of cells; Figure B shows the analysis of the expression of P-Met, Met, and EpCAM in four types of cells by Western blotting after treatment with HGF; Figure C shows the SERS images of four types of cells after treatment with HGF;

[0041] Figure 12 SERS imaging based on the HGF / Met signaling pathway between Breast CAFs or MCF-10A cells and MCF-7 cells in Example 9 of the present invention; Figure A shows a schematic diagram of the co-culture of MCF-7 cells with Breast CAFs or MCF-10A cells, and Figure B shows the SERS images of MCF-7 cells before and after co-culture with Breast CAFs or MCF-10A cells; Figure C shows the detection of the expression of P-Met, Met, and EpCAM in MCF-7 cells in Figure B by Western blotting;

[0042] Figure 13 SERS imaging based on the HGF / Met signaling pathway between BMSC cells and MCF-7 cells in Example 9 of the present invention; wherein, Figure A shows a schematic diagram of the co-culture of MCF-7 cells with BMSC cells, Figure B shows the SERS images of MCF-7 cells before and after co-culture with BMSC cells, and Figure C shows the detection of the expression of P-Met, Met, and EpCAM in MCF-7 cells in Figure B by Western blotting;

[0043] Figure 14 SERS imaging based on Amivantamab targeting and binding to Met to inhibit the HGF / Met signaling pathway in Example 10 of the present invention; Figure A shows the schematic diagram of Amivantamab targeting and binding to Met; Figure B shows the SERS images of MCF-7 cells before and after treatment with HGF, and after treatment with Amivantamab followed by HGF; Figure C shows the detection of the expression of P-Met, Met, and EpCAM in MCF-7 cells in Figure B by Western blotting. Detailed implementation manners

[0044] Combined with the following specific examples and drawings, the present invention will be further described in detail. The following examples are only illustrative, and the protection scope of the present invention is not limited thereto.

[0045] The nucleotide chains involved in the following examples were all synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd. The following preferred designs were adopted for the membrane proteome anchoring chain, recognition probe, SERS probe, and auxiliary signal amplification reagent in the specific kit: The membrane proteome anchoring chains T-Met-1 and T-Met-2 can trigger proximity hybridization when the Met protein dimerizes; the recognition probe RP was prepared by simultaneously anchoring the DNA single strand SYL3C and the DNA single strand Linker on 30 nm AuNP: Among them, the DNA single strand c-aptamer is connected to the DNA single strand SYL3C, and the first RP hairpin H1 and the second RP hairpin H2 are connected to the DNA single strand Linker; the SERS probe SERS tag was prepared by anchoring the SERS hairpin H* and the Raman molecule DTNB on 30 nm AuNP; and the DNA sequences designed in the membrane proteome anchoring chain, recognition probe, SERS probe, and auxiliary signal amplification reagent all adopted the preferred designs shown in Table 1 below in the specific implementation manner:

[0046] Table 1: Specific sequence design

[0047] Sequence Name Sequence Number Specific Sequence T-Met-1 SEQ ID NO: 1 5’-TGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTTTTTTTTTTTTTTTTAGTGAGGTGGAACATTA-3’ T-Met-2 SEQ ID NO: 2 5’-TCAAGTATGCCAAAGCCCTCACTTTTTTTTTTTTTTTTTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCT-3’ Target SEQ ID NO: 3 5’-TCAAGTATGCCAAAGGGAACATTA-3’ H1 SEQ ID NO: 4 5’-GACGACTAATAAGATTAATGCCCACTACAGAGGTTGCGTCTGGTAGTGTTGGATGCTCAGACGCAACCTCT-3’ H2 SEQ ID NO: 5 5’-TGTCCTAAGATAATCTCGAATTCCGTCTGAGCATCCAACACTACCAGACGCAACCTCTGTAGTGTTGGATGCTGGGTC-3’ H* SEQ ID NO: 6 5’-SH-TTTTTTGACCCAGCATCCAAGTACCGAGrArUAATGTTCCGATTTAATGCTGGGTC-3’ SYL3C SEQ ID NO: 7 5’-SH-TTTTTTTTTCACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3’ SYL3C-c SEQ ID NO: 8 5’-CAGGCCAACCCCCCATGACAACGTGGGACAGACGCAACCTCTGTAGTGAAAAAAAAA-3’ c-aptamer SEQ ID NO: 9 5’-CAGACGCAACCTCTGTAGTG-3’ Linker SEQ ID NO: 10 5’-ACATTAATCTTATTAGTCGTCTTTTTTTTTTTTTTCGAGATTATCTTAGGACAGCAGTTGAGGTAATA-SH-3’ DNAzyme SEQ ID NO: 11 5’-GCCTAATGTTCCCTTTGGCATACTTGATCAGGAACATTAGGCTAGCTACAACGACTCGGTAC-3’

[0048] The sources, preservation, and culture methods of the test cells involved in the following examples are specifically as follows: Human breast cancer cells (MCF-7), human breast cancer cells (SK-BR-3), and human normal liver cells (LO2) were purchased from KeyGen Biotech Co., Ltd. (Nanjing, China), bone marrow-derived mesenchymal stem cells (BMSC) were purchased from Cyagen Biosciences Inc. (Suzhou, China), human normal mammary epithelial cells (MCF-10A) were purchased from Yifeixue Co., Ltd. (Nanjing, China), and breast cancer-associated fibroblasts (Breast CAFs) were purchased from Youlike Biotechnology Co., Ltd. (Shanghai, China). MCF-7 cells, SK-BR-3 cells, LO2 cells, and BMSC cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin, 100 U / mL), and MCF-10A cells and breast cancer-associated fibroblasts were cultured in their respective special media. The cells were all cultured in a cell incubator according to the instructions; the HGF inhibitor was a polyclonal antibody against hepatocyte growth factor (HGF), purchased from Wuhan Yunke Biotechnology Co., Ltd., product number PAA047Hu01;

[0049] Example 1: Gel electrophoresis verification of the feasibility characterization of the sensing mechanism

[0050] To verify the feasibility of the proposed mechanism for simultaneous detection of specific membrane protein markers and membrane protein dimers based on SERS imaging, the nucleic acid interactions during the sensing process were characterized by gel electrophoresis. First, 2 μL of each 10 μM DNA sample in Table 1 was dissolved in 18 μL of 1×PBS buffer to ensure that the final concentration of each DNA sample was 1 μM. The above DNA samples were loaded and mixed in a centrifuge tube, and the hybridization reaction was carried out at 37°C for 3 h. Then, 5 μL of the hybridized DNA sample was mixed with 1 μL of 6×DNA Loading Buffer, and the mixture was added to a 10% polyacrylamide gel. Using a Bio-Rad electrophoresis apparatus, in 1×TBE buffer, a constant voltage of 100 V was applied and the electrophoresis was run for 65 min. Then, it was stained with 4SRed Plus Nucleic Acid Stain for 15 min and imaged through the GeneSys system (Syngene, UK).

[0051] As Figure 3 shown is the gel electrophoresis characterization diagram simulating the working mechanism of nucleic acid hybridization in the present invention. To simulate the T-Met double-stranded T structure formed by proximity hybridization after membrane protein dimerization, a synthetic Target that is partially complementary and longer relative to the part where T-Met hybridizes with DNAzyme was used. The specific simulation sequence is shown in the Target sequence in Table 1;

[0052] The results are as Figure 3As shown, the bands of SYL3C and c-aptamer can be clearly seen in lanes 1 and 2, respectively. The slowly migrating band in lane 3 indicates the hybridization of SYL3C with c-aptamer. In lane 4, the bright and fast-migrating band belongs to H1 with a single hairpin structure, while the faint and slow-migrating band corresponds to the self-hybridization band of two H1s. A similar phenomenon was also observed in the H2 band of lane 5. These bands indicate that a single H1 or H2 has a stable hairpin structure, while a very small amount of H1 or H2 can undergo self-hybridization. The bands of the mixture of H1 and H2 in lane 6 are similar to those of H1 (lane 4) or H2 (lane 5) alone, indicating that there is little or no hybridization between H1 and H2 at room temperature. After adding c-aptamer to the mixture of H1 and H2, the bright band in lane 7 indicates that c-aptamer can open the H2 hairpin structure to form a high-yield H1-H2 double-stranded structure and release c-aptamer, demonstrating the successful progress of the CHA reaction. The band of Linker can be clearly seen in lane 8. The slower-migrating bands in lanes 9 and 10 relative to lane 8 indicate the hybridization of Linker with H1 or H2. The even slower-migrating band in lane 11 indicates the hybridization of Linker with both H1 and H2. When c-aptamer is added, the band generated in lane 12 migrates slightly faster than that in lane 11, demonstrating the successful progress of the CHA reaction. The bands of Target, DNAzyme, and H* can be clearly seen in lanes 13, 14, and 16, respectively. The slowly migrating band in lane 15 indicates the hybridization of Target with DNAzyme. The bands of the mixture of DNAzyme and H* in lane 17 are similar to those of DNAzyme (lane 14) or H* (lane 16) alone, indicating that a single DNAzyme cannot cleave H*. After adding Mg 2+ to the mixture of DNAzyme and H*, the bands in lane 18 are similar to those in lane 17, indicating that DNAzyme cannot cleave H* under the action of Mg 2+ either, because the hairpin structure of DNAzyme effectively inhibits its cleavage activity at this time. After adding Target and Mg 2+ to the mixture of DNAzyme and H*, the hairpin-structured DNAzyme in lane 19 can be opened by Target and can effectively cleave H* with the assistance of Mg 2+ . At the same time, mixing Linker, H1, H2, c-aptamer, Target, DNAzyme, H*, Mg 2+, a band with slow migration appeared in lane 20 relative to lane 21 (i.e., lane 12), indicating that the cleaved part of H* could hybridize with the unhybridized part of H2 after reacting with CHA. The above results jointly demonstrated the feasibility and specificity of this sensing strategy based on nucleic acid hybridization mechanism.

[0053] Example 2: Preparation and Characterization of Recognition Probe RP and SERS Probe SERS tag

[0054] Preparation of recognition probe RP: The recognition probe RP was prepared by simultaneously modifying 30 nm AuNP with two parts. Among them, 16.7 μL of a solution containing 11 μM SYL3C and 10 μM c-aptamer and 83.3 μL of a solution containing 11 μM Linker, 10 μM H1 and H2 were incubated at room temperature for 6 h respectively. The two parts of the solution were mixed, 1 mL of 30 nm AuNP was added and incubated at room temperature for 12 h, and then aging treatment was carried out, that is, 10 μL, 20 μL, 30 μL, 40 μL of 2 M NaCl solution were added in portions every 30 min to make the final concentration of NaCl 0.2 M, and then gently shaken at room temperature for 12 h, centrifuged and washed 3 times with PBS (6000 rpm, 20 min), and the precipitate was resuspended with 100 μL of PBS to obtain the recognition probe RP, which was stored at 4°C for later use.

[0055] Preparation of SERS probe SERS tag: The SERS probe SERS tag was prepared by co-modifying 30 nm AuNP with H* and DTNB. Specifically, 100 μL of a solution containing 10 μM H* was taken, 1 mL of 30 nm AuNP was added and incubated at room temperature for 12 h, and then after the same aging treatment as above, 20 μL of 100 μM DTNB was added and incubated at room temperature for 3 h, centrifuged and washed 3 times with PBS (6000 rpm, 20 min), and the precipitate was resuspended with 100 μL of PBS to obtain the SERS probe SERS tag, which was stored at 4°C for later use.

[0056] The characterization results of the normalized absorption spectrum and dynamic light scattering particle size during the preparation of the recognition probe RP and the SERS probe SERS tag are as follows: As Figure 4A shown, after the surface of 30 nm AuNP was modified with Linker, H1, H2, or SYL3C, c-aptamer, the surface plasmon resonance (SPR) peak of 30 nm AuNP at 523.5 nm shifted to 525.5 nm; correspondingly, as Figure 4BAs shown, the average hydrodynamic diameter of the modified AuNP increased from 44.3 nm to 67 nm or 62.5 nm; when AuNP was co-modified with Linker, H1, H2, SYL3C, and c-aptamer, as Figure 4A shown, the SPR of 30 nm AuNP red-shifted from 523.5 nm to 526 nm; correspondingly, as Figure 4B shown, the average hydrodynamic diameter of the co-modified AuNP increased to 80.8 nm, indicating the successful preparation of the recognition probe RP; similarly, as Figure 4C shown, after the surface of AuNP was modified with H*, the SPR peak of 30 nm AuNP at 523.5 nm shifted to 525 nm; correspondingly, as Figure 4D shown, the average hydrodynamic diameter increased from 44.3 nm to 61.5 nm; after the surface of AuNP was modified with H* and further modified with DTNB, as Figure 4C shown, the SPR peak red-shifted to 526 nm; correspondingly, as Figure 4D shown, the average hydrodynamic diameter increased to 70 nm, indicating the successful preparation of the SERS probe SERS tag.

[0057] Example 3: Assembly of AuNP Probes in Buffer to Form Network Nanostructures

[0058] After mixing an equal amount of 30 μL of RPs and SERS tags in Example 2, 940 μL of a 1×PBS solution containing Target, DNAzyme, SYL3C-c, and Mg 2+ was added, and then incubated with gentle shaking at 37 °C for 2 h to assemble a probe network nanostructure containing multiple 30 nm AuNP. The probe network nanostructure without centrifugation and washing was characterized by absorption spectroscopy and scanning electron microscopy. The SERS spectrum of the probe network nanostructure was collected by a Renishaw system. The particle size of the nanoparticles was measured by dynamic light scattering DLS after centrifugation and washing at 6000 rpm for 20 min.

[0059] The following are the normalized absorption spectra, dynamic light scattering particle sizes, SEM, and SERS characterization results of the assembled structures at different stages of the probe network nanostructure: As Figure 5A shown, the SPR peak of the mixture of RPs and SERS tags is at 526 nm, at the same position as the SPR peaks of RPs and SERS tags; correspondingly, as Figure 5B shown, the average hydrodynamic diameter of the mixture of RPs and SERS tags is 74.7 nm, in the middle of the average hydrodynamic diameters of RPs and SERS tags; when DNAzyme and Mg 2+After that, as Figure 5A shown, the SPR peak was still at 526 nm; correspondingly, as Figure 5B shown, the average hydrodynamic diameter was still 74 nm; correspondingly, as Figure 5C shown, the SEM image presented good monodispersity; as Figure 5D shown, the SERS characterization presented a very weak SERS signal; after further adding SYL3C-c or Target, there were no obvious changes in the absorption spectrum, SEM, and SERS characterization, and the dynamic light scattering particle size slightly increased due to slight assembly; after adding SYL3C-c and Target simultaneously, as Figure 5A shown, the SPR peak red-shifted to 531 nm; as Figure 5B shown, the average hydrodynamic diameter increased to 121.6 nm; as Figure 5C shown, the SEM image presented an obvious 30Au-30Au network nanostructure; as Figure 5D shown, the SERS characterization presented an obviously enhanced SERS signal; these results confirmed that AuNP could be effectively assembled due to the dimerization of Met protein and the triggering of EpCAM protein.

[0060] In addition, the finite-difference time-domain FDTD (Lumerical FDTD Solutions 2018) simulation theoretically verified the obvious electromagnetic field enhancement of the network nanostructure. For the networked nanostructure, according to the length of the DNA strand assembling two particles, the gap distance between 30 nm AuNPs was estimated to be 15 nm. The local electromagnetic properties of the networked nanostructure were evaluated using a full-field scattered-field light source with an excitation wavelength of 633 nm. The grid size of the simulation region was set to 0.5 nm. As Figure 6 shown in Figures A and B of

[0061] Example 4: Cytotoxicity test

[0062] The CCK-8 detection method was used to detect the cytotoxicity of the nanoprobe. Specifically, 1×10 4MCF-7 cells were cultured for 24 h, and then HGF, HGF + DNA-like, HGF + DNA-like + RPs, and HGF + DNA-like + RPs + SERS tags at working concentrations were added into the well plates and incubated for 4 h. Among them, the working concentration of HGF was 100 ng / mL. The above DNA-like refers to T-Met-1, T-Met-2, and DNAzyme, and the concentrations of all three were 100 nM. The concentrations of RP and SERS tag were both 0.5 nM. Subsequently, 10 μL of CCK-8 detection reagent was added to each well and incubated for 3 h (the optimal reaction time of CCK-8 was subject to the specific color development degree of the cells). Finally, after gently mixing on a shaker for 10 min, readings were taken. Cells cultured in DMEM medium containing 10% fetal bovine serum (FBS) with the same treatment were used as the control group. By measuring the absorbance at 450 nm, the cytotoxicity of the detection probe was determined.

[0063] As Figure 7 shown, at the working concentrations, HGF, HGF + DNA, HGF + DNA + RPs, and HGF + DNA + RPs + SERS tags had no obvious cytotoxicity to MCF-7 cells.

[0064] Example 5: Time-dependent SERS imaging comparison test of membrane protein dimerization on living cells

[0065] Hepatocyte growth factor (HGF) can activate the HGF / Met signaling pathway and trigger pro-cancer signaling cascades by inducing the dimerization of its receptor Met, inducing tumor cell proliferation, angiogenesis, and cell invasion. To observe and monitor membrane protein dimers on specific living tumor cells, human breast cancer cells MCF-7 positive for Met and EpCAM were specifically selected as the cell model. MCF-7 cells treated with HGF were first co-incubated with T-Met-1 and T-Met-2, then co-incubated with DNAzyme and Mg 2+ co-incubated, and finally RP and SERS tag were added at an interval of 30 min for SERS imaging. The specific operations of this Example 5 are as follows:

[0066] Step S501: Culture MCF-7 cells in DMEM medium containing 10% FBS until they reach a density of 60%, and then place the cells in DMEM medium without 10% FBS and 1% antibiotics (penicillin / streptomycin) for starvation for 24 h. Perform the following three treatments: 1) No treatment; 2) Dimer inducer treatment: Add 100 ng / mL of dimer inducer HGF and culture for 30 min; 3) Inhibitor treatment: First incubate 100 ng / mL of dimer inducer HGF with 2 μg / mL of HGF inhibitor for 1 h, and then incubate the mixture with the cells for 30 min.

[0067] Step S502: Co-incubate the cells treated in Step S501 with 100 nM T-Met-1 and T-Met-2 for 20 min; after washing three times with PBS, co-incubate the cells with 100 nM DNAzyme and 1 mM Mg 2+ for 20 min. After washing three times with PBS, add 0.5 nM of RP and SERS tag and co-incubate for 30 min, and then perform SERS imaging on the cells at different time points using a Renishaw Raman system. The SERS imaging results Figure 8 are shown as follows;

[0068] Figure 8 Figure A in it shows the SERS imaging of Met protein dimerization over time, and Figure B shows the time-dependent SERS signal intensity curve of the corresponding Met protein dimer. The DTNB SERS signal at the position of 1331 cm -1 was detected after 30 min of incubation, and the SERS signal intensity reached saturation after 2 h. As a control, as Figure 8 shown in Figures C, D, E, and F in it, the SERS imaging of Met protein dimerization over time of untreated MCF-7 cells and MCF-7 cells co-treated with 0.1 μg / mL of HGF and 2 μg / mL of HGF inhibitor only detected very weak SERS signals, proving that the detected SERS signals were not obtained by non-specific assembly of SERS probes. The above results indicate that the dimerization process of Met protein on MCF-7 cells can be monitored by SERS signals. It should be noted that due to slight changes in the cells during the signal acquisition process and the movement of the assembled bodies entering the cells, this leads to certain changes in the position of the SERS signals.

[0069] Example 6: Final SERS imaging comparison test for detecting membrane protein dimerization on living cells

[0070] In Example 6, the following membrane protein dimerization detection and SEM characterization of AuNP on living cells were respectively performed on three groups of cells treated in Step S501 of Example 5:

[0071] Detection of membrane protein dimerization: Incubate three groups of cells with 100 nM of membrane protein targeting strands (including T-Met-1 and T-Met-2) in 1 mL of DMEM medium without FBS for 20 min to allow the membrane protein targeting strands to bind to Met proteins on the cell membrane. After washing three times with PBS, incubate the cells with 100 nM DNAzyme and 1 mM Mg 2+ in 1 mL of DMEM medium without FBS for 20 min. After washing three times with PBS, incubate the cells with 0.5 nM RP and SERS tag. After 3 h, rinse the cells three times with PBS to remove free and non-specifically deposited probes, and then perform SERS imaging of the final results of membrane protein dimerization detection on live cells.

[0072] Figure 9 Figure A in [reference] shows the SERS imaging of untreated MCF-7 cells, MCF-7 cells treated with 0.1 μg / mL HGF, and MCF-7 cells treated with a combination of 0.1 μg / mL HGF and 2 μg / mL HGF inhibitor in the present invention. Cells without HGF stimulation had almost no SERS signal of DTNB, such as the untreated group in Figure A, indicating that Met proteins mainly exist in monomer form on the cell membrane in the natural state. HGF-induced MCF-7 cells, such as the HGF group in Figure A, showed obvious SERS signals of DTNB due to the formation of network nanostructures triggered by Met protein dimerization. In addition, once Met protein dimerization was inhibited by the HGF inhibitor, such as the HGF HGF-Inhibitor group in Figure A, the SERS signal of DTNB was hardly detectable. Figure 9 Figure B in [reference] shows the detection of Met phosphorylation levels in cells under different treatments by Western blotting. Compared with untreated cells, the Met phosphorylation (phospho-Met, P-Met) level in HGF-treated cells increased significantly. However, after pretreatment with the HGF inhibitor, the expression of phosphorylated Met decreased. These results are consistent with the widely accepted hypothesis of the HGF / Met signaling pathway mechanism, that is, Met protein dimerization induces its autophosphorylation. Therefore, the above research shows that the SERS detection method proposed in the present invention can highly sensitively detect membrane protein dimers.

[0073] Example 7: SEM Characterization of AuNPs on MCF-7 Cells under the Action of Met Protein Dimerization

[0074] To characterize the assembly and morphology of probes on the surface of live cells, the cells were fixed and then subjected to scanning electron microscopy testing. The specific testing steps are as follows:

[0075] The MCF-7 cells were cultured in DMEM medium containing 10% FBS until they reached 60% confluence, and then the cells were starved for 24 h in DMEM medium without 10% FBS and 1% antibiotics (penicillin / streptomycin); they were divided into four treatment groups, one of which was untreated and served as the RPs+SERS tags group; the other three groups were all treated with a dimer inducer, that is, 100 ng / mL of the dimer inducer HGF was added and cultured for 30 min, and these three groups were respectively labeled as the SERS tags+HGF group, the RPs+HGF group, and the RPs+SERS tags+HGF group;

[0076] The four groups of cells were respectively incubated with 100 nM of membrane protein targeting strands (including T-Met-1 and T-Met-2) in 1 mL of DMEM medium without FBS for 20 min to allow the membrane protein targeting strands to bind to the Met protein on the cell membrane; after washing three times with PBS, the cells were incubated with 100 nM of DNAzyme and 1 mM Mg 2+ in 1 mL of DMEM medium without FBS for 20 min; after washing three times with PBS, among them, the cells in the SERS tags+HGF group were only incubated with 0.5 nM of SERS tag, the RPs+HGF group was only incubated with 0.5 nM of RP, and the RPs+SERS tags+HGF group and the RPs+SERS tags group were incubated with 0.5 nM of RP and SERS tag; after 3 h, the cells were rinsed three times with PBS to remove free and non-specifically deposited probes, and then SERS imaging of the final results of membrane protein dimer detection was performed on the live cells.

[0077] The treated cells were washed three times with PBS, 500 μL of trypsin-EDTA was added to digest the cells in the confocal dish at 37 °C for 30 s, and then 500 μL of DMEM complete culture medium containing 10% fetal bovine serum was added to terminate the digestion. The cells at the bottom of the dish were gently scraped with a cell scraper to resuspend the cells. Subsequently, the resuspended solution was centrifuged at 1000 rpm for 5 min to remove the broken cells and small molecule impurities in the supernatant. Thereafter, 500 μL of 2.5% glutaraldehyde solution was added to resuspend the cells, and they were fixed overnight in a refrigerator at 4 °C, and then dehydrated successively with 1 mL of ethanol solutions with different concentrations (30%, 50%, 70%, and 90%) from high concentration to low concentration, each dehydration was carried out at 4 °C for 10 min. After centrifuging the cells to remove the excess ethanol solution, the fixed cells were gently dropped on the surface of a clean silicon wafer to obtain cell samples for SEM characterization, and the characterization results are as Figure 10 shown, Figure 10The SERS tags + HGF group represents the MCF-7 cell group without RPs treatment, the RPs + HGF group represents the MCF-7 cells without SERS tags treatment, the RPs + SERS tags group represents the MCF-7 cell group without HGF treatment, and the RPs + SERS tags + HGF group represents the normally treated MCF-7 cell group;

[0078] As Figure 10 Shown are the SEM images of 30 nm AuNP on MCF-7 cells in different treatment groups. As in the SERS tags + HGF group, on the MCF-7 cell membrane without RPs treatment, due to the small size of 30 nm AuNP particles, they are easily non-specifically adsorbed and thus sparsely deposited on the cell membrane. As in the RPs + HGF group, on the MCF-7 cell membrane without SERS tags treatment, in addition to non-specifically adsorbed particles, SYL3C on RPs can specifically recognize and bind to the receptor protein EpCAM randomly distributed on the cell membrane, so more particles are deposited on the cell membrane. As in the RPs + SERS tags group, when there is no HGF treatment, due to the small amount of Met protein dimers originally present on the MCF-7 cell membrane, a small amount of reticular 30Au-30Au nanostructures are assembled on the cell membrane. As in the RPs + SERS tags + HGF group, when RPs, SERS tags, and HGF are combined for treatment, a large amount of reticular 30Au-30Au nanostructures are assembled on the cell membrane. The characterization results of SEM strongly support the accuracy of the SERS results.

[0079] Example 8: SERS imaging comparison test of different cells

[0080] Precisely identifying specific tumor cells in a complex tumor microenvironment and highly sensitively detecting the membrane protein dimerization of specific cells is of great significance. Here, to verify the highly specific SERS sensing strategy proposed by the present invention for precisely identifying tumor cells through specific markers existing in tumor cells themselves, four different cells were selected as cell models. As Figure 11 shown in Figure A in, human breast cancer cell MCF-7 is positive for both Met and EpCAM, human breast cancer cell SK-BR-3 is negative for Met and positive for EpCAM, human normal liver cell LO2 is positive for Met and negative for EpCAM, and human normal mammary epithelial cell MCF-10A is negative for both Met and EpCAM; The four cells shown in Figure A in were treated respectively according to the treatment method of the HGF treatment group in Example 5, and detected according to the specific detection process of membrane protein dimer in Example 6. Figure 11 This was confirmed by Western blotting of the four cells after HGF treatment shown in Figure B in. As Figure 11 Figure 11 ​SERS imaging of four types of cells after HGF treatment as shown in Figure C in the text indicates that obvious SERS signals were detected only in MCF-7 cells that were positive for both Met and EpCAM, demonstrating that the highly specific SERS sensing strategy proposed in the present invention can provide an effective approach for the specific and accurate identification of tumor cells.

[0081] Example 9: SERS Imaging of Signal Transduction between Cancer Cells and Other Cells

[0082] In this example, a Transwell system was used for co-culture experiments of donor-recipient cells. The Transwell system includes a culture plate and a Transwell chamber. The Transwell chamber forms the upper chamber, and the culture plate forms the lower chamber. Donor cells (BMSC, MCF-10A, or Breast CAFs) were seeded in the upper chamber, and recipient cells (MCF-7) were seeded in the lower chamber. The two chambers were separated by a polycarbonate membrane. Since the polycarbonate membrane is permeable, the components of the donor cells in the upper chamber can affect the recipient cells in the lower chamber, thereby enabling the study of the effects of donor cells on the growth and migration of recipient cells. Specifically, MCF-7 cells were seeded in a 6-well plate and cultured in DMEM medium containing 10% FBS for 24 h, and then starved in DMEM medium without FBS for 24 h. At the same time, BMSC, MCF-10A, and Breast CAFs were respectively placed in chambers with a pore size of 0.4 μm and cultured using DMEM culture medium without FBS, and the culture density was 5.0×10 4 cells / mL. After co-culture for 24 h, MCF-7 cells were incubated with 100 nM T-Met-1 and T-Met-2 for 20 min, washed 3 times with PBS, and then incubated with 1 mM Mg 2+ and 100 nM DNAzyme for 20 min, and washed 3 times with PBS. In terms of SERS imaging, MCF-7 cells were incubated for 3 h after adding 0.5 nM RP and SERS tag, and data was collected by a Renishaw system.

[0083] Fibroblasts are an important component of the solid tumor stroma. Cancer-associated fibroblasts (CAFs) are different from fibroblasts in normal tissues and can promote tumor progression at all stages of the tumor. Breast cancer-associated fibroblasts (Breast CAFs) are a key stromal cell type in the breast cancer tumor microenvironment (TME). Previous studies have shown that Breast CAFs can promote the proliferation and metastasis of breast cancer cells by secreting a large number of growth factors (including HGF), cytokines, and extracellular matrix. Here, in the present invention, Breast CAFs and breast cancer cells MCF-7 were co-cultured in an invasion chamber to observe the HGF / Met signaling pathway between Breast CAFs and MCF-7 cells as Figure 12 shown in Figure A of Figure 12 ; in SERS imaging, as a control, MCF-7 cells cultured alone and MCF-7 cells co-cultured with MCF-10A cells, as shown in Figure B of Figure 12 , produced only very weak SERS signals, while enhanced SERS signals were detected in MCF-7 cells co-cultured with Breast CAFs, which clearly indicates that the proposed SERS sensing strategy can highly sensitively detect the HGF / Met signaling pathway; as Figure 13 shown in Figure A of Figure 13 , MCF-7 cells and BMSC cells were co-cultured; the SERS image in Figure B of Figure 13 showed that obvious SERS signals could be observed in MCF-7 cells co-cultured with BMSC cells, indicating that BMSC cells can regulate the physiological state of MCF-7 cells through the HGF / Met signaling pathway; the Western blot analysis shown in Figure C of

[0084] further confirmed this. Imaging the dimerization of cell membrane proteins induced by stem cell secretions by SERS technology reveals the key role of stem cells in biological regulation.

[0084] The above results indicate that the SERS sensing strategy proposed in the present invention is a convenient, intuitive, and reliable method that can accurately identify signaling pathways through membrane protein dimers in a complex cell environment and successfully apply this concept to the visual monitoring of intercellular signal transduction in the process of cell communication.

[0085] Example 10: Evaluation of the inhibitory effect of a targeted drug on receptor dimerization by SERS imaging

[0086] Amivantamab is a bispecific antibody targeting epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (Met). Especially in the research on the HGF / Met signaling pathway, as Figure 14 shown in Figure A of Figure 14 , Amivantamab effectively inhibits HGF-induced Met dimerization by directly binding to the Met receptor, blocking the activation of the HGF / Met signaling pathway at the source. In this invention, the therapeutic effect of Amivantamab on blocking the HGF / Met signaling pathway was evaluated by SERS imaging technology. Specifically, MCF-7 cells were cultured in DMEM medium containing 10% FBS until they reached a density of 60%, and then the cells were starved for 24 h in DMEM medium without 10% FBS and 1% antibiotics (penicillin / streptomycin), followed by different comparative treatments: (1) no treatment; (2) dimer inducer treatment: adding 100 ng / mL of the dimer inducer HGF and culturing for 30 min; (3) drug treatment, and the drug treatment steps were: incubating the antibody Amivantamab (500 μg / mL) with the cells for 6 h, and then adding HGF (100 ng / mL) and incubating for 30 min; Detection was carried out according to the specific detection process of membrane protein dimerization in Example 6, as Figure 14 shown in Figure B of Figure 14 . Obvious SERS signals were detected in MCF-7 cells treated with HGF, while only very weak SERS signals were generated in MCF-7 cells treated with Amivantamab first and then with HGF, which clearly demonstrated that Amivantamab effectively inhibited HGF-induced Met protein dimerization by binding to the Met protein. As Figure 14 further confirmed by the Western blotting shown in Figure C of Figure 14 . The above results indicate that the SERS sensing strategy proposed in this invention can highly sensitively and real-time monitor the HGF / Met signaling pathway, providing a reliable basis for the therapeutic effect of Amivantamab targeting and binding to the Met receptor.

Claims

1. A kit for simultaneously detecting a specific membrane protein marker and a membrane protein dimer, characterized in that, The kit includes: a membrane proteome anchoring chain, a recognition probe, a SERS probe, and an auxiliary signal amplification reagent; The membrane proteome anchoring chain includes two membrane protein anchoring chains T-Met-1 and T-Met-2, which are respectively used to anchor two receptor protein monomers on the first membrane protein Met, namely Met-1 and Met-2. The T-Met-1 and T-Met-2 can perform proximity hybridization when the two receptor protein monomers Met-1 and Met-2 dimerize. The T-Met-1 and T-Met-2 each sequentially include: an aptamer sequence, a free sequence, a hybridization sequence, and a trigger sequence; the free sequence is a T base sequence of 10-20 nt; the aptamer sequences of T-Met-1 and T-Met-2 are the same and are used to target and bind Met-1 and Met-2; the hybridization sequences of T-Met-1 and T-Met-2 are 6-8 mutually complementary base sequences; the aptamer sequence can bind to the receptor protein monomer on the cell membrane. When the first membrane protein Met forms a dimer under the action of the dimerization inducer HGF, the two membrane protein anchoring chains T-Met-1 and T-Met-2 can form a double-stranded T structure through complementary hybridization of the hybridization sequences; The recognition probe RP is prepared by simultaneously anchoring a first probe and a second probe on AuNPs with a diameter range of 20-50 nm. The first probe is a structure formed by anchoring and connecting the thiol-modified DNA single strand SYL3C to the DNA single strand c-aptamer through base complementary pairing; the second probe is a structure formed by anchoring the first RP hairpin H1 and the second RP hairpin H2 to the thiol-modified DNA single strand Linker through base complementary pairing; the DNA single strand SYL3C can specifically recognize and bind to the second membrane protein and release the DNA single strand c-aptamer; the first RP hairpin H1 and the second RP hairpin H2 are both assembled from DNA sequences, can initiate a CHA reaction after the release of the DNA single strand c-aptamer, form a stable double-stranded structure through a toehold-mediated strand displacement reaction, and further release the DNA single strand c-aptamer; The SERS probe, namely the SERS tag, is prepared by anchoring a SERS hairpin and a Raman molecule on AuNPs with a diameter range of 20-50 nm. The SERS hairpin H* is assembled from a DNA sequence; The auxiliary signal amplification reagent is used to assist signal amplification during dimerization. The auxiliary signal amplification reagent is a single-stranded DNAzyme with a hairpin structure. The single-stranded DNAzyme can specifically recognize the trigger sequence of the membrane protein anchoring strand, that is, form a complex with the double-stranded T-shaped structure, and cut the SERS hairpin H* under the action of Mg 2+ to cut the SERS hairpin H*; The unhybridized part of the H2 sequence can hybridize with the cleaved SERS hairpin H*, thereby enabling the assembly of the recognition probe and the SERS probe.

2. The kit for simultaneously detecting a specific membrane protein marker and a membrane protein dimer according to claim 1, wherein, The auxiliary signal amplification reagent further includes Mg 2+ .

3. The simultaneous detection kit for a specific membrane protein marker and a membrane protein dimer according to claim 1, wherein The Raman molecule is selected from any one of 4-MBA, DTNB, or 2-MBT.

4. The kit for simultaneously detecting a specific membrane protein marker and a membrane protein dimer according to claim 1, wherein The molar ratio of the recognition probe RP to the SERS probe is 1:

1.

5. The simultaneous detection kit for a specific membrane protein marker and a membrane protein dimer according to claim 1, wherein The hybridization sequences of T-Met-1 and T-Met-2 are 7 mutually complementary base sequences.

6. The simultaneous detection kit for a specific membrane protein marker and a membrane protein dimer according to claim 1, characterized in that, The preparation steps of the recognition probe RP include: separately incubating the solutions containing single-stranded DNA SYL3C, single-stranded DNA c-aptamer, and the solution containing single-stranded DNA Linker, the first RP hairpin H1, and the second RP hairpin H2 at room temperature for 4 h each. Incubate the two parts of the solution with 30 nm AuNP at room temperature for 5 h, and then perform probe aging treatment, that is, add different gradient volumes of 2 M NaCl to the above solution at intervals of 30 min each time, so that the final concentration of NaCl is 0.2 M. Gently shake the mixture at room temperature for 6 - 12 h, centrifuge and wash with PBS at 6000 rpm for 20 min. After centrifuging and washing three times, resuspend the precipitate in PBS to obtain the recognition probe RP.

7. A kit for simultaneously detecting a specific membrane protein marker and a membrane protein dimer according to claim 6, characterized in that, The preparation steps of the SERS probe SERS tag include: incubating the SERS hairpin H* with 30 nm AuNP at room temperature for 6 - 12 h. After performing the same aging treatment as above, incubate the Raman molecule DTNB with the AuNP modified with SERS hairpin H* at room temperature for 3 h, centrifuge and wash with PBS at 6000 rpm for 20 min. After centrifuging and washing three times, resuspend the precipitate in PBS to obtain the SERS probe SERS tag.

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