Dual-mode breast cancer typing method and detection method based on electron and chiral transfer probe

Abstract

The invention provides a dual-mode breast cancer typing method based on an electronic and chiral transfer probe and a detection method. The method comprises the following steps: constructing a cBDP-Cy5 bimolecular probe with electron and chiral transfer, wherein the probe is of a coupled structure formed by cBDP and Cy5 chromophores through DNA complementary pairing; based on the coupling and decoupling characteristics of the cBDP and the Cy5, constructing two signal amplification type DNA loops with dual modes; molecular typing is carried out on breast cancer based on two signal amplification type DNA loops. The invention provides a new strategy for precise molecular typing of tumors, has the characteristics of simplicity and convenience in operation and rapidness in detection, and has important application value in the fields of tumor marker detection and personalized treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a dual-mode breast cancer typing method and detection method based on electron and chiral transfer probes. Background Art

[0002] Most malignant tumors are highly heterogeneous, showing high diversity and differences in molecular characteristics, clinical manifestations, treatment sensitivity, prognosis, etc. Clinically, molecular typing of tumors can further explore the heterogeneity within each subtype and perform correlation analysis with clinical characteristics, treatment responses, prognosis outcomes, etc. Therefore, accurate diagnosis of tumor molecular typing is of great significance. Currently, the methods for tumor typing mainly include immunohistochemistry and fluorescence in situ hybridization. Although these two methods have been widely used clinically, they still face problems such as complex operations, long time consumption, and strong subjectivity in result interpretation. In recent years, some emerging methods have also been developed for the detection of tumor molecular typing, such as single-cell sequencing, circulating tumor typing in liquid biopsy, and spatial omics, etc. However, their high costs and complex data analysis processes have limited clinical promotion to a certain extent. Therefore, developing new, simple, rapid, and accurate molecular typing detection methods is an urgent need.

[0003] Currently, the luminescence or surface plasmon resonance characteristics of nanoparticles are often used as optical detection signals. For example, chiral assemblies of gold nanoparticles have been used for the detection of membrane receptors through specific structural recognition and changes in plasmon resonance signals. Quantum dots and upconversion nanoparticles achieve multiplex detection through different emission wavelengths and enhanced luminescence signals. However, such materials may still face difficulties such as biocompatibility, surface modification stability, and batch-to-batch reproducibility. On the other hand, although organic fluorescent molecular probes have good biocompatibility, they are mostly limited to the analysis mode of a single spectral channel and are prone to background interference and generate false positive and false negative signals in the molecular diagnosis of complex diseases such as tumors. In contrast, the analysis strategy based on multi-spectral channels can significantly improve the accuracy and reliability of detection results through cross-validation of different characteristic optical signals. Therefore, creating a signal detection and amplification system with stable structure, precise controllability, and multi-channel spectral response is crucial for the development of tumor molecular typing methods. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a dual-mode breast cancer typing method and detection method based on electron and chiral transfer probes, which is simple to operate, has a rapid reaction, realizes signal amplification within 1 hour, and can provide a new means for breast cancer molecular typing.

[0005] To achieve the above invention purpose, the present invention adopts the following technical solutions:

[0006] A dual-mode breast cancer typing method based on electron and chiral transfer probes, comprising the following steps:

[0007] Step S1, construct a cBDP-Cy5 bimolecular probe with electron and chiral transfer, where the probe forms a coupling structure by DNA complementary pairing of cBDP and Cy5 chromophores;

[0008] Step S2, based on the coupling and decoupling characteristics of cBDP and Cy5, construct two signal-amplifying DNA circuits with dual modes;

[0009] Step S3, perform molecular typing of breast cancer based on the two signal-amplifying DNA circuits.

[0010] A detection method based on the dual-mode breast cancer typing method of the present invention, comprising the following steps:

[0011] Step (1) Obtain a breast cancer cell sample to be tested, inoculate the cells in a 96-well plate at a density of 10,000 cells per well, and incubate overnight at 37 °C to ensure cell adhesion;

[0012] Step (2) Use the dual-mode detection system to detect the expression status of ER and HER2 in the sample;

[0013] Step (3) Type the breast cancer cells according to the expression characteristics of ER and HER2.

[0014] Adopting the above technical solution, the beneficial effects of the present invention are as follows:

[0015] The system constructed by the present invention can not only effectively perform molecular subtypes on four breast cancer cell lines, but also semi-quantitatively analyze the expression levels of ER and HER2.

[0016] The cBDP-Cy5 dimer probe adopted by the present invention can generate dual response signals of CD spectrum and fluorescence spectrum during dissociation and formation, and significantly improves the accuracy and reliability of the detection results through the cross-validation of dual-channel data.

[0017] The present invention utilizes the programmability of DNA molecules and the ability to easily modify organic chromophores to obtain rich optical information including polarization state, intensity, and wavelength, etc., for the analysis and detection of various disease markers. Combining deep learning algorithms to perform feature extraction and intelligent analysis on spectral data is expected to provide a new technical path for the early and accurate diagnosis of tumors. Description of the Drawings

[0018] Att Figure 1 is a diagram of the interaction mechanism between two dye molecules and a schematic diagram of the designed DNA circuit for sensing and analyzing two receptors on cells.

[0019] Appendix Figure 2 It is a schematic diagram for screening the optimal cBDP-Cy5 dimer using DNA technology and exploring its mechanism using spectroscopic characterization technology.

[0020] Appendix Figure 3 It is a schematic diagram for verifying two DNA circuits using ATP and thrombin as model molecules.

[0021] Appendix Figure 4 It is a schematic diagram for verifying the sensing analysis performance of two DNA circuits of the present invention on four different breast cancer cells.

[0022] Appendix Figure 5 It is a statistical chart related to evaluation factors for detecting ER and HER2 in four breast cancer molecular subtype model cell lines (MCF-7, ZR-75-1, SK-BR-3, and MDA-MB-231) using the developed DNA circuit sensing system, and determining the expression status of ER and HER2 in four breast cancer cell lines using the traditional ICC method. Detailed implementation manners

[0023] A dual-mode breast cancer typing method based on electron and chiral transfer probes of the present invention includes constructing a cBDP-Cy5 bimolecular probe and a DNA circuit with signal amplification function. The cBDP-Cy5 bimolecular probe has high-efficiency electron, chiral, and fluorescence energy transfer characteristics. Through the cascade reaction of the DNA circuit system, the dissociation and formation of the bimolecule can be controlled, realizing the amplified output of CD and fluorescence dual-mode optical signals. By binding the DNA circuit with specific DNA aptamers, the detection of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) in breast cancer cells can be completed within 1 hour, and the rapid classification of four breast cancer molecular subtypes, namely Luminal-A type, Luminal-B type, HER2-enriched type, and triple-negative type, can be achieved through the spectral response characteristics of the receptors. The present invention provides a new strategy for accurate tumor molecular typing, has the characteristics of simple operation and rapid detection, and has important application value in the fields of tumor marker detection and personalized treatment.

[0024] The present invention will be described in detail below in conjunction with specific embodiments and the accompanying drawings.

[0025] A dual-mode breast cancer typing method based on electron and chiral transfer probes includes the following steps:

[0026] Step S1, construct a cBDP-Cy5 bimolecular probe with electron and chiral transfer, and the probe forms a coupling structure by DNA complementary pairing of cBDP and Cy5 chromophores;

[0027] Step S2: Based on the characteristics of the coupling and decoupling of cBDP and Cy5, two signal-amplified DNA circuits with dual modes are constructed.

[0028] Step S3: Based on the two signal-amplified DNA circuits, molecular typing of breast cancer is performed.

[0029] Appendix Figure 1 It is a schematic diagram of the interaction mechanism between two dye molecules and the sensing analysis of two receptors on cells by the designed DNA circuit. Figure A shows the molecular structures of cBDP and Cy5 molecules, the interaction between the two molecules, and the signal responses of cBDP monomers and dimers under different light sources. Figure B is a schematic diagram of the sensing analysis of two receptors on cells by designing two DNA circuits.

[0030] The construction process of the cBDP-Cy5 bimolecular probe of the present invention is as follows: using DNA technology to screen the optimal cBDP-Cy5 dimer; precisely constructing cBDP-Cy5 dimers with different spatial positions and different distances by DNA, and characterizing the constructed dimers by CD, fluorescence, etc., and screening out the required cBDP-Cy5 bimolecular probe.

[0031] Each DNA circuit of the present invention consists of three parts, including: a DNA initiation strand, a DNA fuel strand, and a signal probe molecule; DNA circuit 1 causes the dissociation of the cBDP-Cy5 probe, and DNA circuit 2 promotes the formation of the cBDP-Cy5 bimolecular probe.

[0032] The present invention uses ATP and thrombin as model molecules to verify the performance of the two designed DNA circuits respectively.

[0033] The present invention applies the designed DNA circuit to the detection of ER and HER2 receptors on four breast cancer cells, and performs molecular typing on the four breast cancer cells by combining the expression status of ER and HER2.

[0034] The present invention performs detection through two modes of circular dichroism and fluorescence spectroscopy.

[0035] A detection method based on a dual-mode breast cancer typing method, comprising the following steps:

[0036] Step (1): Obtain a breast cancer cell sample to be tested, inoculate the cells in a 96-well plate at a density of 10,000 cells per well, and incubate overnight at 37 °C to ensure cell adhesion.

[0037] Step (2): Use the dual-mode detection system to detect the expression status of ER and HER2 in the sample.

[0038] Step (3) classifies breast cancer cells according to the expression characteristics of ER and HER2.

[0039] The breast cancer cell types are:

[0040] Luminal-A type: ER positive and HER2 negative;

[0041] Luminal-B type: ER positive and HER2 positive;

[0042] HER2 overexpression type: ER negative and HER2 positive;

[0043] Triple-negative type: ER negative and HER2 negative.

[0044] The present invention provides a dual-mode breast cancer typing method based on electron and chiral transfer probes, including the construction and screening of probes, the design of signal-amplified DNA circuits, and their application in breast cancer typing.

[0045] Example 1: Construction and screening of probes

[0046] In this example, DNA nanotechnology is used to precisely regulate the spatial sites of chromophores. Specifically, cBDP and Cy5 are used as chromophores, and a series of molecular coupling structures are constructed through DNA complementary pairing. The constructed probes are characterized by various spectroscopic techniques such as fluorescence spectroscopy and CD to analyze their intermolecular interaction mechanisms. Finally, a bimolecular probe with both efficient electron and chiral transfer characteristics is obtained, as shown in the appendix Figure 2 .

[0047] Appendix Figure 2 shows the screening of the optimal cBDP-Cy5 dimer using DNA technology and the exploration of its mechanism using spectroscopic characterization techniques. Figure A shows 7 cBDP-Cy5 dimers with dye molecules constructed by DNA in different positions or at different distances. Figures B and C are the CD and fluorescence spectra of 4 cBDP-Cy5 dimers in different positions. Figures D and E are the CD and fluorescence spectra of 4 cBDP-Cy5 dimers in different distances.

[0048] The DNA strand sequences modifying the cBDP and Cy5 chromophores are shown in Table 1, and different structures are obtained by annealing the specific DNA strands in TE buffer according to the annotations.

[0049] The concentration of the above cBDP-Cy5 dimer in the sample is 1 μM, and the volume is 400 μL. The reaction buffer includes 10 mM Tris (pH = 8.0), 150 mM NaCl, 5 mM KCl, and the annealing program is a gradient cooling from 60 °C to 25 °C for one and a half hours.

[0050] The above dimers were characterized by fluorescence spectroscopy and CD. Among them, the structure d-0bp has the characteristics of efficient CD enhancement and fluorescence transfer, which confirms the electron transfer between cBDP and Cy5. This structure was selected as the detection probe.

[0051] Example 2: Construction and Performance Verification of Signal-Amplifying DNA Circuits

[0052] In this example, two DNA circuits with signal amplification functions were designed and constructed. The DNA circuits can control the coupling and decoupling processes of cBDP and Cy5. Taking ATP and thrombin as model molecules, the detection performances of the two DNA circuits were verified respectively. The experimental results show that the constructed DNA circuits can achieve sensitive detection of biomolecules through CD and fluorescence dual spectra, as shown in the appendix Figure 3 .

[0053] Appendix Figure 3 It is a schematic diagram for verifying two DNA circuits using ATP and thrombin as model molecules. Among them, Figure A is a schematic diagram of DNA circuit 1 triggered by ATP molecules to initiate the dissociation of the cBDP-Cy5 dimer. Figure B is a schematic diagram of DNA circuit 2 triggered by thrombin to initiate the formation of the cBDP-Cy5 dimer. Figure C is a bar chart of the fluorescence intensity at 516 nm (left) and the CD intensity at 514 nm (right) of DNA circuit 1 under different molecular inputs. Figures D and E are the CD spectra and fluorescence spectra of DNA circuit 1 in the presence of different concentrations of ATP molecules. Figure F is a bar chart of the fluorescence intensity at 516 nm (left) and the CD intensity at 514 nm (right) of DNA circuit 2 under different protein inputs. Figures G and H are the CD spectra and fluorescence spectra of DNA circuit 2 in the presence of different concentrations of thrombin. Figures I–K are the time-dependent CD intensity changes of DNA circuit 1 and DNA circuit 2 at 514 nm.

[0054] The DNA circuit designed in the present invention mainly includes three parts, including the primer strands (T1, T2), DNA hairpins as fuel strands (H1-1, H2-1, H1-2, H2-2), and signal reporter molecules (probe-1 and probe-2, which are respectively the cBDP-Cy5 dimer formed by binding of DNA double strands and the cBDP monomer coupled to a single-stranded DNA). When the primer strand T is added, T first opens the fuel strand H1, exposing the binding domain of H1, and then opens H2 and forms H1·H2. At this time, T is displaced by H2 and released, and can continue to participate in the next cycle of reactions. Such multiple cycles ultimately achieve signal amplification. In DNA circuit 1, H1-1·H2-1 dissociates the cBDP-Cy5 dimer through strand displacement reaction; while in DNA circuit 2, the binding domain exposed on the intermediate product H1-2·H2-2 hybridizes with probe-2 to form a cBDP-Cy5 dimer. In this specific system, the CD and fluorescence signal change trends of the two DNA circuits at the characteristic wavelength positions (the absorption and emission wavelengths of cBDP) are opposite, serving as a highly contrasting dual-channel optical signal output.

[0055] The DNA sequences used above are shown in Table 2. Specifically, in DNA circuit 1, the ATP aptamer first anneals and hybridizes with ATP-T within the temperature range of 60°C to 25°C. Subsequently, different concentrations of ATP molecules (1 μM of H1-ATP, H2-1, and cBDP-Cy5 dimer) are mixed and incubated at 37°C for 1 hour, and then spectroscopic tests are performed as the experimental group. The mixture without ATP molecules serves as the control group. In DNA circuit 2, Thrombin-apt-T and Thrombin-inh are first annealed and hybridized within the temperature range of 60°C to 25°C, and then different concentrations of thrombin (1 μM of H1-2, H2-2, ssDNA-cBDP) are mixed. After incubation at 37°C for 1 hour, spectroscopic detection is performed as the experimental group, and the mixture without thrombin molecules serves as the control group.

[0056] Example 3: Typing detection of breast cancer cell lines

[0057] In this example, the above d-0bp structure probe and signal-amplified DNA circuit are used to detect and analyze different types of breast cancer cell lines. See Appendix Figure 4 Appendix Figure 4It is the performance verification of the sensing analysis of two DNA circuits of the present invention on four different breast cancer cells. Among them, Figure A is a schematic diagram of DNA circuits 1 and 2 for the sensing analysis of the cell membrane surface receptors ER and HER2 of breast cancer respectively. Figures B–E are CD spectrograms of DNA circuit 1 and DNA circuit 2 for the sensing of ER (upper) and HER2 (lower) in MCF-7, ZR-75-1, SK-BR-3 and MDA-MB-231 cell lines respectively. Figures F–I are fluorescence spectrograms of DNA circuit 1 and DNA circuit 2 for the sensing of ER (upper) and HER2 (lower) in MCF-7, ZR-75-1, SK-BR-3 and MDA-MB-231 cell lines respectively. Figures J–M are the statistical analyses of the CD intensity at 514 nm and the fluorescence intensity at 516 nm of ER and HER2 in the four cell lines respectively according to the data in Figures B–I.

[0058] A detection method based on a dual-mode breast cancer typing method, the specific steps are as follows:

[0059] Select breast cancer cell lines with different estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) expression statuses as detection objects;

[0060] The DNA sequences used are shown in Table 3. Specifically, to analyze ER using DNA circuit 1, first hybridize the ER aptamer with ER-T to prevent non-specific signal leakage caused by the accidental activation of DNA circuit 1 in the absence of ER. Subsequently, H1-ER, H2-1, the cBDP-Cy5 dimer, and the primer strand hybrid were respectively mixed in four different breast cancer cell lines. After incubation at 37 °C for 1 hour, the samples were characterized using CD and fluorescence spectroscopy.

[0061] To detect HER2 using DNA circuit 2, first hybridize Thrombin-aptamer-T with Thrombin-inh, and then add H1-2, H2-2, and ssDNA-cBDP-3 to four different breast cancer cell lines respectively. After incubation at 37 °C for 1 h, CD and fluorescence spectroscopy analyses were performed for spectral characterization.

[0062] Using the constructed detection system, distinguish the expression statuses of ER and HER2 in different cell lines through spectral signal responses. If the receptor is positively expressed in the cell line, the signal change of the cBDP characteristic peak in the CD and fluorescence spectra is relatively large; if the receptor is negatively expressed in the cell line, the signal change of the cBDP characteristic peak in the CD and fluorescence spectra is relatively small.

[0063] According to the expression characteristics of ER and HER2, classify breast cancer cell lines into the following four molecular subtypes:

[0064] Luminal-A type (ER positive, HER2 negative)

[0065] Luminal-B type (ER positive, HER2 positive)

[0066] HER2 overexpression type (ER negative, HER2 positive)

[0067] Triple-negative type (ER negative, HER2 negative)

[0068] Example 4: Statistics of ER and HER2 evaluation factors in four breast cancer cell lines

[0069] To quantitatively measure the expression levels of ER and HER2 in the measured breast cancer cells with specific numbers, fluorescence transfer efficiency (E F ) and CD enhancement efficiency (E CD ) were used as evaluation factors, and their definitions are as follows:

[0070]

[0071] Where F cBDP is the fluorescence emission peak intensity of cBDP at 516 nm; F dimer is the fluorescence emission peak intensity of cBDP-Cy5 dimer at 516 nm; CD cBDP is the CD signal intensity of cBDP at 514 nm; CD dimer is the CD signal intensity of cBDP-Cy5 dimer at 514 nm. For the convenience of comparison, all calculation results were taken as their absolute values. Further statistical analysis was performed on the spectral data of ER and HER2 in four different molecular subtype cell lines, as Figure 5 shown. By comparing the values of the two evaluation indicators E F and E CD in the samples, the receptor with a higher evaluation indicator value is the positively expressed receptor, that is, ER in MCF-7 and ZR-75-1 cell lines and HER2 in SK-BR-3 and ZR-75-1 cell lines; while the receptor with a lower evaluation indicator value is the negatively expressed receptor, that is, ER in SK-BR-3 and MDA-MB-231 cell lines and HER2 in MCF-7 and MB-MDA-231 cell lines.

[0072] As can be seen from the above examples, the dual-spectrum detection system constructed by the present invention can not only achieve precise regulation of DNA nanoprobes, but also improve the detection sensitivity through signal-amplified DNA circuits, showing good application effects in breast cancer cell typing. Those skilled in the art can make various non-substantive improvements and modifications to the present invention under the inspiration of the technical solution of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

[0073] Table 1

[0074]

[0075] Note: a-Cy5, a-cBDP hybridize to form the a structure; b-Cy5 hybridizes with b-cBDP to form the b structure; b-cBDP hybridizes with c-Cy5 to form the c structure; d-0bp-cBDP hybridizes with d-0bp-Cy5 to form the d-0bp structure; d-6bp-cBDP hybridizes with d-6bp-Cy5 to form the d-6bp structure; d-6bp-cBDP hybridizes with d-14bp-Cy5 to form the d-14bp structure; d-6bp-cBDP hybridizes with d-18bp-Cy5 to form the d-18bp structure.

[0076] Table 2

[0077]

[0078] Table 3

[0079]

Claims

1. A dual-mode breast cancer typing method based on electron and chiral transfer probes, characterized in that: The steps include: Step S1, constructing a cBDP-Cy5 bimolecular probe with electron and chirality transfer, wherein the probe is formed by a coupling structure formed by cBDP and Cy5 chromophores through DNA complementary pairing; Step S2, based on the coupling and decoupling characteristics of cBDP and Cy5, two signal amplification DNA circuits with dual modes are constructed; Step S3: molecularly classify breast cancer based on two signal amplification DNA circuits.

2. The dual-mode breast cancer typing method according to claim 1, characterized in that: The construction process of the cBDP-Cy5 bimolecular probe is as follows: using DNA technology to screen the optimal cBDP-Cy5 dimer; using DNA to accurately construct cBDP-Cy5 dimers at different spatial positions and different distances, and characterizing the constructed dimers by CD, fluorescence, etc., and screening out the desired dimers.

3. The dual-mode breast cancer typing method according to claim 1, characterized in that: Each DNA loop consists of three parts, including: DNA initiator chain, DNA fuel chain and signal probe molecule; DNA loop 1 leads to the dissociation of cBDP-Cy5 probe, and DNA loop 2 promotes the formation of cBDP-Cy5 bimolecular probe.

4. The dual-mode breast cancer typing method according to claim 1, characterized in that: ATP and thrombin were used as model molecules to verify the performance of the two designed DNA circuits.

5. The dual-mode breast cancer typing method according to claim 1, characterized in that: The designed DNA circuit was used to detect ER and HER2 receptors on four breast cancer cells, and the four breast cancer cells were molecularly classified based on the expression status of ER and HER2.

6. The dual-mode breast cancer typing method according to claim 1, characterized in that: The detection was carried out in two modes: CD spectroscopy and fluorescence spectroscopy.

7. A detection method based on the dual-mode breast cancer typing method according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step (1) obtaining a breast cancer cell sample to be tested, seeding the cells in a 96-well plate at a density of 10,000 cells per well, and incubating at 37° C. overnight to ensure cell adhesion; Step (2) detecting the expression status of ER and HER2 in the sample using the dual-mode detection system; Step (3) classifies breast cancer cells according to the expression characteristics of ER and HER2.

8. The detection method according to claim 7, characterized in that: Breast cancer cell types are: Luminal-A type: ER positive and HER2 negative; Luminal-B: ER-positive and HER2-positive; HER2 overexpression type: ER negative and HER2 positive; Triple negative: ER negative and HER2 negative.

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