Targeted in-situ self-assembly FRET signal amplification probe system and application

Abstract

The invention relates to a targeted in-situ self-assembly FRET (Fluorescence Resonance Energy Transfer) signal amplification probe system and application. The targeted in-situ self-assembly FRET signal amplification probe system comprises an amplification probe system I and an amplification probe system II, and the amplification probe system I comprises an activated Aptamer fluorescent probe I containing an EGFR aptamer sequence, an FRET signal amplification chain H1, an FRET signal amplification chain H2 and a beta-cyclodextrin polymer; the amplification probe system II comprises an activated Aptamer fluorescent probe II containing an HER2 aptamer sequence, an FRET (Fluorescence Resonance Energy Transfer) signal amplification chain H3, an FRET signal amplification chain H4 and a beta-cyclodextrin polymer. The targeted in-situ self-assembly FRET signal amplification probe system simultaneously realizes effective distinguishing of lung adenocarcinoma tissues from normal lung tissues, wherein the lung adenocarcinoma tissues are derived from an A549 lung adenocarcinoma mouse transplantation tumor model, and provides new technical support for early auxiliary diagnosis, curative effect monitoring and prognosis evaluation of lung cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of cell membrane surface receptor protein detection, and specifically relates to a targeted in-situ self-assembly FRET signal amplification probe system and application thereof. Background Art

[0002] Cell membrane surface receptor proteins are not only important components of cells, but also can play a vital role in the basic life activities of cells such as cell proliferation, differentiation, signal transduction, immune recognition, etc. by recognizing various components of the extracellular matrix as their ligands. In addition, many human diseases are related to abnormal expression of receptor proteins and protein-protein interactions. In particular, oligomerization of cell membrane surface receptor proteins is generally considered to be the first step in intracellular signal transduction. Abnormal receptor protein oligomerization can induce phosphorylation, activate downstream key signaling pathways, and ultimately lead to tumor formation and metastasis.

[0003] Epidermal growth factor receptor (EGFR, also known as HER1) is a transmembrane glycoprotein widely distributed on the surface of epithelial cells. As a key regulator of cell behavior, it is considered to be an important cancer-related protein marker. EGFR-mediated signaling pathways play an important role in tumor cell proliferation, migration, and differentiation, and its overexpression is also associated with the occurrence and development of many tumor cells, including non-small cell lung cancer and breast cancer. Therefore, EGFR has become an important therapeutic target for non-small cell lung cancer, breast cancer, and gastroesophageal cancer, and is related to the treatment of various other malignant tumors. However, the therapeutic effect of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) targeting EGFR is not ideal in some cases, and it shows drug resistance. Studies have found that EGFR can not only homodimerize to activate intracellular signal transduction processes, but also heterodimerize with other members of the HER family, such as human epidermal growth factor receptor-2 (HER2), and play a role in the carcinogenic signaling pathway of the HER family, thereby affecting the patient's sensitivity to drugs, which will directly affect the effect of targeted therapy and the choice of treatment options. At the same time, lung cancer, as a common malignant tumor, has become a major public health problem in my country and even around the world. The 5-year relative survival rate of lung cancer patients is about 20%, which can be significantly improved by early detection and standardized treatment. Therefore, efficient and accurate detection of the dimerization state of EGFR on the cell membrane surface has important clinical guidance value for early auxiliary diagnosis, efficacy monitoring and prognosis evaluation of lung cancer.

[0004] At present, immunohistochemistry (IHC) is still the most commonly used method for detecting cell surface receptor proteins in clinical pathology laboratories. IHC uses the principle of specific recognition between antigens and antibodies. Through the colorimetric agents (such as fluorescent dyes, enzymes, etc.) labeled on antibodies, the localization, qualitative and quantitative studies of the corresponding proteins can be achieved. However, immunohistochemistry still has some limitations, such as reliance on multi-step antigen-antibody recognition, complex antibody modification processes, and most importantly, these techniques can only be used to analyze the overall expression level of receptor proteins in situ, but cannot accurately reflect their dimerization state. The most commonly used method to study the protein dimerization process is fluorescent protein genetic modification. By fusing fluorescent protein tags with target proteins of interest and combining them with advanced fluorescence imaging technology, in situ visualization studies of protein-protein interactions and dimerization states can be achieved. These imaging methods have been successfully applied to dimerization imaging studies of fibroblast growth factor receptors and Met receptors. However, to date, these methods involve gene manipulation, and their gene modification may affect the efficiency of receptor dimerization or interfere with other biological processes in cells, and have limitations such as being time-consuming and unpredictable.

[0005] Proximity ligation technology can be used as an alternative, non-genetic method to analyze the interaction and dimerization state between cell surface protein receptors. This technology refers to a pair of oligonucleotide probes (called proximity probes) labeled with affinity ligands, which simultaneously recognize and bind to the target molecule. The proximity effect between the two molecules is generated due to the close distance and triggers the subsequent reaction, thereby giving a detection signal; when the target is not present, the proximity probes cannot approach each other to trigger the subsequent reaction. This technology provides an alternative solution for the study of the interaction and dimerization state of cell surface protein receptors. In the design of proximity ligation technology, the choice of affinity ligands on proximity probes is crucial, which often has a decisive influence on the specificity of the entire method. In recent years, nucleic acid aptamers (Aptamers, Apt) have been called "chemical antibodies" because of their high affinity and high specificity, and their applications in biological analysis and imaging have received widespread attention. Compared with antibodies, Aptamers have many advantages due to their chemical nature of nucleic acid molecules, such as small size, low immunogenicity, easy synthesis and modification, stable properties, low production cost, and a wide target range. As a non-genetic engineering strategy, proximity ligation technology combined with Aptamer has achieved imaging research on dimerization of cell membrane receptor proteins. However, the above non-genetic engineering strategy still has the following three limitations that need to be further improved: First, it is impossible to achieve synchronous detection of cell membrane receptor homodimers and heterodimers on the same living cell surface, which has important guiding value for the evaluation of tumor occurrence and development; second, in general, the content of homodimers / heterodimers formed by cell surface receptor dimerization is low, and its detection sensitivity cannot effectively achieve in situ imaging analysis of low-abundance homodimers / heterodimers; third, the "always on" signal mode, when applied to cell or tissue imaging analysis, has high imaging background and needs to be washed to remove unbound or weakly bound signal probes, which prolongs the diagnosis time. Therefore, constructing a signal amplification strategy to improve imaging contrast, improve detection sensitivity, expand new diagnostic models for lung cancer, and achieve synchronous detection and imaging analysis of low-abundance homodimers and heterodimers on the same living cell surface will effectively promote the development of new methods and technologies for early diagnosis of lung cancer. Summary of the invention

[0006] The first purpose of the present invention is to provide a targeted in situ self-assembled FRET signal amplification probe system to solve the technical problems that the existing detection technology cannot achieve the synchronous detection of cell membrane receptor homodimers and heterodimers of the same living cell, has low sensitivity and long detection time.

[0007] The second object of the present invention is to provide an application of a targeted in-situ self-assembled FRET signal amplification probe system.

[0008] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0009] A targeted in situ self-assembled FRET signal amplification probe system comprises an amplification probe system 1 and an amplification probe system 2, wherein the amplification probe system 1 comprises an activated Aptamer fluorescent probe 1 containing an EGFR aptamer sequence, a FRET signal amplification chain H1, a FRET signal amplification chain H2, and a β-cyclodextrin polymer; the amplification probe system 2 comprises an activated Aptamer fluorescent probe 2 containing a HER2 aptamer sequence, a FRET signal amplification chain H3, a FRET signal amplification chain H4, and a β-cyclodextrin polymer.

[0010] Furthermore, the activated Aptamer fluorescent probe 1 containing the EGFR aptamer sequence includes probe R1 and probe R2, and the activated Aptamer fluorescent probe 2 containing the HER2 aptamer sequence includes probe R2 and probe R3; the nucleotide sequences of the probes R1, R2 and R3 are shown in SEQ ID NO.1-3.

[0011] Furthermore, the 5′ end of the probe R1 is modified with Dabcyl, and the 3′ end of the probe R1 is modified with FAM; the 5′ end of the probe R2 is modified with FAM, and the 3′ end of the probe R2 is modified with Dabcyl; the 5′ end of the probe R3 is modified with BHQ2, and the 3′ end of the probe R3 is modified with Cy3.

[0012] Furthermore, the nucleotide sequences of the FRET signal amplification chain H1, the FRET signal amplification chain H2, the FRET signal amplification chain H3, and the FRET signal amplification chain H4 are as shown in SEQ ID NO.4-7; the FRET signal amplification chain H1 is modified with FAM, the FRET signal amplification chain H2 is modified with Cy5, the FRET signal amplification chain H3 is modified with Cy3, and the FRET signal amplification chain H4 is modified with Cy7.

[0013] Furthermore, the concentration ratio of the FRET signal amplification chain H1 to the FRET signal amplification chain H2 is 1:1, and the concentration ratio of the FRET signal amplification chain H3 to the FRET signal amplification chain H4 is 1:1.

[0014] A targeted in situ self-assembled FRET signal amplification probe system is used to achieve simultaneous imaging of cell membrane EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the same living cell surface.

[0015] Furthermore, the steps of EGFR / EGFR homodimer imaging and EGFR / HER2 heterodimer synchronous imaging include:

[0016] S1: Construction of activated Aptamer fluorescent probe 1 containing EGFR aptamer sequence and activated Aptamer fluorescent probe 2 containing HER2 aptamer sequence;

[0017] S2: co-incubating the cells with activated Aptamer fluorescent probe 1 containing EGFR aptamer sequence and activated Aptamer fluorescent probe 2 containing HER2 aptamer sequence, and then adding FRET signal amplification chain H1, FRET signal amplification chain H2, FRET signal amplification chain H3, FRET signal amplification chain H4, and β-cyclodextrin polymer for continued incubation;

[0018] S3: Observe the fluorescence signal.

[0019] Beneficial effects of the present invention:

[0020] The targeted in situ self-assembled FRET signal amplification probe system constructed by the present invention can achieve the purpose of effectively distinguishing two cell lines, human non-small cell lung cancer cells (A549) and human bronchial epithelial cells (BEAS-2B), by simultaneously identifying EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the cell membrane surface. The targeted in situ self-assembled FRET signal amplification probe system can also effectively distinguish lung adenocarcinoma tissue derived from the A549 lung adenocarcinoma mouse transplant tumor model from normal lung tissue, providing new technical support for early auxiliary diagnosis, efficacy monitoring and prognosis evaluation of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the principle of synchronous in situ imaging of the targeted in situ self-assembled FRET signal amplification probe system in Example 1 for detecting EGFR, HER2 monomers and EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the cell membrane surface;

[0022] Figure 2 The fluorescence spectrum results of the probe R1, probe R2, and probe R3 in Example 1 after incubation with A549 positive cells and BEAS-2B control cells, wherein A is R1, B is R2, and C is R3;

[0023] Figure 3 The gel electrophoresis characterization diagrams of HCR reaction system 1 and HCR reaction system 2 in Example 1, wherein A is the electrophoresis result diagram of HCR reaction system 1, and B is the electrophoresis result diagram of HCR reaction system 2;

[0024] Figure 4 The fluorescence spectra of the amplified probe system 1 and the amplified probe system 2 in Example 1 are characterized, wherein A is the amplified probe system 1 and B is the amplified probe system 2;

[0025] Figure 5The fluorescence spectrum of the enlarged probe system 1 is shown in Figure 1, where A is the fluorescence spectrum and B is the fluorescence intensity comparison diagram;

[0026] Figure 6 The fluorescence spectrum of the enlarged probe system 2, where A is the fluorescence spectrum and B is the fluorescence intensity comparison diagram;

[0027] Figure 7 It is a fluorescence intensity diagram of different reaction times of HCR reaction system 1 and HCR reaction system 2 in Example 1, wherein A is HCR reaction system 1 and B is HCR reaction system 2;

[0028] Figure 8 The fluorescence spectrum characterization diagrams of different S1 / H1 / H2 concentration ratios in the HCR reaction system 1 in Example 1, wherein A is the fluorescence spectrum diagram of different concentration ratios of S1 / H1 / H2, and B is the trend diagram of the fluorescence intensity value of Cy5 at 668nm;

[0029] Fig. 9 The fluorescence spectrum characterization diagrams of different S2 / H3 / H4 concentration ratios in the HCR reaction system 2 in Example 1, wherein A is the fluorescence spectrum diagram of different concentration ratios of S2 / H3 / H4, and B is the trend diagram of the fluorescence intensity value of Cy7 at 772nm;

[0030] Fig.10 This is a diagram showing the effect of α-CD, β-CD, γ-CD, and polyβ-CD on enhancing FRET signal amplification.

[0031] Fig.11 The laser confocal imaging images (40×) of the probe R1 targeting and recognizing A549 cells in Example 1, wherein A is the laser confocal imaging result image of the probe R1 imaging the EGFR monomer on the membrane surface of A549 cells, B is the laser confocal imaging result image of the probe R1 imaging the EGFR monomer on the membrane surface of BEAS-2B cells, and C is the fluorescence intensity comparison image after the images are quantified using Image J software;

[0032] Fig.12 The laser confocal imaging images (40×) of the probe R2 targeting and recognizing A549 cells in Example 1, wherein A is the laser confocal imaging result image of the probe R2 imaging the EGFR monomer on the membrane surface of A549 cells, B is the laser confocal imaging result image of the probe R2 imaging the EGFR monomer on the membrane surface of BEAS-2B cells, and C is the fluorescence intensity comparison image after the images are quantified using Image J software;

[0033] Fig.13The laser confocal imaging images (40×) of the probe R3 targeting and recognizing A549 cells in Example 1, wherein A is the laser confocal imaging result image of the probe R3 imaging the HER2 monomer on the membrane surface of A549 cells, B is the laser confocal imaging result image of the probe R3 imaging the HER2 monomer on the membrane surface of BEAS-2B cells, and C is the fluorescence intensity comparison image after the images are quantified using Image J software;

[0034] Fig.14 The targeted in situ self-assembled FRET signal amplification probe system is used for the simultaneous imaging of EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the A549 cell membrane surface (40×);

[0035] Fig.15 The targeted in situ self-assembled FRET signal amplification probe system is used for synchronous imaging of EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the membrane surface of BEAS-2B cells (40×);

[0036] Fig.16 This is a laser confocal imaging image (10×) of tissue slices using the targeted in situ self-assembled FRET signal amplification probe system, where A is a tumor tissue slice and B is a normal lung tissue slice;

[0037] Fig.17 are the pathological staining results of tumor tissue and normal lung tissue, wherein A is the HE staining image of tumor tissue, B is the IHC staining result image of tumor tissue using EGFR as a biomarker, C is the IHC staining result image of tumor tissue using HER2 as a biomarker, D is the HE staining image of normal lung tissue, E is the IHC staining result image of normal lung tissue using EGFR as a biomarker, and F is the IHC staining result image of normal lung tissue using HER2 as a biomarker. DETAILED DESCRIPTION

[0038] The preparation method of β-cyclodextrin polymer (polyβ-CD) comprises the following steps:

[0039] 1. Synthesis of poly-β-CD: ① Dissolve 10 g of β-cyclodextrin (β-CD) in 15 mL of 15 wt% NaOH aqueous solution, and keep the reaction temperature at 35 °C; ② After stirring for 2 h, add 2 mL of toluene and continue stirring for 2 h; ③ After the β-CD hydrophobic cavity is fully embedded with toluene, slowly add 3.8 mL of epichlorohydrin and react for 3 h to form a cross-linked polymer crude product of β-CD.

[0040] 2. Purification of poly-β-CD: ① Use 200 mL of isopropanol to pre-precipitate the cross-linked polymer crude product obtained after the reaction, collect the precipitate, dissolve it with water and adjust the pH to neutral; ② Use a dialysis membrane Mw = 8000 to dialyze the product to remove oligomers and uncross-linked monomers, change the water every 3 hours, and dialyze for a total of 2 days. After freeze-drying the dialyzed product, label it and store it at -20°C.

[0041] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0042] The nucleic acid chains used in the present invention are all synthesized by Shanghai Sangon Biotechnology Co., Ltd. The nucleotide sequences of the nucleic acid chains used in the present invention are shown in Table 1.

[0043] Table 1 Nucleic acid names and base sequences

[0044]

[0045]

[0046] Example 1

[0047] The preparation method of the targeted in situ self-assembled FRET signal amplification probe system of this embodiment comprises the following steps:

[0048] 1. Preparation of main solutions

[0049] 1) Preparation of PBS buffer

[0050] Purchase bagged PBS powder and pour it into a beaker with a little ultrapure water (Milli-Q water), stir with a rotor and magnetic stirrer until dissolved, then transfer it to a volumetric flask, repeatedly add Milli-Q water to the beaker to wash and add it to the volumetric flask to make up the volume, shake it up and down to mix the solution evenly, finally dispense it into clean glass bottles, sterilize it under high pressure, seal it, and store it in a refrigerator at 4°C. The concentration of PBS buffer is 0.01mol / L.

[0051] (2) Preparation of cell culture medium

[0052] Take the purchased 500mL bottle of 1640 cell culture medium, use a pipette to draw 55.6mL of fetal bovine serum and add it, shake it up and down to mix the solution evenly, and transfer it to a 4℃ refrigerator for storage. Note that the name and date should be marked on the centrifuge tube at the same time, and it should be used as soon as possible.

[0053] (3) Preparation of hybridization buffer

[0054] Dissolve 1.2114g of Tris, 1.0161g of MgCl2·6H2O and 2.9220g of NaCl in 450mL of high-pressure water in a beaker using a magnetic stirrer, adjust the pH to 7.4 using a pH meter, transfer to a volumetric flask and continue to adjust the volume to 500mL with high-pressure water, and mix the solution evenly by shaking up and down. The final concentration of Tris in the hybridization buffer is 20mmol / L, the final concentration of NaCl is 0.1mol / L, and the final concentration of MgCl2·6H2O is 10mmol / L.

[0055] (4) Preparation of binding buffer

[0056] Dissolve 0.5000g of BSA, 0.5083g of MgCl2·6H2O and 2.7500g of glucose in a small amount of PBS in a beaker using a magnetic stirrer, and finally adjust the volume to 500mL in a volumetric flask, and mix the solution evenly by shaking up and down. The final concentration of BSA in the binding buffer is 1g / L, the final concentration of MgCl2·6H2O is 5mmol / L, and the final concentration of glucose is 25mmol / L.

[0057] (5) Preparation of 10×TBE buffer

[0058] Pour the purchased 10×TBE powder into a beaker with a little Milli-Q water, stir with a rotor and magnetic stirrer until dissolved, then transfer to a volumetric flask, repeatedly add Milli-Q water to the beaker to wash and add to the volumetric flask to make up the volume, shake up and down to mix the solution evenly, and finally dispense into clean glass bottles, sterilize under high pressure, seal and store in a refrigerator at 4°C. 10×TBE buffer includes 890mmol / L Tris-boric acid and 20mmol / L ethylenediaminetetraacetic acid (EDTA).

[0059] (6) Preparation of electrophoresis buffer

[0060] Take an appropriate amount of pre-prepared 10×TBE electrophoresis buffer and dilute it with Milli-Q water to 1×TBE electrophoresis buffer. Mix the solution evenly by shaking it up and down. Note that the electrophoresis buffer should be replaced and remade in time after being reused 3 times. 1×TBE electrophoresis buffer contains 89mmol / L Tris-boric acid and 2mmol / L EDTA.

[0061] (7) Preparation of Nucleic Acid Chain Stock Solution

[0062] Centrifuge the light-proof tube containing the dry powder of nucleic acid chain purchased from Bio-Tech at 4000r / min for 2min to make the powder settle to the bottom of the tube, then add the required volume of TE buffer according to the prompt, fully dissolve and mix the nucleic acid chain on a mixer, and finally obtain a nucleic acid chain mother solution with a concentration of 10μmol / L. After the nucleic acid chain mother solution is divided and stored in a -20℃ refrigerator in the dark.

[0063] 2. Construction of targeted in situ self-assembled FRET signal amplification probe system

[0064] 2.1 Investigation of activation performance and targeted recognition performance of activated Aptamer fluorescent probe

[0065] Take the activated Aptamer fluorescent probe nucleic acid chain master solution (R1, R2, R3) labeled with fluorescent group, place it in a 95℃ constant temperature water bath, take it out after 5 minutes, and then slowly cool it down at room temperature to complete the annealing operation; dilute the nucleic acid chain master solution to 1μmol / L with PBS buffer, mix probe R1, probe R2, probe R3 with A549 cells respectively, set up a blank control group (containing only activated Aptamer fluorescent probe and binding buffer), and incubate it in a 37℃ constant temperature metal oscillator at 400r / min for 30 minutes before fluorescence spectroscopy characterization. The specific operation steps are as follows.

[0066] According to the optimal emission and optimal excitation wavelengths of the corresponding fluorescent groups, take R1 as an example: take 50 μL of the mixed solution of R1 and A549 cells incubated in a micro-cuvette, set the excitation wavelength to 492nm, the slit width to 1.5nm, and the scanning collection range of the emission wavelength is 500-550nm; R2 operation steps are the same as above; R3 sets the excitation wavelength to 554nm, the slit width to 1.5nm, and the scanning collection range of the emission wavelength is 560-650nm, and obtains its corresponding fluorescence emission spectra. In this way, it is determined whether the activated Aptamer fluorescent probe can target and label the corresponding cell membrane surface receptors and activate the conformational phase transition to restore fluorescence.

[0067] 2.2 Preparation and characterization of FRET signal amplification probe system

[0068] 2.2.1 Gel electrophoresis of HCR signal amplification probe

[0069] Take an appropriate amount of HCR signal amplification probe and auxiliary chain to complete the annealing operation; adjust the A1 and A2 nucleic acid chain mother solutions to 4 μmol / L, and mix the auxiliary chain S1 and the HCR signal amplification probe (taking S1, A1, A2 as an example) at a concentration ratio of 1:4:4 to obtain a mixed solution, and control the final concentration of the auxiliary chain S1 added to the mixed solution to be 200nmol / L, and incubate at 400r / min in a 37°C constant temperature metal oscillator for 30min, and finally characterize using 15% non-denaturing polyacrylamide gel electrophoresis. The nucleotide sequence of the auxiliary chain S1 is shown in SEQ ID NO.8. The nucleotide sequences of A1 and A2 are shown in SEQ ID NO.15 and 16.

[0070] 2.2.2 Fluorescence spectrum of FRET signal amplification probe

[0071] The spectral performance of the two pairs of FRET fluorescent groups selected was explored: an appropriate amount of DNA bridge nucleic acid chain labeled with corresponding fluorescent groups was annealed and diluted to 100 nmol / L with hybridization buffer; three DNA single strands (B2-Cy5, B3-Cy7, FAM-B1-Cy3) were mixed at a concentration ratio of 1:1:1, and then incubated at 400r / min for 30min in a 37℃ constant temperature metal oscillator to form double-stranded DNA, and fluorescence spectral characterization was performed. The nucleotide sequences of the three DNA single strands (B2-Cy5, B3-Cy7, FAM-B1-Cy3) are shown in SEQ ID NO.10-12.

[0072] 2.2.3 Optimization of FRET signal amplification probe system conditions

[0073] Optimization of HCR reaction time: FRET signal amplification in the present invention is achieved by HCR reaction, so it is necessary to control the HCR reaction time. Too short reaction time may reduce the detection sensitivity, and too long time will waste time. Therefore, 7 experimental groups with gradient reaction time of 5min, 15min, 30min, 60min, 90min, 120min, and 150min are set, and three parallel experiments are set in each group. Take an appropriate amount of FRET signal amplification chain and the single nucleic acid chain mother liquor of auxiliary chain S1 and auxiliary chain S2, complete the annealing operation, and then the single nucleic acid chain mother liquor of the corresponding FRET signal amplification chain and the auxiliary chain (taking S1, H1, H2 as an example) are mixed according to a concentration ratio of 1: 4: 4 to obtain a mixed solution, and the final concentration of the auxiliary chain S1 added in the mixed solution is controlled to be 100nmol / L, and the corresponding gradient time is incubated at 400r / min in a 37°C constant temperature metal oscillator.

[0074] Optimization of HCR reaction concentration ratio: The ratio of auxiliary chain and HCR amplification chain added to the reaction system is also a key factor affecting the sensitivity of the experiment. Therefore, 6 experimental groups with gradient concentrations of auxiliary chain and FRET signal amplification probe were set. Taking S1 / H1 / H2 as an example: after annealing the single-stranded nucleic acid probe mother chain, a gradient concentration ratio was set, with a total of 6 groups of experiments, namely, the concentration ratios of S1:H1:H2 were 1:2:2, 1:4:4, 1:6:6, 1:8:8, 1:10:10, 1:15:15, where the final concentration of the auxiliary chain S1 added to the system was controlled to be 100nmol / L. Incubate at 400r / min for 30min in a 37℃ constant temperature metal oscillator, and finally use a multifunctional microplate reader for fluorescence spectroscopy characterization. At the same time, the fluorescence intensity of the receptor fluorescent group in each experimental group at the optimal emission wavelength was quantified to determine the optimal concentration ratio.

[0075] 2.2.4 Investigation of the effect of cyclodextrin on FRET signal amplification

[0076] Studies have shown that cyclodextrin can encapsulate substances in its hydrophobic cavity through host-guest interaction, thereby changing certain properties of the substance. The present invention explores the effects of four cyclodextrins on the FRET effect and screens out cyclodextrins that can enhance the fluorescence intensity of FRET receptors, thereby further improving the sensitivity of the method. The specific experimental steps are as follows.

[0077] Take the amplification probe system 1 as an example: take an appropriate amount of single nucleic acid chain master solution, anneal it, and first construct T1 / R1 / R2 / H1 / H2. The four cyclodextrins (α-CD, β-CD, γ-CD, polyβ-CD) monomer concentration is 10mmol / L, and they are mixed with T1 / R1 / R2 / H1 / H2 (the concentration ratio is 1:1:1:4:4, and the overall concentration is expressed by the concentration of the contained T1 chain). By adjusting the high-span gradient concentration ratio of the four cyclodextrins (1:200, 1:2000, 1:10000, 1:50000), different experimental groups are set up, and three parallel experiments are set up in each group. After incubation at 400r / min in a 37°C constant temperature metal oscillator for 30min, fluorescence spectroscopy characterization is performed.

[0078] 2.3 Study on the effect of targeted in situ self-assembled FRET signal amplification probe system on different cell lines

[0079] 2.3.1 Activated Aptamer fluorescent probe for imaging of EGFR / HER2 monomers on cell membrane surface

[0080] A549 and BEAS-2B cells were incubated with activated Aptamer fluorescent probes, and laser confocal microscopy was used to observe whether fluorescence was generated on the cell membrane surface, thereby determining whether the probe bound to the corresponding membrane protein receptor on the cell surface and whether the conformation changed to restore fluorescence.

[0081] 2.3.2 Targeted in situ self-assembled FRET signal amplification probe system for simultaneous imaging of EGFR / EGFR and EGFR / HER2 dimers on the cell membrane surface

[0082] The FRET signal amplification probe system was used to simultaneously image two homologous / heterodimers on the surface of two cell membranes. A549 and BEAS-2B cells with good growth conditions were co-incubated with the two FRET signal amplification probe systems, and the two FRET effects on the corresponding cell membrane surfaces were simultaneously observed using a laser confocal microscope to verify whether the two probe systems could simultaneously image the two cell surface dimers without affecting each other.

[0083] 2.4 Study on the effect of targeted in situ self-assembled FRET signal amplification probe system on different tissues

[0084] In order to explore the imaging recognition ability of the FRET signal amplification probe system in different tissues, A549 cells were used to construct a lung adenocarcinoma mouse transplant tumor model, and tumor tissue was obtained by subcutaneous tumor formation in nude mice as the experimental group, while healthy nude mouse lung tissue slices were used in the control group.

[0085] Use a pipette to drop 60 μL of PBS on the surface of the frozen section and soak for 3 minutes. Then use filter paper to absorb the liquid around the tissue, and add 50 μL of the mixed solution of the FRET signal amplification probe system with adjusted probe ratio on the tissue section. Incubate in the dark for 15 minutes, cover with a coverslip, and observe under a laser confocal microscope.

[0086] 3. Results

[0087] 3.1 Construction and related characterization of targeted in situ self-assembled FRET signal amplification probe system

[0088] 3.1.1 Fluorescence spectroscopy characterization of activation performance and target recognition performance of activated Aptamer fluorescent probe

[0089] Fluorescence spectroscopy was used to verify the ability of activated Aptamer fluorescent probe to target the corresponding membrane receptors. Figure 2 Take R1 as an example (as shown in Figure 2As shown in A), the fluorescence spectrum shows that when excited by the optimal excitation wavelength of FAM at 492nm, the solution containing only the R1 probe has no obvious fluorescence emission peak in the collection band of 500-550nm. This is because the designed R1 probe is of hairpin type and is labeled with a fluorescent group and a quenching group (FAM and Dabcy1) at the end of the DNA single strand. When the hairpin is not activated, it is in a fluorescence quenching state. After incubating the R1 probe with A549 positive cells and BEAS-2B control cells, respectively, corresponding fluorescence peaks appeared in both cell solutions. This is because the hairpin probe contains an aptamer sequence, which can serve as an "activation" signal when encountering the corresponding membrane receptor, inducing the hairpin conformational transition and thus restoring fluorescence. And after normalizing the fluorescence signal value (the highest fluorescence signal value is assigned to 1), it can be seen that the fluorescence intensity of the positive cells is significantly higher than that of the control cells. The reason is that the positive cells express more EGFR than the negative cells. The results of probes R2 and R3 are similar to those of probe R1 (such as Figure 2 The above results show that the constructed activated Aptamer fluorescent probe can successfully target and bind to the corresponding membrane receptors to produce activated fluorescence.

[0090] 3.1.2 Preparation and characterization of FRET signal amplification probe system

[0091] 3.1.2.1 Gel electrophoresis characterization of HCR signal amplification probe

[0092] In order to verify whether the designed HCR signal amplification probe can be successfully hybridized, polyacrylamide gel electrophoresis was used for characterization. Figure 3 As shown in A), the lanes of DNA single strands S1, A1 and A2 all show only one clear band; when only A1 and A2 chains are mixed without the presence of the starter chain S1, there is no obvious two-strand hybridization amplification phenomenon (lane 5); in lane 6, two nucleic acid bands appear after the addition of S1 chain to A1 chain, indicating that S1 can effectively open the A1 hairpin; only after the three chains are fully added to the reaction system, waterfall-like nucleic acid electrophoresis bands can be seen in the lanes (lanes 7-8). The electrophoresis results of S2, A3, and A4 are the same (as shown in Figure 3 B). The above results indicate that the HCR signal amplification probe can be normally assembled in the solution to cause HCR amplification. The nucleotide sequences of A3 and A4 are shown in SEQ ID NO.17-18.

[0093] 3.1.2.2 Fluorescence spectroscopy characterization of FRET signal amplification probe

[0094] Fluorescence spectroscopy was used to verify whether the two pairs of FRET fluorescent dyes (FAM / Cy5, Cy3 / Cy7) screened could effectively produce FRET. Taking the FRET pair (FAM / Cy5) as an example, Figure 4As shown in Figure A, the excitation wavelength is set to 492nm. At this time, the collection range of the emission wavelength is 500-750nm. It can be seen that the donor fluorescent group is significantly reduced, and the acceptor fluorescence peak appears (as shown by the black arrow in the figure), indicating that effective FRET can occur between FAM / Cy5; similarly, the fluorescence peak of Cy7 can also be observed under 540nm excitation for the FRET pair (Cy3 / Cy7) (as shown in Figure A). Figure 4 B), indicating that FRET occurs. The above results show that FAM and Cy5, Cy3 and Cy7 can undergo FRET and can be used for subsequent experiments.

[0095] Furthermore, the fluorescence spectrum of the overall fluorescence performance of the FRET signal amplification probe system was characterized in the solution to examine the influence of each logical component in the probe system on fluorescence. The FRET phenomenon was used to determine whether the probes were successfully hybridized in sequence to form a FRET signal amplification double-stranded DNA complex. Taking the amplification probe system 1 as an example: the five DNA single strands (T1, R1, R2, H1, H2) were respectively constructed into experimental groups in a ratio of 1:1:1:10:10. Only in the experimental group where all five DNA single strands (T1, R1, R2, H1, H2) were added, FERT occurred, that is, the FAM fluorescence peak decreased and the Cy5 fluorescence peak increased. Figure 5 It can be seen that the fluorescence intensity values ​​at the receptor fluorescence peak are quantified and compared, and it is obvious that the receptor fluorescence intensity of the T1 / R1 / R2 / H1 / H2 group is significantly higher than that of the other control groups. Figure 6 It can be seen that although no obvious Cy7 fluorescence peak can be observed in the spectrum (probably because the fluorescence intensity value of the donor fluorescence peak is too high, resulting in the failure of the Cy7 fluorescence peak to appear), the decrease in the donor fluorescence peak and the quantification of the fluorescence intensity at the optimal emission wavelength of the acceptor fluorescent group show that the fluorescence intensity of the T2 / R2 / R3 / H3 / H4 group is significantly higher than that of other control groups, indicating that FRET has successfully occurred. The above results show that each logical component in the constructed FRET signal amplification probe system is indispensable and has the characteristics of reducing background signals without washing.

[0096] 3.1.2.3 Optimization results of FRET signal amplification probe system

[0097] In order to explore the effects of different reaction conditions on the FRET signal amplification probe system and find out the optimal reaction conditions to achieve the best detection effect, the reaction conditions were optimized.

[0098] (1) Optimization of HCR reaction time, such as Figure 7As shown in A, by collecting the fluorescence intensity values ​​of Cy5 at 668 nm and plotting them, and normalizing the fluorescence signal values, it can be seen that the HCR reaction rate of HCR reaction system 1 (S1, H1, H2) is relatively fast, reaching the maximum value quickly after 5 minutes, and then the overall fluorescence trend remains stable within 150 minutes, indicating that its amplification is rapid and stable; Figure 7 The HCR reaction system 2 (S2, H3, H4) shown in B was plotted by collecting the fluorescence intensity values ​​of Cy7 at 772 nm. It can be seen that the fluorescence intensity increased rapidly after 5 minutes, and then the overall fluorescence trend remained stable within 120 minutes, indicating that the amplification was rapid and stable. Thereafter, the fluorescence intensity decreased slightly, so 30 minutes was selected as the HCR amplification reaction condition for subsequent experiments.

[0099] (2) Optimization of HCR reaction concentration ratio. The ratio of auxiliary chain and HCR amplification chain added to the reaction system is also a key factor affecting the amplification effect. Set the gradient concentration ratio separately, and a total of 6 experimental groups (taking S1 / H1 / H2 as an example) are as follows:

[0100] The concentration ratio of S1:H1:H2 is 1:2:2;

[0101] The concentration ratio of S1:H1:H2 is 1:4:4;

[0102] The concentration ratio of S1:H1:H2 is 1:6:6;

[0103] The concentration ratio of S1:H1:H2 is 1:8:8;

[0104] The concentration ratio of S1:H1:H2 is 1:10:10;

[0105] The concentration ratio of S1:H1:H2 is 1:15:15;

[0106] like Figure 8 As shown in the fluorescence spectrum, it can be seen that with the increase of concentration ratio, the fluorescence intensity of the fluorescence peak at Cy5 continues to increase. The fluorescence intensity is further quantified. The results show that the fluorescence intensity gradually increases before the S1:H1:H2 concentration ratio is 1:10:10, and then the growth trend gradually slows down. The same concentration ratio experimental group is set to explore the amplification effect of S2 / H3 / H4. Fig. 9 The results showed that the fluorescence intensity of the fluorescence peak at Cy7 continued to increase, and the fluorescence quantification graph also showed that the fluorescence intensity continued to increase with the increase in the concentration ratio. Based on the above results, the S2:H3:H4 concentration ratio of 1:10:10 was used as the optimal HCR amplification concentration ratio for subsequent experiments.

[0107] 3.1.2.4 Cyclodextrin enhances FRET signal amplification effect

[0108] In order to further improve the FRET signal amplification effect of the probe system, the effects of four cyclodextrins (α-CD, β-CD, γ-CD, polyβ-CD) on the reaction system were investigated, and cyclodextrins that can enhance the fluorescence intensity of FRET receptors were screened out, thereby improving the sensitivity of the detection method. Five experimental groups (taking α-CD as an example, and the concentration ratio of T1 / R1 / R2 / H1 / H2 is 1:1:1:4:4) were set up as follows.

[0109] The control group was a DNA chain group containing only T1 / R1 / R2 / H1 / H2;

[0110] 1:200 means the concentration ratio of T1 / R1 / R2 / H1 / H2 to α-CD is 1:200;

[0111] 1:2000 means the concentration ratio of T1 / R1 / R2 / H1 / H2 to α-CD is 1:2000;

[0112] 1:10000 means the concentration ratio of T1 / R1 / R2 / H1 / H2 to α-CD is 1:10000;

[0113] 1:50000 means the concentration ratio of T1 / R1 / R2 / H1 / H2 to α-CD is 1:50000;

[0114] Fluorescence spectrum measurement was performed in a multifunctional microplate reader, with the excitation wavelength set to 480 nm and the collection band set to 506-700 nm. The fluorescence spectrum of each experimental group was obtained and the fluorescence intensity value of the acceptor fluorescent group at the optimal fluorescence emission peak was further quantified and plotted ( Fig.10 ), the results showed that polyβ-CD showed an enhancement effect on the fluorescence value under different ratio concentration conditions, and the enhancement effect was stronger than that of the other three cyclodextrins. When the concentration ratio was 1:200, the enhancement effect of polyβ-CD was the best (1.21 times). Therefore, polyβ-CD was used as a fluorescence enhancer in subsequent experiments.

[0115] 3.2 Targeted in situ self-assembled FRET signal amplification probe system for different cell lines

[0116] 3.2.1 Laser confocal imaging of activated Aptamer fluorescent probes on cell membrane surface monomers

[0117] In order to visualize the expression of corresponding membrane proteins on the cell membrane surface, laser confocal microscopy was used to perform fluorescence imaging of three activated Aptamer fluorescent probes (R1, R2, and R3) after incubation with the two cells for 2 hours, and the acquired images were quantified using Image J software. The imaging results of the two cells with R1 probe are shown in Figure 2. Fig.11As shown, under 488nm excitation, the fluorescence of the FAM channel (496-570nm) was collected, and the fluorescence was given a green pseudo-color. The outline of the cells can be clearly seen in A549 cells, while only fuzzy cell boundaries can be seen in the control cells BEAS-2B. After quantifying the acquired images using Image J software, it can be seen that the fluorescence intensity of A549 cells is significantly higher than that of BEAS-2B cells. The imaging results of R2 and R3 probes are similar ( Fig.12 , Fig.13 ). The above results show that the constructed activated Aptamer fluorescent probe has the ability to directly image the corresponding membrane receptor monomers on the cell membrane surface, and no obvious probe internalization phenomenon was observed within 2 hours. At the same time, the quantitative results of the image show that the activated Aptamer fluorescent probe can effectively identify two different cell lines.

[0118] 3.2.2 Targeted in situ self-assembled FRET signal amplification probe system for simultaneous imaging of EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the cell membrane

[0119] The effects of amplification probe system 1 and amplification probe system 2 on the synchronous imaging of EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the A549 or BEAS-2B cell membrane were investigated. Amplification probe system 1 and amplification probe system 2 were co-incubated with cells at the same time, and the fluorescence of four channels, FAM, Cy5, Cy3, and Cy7, were collected respectively. The results are shown in Fig.14 and Fig.15 As shown, only the FAM, Cy5, Cy3, and Cy7 channels in A549 cells show obvious fluorescence signals, while no obvious fluorescence signals are seen in BEAS-2B cells. The appearance of fluorescence signals in the receptor fluorescence channels (Cy5 and Cy7) indicates that the amplification probe system 1 and the amplification probe system 2 can simultaneously image the EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the surface of living cell membranes without affecting each other, and can thereby distinguish the two types of cells.

[0120] 3.3 Targeted in situ self-assembled FRET signal amplification probe system for differentiation of lung adenocarcinoma tissue and normal lung tissue

[0121] Lung adenocarcinoma tumor tissue was used as the experimental group, and normal lung tissue was used as the control group. The FRET signal amplification probe system was incubated with the frozen tissue sections in the dark for 15 minutes. The fluorescence of the FAM channel (496-570nm) and the Cy5 channel (600-700nm) was collected at 488nm excitation; the fluorescence of the Cy3 channel (560-600nm) and the Cy7 channel (700-800nm) was collected at 552nm excitation. The results are shown in Figure 2. Fig.16As shown in the figure, after incubation of normal lung tissue with the targeted in situ self-assembled FRET signal amplification probe system, only weak fluorescence signals were shown in the Cy5 channel and the Cy7 channel, while the laser confocal imaging results of tumor tissue showed bright fluorescence signals in both the Cy5 channel and the Cy7 channel. Pathological results HE staining showed ( Fig.17 A), the tumor tissue showed dense and disordered growth, the nucleus was clearly stained blue, and other parts such as the cytoplasm were red to varying degrees; IHC staining results showed that on the tumor tissue sections, the nucleus was dark blue, and brown-yellow EGFR ( Fig.17 B) HER2( Fig.17 C) positive staining markers, and the positive staining markers in tumor tissues are higher than those in normal lung tissues. The above results show that the two biomarkers are overexpressed in tumor tissues. The results of pathological HE staining and IHC staining are also consistent with the results of imaging tissues by the FRET signal amplification probe system. The above research results show that the FRET signal amplification probe system has the property of visually distinguishing different tissues at the tissue level.

Claims

1. A targeted in situ self-assembled FRET signal amplification probe system, characterized in that: It includes an amplification probe system 1 and an amplification probe system 2. The amplification probe system 1 includes an activated Aptamer fluorescent probe 1 containing an EGFR aptamer sequence, a FRET signal amplification chain H1, a FRET signal amplification chain H2, and a β-cyclodextrin polymer; the amplification probe system 2 includes an activated Aptamer fluorescent probe 2 containing a HER2 aptamer sequence, a FRET signal amplification chain H3, a FRET signal amplification chain H4, and a β-cyclodextrin polymer.

2. The targeted in situ self-assembled FRET signal amplification probe system according to claim 1, characterized in that: The activated Aptamer fluorescent probe 1 containing the EGFR aptamer sequence includes probe R1 and probe R2, and the activated Aptamer fluorescent probe 2 containing the HER2 aptamer sequence includes probe R2 and probe R3; the nucleotide sequences of the probes R1, R2 and R3 are shown in SEQ ID NO.1-3.

3. The targeted in situ self-assembled FRET signal amplification probe system according to claim 1 or 2, characterized in that: The 5′ end of the probe R1 is modified with Dabcyl, and the 3′ end of the probe R1 is modified with FAM; the 5′ end of the probe R2 is modified with FAM, and the 3′ end of the probe R2 is modified with Dabcyl; the 5′ end of the probe R3 is modified with BHQ2, and the 3′ end of the probe R3 is modified with Cy3.

4. The targeted in situ self-assembled FRET signal amplification probe system according to claim 1, characterized in that: The nucleotide sequences of the FRET signal amplification chain H1, the FRET signal amplification chain H2, the FRET signal amplification chain H3, and the FRET signal amplification chain H4 are shown in SEQ ID NO.4-7; the FRET signal amplification chain H1 is modified with FAM, the FRET signal amplification chain H2 is modified with Cy5, the FRET signal amplification chain H3 is modified with Cy3, and the FRET signal amplification chain H4 is modified with Cy7.

5. The targeted in situ self-assembled FRET signal amplification probe system according to claim 1, characterized in that: The concentration ratio of the FRET signal amplification chain H1 to the FRET signal amplification chain H2 is 1:1, and the concentration ratio of the FRET signal amplification chain H3 to the FRET signal amplification chain H4 is 1:

1.

6. An application of the targeted in situ self-assembled FRET signal amplification probe system as claimed in claim 1 to achieve synchronous imaging of cell membrane EGFR / EGFR homodimers and EGFR / HER2 heterodimers on the same living cell surface.

7. The use of the targeted in situ self-assembled FRET signal amplification probe system according to claim 6, characterized in that: The steps of EGFR / EGFR homodimer imaging and simultaneous imaging of EGFR / HER2 heterodimers include: S1: Construction of activated Aptamer fluorescent probe 1 containing EGFR aptamer sequence and activated Aptamer fluorescent probe 2 containing HER2 aptamer sequence; S2: co-incubating the cells with activated Aptamer fluorescent probe 1 containing EGFR aptamer sequence and activated Aptamer fluorescent probe 2 containing HER2 aptamer sequence, and then adding FRET signal amplification chain H1, FRET signal amplification chain H2, FRET signal amplification chain H3, FRET signal amplification chain H4, and β-cyclodextrin polymer for continued incubation; S3: Observe the fluorescence signal.

Images