Surface-enhanced fluorescence coding nanoprobe as well as preparation method and application thereof

Abstract

The invention relates to a surface-enhanced fluorescence coding nanoprobe as well as a preparation method and application thereof. The preparation method of the surface-enhanced fluorescence coding nano-probe comprises the following steps: modifying an anchoring chain to the surface of a fluorescence coding nano-particle through an amidation reaction of carboxyl and amino to obtain the fluorescence coding nano-particle modified with the anchoring chain; and then self-assembling the H1 chain, the H2 chain and the recognition chain of the tumor biomarker to the surface of the fluorescence coding nano-particle modified with the anchoring chain through complementary base pairing to obtain the fluorescence coding nano-particle. According to the invention, beta-CD with fluorescence sensibilization characteristics is introduced into the two functional areas, so that high-sensitivity and high-contrast synchronous imaging of a plurality of mRNA and proteins related to lung cancer in living single cells is realized, and the method is successfully used for monitoring change of expression quantity of EGFR mRNA and proteins after siRNA interference; therefore, a novel method is provided for synchronous analysis and imaging of various biological molecules related to lung cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-biomaterials, and in particular relates to a surface-enhanced fluorescence coded nano-probe and a preparation method and application thereof. Background Art

[0002] As a serious public health problem, cancer has attracted increasing attention from the world. It is complex and heterogeneous, with characteristics such as abnormal cell differentiation and proliferation, invasiveness and metastasis. Today, cancer has become the leading cause of death worldwide, with 20 million new cases and 9.7 million deaths in 2022. Lung cancer is the most common cancer and the most common cause of cancer death. Lung cancer has a high incidence rate, ranking second among all cancers, and its mortality rate ranks first. More than 1.8 million people die from lung cancer each year worldwide, and it is expected that the number of deaths from lung cancer will increase to 3 million in 2035. The 5-year survival rate of lung cancer patients is less than 20%, while early detection and standardized treatment can increase it to 70%-80%. Traditional diagnostic methods for lung cancer used pathology, spiral CT, cytology, etc., but most lung cancer patients had no typical symptoms in the early stage, and the disease had progressed to the middle and late stages when diagnosed, and even metastasized to other tissues, which greatly increased the misdiagnosis and missed diagnosis rates. Therefore, the trend of developing more reliable lung cancer diagnostic methods should be committed to the accurate detection of biomarkers at the molecular level. Studies have shown that the occurrence and development of lung cancer is related to a variety of mRNA and protein markers in cells. However, there is currently no method to clearly diagnose specific biomarkers for lung cancer. Therefore, the in situ joint detection of multiple mRNA and protein markers in cells is of great significance for the early auxiliary diagnosis of lung cancer. By jointly analyzing multiple mRNAs and their expressed proteins, higher accuracy can be obtained than when analyzing a single target, which plays an important role in precision medicine, efficacy monitoring and prognosis evaluation.

[0003] Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein widely distributed on the surface of epithelial cells. As a key regulator of cell behavior, EGFR 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. Overexpression of its mRNA and protein is associated with the occurrence and development of many tumors, including non-small cell lung cancer, breast cancer, and colorectal cancer. Programmed death ligand 1 (PD-L1) is a major immune checkpoint protein embedded in the tumor cell membrane. It evades the recognition of the immune system through the interaction between PD-L1 and its programmed death receptor 1, thereby achieving immune escape of tumor cells and promoting the growth of tumor cells. Therefore, it is a key protein in cancer diagnosis and immunotherapy. At present, PD-L1 has been used as an important biomarker to guide clinical diagnosis and medication. Many studies have shown that PD-L1 mRNA and protein are highly expressed in lung cancer. PD-L1 is a potential ideal tumor marker in auxiliary diagnosis of lung cancer. Vascular endothelial growth factor (VEGF) is an important vascular endothelial growth factor that is overexpressed in tumors. It promotes tumor progression and metastasis by promoting endothelial cell growth, survival and migration and regulating vascular permeability. It is considered to be a key biomarker for a variety of cancers, including lung cancer. Clinical studies have shown that increased levels of VEGF mRNA and protein are associated with the occurrence and development of a variety of cancers, including pancreatic cancer, ovarian cancer, non-small cell lung cancer and breast cancer. Therefore, the in situ combined detection of EGFR, VEGF and PD-L1 mRNA and corresponding proteins in living cells will play an important role in promoting the early auxiliary diagnosis of lung cancer.

[0004] Fluorescent probe optical imaging, with its high sensitivity, non-invasiveness, visualization and high spatiotemporal resolution, provides a good molecular information visualization and quantification method for biomedical research and clinical research, and has become one of the most promising imaging technologies in the biomedical field. Fluorescent probes can generally be divided into two categories: "always-on" fluorescent probes and activated ("turn-on") fluorescent probes. Fluorescent imaging based on "always-on" fluorescent probes is a common strategy for bioimaging and biosensing. However, when the "always on" signal mode is applied to the imaging and targeted positioning of targets in cells, there are shortcomings such as high imaging background and the need to wash and remove weakly bound probes, which prolongs the diagnosis time. In addition, when the "always on" signal mode is detected, the "always-on" fluorescent probe will emit fluorescence regardless of the presence or absence of the target. The "turn-on" fluorescent probe can realize the transition of the detection signal from "no" to "yes" when the target (reactive oxygen, pH, metal ions, tumor-related receptors or biological enzymes, etc.) is present. It has the advantages of no washing and low background signal, which can effectively improve the imaging signal-to-background ratio, reduce the detection limit, and improve the detection performance of biosensors. Therefore, a large number of "turn-on" fluorescent probes have been constructed by researchers to detect biomolecules, inorganic molecules, and metal ions in vitro and in vivo, which provides an important reference for the design of activated fluorescent nanoprobes. Fluorescence imaging is a powerful technology that can reveal the spatial information of protein and nucleic acid molecules in cells. However, the spectral overlap of fluorescent groups limits the number of targets that can be detected simultaneously by conventional fluorescent probe optical imaging technology (no more than 5 targets). Summary of the invention

[0005] The first object of the present invention is to provide a surface-enhanced fluorescence coded nanoprobe to solve the technical problem that the number of targets to be detected simultaneously by the existing fluorescent probe optical imaging technology is small.

[0006] The second object of the present invention is to provide a method for preparing a surface enhanced fluorescence coded nanoprobe.

[0007] The third object of the present invention is to provide an application of a surface enhanced fluorescence coded nanoprobe.

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

[0009] A preparation method of a surface enhanced fluorescence coded nanoprobe comprises the following steps: modifying an anchor chain to the surface of a fluorescence coded nanoparticle by an amidation reaction of a carboxyl group and an amino group to obtain a fluorescence coded nanoparticle modified with an anchor chain, and then self-assembling an H1 chain, an H2 chain and a recognition chain of a tumor biomarker to the surface of the fluorescence coded nanoparticle modified with an anchor chain by base complementary pairing, so as to obtain; the recognition chain of the tumor biomarker comprises an EGFR mRNA recognition chain, an EGFR protein recognition chain, a PD-L1 mRNA recognition chain, a PD-L1 protein recognition chain, a VEGF mRNA recognition chain and a VEGF protein recognition chain, the fluorescence coded nanoparticle is coated with a dye by β-cyclodextrin, the dye is one or more of a fluorescein sodium solution and a rhodamine B solution, the dye is divided into six types: A dye, B dye, C dye, D dye, E dye and F dye, the content of the fluorescein sodium solution in the dye A to the dye F gradually decreases, and the content of the rhodamine B solution gradually increases; the EGFR mRNA recognition chain, the EGFR protein recognition chain, the PD-L1 mRNA recognition chain, the PD-L1 protein recognition chain, the VEGF mRNA recognition chain and the VEGF protein recognition chain, the fluorescence coded nanoparticle is coated with a dye by β-cyclodextrin, the dye is one or more of a fluorescein sodium solution and a rhodamine B solution, the dye is divided into six types: A dye, B dye, C dye, D dye, E dye and F dye, the content of the fluorescein sodium solution in the dye A to the dye F gradually decreases, and the content of the rhodamine B solution gradually increases; The mRNA recognition chain, the PD-L1 protein recognition chain, the VEGF mRNA recognition chain, and the VEGF protein recognition chain are randomly added to one of the A dyes and the F dyes.

[0010] Furthermore, the nucleotide sequence of the H1 chain is shown in SEQ ID NO.1, the nucleotide sequence of the H2 chain is shown in SEQ ID NO.2, and the nucleotide sequence of the anchor chain is shown in SEQ ID NO.3; the nucleotide sequence of the EGFR mRNA recognition chain is shown in SEQ ID NO.4, the nucleotide sequence of the EGFR protein recognition chain is shown in SEQ ID NO.5, the nucleotide sequence of the PD-L1 mRNA recognition chain is shown in SEQ ID NO.6, the nucleotide sequence of the PD-L1 protein recognition chain is shown in SEQ ID NO.7, the nucleotide sequence of the VEGF mRNA recognition chain is shown in SEQ ID NO.8, and the nucleotide sequence of the VEGF protein recognition chain is shown in SEQ ID NO.9.

[0011] Furthermore, the A dye is a sodium fluorescein solution, the F dye is a rhodamine B solution, and the molar ratios of the sodium fluorescein solution to the rhodamine B solution in the B dye, C dye, D dye, and E dye are 5:1, 4:2, 3:3, and 1:5, respectively; the A dye corresponds to the EGFR mRNA recognition chain, the B dye corresponds to the EGFR protein recognition chain, the C dye corresponds to the PD-L1 protein recognition chain, the D dye corresponds to the PD-L1 mRNA recognition chain, the E dye corresponds to the VEGF protein recognition chain, and the F dye corresponds to the VEGF mRNA recognition chain.

[0012] Furthermore, the method comprises the following steps: carboxylating the fluorescently coded nanoparticles to obtain carboxylated fluorescently coded nanoparticles, dispersing the carboxylated fluorescently coded nanoparticles in a 2-morpholineethanesulfonic acid buffer, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysulfosuccinimide, and an anchor chain to react at 20-30° C. for 4-5 hours to obtain fluorescently coded nanoparticles modified with an anchor chain, dispersing the fluorescently coded nanoparticles modified with an anchor chain in a hybridization buffer, adding the H1 chain and the H2 chain to react at 30-40° C. for 20-40 minutes, and then adding the recognition chain of the tumor biomarker and dispersing them in the hybridization buffer solution to obtain the method.

[0013] Furthermore, the mass ratio of the carboxylated fluorescent coded nanoparticles to the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:6, the mass ratio of the carboxylated fluorescent coded nanoparticles to the N-hydroxysulfosuccinimide is 1:6, 40-60 μL of the anchor chain is added per mg of the carboxylated fluorescent coded nanoparticles, 10-20 μL of the H1 chain is added per mg of the carboxylated fluorescent coded nanoparticles, and 10-20 μL of the H 2 chains, 20-40 μL of the tumor biomarker recognition chain is added to each mg of the carboxylated fluorescent coded nanoparticles; the concentration of the anchor chain is 9-11 μmol / L, the concentration of the H1 chain is 9-11 μmol / L, the concentration of the H2 chain is 9-11 μmol / L, and the concentration of the tumor biomarker recognition chain is 9-11 μmol / L; the carboxylated fluorescent coded nanoparticles are prepared by carboxylating the fluorescent coded nanoparticles using tetraethyl orthosilicate and carboxyethylsilanetriol sodium salt.

[0014] Furthermore, the preparation method of the H2 chain is: dissolving the H chain modified with dibenzocyclooctyne in triethylammonium acetate to obtain solution one, dissolving azide-modified β-cyclodextrin in dimethyl sulfoxide to obtain solution two, mixing equal volumes of solution one and solution two, reacting at 20-30°C for 10-14h, then fixing the volume and concentrating to obtain the product; the nucleotide sequence of the H chain is shown in SEQ ID NO.10, and the concentration ratio of the H chain to the azide-modified β-cyclodextrin is 1:5.

[0015] Furthermore, the preparation method of the fluorescent coded nanoparticles is: one of dye A, dye B, dye C, dye D, dye E, and dye F is mixed with equal volumes of β-cyclodextrin solution, and then ultrasonically obtained an inclusion compound for use, cyclohexane, n-hexanol and Triton X-100 are mixed to obtain a mixed solution, the inclusion compound is added dropwise to the mixed solution, and the mixture is reacted in the dark at room temperature for 20-40 minutes; then, tetraethyl orthosilicate is added dropwise, stirred for 20-40 minutes, and then ammonia water is added dropwise, and stirred at room temperature for 24-30 hours to obtain the obtained nanoparticles.

[0016] Furthermore, the concentration of the β-cyclodextrin solution is 1×10 -2 -3×10 -2 mol / L, the volume ratio of the β-cyclodextrin solution to the cyclohexane is 4:125, the volume ratio of the β-cyclodextrin solution to the n-hexanol is 3:20, the volume ratio of the β-cyclodextrin solution to Triton X-100 is 2:15, the volume ratio of the β-cyclodextrin solution to the tetraethyl orthosilicate is 12:5, the volume ratio of the β-cyclodextrin solution to the ammonia water is 12:5, and the volume ratio of the β-cyclodextrin solution to the dye is 1:1.

[0017] A surface enhanced fluorescence coded nanoprobe is prepared by using the above-mentioned preparation method of the surface enhanced fluorescence coded nanoprobe.

[0018] A surface-enhanced fluorescence encoded nanoprobe for simultaneous in situ detection of EGFR mRNA, EGFR protein, PD-L1 mRNA, PD-L1 protein, VEGF mRNA, VEGF protein and imaging in single living cells.

[0019] Beneficial effects of the present invention:

[0020] The present invention successfully constructed a new type of surface-enhanced fluorescence encoded nanoprobe (SFENP) based on the fluorescence encoding principle and the fluorescence sensitization performance of β-cyclodextrin, realizing high-contrast synchronous imaging of three mRNAs (EGFR, VEGF and PD-L1) and three proteins (EGFR, VEGF and PD-L1) related to lung cancer in living single cells, and successfully used it to monitor the changes in EGFR mRNA and protein expression after siRNA interference, thus providing a new method for the synchronous analysis and imaging of multiple biological molecules related to lung cancer, which is helpful for the early auxiliary diagnosis of lung cancer.

[0021] The surface-enhanced fluorescence coded nanoprobe of the present invention consists of two functional areas: 1. Fluorescence coded nanoparticles loaded with 6 different proportions of sodium fluorescein and rhodamine B for qualitative analysis. The presence of β-CD in the fluorescence coded nanoparticles leads to enhanced fluorescence signals in the qualitative coding functional area; 2. Activated nucleic acid self-assembly modules anchored on the surface of the fluorescence coded nanoparticles can activate the fluorescence of near-infrared fluorescent dyes when targets are present, triggering the surface-anchored β-cyclodextrin to mediate the sensitization of the fluorescent dyes through host-guest interaction for quantitative detection.

[0022] The present invention applies β-cyclodextrin with fluorescence sensitization performance to SFENP, and the two functional regions of β-cyclodextrin simultaneously realize fluorescence sensitization. The β-cyclodextrin present in the fluorescence-encoded nanoparticles makes the fluorescence signal of the qualitative coding functional region stronger; the activated nucleic acid self-assembly module anchored on the surface of the fluorescence-encoded nanoparticles can activate Cy5.5 fluorescence when a target exists, triggering the surface-anchored β-cyclodextrin to mediate Cy5.5 fluorescence sensitization through host-guest interaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the construction principle of fluorescent coded nanoparticles in Example 1;

[0024] Figure 2 The experimental principle diagram of the surface-enhanced fluorescence coded nanoprobe for detecting mRNA and protein in Example 1, wherein A is a schematic diagram of the preparation of the surface-enhanced fluorescence coded nanoprobe, and B is a schematic diagram of the surface-enhanced fluorescence coded nanoprobe for detecting multiple mRNAs and proteins;

[0025] Figure 3 It is a fluorescence spectrum of No. 1 fluorescent coded nanoparticle-No. 6 fluorescent coded nanoparticle in Example 1, wherein A is No. 1 fluorescent coded nanoparticle, B is No. 4 fluorescent coded nanoparticle, C is No. 6 fluorescent coded nanoparticle, and D is No. 1 fluorescent coded nanoparticle-No. 6 fluorescent coded nanoparticle;

[0026] Figure 4 It is the fluorescence spectra of the supernatant of No. 1 fluorescent coded nanoparticles, No. 4 fluorescent coded nanoparticles, and No. 6 fluorescent coded nanoparticles in Example 1, wherein A is No. 1 fluorescent coded nanoparticles, B is No. 4 fluorescent coded nanoparticles, and C is No. 6 fluorescent coded nanoparticles;

[0027] Figure 5 The fluorescence spectra of the supernatant of No. 1 fluorescent coded nanoparticles, No. 4 fluorescent coded nanoparticles, and No. 6 fluorescent coded nanoparticles after being placed for different time periods in Example 1, wherein A is No. 1 fluorescent coded nanoparticles, B is No. 4 fluorescent coded nanoparticles, and C is No. 6 fluorescent coded nanoparticles;

[0028] Figure 6The light stability of the fluorescent coded nanoparticle No. 1, the fluorescent coded nanoparticle No. 4, and the fluorescent coded nanoparticle No. 6 in Example 1, wherein A is the fluorescent coded nanoparticle No. 1, B is the fluorescent coded nanoparticle No. 4, and C is the fluorescent coded nanoparticle No. 6;

[0029] Figure 7 The fluorescence spectrum of the fluorescently encoded nanoparticles modified with the anchoring chain in Example 1;

[0030] Figure 8 The fluorescence spectra of FENP1, FENP4 and FENP6 in Example 1, wherein A is FENP1, B is FENP4 and C is FENP6;

[0031] Fig. 9 : are fluorescence spectra of the FENPs in Example 1 in response to target mRNA, wherein A is the fluorescence spectrum of FENP1 to EGFR mRNA, B is the fluorescence spectrum of FENP4 to PD-L1 mRNA, and C is the fluorescence spectrum of FENP6 to VEGF mRNA;

[0032] Fig.10 It is a characterization diagram of the fluorescence sensitization performance of SFENP1, SFENP4 and SFENP6 in Example 1, wherein A is SFENP1, B is SFENP4, and C is SFENP6;

[0033] Fig.11 This is a graph showing the effect of SFENP4 on cell viability in Example 1;

[0034] Fig.12 These are laser confocal images of SFENP1-SFENP6 in Example 1 after incubation with A549 cells respectively;

[0035] Fig.13 The fluorescence intensity and ratio distribution diagram of SFENP1-SFENP6 in A549 cells in Example 1, wherein A is the fluorescence intensity diagram and B is the fluorescence intensity ratio distribution diagram

[0036] Fig.14 This is a laser confocal image of SFENP1-SFENP6 in Example 1 after incubation with A549 cells simultaneously;

[0037] Fig.15 These are laser confocal imaging images of SFENP1 and SFENP2 in Example 1, respectively, detecting changes in EGFR mRNA and EGFR protein expression levels after siRNA interference. DETAILED DESCRIPTION

[0038] Azide-modified β-cyclodextrin (N3-β-cyclodextrin) was purchased from Tokyo Chemical Industry Co., Ltd.

[0039] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0040] The nucleic acids used in the present invention were purchased from Shanghai Sangon Biotechnology Co., Ltd. The nucleic acid sequences are shown in Table 1.

[0041] Table 1 Nucleic acid names and base sequences

[0042]

[0043]

[0044] Example 1

[0045] The preparation method of the surface enhanced fluorescence coded nanoprobe of this embodiment comprises the following steps:

[0046] 1. Preparation of main solution

[0047] 1) Preparation of PBS buffer: Pour the PBS powder bag into a 1000mL beaker, add Milli-Q water, stir with a glass rod until the PBS powder is dissolved, and then transfer to a 2000mL volumetric flask. Rinse the beaker with Milli-Q water several times, and transfer all of it into the volumetric flask, adjust the volume, mix well to obtain PBS buffer, and dispense the obtained PBS buffer into a 1000mL glass bottle sterilized by autoclave. Finally, sterilize the dispensed PBS buffer by autoclave, seal it, and place it in a 4°C refrigerator for later use. The concentration of PBS buffer is 0.01mol / L, and the pH is 7.2-7.4.

[0048] 2) Preparation of hybridization buffer: 11.2114g of tris (hydroxymethyl) aminomethane (Tris), 1.0165g of magnesium chloride hexahydrate (MgCl2·6H2O) and 2.9220g of sodium chloride (NaCl) were placed in a beaker, 450mL of Milli-Q water was added and stirred with a glass rod, then the pH of the solution was adjusted to 7.4 with 0.1mol / L hydrochloric acid (HCl), then transferred to a 500mL volumetric flask, fixed to volume and mixed upside down to obtain the final hybridization buffer. Sterilize under high pressure, seal after leaving at room temperature, and place in a 4°C refrigerator for use. The pH of the hybridization buffer is 7.4.

[0049] 3) 2×10 -3 1 mol / L fluorescein sodium solution: Place 0.2350 g of fluorescein sodium in a beaker, add 200 mL of MilliQ water, stir thoroughly with a glass rod to fully dissolve the fluorescein sodium, then transfer to a 250 mL volumetric flask to make up the volume, mix thoroughly up and down to obtain a fluorescein sodium solution, dispense the obtained fluorescein sodium solution into 50 mL imported sterile centrifuge tubes, seal them, and store them in a 4°C refrigerator for later use.

[0050] 4) 2×10 -3 mol / L Rhodamine B solution: Place 0.1198 g of Rhodamine B in a beaker, add 200 mL of MilliQ water, stir thoroughly with a glass rod to fully dissolve the Rhodamine B, then transfer to a 250 mL volumetric flask to make up the volume, mix up and down to obtain the Rhodamine B solution, dispense the Rhodamine B solution into 50 mL imported sterile centrifuge tubes, seal them, and store them in a 4°C refrigerator for later use.

[0051] 5) Preparation of 0.1 mol / L 2-(4-Morpholino)ethanesulfonic acid (MES) buffer: Weigh 5 g of MES monohydrate and dissolve it in water. Adjust the pH to 5.65, make up to 1000 mL, and place in a refrigerator at 4°C for later use.

[0052] 6) Preparation of nucleic acid chain master solution: centrifuge tubes containing nucleic acid chain dry powder at room temperature at 4000r / min for 1min to make the nucleic acid chain dry powder fall off the tube wall to the bottom, then add the corresponding volume of Milli-Q water or buffer according to the instructions provided by Shanghai Sangon Biotechnology Co., Ltd., and mix on a vortex mixer for 1-2min to obtain a nucleic acid chain master solution with a concentration of 10μmol / L. Finally, transfer the nucleic acid chain master solution to a -20℃ refrigerator for storage, and mark the nucleic acid chain name, date, and user, etc.

[0053] 2. Preparation of Fluorescent Encoded Nanoparticles

[0054] 1) Preparation of fluorescent coded nanoparticles (Flu / RB@β-CD@SiO2): The flask was cleaned with 5% hydrofluoric acid and then with Milli-Q water before use to remove the nucleation sites that may be brought by the flask. The specific steps for preparing fluorescent coded nanoparticles by oil-in-water reverse microemulsion method are as follows: Taking fluorescent coded nanoparticle No. 1 as an example: First, 480 μL of 2×10 -3 mol / L Flu solution and 480 μL of 2×10 -2mol / L β-CD solution was mixed in equal volumes, and after ultrasonication for 5 minutes, Flu@β-CD inclusion complex was formed for use. 7.5mL cyclohexane, 1.6mL n-hexanol and 1.8mL Triton X-100 were added to the flask to obtain a mixed solution, stirred for 5 minutes until the mixed solution was clear, and then 480μL Flu@β-CD inclusion complex was added dropwise as the dispersed phase, stirred for 30 minutes at room temperature in the dark to form a stable oil-in-water microemulsion system, and then 100μL of tetraethyl orthosilicate was added dropwise, stirred for 30 minutes, and then 100μL of ammonia water was added dropwise, and stirred at room temperature for 24 hours to form SiO2 nanoparticles. When the reaction was completed, demulsification was performed with anhydrous ethanol, and then the particles were dispersed in Milli-Q water after centrifugation and washing with anhydrous ethanol and water for several times, and then stored at 4℃ for use.

[0055] 2) The preparation method of No. 2 fluorescent coded nanoparticles-No. 6 fluorescent coded nanoparticles is roughly the same as the preparation method of No. 1 fluorescent coded nanoparticles. The difference between the preparation method of No. 2 fluorescent coded nanoparticles-No. 6 fluorescent coded nanoparticles and the preparation method of No. 1 fluorescent coded nanoparticles is that No. 2 fluorescent coded nanoparticles are prepared by mixing 400 μL of Flu solution with 80 μL of RB solution and then mixing with 480 μL of β-CD solution; No. 3 fluorescent coded nanoparticles are prepared by mixing 320 μL of Flu solution with 160 μL of RB solution was mixed and then mixed with 480 μL of β-CD solution; No. 4 fluorescent coded nanoparticles were mixed with 240 μL of Flu solution and 240 μL of RB solution and then mixed with 480 μL of β-CD solution; No. 5 fluorescent coded nanoparticles were mixed with 80 μL of Flu solution and 400 μL of RB solution and then mixed with 480 μL of β-CD solution; No. 6 fluorescent coded nanoparticles were mixed with 480 μL of RB solution and 480 μL of β-CD solution.

[0056] The construction principle of fluorescent coded nanoparticles is as follows Figure 1As shown, the fluorescent coding nanoparticle carrier is SiO2, which is prepared by the oil-in-water reverse microemulsion method. Flu, RB and β-CD are first ultrasonically treated to form a Flu / RB@β-CD inclusion complex, and then the Flu / RB@β-CD inclusion complex is used as the aqueous phase to synthesize Flu / RB@β-CD@SiO2 nanoparticles by the reverse microemulsion method. By adjusting the ratio of added Flu and RB, fluorescent coding nanoparticles with different codes can be obtained. The molar ratio of Flu solution to RB solution in No. 1 fluorescent coding nanoparticle is 6:0, the molar ratio of Flu solution to RB solution in No. 2 fluorescent coding nanoparticle is 5:1, the molar ratio of Flu solution to RB solution in No. 3 fluorescent coding nanoparticle is 4:2, the molar ratio of Flu solution to RB solution in No. 4 fluorescent coding nanoparticle is 3:3, the molar ratio of Flu solution to RB solution in No. 5 fluorescent coding nanoparticle is 1:5, and the molar ratio of Flu solution to RB solution in No. 6 fluorescent coding nanoparticle is 0:6.

[0057] 3. Construction of surface-enhanced fluorescence coded nanoprobes

[0058] 3.1 Construction principle of surface-enhanced fluorescence coded nanoprobes

[0059] Preparation of surface-enhanced fluorescence coded nanoprobe (SFENP) First, the anchor chain is modified to the surface of Flu / RB@β-CD@SiO2 fluorescence coded nanoparticles through the amidation reaction of carboxyl and amino groups, and then the H1 chain, H2 chain and the recognition chain of tumor biomarkers are self-assembled to the surface of the fluorescence coded nanoparticles through base complementary pairing, thereby preparing the surface-enhanced fluorescence coded nanoprobe. The specific principle is as follows Figure 2 shown.

[0060] 3.2 Determination of the molar concentration of fluorescently encoded nanoparticles

[0061] In order to obtain the molar concentration of the fluorescently encoded nanoparticles, it is necessary to first obtain the density of the light-encoded nanoparticles. The density ρ of the fluorescently encoded nanoparticles is measured by constructing a density gradient using tribromoethane and acetone: first, to obtain a series of density gradients, different volumes of tribromoethane and acetone can be mixed to obtain a mixed solution, and then 2 mg of fluorescently encoded nanoparticles are added to each mixed solution, which is fixed on a horizontal shaker and mixed for 20 minutes, and then allowed to stand at room temperature for 10 minutes to observe the sinking and floating of the fluorescently encoded nanoparticles in each solution. The present invention takes No. 4 fluorescently encoded nanoparticle Flu / RB@β-CD@SiO2 as an example.

[0062] 3.3 Covalent connection of fluorescent coded nanoparticles to anchor chains

[0063] 3.3.1 Preparation of carboxylated fluorescent coded nanoparticles

[0064] 50 μL of tetraethyl orthosilicate (TEOS) and 50 μL of carboxyethylsilanetriol sodium salt (CEOS) were added dropwise to the fluorescent coded nanoparticles, and stirred in the dark for 12 h at room temperature for carboxylation. When the reaction was completed, the emulsification was performed with anhydrous ethanol, and then the particles were washed several times with anhydrous ethanol and water respectively by centrifugation, dispersed in Milli-Q water, and stored at 4°C for later use.

[0065] 3.3.2 Covalent connection reaction process between fluorescent coded nanoparticles and anchor chains

[0066] 1 mg of carboxylated fluorescent coded nanoparticles was taken and washed three times with 0.1 mol / L MES (pH=5.65) buffer, and Flu / RB@β-CD@SiO2 was redispersed in 550 μL MES buffer, and then 6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 6 mg of N-hydroxysulfosuccinimide (Sulfo-NHS) were added respectively, mixed on a vortex mixer, and then 50 μL of 10 μmol / L anchor chain was added, and the mixture was reacted at 25°C on a constant temperature oscillator for 4.5 h; when the reaction was completed, the mixture was washed three times with hybridization buffer to remove the unreacted anchor chain, and the fluorescent coded nanoparticles modified with the anchor chain were obtained after centrifugation. The fluorescent coded nanoparticles modified with the anchor chain were redispersed in 500 μL hybridization buffer to obtain a dispersion of fluorescent coded nanoparticles modified with the anchor chain, which was stored for later use.

[0067] 3.4 Preparation of surface-enhanced fluorescence coded nanoprobes

[0068] 3.4.1 Synthesis of H2 Chain

[0069] The synthesis of H2 chain was carried out by click chemistry reaction of dibenzocyclooctyne (DBCO) and azide (N3). The H chain modified with DBCO was dissolved in 0.1 mol / L triethylamine acetate (pH=7.4) to obtain solution one, and then the azide-modified β-cyclodextrin was dissolved in dimethyl sulfoxide (DMSO) to obtain solution two. 150 μL of each of solution one and solution two were mixed to obtain a suspension, and the formed suspension was placed in a constant temperature oscillator at 25°C for 12 hours. After the reaction was completed, it was fixed to 400 μL with Milli-Q water. Subsequently, it was ultrafiltered and concentrated several times to collect the synthesized H2 chain. Solution two was prepared and used immediately, and the concentration ratio of H chain to azide-modified β-cyclodextrin was 1:5.

[0070] 3.4.2 Preparation process of surface-enhanced fluorescence encoded nanoprobe (SFENP)

[0071] The specific preparation process is described by taking SFENP1 as an example, that is, the fluorescent coded nanoparticles used are fluorescent coded nanoparticles No. 1. Take 1 mg of carboxylated fluorescent coded nanoparticles, wash them three times with 0.1 mol / L MES (pH = 5.65) buffer, and then redisperse them in 500 μL MES buffer, then add 6 mg of EDC and 6 mg of Sulfo-NHS, mix them on a vortex mixer, and then add 50 μL of 10 μmol / L anchor chain, and react on a constant temperature oscillator at 25°C for 4.5 hours; when the reaction is completed, wash it 3 times with hybridization buffer to remove the unreacted anchor chain, and the centrifugal washing product is the fluorescent coded nanoparticles modified with the anchor chain, and the fluorescent coded nanoparticles modified with the anchor chain are redispersed in 500 μL of hybridization buffer; then add 15 μL of 10 μmol / L H1 chain and H2 chain, respectively, and react on a constant temperature oscillator at 37°C for 30 minutes. After the reaction is completed, wash it 3 times with hybridization buffer, and then add 30 μL of 10 μmol / L EGFR The mRNA recognition chain is obtained, and finally SFENP1 is redispersed in 500 μL of hybridization buffer solution for use.

[0072] The preparation methods of SFENP2, SFENP3, SFENP4, SFENP5 and SFENP6 are the same as SFENP1. The recognition chain added to SFENP2 is the EGFR protein recognition chain, and SFENP2 uses No. 2 fluorescent coding nanoparticles; the recognition chain added to SFENP3 is the PD-L1 protein recognition chain, and SFENP3 uses No. 3 fluorescent coding nanoparticles; the recognition chain added to SFENP4 is the PD-L1 mRNA recognition chain, and SFENP4 uses No. 4 fluorescent coding nanoparticles; the recognition chain added to SFENP5 is the VEGF protein recognition chain, and SFENP5 uses No. 5 fluorescent coding nanoparticles; the recognition chain added to SFENP6 is the VEGFmRNA recognition chain, and SFENP6 uses No. 6 fluorescent coding nanoparticles.

[0073] Surface-enhanced fluorescence-encoded nanoprobes for high-contrast imaging of multiple mRNAs and proteins in living single cells

[0074] Based on the principle of fluorescence encoding and β-CD fluorescence sensitization, SFENP was constructed for high-contrast imaging of three mRNAs (EGFR, VEGF, and PD-L1) and three proteins (EGFR, VEGF, and PD-L1) related to lung cancer. SFENP itself can effectively enter cells through endocytosis, avoiding pretreatment processes such as fixation of cells, and can realize imaging analysis of targets in living cells. The principle of SFENP to detect multiple mRNAs and proteins is as follows Figure 2 shown.

[0075] When the surface-enhanced fluorescence encoded nanoprobe recognizes the target, the EGFR mRNA recognition chain (labeled with the fluorescence quencher group BHQ-3) on the surface of SFENP1 specifically recognizes the EGFRmRNA in the cytoplasm through the principle of base complementary pairing. Since the stability of the mRNA / recognition chain complex is greater than the stability of the EGFR mRNA recognition chain's own hairpin, the fluorescence quencher group BHQ-3 labeled on the EGFR mRNA recognition chain is away from the near-infrared fluorescent group Cy5.5 labeled on the H1 chain, so that the fluorescence of Cy5.5 is activated. The EGFR protein recognition chain on the surface of SFENP2 is designed from the EGFR protein aptamer and can specifically recognize the EGFR protein on the cell membrane and in the cytoplasm. The stability of the EGFR protein recognition chain binding to the protein is greater than the stability of the EGFR protein recognition chain's own hairpin, which causes the fluorescence quencher group BHQ-3 labeled on the EGFR protein recognition chain to be away from the near-infrared fluorescent group Cy5.5 labeled on the H1 chain, so that the fluorescence of Cy5.5 is activated. The main consideration for labeling Cy5.5 on the H1 chain is that its excitation and emission wavelengths are both greater than 600nm, which can effectively filter the influence of the spontaneous background fluorescence of the organism, thereby effectively reducing the background signal and improving the imaging contrast and sensitivity during cell imaging. Based on the same principle, the joint detection of SFENP1 on EGFR mRNA, SFENP2 on EGFR protein, SFENP3 on PD-L1 protein, SFENP4 on PD-L1 mRNA, SFENP5 on VEGF protein, and SFENP6 on VEGF mRNA in the same living cell can be achieved, that is, the multi-target joint detection system composed of the SFENP probe series can be used to achieve high-contrast imaging of 3 mRNAs and 3 proteins in living cells.

[0076] The six SFENPs were incubated with A549 cells respectively, and laser confocal images were collected to decode the six targets. The specific operation steps are as follows: Flu bright spots in the 488 channel and the corresponding RB bright spots in the 552 channel of the laser confocal image were collected, and the fluorescence values ​​of the corresponding bright spots were obtained by processing with Image J software, so that the fluorescence intensity range and encoding ratio range corresponding to Flu and RB in the six probes can be obtained, thereby achieving qualitative analysis of the six targets in the cell. Then, the six SFENPs were incubated with A549 cells at the same time, and laser confocal images were collected to decode the six targets. The specific operation steps are as follows: Cy5.5 bright spots in the 638 channel, Flu bright spots in the 488 channel, and RB bright spots in the 552 channel of the laser confocal image were collected, and Cy5.5 bright spots were used as signal points. All bright spots in the Cy5.5 channel were obtained by Image J, and one bright spot was used as a target. The fluorescence intensity value and ratio of the Cy5.5 bright spot in the corresponding Flu and RB channels were analyzed to confirm the type of the target.

[0077] 5. Results

[0078] 5.1 Characterization of Fluorescence-Encoded Nanoparticles

[0079] 5.1.1 Fluorescence spectroscopy characterization

[0080] After synthesizing SiO2 nanoparticles by reverse microemulsion method, the fluorescence spectra of the particles Flu / RB@SiO2 that only encapsulate the dye and the fluorescence coded nanoparticles Flu / RB@β-CD@SiO2 formed by encapsulating the dye are shown in Figure 3 As shown. Figure 3 A shows that compared with Flu / RB@SiO2 (Flu solution / RB solution molar ratio 6:0), the Flu fluorescence in No. 1 Flu / RB@β-CD@SiO2 is enhanced by about 1.3 times; Figure 3 It can be seen from C that compared with Flu / RB@SiO2 (Flu solution / RB solution volume ratio 0:6), the RB fluorescence in No. 6 Flu / RB@β-CD@SiO2 is enhanced by about 1.2 times; Figure 3 As can be seen in B, compared with Flu / RB@SiO2 (Flu / RB molar ratio 3:3), the fluorescence of Flu and RB in No. 4 Flu / RB@β-CD@SiO2 (Flu / RB=3:3) is enhanced.

[0081] The fluorescence spectra of the six prepared Flu / RB@β-CD@SiO2 fluorescent coding nanoparticles are shown in Figure 3D. As the Flu / RB feed ratio decreases, the Flu fluorescence intensity gradually decreases and the RB fluorescence intensity gradually increases. By adjusting the Flu / RB feed ratio, the fluorescence intensity of the 6 prepared Flu / RB@β-CD@SiO2 also changes and can be clearly distinguished, indicating that the 6 prepared Flu / RB@β-CD@SiO2 have coding properties, laying the foundation for the subsequent preparation of surface-enhanced fluorescence coded nanoprobes. No. 4 Flu / RB@β-CD@SiO2 was taken for transmission electron microscopy. No. 4 Flu / RB@β-CD@SiO2 was spherical, uniform in size and well dispersed, with a particle size of about 60nm. The hydrated particle size of the 6 prepared fluorescent coded nanoparticles was measured using a dynamic light scattering nanoparticle size analyzer. The results showed that the hydrated particle size of the 6 fluorescent coded nanoparticles was basically around 90nm.

[0082] 5.1.2 Dye leakage

[0083] After different types of fluorescent coded nanoparticles were placed for 0.5 days, the same volume and concentration of Flu / RB@SiO2 and Flu / RB@β-CD@SiO2 were ultrafiltrated and the fluorescence of the ultrafiltrate was measured. The results are as follows: Figure 4 As shown. The fluorescence intensity of Flu in No. 1 Flu / RB@β-CD@SiO2 filtrate is about 66.65% of that of Flu@SiO2 filtrate, and the fluorescence intensity of RB in No. 1 Flu / RB@β-CD@SiO2 filtrate is about 70.68% of that of RB@SiO2 filtrate; while the fluorescence intensity of Flu in No. 4 Flu / RB@β-CD@SiO2 filtrate is about 88.06% of that of Flu / RB@SiO2 filtrate, and the fluorescence intensity of RB is about 65.59% of that of Flu / RB@SiO2 filtrate. It can be seen that the fluorescent coded nanoparticles formed by β-CD encapsulating the dye can significantly reduce the leakage of dye molecules.

[0084] In order to further investigate the dye leakage of Flu / RB@β-CD@SiO2, different types of Flu / RB@β-CD@SiO2 fluorescent coding nanoparticles were dispersed in PBS. After 1, 3 and 5 days, ultrafiltration centrifugation was performed and the fluorescence of the filtrate was measured. The results are shown in Figure 5 As shown in Figure 5, the fluorescence of the filtrate of the three types of Flu / RB@β-CD@SiO2 fluorescent coding nanoparticles fluctuated slightly from the first day to the fifth day, and the fluorescence spectrum graphs were consistent without obvious changes, indicating that the prepared Flu / RB@β-CD@SiO2 fluorescent coding nanoparticles have good anti-dye leakage performance.

[0085] 5.1.3 Photostability

[0086] Different types of Flu / RB@β-CD@SiO2 were irradiated under a 7W handheld UV lamp for 0, 10, 20, 30, 40, 50 and 60 min, and then the fluorescence values ​​F of the best emission points of different types of Flu / RB@β-CD@SiO2 were measured. The fluorescence value of the best emission point at 0 min was taken as F0, and F / F0 was obtained. The results are shown in Figure 6 As shown in the figure, under ultraviolet light, with the increase of time, the fluorescence intensity of Flu / RB@SiO2 and No. 1 Flu / RB@β-CD@SiO2 and No. 4 Flu / RB@β-CD@SiO2 particles decreased, but it is obvious that the decline trend of Flu / RB@β-CD@SiO2 is lower than that of Flu / RB@SiO2, indicating that Flu / RB@β-CD@SiO2 fluorescent encoding nanoparticles have high photostability. It may be that after β-CD is wrapped around the dye, it can effectively resist ultraviolet light bleaching and enhance its photostability.

[0087] 5.2 Characterization of surface-enhanced fluorescence coded nanoprobes

[0088] 5.2.1 Covalent attachment of fluorescent coded nanoparticles to anchor chains

[0089] 5.2.1.1 Determination of the molar concentration of fluorescent coded nanoparticles

[0090] The concentration of nanoparticles is generally described as mass concentration (mg / mL). The mass of nanoparticles in a liquid can be obtained by vacuum freeze-drying. However, to obtain the molar concentration of nanoparticles, it is necessary to first determine the density ρ of the nanoparticles. Combined with the radius r of the nanoparticles, the mass m of a single nanoparticle can be calculated, and then the number of nanoparticles contained in a certain mass (M) of nanoparticles can be further calculated, and finally the molar concentration of the nanoparticles can be obtained. In view of this, the density ρ of the fluorescent coded nanoparticles is first measured. The density of the mixed liquid is greater than 2.06 g.cm -3 The particles are floating and less than 1.7g.cm -3 When the particle is in a sinking state, and at 1.88g.cm -3 The particles are in a suspended state, so the density of the synthesized fluorescent coded nanoparticles is about 1.88 g.cm -3 According to this density and the radius r obtained by transmission electron microscopy, according to 4 / 3πr 3 ·ρ·N A =m (m: mass of fluorescent encoded nanoparticles, r: radius of fluorescent encoded nanoparticles, N: Avogadro constant, ρ: density of fluorescent encoded nanoparticles), based on which it can be calculated that the molar concentration of 1 mg / mL fluorescent encoded nanoparticles is about 7.8 nmol / L.

[0091] 5.2.1.2 Characterization of covalent attachment of fluorescent coded nanoparticles to anchor chains

[0092] Hoechst33258 fluorescent dye is often used for quantitative detection of double-stranded DNA. Hoechst33258 fluorescent dye can specifically enter the AT base pair minor groove in double-stranded DNA and show a strong fluorescence emission peak at 460nm, while there is no fluorescence signal at 460nm in single-stranded DNA. Based on this, the connection between the fluorescent encoded nanoparticles and the anchor chain was characterized, and the fluorescence emission spectrum of each experimental group at 460nm was scanned with a fluorescence spectrometer. The results are as follows Figure 7 As shown: Hoechst33258 dye solution alone, Hoechst33258 dye added to anchor chain or complementary chain, and Hoechst33258 dye added to fluorescent coding nanoparticles modified with anchor chain all have no obvious fluorescence emission peak at 460nm, but when the fluorescent coding nanoparticles modified with anchor chain are hybridized with complementary chain, Hoechst33258 fluorescent dye is added, and an obvious fluorescence emission peak is presented at about 460nm, indicating that the anchor chain can be successfully covalently modified to the surface of fluorescent coding nanoparticles through the amide reaction between carboxyl and amino groups. The nucleotide sequence of the complementary chain is shown in SEQ ID NO.11.

[0093] 5.2.1.3 Modification amount of anchor chain on fluorescent coded nanoparticles

[0094] The amount of anchor chain modification on the fluorescent coded nanoparticles can be determined by measuring the absorbance (260 nm) of the added complementary chain and the remaining complementary chain in the supernatant. The difference in OD values ​​between the complementary chain in the supernatant and the added complementary chain was measured to be 0.05±0.002. Based on this, it was calculated that 1 mg of carboxylated Flu / RB@β-CD@SiO2 fluorescent coded nanoparticles was connected to 0.2 nmol anchor chain through amide reaction. Combined with the result that the molar concentration of 1 mg / mL fluorescent coded nanoparticles is about 7.8 nmol / L, it was converted to about 26 anchor chains that can be modified on the surface of 1 fluorescent coded nanoparticle.

[0095] 5.2.1.4 Characterization of H2 chain synthesis

[0096] The synthesized H2 chain was characterized by biomass spectrometry. It is known that the theoretical molecular weight of the H2 chain should be 14052.8 Da, while the actual measured molecular weight of the H2 chain was 14052.7 Da, indicating that the β-CD modified H chain has been successfully prepared.

[0097] 5.2.2 Characterization results of surface-enhanced fluorescence coded nanoprobes

[0098] 5.2.2.1 Hydration particle size characterization results

[0099] Compared with Flu / RB@β-CD@SiO2, the hydrated particle size of the six SFENPs increased to about 140 nm, indicating that the nucleic acids had been successfully self-assembled onto the surface of the nanoparticles, thereby producing the six SFENPs.

[0100] 5.2.2.2 Transmission electron microscopy characterization results

[0101] Low-voltage transmission electron microscopy characterization of SFENP4 showed that its particle size increased to about 65 nm, which together with the above-mentioned hydrated particle size results proved that SFENP had been successfully prepared.

[0102] 5.2.2.3 Fluorescence spectroscopy characterization results

[0103] Flu / RB@β-CD@SiO2 No. 1, Flu / RB@β-CD@SiO2 No. 4, and Flu / RB@β-CD@SiO2 No. 6 were selected to gradually verify the synthesis of FENPs. The results are shown in Figure 8 As shown. When the anchor chain is modified onto Flu / RB@β-CD@SiO2, there is no fluorescence emission peak; when the H1 chain hybridizes with the anchor chain, a strong fluorescence emission peak appears, proving that the H1 chain and the anchor chain are successfully hybridized; then the recognition chain of the tumor biomarker hybridizes with the H1 chain, and the quenching group quenches the fluorescence of the fluorescent group on the H1 chain, and the fluorescence emission peak disappears, indicating that the recognition chain of the tumor biomarker is successfully combined with the H1 chain, proving that three types of FENPs have been successfully prepared, namely FENP1 (Flu solution, RB solution molar ratio 6:0), FENP4 (Flu solution, RB solution molar ratio 6:0) and FENP6 (Flu solution, RB solution molar ratio 6:0). The difference between FENP and SFENP is that FENP is not connected to the H2 chain.

[0104] The three FENPs were incubated with their target mRNAs respectively and the fluorescence spectra were scanned. The results were as follows Fig. 9 As shown. When the three FENPs were scanned separately, there was no fluorescence emission peak; when FENPs were combined with the target mRNA, strong fluorescence emission peaks appeared. This proves that the target can open the recognition chain of the tumor biomarker of FENP and restore the fluorescence, proving that FENP can respond to the corresponding mRNA in the solution.

[0105] Flu / RB@β-CD@SiO2 No. 1, Flu / RB@β-CD@SiO2 No. 4, and Flu / RB@β-CD@SiO2 No. 6 were selected to prepare FENPs and SFENPs, respectively, and the corresponding targets were detected and the fluorescence spectra were scanned. The results are shown in Fig.10As shown in the figure, the fluorescence intensity of the three SFENPs was significantly stronger than that of FENP, among which SFENP1 was enhanced by about 1.7 times compared with FENP1, SFENP4 was enhanced by about 1.6 times compared with FENP4, and SFENP6 was enhanced by 1.7 times compared with FENP6. This indicates that the H2 chain enhances the fluorescence of the recognition chain of the tumor biomarker after recognizing the target, proving the successful preparation of SFENP.

[0106] 5.3 Results of Optimization of Response Performance of Surface-Enhanced Fluorescence Encoded Nanoprobes in Solution

[0107] 5.3.1 Optimization of the ratio of H1 chain to H2 chain

[0108] Take No. 1 Flu / RB@β-CD@SiO2 (Flu / RB=6:0), adjust the volume ratio of H1:H2 to 1:1, 1:2 and 1:3 to prepare SFENP1, and detect the fluorescence spectrum when the target is present. The results show that compared with the nanoprobes with only H1 chain added, the fluorescence of the three nanoprobes with H1:H2=1:1, 1:2 and 1:3 are all enhanced, and the fluorescence enhancement effect of H1:H2=1:1 is the most obvious, which is enhanced by about 1.7 times. Therefore, H1:H2=1:1 is selected as the optimal ratio for preparing surface enhanced fluorescence coded nanoprobes.

[0109] 5.3.2 Optimization of fluorescence recovery time

[0110] The results of fluorescence recovery time of different types of SFENPs hybridized with the target showed that the fluorescence intensity of SFENP1 hybridized with EGFR mRNA, SFENP4 hybridized with PD-L1 mRNA and SFENP6 hybridized with VEGF mRNA was the strongest at 30 minutes, and the results of the three SFENPs were consistent, so 30 minutes was selected as the optimal fluorescence recovery time for SFENP detection of the target.

[0111] 5.3.3 Optimization of surface-enhanced fluorescence coded nanoprobe dosage

[0112] Taking SFENP1, SFENP4 and SFENP6 as examples, different concentrations of EGFR, PD-L1 and VEGF mRNA were detected in the solution to optimize the dosage of the three SFENPs. The results showed that the results of SFENP1 and SFENP6 were consistent, and the fluorescence intensity was the strongest when the probe concentration was 100nmol / L; the fluorescence intensity was the strongest when the concentration of SFENP4 was 200nmol / L. Therefore, when detecting the target in the solution, 100nmol / L was used for SFENP1 and SFENP6, and 200nmol / L was used for SFENP4.

[0113] 5.4 Surface-enhanced fluorescence-encoded nanoprobes for high-contrast imaging of multiple mRNAs and proteins in living single cells

[0114] 5.4.1 Observation of A549 cell morphology

[0115] Observation under an inverted microscope showed that A549 cells were growing well, mostly in a spindle shape attached to the bottom of the cell culture flask.

[0116] 5.4.2 Effect of SFENP on A549 cell viability

[0117] The MTT method was used to determine the effect of SFENP on cell viability. Since the six probes have similar compositions except for the different ratios of Flu / RB, No. 4 Flu / RB@β-CD@SiO2 fluorescently encoded nanoparticles and SFENP4 were selected to complete the MTT experiment. The results are shown in Fig.11 After incubating cells with different concentrations of Flu / RB@β-CD@SiO2 and SFENP4 for 24 hours, the cell survival rate was above 80%, which proved that SFENP has good biocompatibility and is conducive to the detection and imaging of multiple mRNAs and proteins in living cells.

[0118] 5.4.3 Coding Capacity of SFENP in Cells

[0119] The six SFENPs were incubated with A549 cells respectively, and the results of laser confocal imaging were shown in Fig.12 As shown. Fig.12 It can be seen that as the Flu / RB feed ratio in SFENP decreases, the Flu fluorescence intensity gradually decreases and the RB fluorescence intensity gradually increases. The Flu and RB fluorescence intensities of the six probes were collected and the corresponding ratios were calculated. The results are shown in Fig.13 As shown in Figure 2, the Flu and RB fluorescence values ​​of the six SFENPs are scattered in different regions, and their ratios can be clearly distinguished, so the six SFENPs can be effectively encoded in A549 cells.

[0120] 5.4.4 Imaging of three mRNAs and three proteins in single cells

[0121] Six SFENPs were incubated with A549 cells simultaneously, and laser confocal imaging of three mRNAs and three proteins in the cells was performed. Fig.14As shown. Fluorescent spots of Flu, RB and Cy5.5 channels were collected respectively, and the fluorescent spots in Cy5.5 channel were taken as the target signal points. Then the laser confocal image was decoded: signal points were selected in the confocal image of Cy5.5 channel using Image J, and one target was considered as one signal point; then the signal points were matched to Flu channel and RB channel, and the fluorescence values ​​and ratios of Flu and RB were obtained, which were matched with the fluorescence values ​​and ratios obtained by incubating the six SFENPs with cells separately, so as to successfully decode and confirm the target type corresponding to the signal point.

[0122] Collect multiple confocal images and decode 100 cells to obtain the expression of the corresponding target in A549 cells. The average copy number of the target mRNA in a single cell is about 36 copies of EGFR mRNA, about 33 copies of PD-L1 mRNA, and about 34 copies of VEGF mRNA. Compared with the results of RT-qPCR detection, the imaging detection results of the expression levels of the three mRNAs are consistent with the trends of the RT-qPCR results. The average number of target proteins in a single cell is about 23 copies of EGFR protein, about 10 copies of PD-L1 protein, and about 18 copies of VEGF protein.

[0123] 5.4.5SFENP monitoring of EGFR mRNA and protein expression changes after siRNA interference

[0124] SFENP monitored the changes in EGFR mRNA and protein expression in single cells after siRNA interference. Fig.15 The signal of Cy5.5 is used as the detection signal of the target. Fig.15 It can be seen that at 0h after siRNA interference in the control group, both EGFR mRNA and EGFR protein were significantly expressed, and there were a large number of Cy5.5 signals in the field of view; after 24h of siRNA interference targeting EGFR, there was almost no Cy5.5 signal in the field of view, indicating that EGFR mRNA was effectively inhibited; and there were still a large number of Cy5.5 signals in the field of view of EGFR protein detection, indicating that there was still a lot of EGFR protein expression at this time. After 48h of siRNA interference targeting EGFR, there was almost no Cy5.5 signal in the field of view, indicating that both EGFR mRNA and protein were effectively inhibited at this time. The above results show that SFENP can be used to monitor changes in mRNA and protein expression in single cells.

Claims

1. A method for preparing a surface-enhanced fluorescence coded nanoprobe, characterized in that: The following steps are involved: The anchor chain is modified to the surface of the fluorescent coding nanoparticle by amidation reaction of carboxyl and amino groups to obtain fluorescent coding nanoparticles modified with the anchor chain, and then the H1 chain, H2 chain and the recognition chain of the tumor biomarker are self-assembled to the surface of the fluorescent coding nanoparticle modified with the anchor chain by base complementary pairing, so as to obtain; the recognition chain of the tumor biomarker includes EGFRmRNA recognition chain, EGFR protein recognition chain, PD-L1 mRNA recognition chain, PD-L1 protein recognition chain, VEGF mRNA recognition chain, VEGF protein recognition chain, the fluorescent coding nanoparticle is coated with a dye by β-cyclodextrin, and the dye is one or more of fluorescein sodium solution and rhodamine B solution. The dye is divided into six types: A dye, B dye, C dye, D dye, E dye and F dye. The content of fluorescein sodium solution in dye A gradually decreases, and the content of rhodamine B solution gradually increases; EGFR mRNA recognition chain, EGFR protein recognition chain, PD-L1 mRNA recognition chain, PD-L1 protein recognition chain, VEGF The mRNA recognition chain and the VEGF protein recognition chain are randomly added to one of the A dye and the F dye.

2. The method for preparing the surface-enhanced fluorescence coded nanoprobe according to claim 1, characterized in that: The nucleotide sequence of the H1 chain is shown in SEQ ID NO.1, the nucleotide sequence of the H2 chain is shown in SEQ ID NO.2, and the nucleotide sequence of the anchor chain is shown in SEQ ID NO.3; the nucleotide sequence of the EGFR mRNA recognition chain is shown in SEQ ID NO.4, the nucleotide sequence of the EGFR protein recognition chain is shown in SEQ ID NO.5, the nucleotide sequence of the PD-L1 mRNA recognition chain is shown in SEQ ID NO.6, the nucleotide sequence of the PD-L1 protein recognition chain is shown in SEQ ID NO.7, the nucleotide sequence of the VEGF mRNA recognition chain is shown in SEQ ID NO.8, and the nucleotide sequence of the VEGF protein recognition chain is shown in SEQ ID NO.

9.

3. The method for preparing the surface-enhanced fluorescence coded nanoprobe according to claim 1, characterized in that: The A dye is a sodium fluorescein solution, the F dye is a rhodamine B solution, and the molar ratios of the sodium fluorescein solution to the rhodamine B solution in the B dye, C dye, D dye, and E dye are 5:1, 4:2, 3:3, and 1:5, respectively; the A dye corresponds to the EGFRmRNA recognition chain, the B dye corresponds to the EGFR protein recognition chain, the C dye corresponds to the PD-L1 protein recognition chain, the D dye corresponds to the PD-L1 mRNA recognition chain, the E dye corresponds to the VEGF protein recognition chain, and the F dye corresponds to the VEGF mRNA recognition chain.

4. The method for preparing the surface-enhanced fluorescence coded nanoprobe according to claim 1, characterized in that: The following steps are involved: The fluorescent coded nanoparticles are carboxylated to obtain carboxylated fluorescent coded nanoparticles, the carboxylated fluorescent coded nanoparticles are dispersed in 2-morpholineethanesulfonic acid buffer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysulfosuccinimide, and anchor chains are added to react at 20-30° C. for 4-5 hours to obtain fluorescent coded nanoparticles modified with anchor chains, the fluorescent coded nanoparticles modified with anchor chains are dispersed in hybridization buffer, the H1 chain and the H2 chain are added to react at 30-40° C. for 20-40 minutes, and then the recognition chain of the tumor biomarker is added and dispersed in the hybridization buffer solution to obtain.

5. The method for preparing the surface-enhanced fluorescence coded nanoprobe according to claim 4, characterized in that: The mass ratio of the carboxylated fluorescent coding nanoparticles to the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:6, the mass ratio of the carboxylated fluorescent coding nanoparticles to the N-hydroxysulfosuccinimide is 1:6, 40-60 μL of anchor chain is added per mg of the carboxylated fluorescent coding nanoparticles, 10-20 μL of the H1 chain is added per mg of the carboxylated fluorescent coding nanoparticles, and 10-20 μL of the H2 chain is added per mg of the carboxylated fluorescent coding nanoparticles. , 20-40 μL of the tumor biomarker recognition chain is added to each mg of the carboxylated fluorescent coded nanoparticles; the concentration of the anchor chain is 9-11 μmol / L, the concentration of the H1 chain is 9-11 μmol / L, the concentration of the H2 chain is 9-11 μmol / L, and the concentration of the tumor biomarker recognition chain is 9-11 μmol / L; the carboxylated fluorescent coded nanoparticles are prepared by carboxylating the fluorescent coded nanoparticles using tetraethyl orthosilicate and carboxyethylsilanetriol sodium salt.

6. The method for preparing the surface-enhanced fluorescence coded nanoprobe according to claim 5, characterized in that: The preparation method of the H2 chain is as follows: dissolving the H chain modified with dibenzocyclooctyne in triethylammonium acetate to obtain solution one, dissolving azide-modified β-cyclodextrin in dimethyl sulfoxide to obtain solution two, mixing equal volumes of solution one and solution two, reacting at 20-30° C. for 10-14 hours, then fixing the volume and concentrating to obtain the H2 chain; the nucleotide sequence of the H chain is shown in SEQ ID NO.10, and the concentration ratio of the H chain to azide-modified β-cyclodextrin is 1:

5.

7. The method for preparing the surface-enhanced fluorescence coded nanoprobe according to claim 1, characterized in that: The preparation method of the fluorescent coded nanoparticles is as follows: one of dye A, dye B, dye C, dye D, dye E, and dye F is mixed with a β-cyclodextrin solution in equal volumes, and then subjected to ultrasonication to obtain an inclusion compound for later use; cyclohexane, n-hexanol, and Triton X-100 are mixed to obtain a mixed solution, the inclusion compound is added dropwise to the mixed solution, and the mixture is reacted in the dark at room temperature for 20-40 minutes; then, ethyl orthosilicate is added dropwise, stirred for 20-40 minutes, and then ammonia water is added dropwise, and stirred at room temperature for 24-30 hours to obtain the obtained nanoparticles.

8. The method for preparing the surface-enhanced fluorescence coded nanoprobe according to claim 7, characterized in that: The concentration of the β-cyclodextrin solution is 1×10 -2 -3×10 -2 mol / L, the volume ratio of the β-cyclodextrin solution to the cyclohexane is 4:125, the volume ratio of the β-cyclodextrin solution to the n-hexanol is 3:20, the volume ratio of the β-cyclodextrin solution to Triton X-100 is 2:15, the volume ratio of the β-cyclodextrin solution to the tetraethyl orthosilicate is 12:5, the volume ratio of the β-cyclodextrin solution to the ammonia water is 12:5, and the volume ratio of the β-cyclodextrin solution to the dye is 1:

1.

9. A surface-enhanced fluorescence coded nanoprobe, characterized in that: The surface enhanced fluorescence coded nanoprobe is prepared by the preparation method of any one of claims 1 to 8.

10. Use of the surface-enhanced fluorescence encoded nanoprobe as claimed in claim 9 for simultaneous in situ detection of EGFR mRNA, EGFR protein, PD-L1 mRNA, PD-L1 protein, VEGF mRNA, VEGF protein and imaging in a single living cell.

Images