Activated supramolecular fluorescent nanoprobe system as well as preparation method and application thereof

Abstract

The invention relates to an activated supramolecular fluorescent nanoprobe system as well as a preparation method and application thereof. The activated type supramolecular fluorescent nanoprobe system comprises a universal activated type supramolecular fluorescent nanoprobe and a specific activated type supramolecular fluorescent nanoprobe, and the universal activated type supramolecular fluorescent nanoprobe comprises a universal molecular beacon and covalently modified beta-cyclodextrin, a nucleotide sequence of the general molecular beacon is designed according to a non-specific sequence of 16S rRNA in bacteria, and the general molecular beacon is modified with a fluorophore I; the specific activated supramolecular fluorescent nanoprobe comprises a specific molecular beacon and covalently modified beta-cyclodextrin, the nucleotide sequence of the specific molecular beacon is designed according to the specific sequence of 16S rRNA of staphylococcus aureus, and the specific molecular beacon is modified with a fluorophore II. Compared with a monochromatic fluorescence signal, the detection accuracy can be improved, and a false positive signal caused by interference factors in a complex environment can be effectively avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and specifically relates to an activated supramolecular fluorescent nanoprobe system, a preparation method and an application thereof. Background Art

[0002] According to existing reports, in addition to the well-known genetic, epigenetic and stromal microenvironmental factors, the host bacterial community, as an important component of the body, is an important mediator of cancer susceptibility and tumor progression, which mainly acts in distal or proximal tumor tissues through indirect pathways (including metabolites and the immune system). Clinical cohort studies have shown that the characteristics of tumor-resident bacteria have a profound impact on cancer risk, pathological type, cancer prognosis and treatment response. In recent years, more and more evidence has shown that many types of cancer (such as colorectal cancer, pancreatic cancer, lung cancer, breast cancer, etc.) contain different bacteria, and these bacteria are mostly in tumor cells and immune cells, and are intracellular bacteria. Among them, the rich and diverse bacteria in breast cancer tumors play an important role in the metastasis of breast cancer.

[0003] In the "2022 Global Latest Cancer Burden Data", the global incidence of breast cancer ranks second. And among the global female cancer patient population, the incidence and mortality of breast cancer rank first, and it is the main cause of cancer death in women. It is well known that breast cancer tumors are prone to metastasis, and about 90% of cancer-related deaths are caused by cancer metastasis. Intracellular bacteria resident in breast cancer tumors, especially intracellular Staphylococcus aureus (referred to as Staphylococcus aureus), which accounts for a high proportion in the breast cancer tumor microbiome, play an important role in the metastasis of breast cancer tumor cells. Since most chemotherapy drugs on the market often target tumor cell growth rather than tumor cell metastasis, intracellular bacteria in tumor tissues will become potential targets for early prevention of metastasis of multiple cancers. However, tumor-resident intracellular bacteria have the characteristics of low biomass. Therefore, it is urgent to develop a co-localization imaging method with strong specificity, high sensitivity, and not easily interfered by the tissue microenvironment for the detection of tumor-resident intracellular bacteria, which effectively promotes the development of new methods for early warning of breast cancer metastasis.

[0004] At present, traditional bacterial identification methods (such as bacterial culture and colony counting) consume a lot of manpower and time, usually requiring long-term culture and screening, and reliable results can only be obtained after several weeks, so they are not suitable for rapid diagnosis. Smear staining can be used to identify Gram-positive or Gram-negative bacteria, and is more suitable for the detection of some bacteria with more special morphology, but it often requires observation and judgment by inspectors, which is somewhat subjective. Serological testing is mainly based on immune response for auxiliary diagnosis, which requires the preparation of antigens and the processing of a large number of serum samples, and due to differences in immune responses among different populations, false negative or false positive results may occur. Quantitative polymerase chain reaction (qPCR) is mostly used for quantitative analysis of bacterial nucleic acids and has high sensitivity, but due to the low biomass of intracellular bacteria in tumor tissues, more biological samples need to be processed to obtain the nucleic acid of the target intracellular bacteria, and in situ detection cannot be achieved. Fluorescence in situ hybridization (FISH) can detect tumor-resident intracellular bacteria in situ, but it often uses the "always on" signal mode, so when used for imaging analysis, it has the disadvantage of low signal-to-noise ratio, and needs to be washed to remove unbound or weakly bound signal probes, which prolongs the diagnosis time. Although molecular beacon technology can reduce the fluorescence background value during bacterial imaging analysis and improve the signal-to-noise ratio to a certain extent, it has the disadvantage of low fluorescence signal in complex biological environments such as cells or tissues, which affects the sensitivity of bacterial detection. Summary of the invention

[0005] The first objective of the present invention is to provide an activated supramolecular fluorescent nanoprobe system to solve the technical problems that the detection of intracellular bacteria in tumor tissues takes a long time and is susceptible to interference factors in a complex environment to generate false positive signals.

[0006] The second object of the present invention is to provide a method for preparing an activated supramolecular fluorescent nanoprobe system.

[0007] The third object of the present invention is to provide an application of an activated supramolecular fluorescent nanoprobe system.

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

[0009] An activated supramolecular fluorescent nanoprobe system, wherein the activated supramolecular fluorescent nanoprobe comprises a universal activated supramolecular fluorescent nanoprobe and a specific activated supramolecular fluorescent nanoprobe, wherein the universal activated supramolecular fluorescent nanoprobe comprises a universal molecular beacon and covalently modified β-cyclodextrin, wherein the nucleotide sequence of the universal molecular beacon is designed according to the non-specific sequence of 16S rRNA in bacteria, and the universal molecular beacon is modified with a first fluorescent group; wherein the specific activated supramolecular fluorescent nanoprobe comprises a specific molecular beacon and covalently modified β-cyclodextrin, wherein the nucleotide sequence of the specific molecular beacon is designed according to the specific sequence of 16S rRNA of Staphylococcus aureus, and the specific molecular beacon is modified with a second fluorescent group, and the fluorescent colors of the first fluorescent group and the second fluorescent group are different.

[0010] Furthermore, the nucleotide sequence of the universal molecular beacon is shown as SEQ ID NO.1, and the nucleotide sequence of the specific molecular beacon is shown as SEQ ID NO.2.

[0011] Furthermore, the first fluorescent group is a 6-FAM fluorescent group, and the second fluorescent group is a Cy3 fluorescent group.

[0012] A method for preparing an activated supramolecular fluorescent nanoprobe system comprises the following steps:

[0013] S1: A universal molecular beacon is designed based on the non-specific sequence of 16S rRNA in bacteria. The universal molecular beacon undergoes a dehydration condensation reaction with DBCO-PEG-NHS to form an intermediate product 1. The intermediate product 1 undergoes a click chemistry reaction with azide-modified β-cyclodextrin to obtain a universal activated supramolecular fluorescent nanoprobe.

[0014] S2: Design a specific molecular beacon based on the specific sequence of 16S rRNA in Staphylococcus aureus, and react the specific molecular beacon with DBCO-PEG-NHS to form an intermediate product 2, and the intermediate product 2 reacts with azide-modified β-cyclodextrin to produce a specific activated supramolecular fluorescent nanoprobe by click chemistry;

[0015] S3: Mixing the universal activated supramolecular fluorescent nanoprobe with the specific activated supramolecular fluorescent nanoprobe to obtain.

[0016] Furthermore, the preparation method of the universal activated supramolecular fluorescent nanoprobe comprises the following steps: mixing a universal molecular beacon nucleic acid chain solution with a DBCO-PEG-NHS solution for overnight reaction, purifying to obtain a CMB-PEG-DBCO solution, mixing an equal volume of the CMB-PEG-DBCO solution with an azide-modified β-cyclodextrin solution, reacting in the dark overnight, and purifying to obtain the solution; the preparation method of the specific activated supramolecular fluorescent nanoprobe comprises the following steps: mixing a specific molecular beacon nucleic acid chain solution with a DBCO-PEG-NHS solution for overnight reaction, purifying to obtain a SMB-PEG-DBCO solution; mixing an equal volume of the SMB-PEG-DBCO solution with an azide-modified β-cyclodextrin solution, reacting in the dark overnight, and purifying to obtain the solution; the DBCO-PEG-NHS solution is obtained by dissolving DBCO-PEG-NHS in a mixed solvent, and the azide-modified β-cyclodextrin solution is obtained by dissolving azide-modified β-cyclodextrin in dimethyl sulfoxide.

[0017] Furthermore, the concentration of the universal molecular beacon nucleic acid chain solution is 50-200 μmol / L, the concentration of the specific molecular beacon nucleic acid chain solution is 50-200 μmol / L, the concentration of the DBCO-PEG-NHS solution is 20-50 mmol / L, and the concentration of the azide-modified β-cyclodextrin solution is 400-600 μmol / L.

[0018] Furthermore, the mixed solvent is a mixed solution of dimethylformamide and acetonitrile, and the volume ratio of the dimethylformamide to the acetonitrile is 1:1.

[0019] Furthermore, the volume ratio of the universal molecular beacon nucleic acid chain solution to the DBCO-PEG-NHS solution is 1:1, and the volume ratio of the specific molecular beacon nucleic acid chain solution to the DBCO-PEG-NHS solution is 1:1.

[0020] Furthermore, the preparation method of the universal molecular beacon nucleic acid chain solution is to dissolve the universal molecular beacon in a sodium carbonate buffer solution; the preparation method of the specific molecular beacon nucleic acid chain solution is to dissolve the specific molecular beacon in a sodium carbonate buffer solution; the concentration of the sodium carbonate buffer solution is 0.1-0.2 mol / L, and the pH of the sodium carbonate buffer solution is 8.0-9.0.

[0021] Application of an activated supramolecular fluorescent nanoprobe system for co-localization imaging of intracellular Staphylococcus aureus.

[0022] Beneficial effects of the present invention:

[0023] Compared with monochromatic fluorescent signals, the present invention uses colocalization signals (the superimposed color of green and red is yellow) to detect Staphylococcus aureus, which can improve the accuracy of detection because the colocalization signals can effectively avoid false positive signals caused by interfering factors (such as enzyme cutting, etc.) in a complex environment.

[0024] The present invention uses Staphylococcus aureus as a model bacterium and 16S rRNA contained in Staphylococcus aureus as a research object, and uses the fluorescence "activation" performance of molecular beacons and the fluorescence sensitization of cyclodextrin to construct an activated supramolecular fluorescent nanoprobe. The probe system is composed of CMB-PEG-β-CD targeting bacteria and SMB-PEG-β-CD specifically targeting Staphylococcus aureus, and has the advantages of strong specificity, high sensitivity, and not easily disturbed by tissue microenvironment.

[0025] The present invention constructs an MCF-7 intracellular Staphylococcus aureus model and an MCF-7 breast cancer mouse transplant tumor model with intracellular Staphylococcus aureus, and verifies the application of the constructed universal activated supramolecular fluorescent nanoprobe and specific activated supramolecular fluorescent nanoprobe to co-localized imaging detection of tumor-resident intracellular Staphylococcus aureus at the cellular and tissue levels, which is expected to provide important support for early warning of breast cancer metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The schematic diagram shows the construction of the activated supramolecular fluorescent nanoprobe system and its application in the detection of tumor-resident intracellular bacteria.

[0027] Figure 2 Schematic diagram of the synthesis route of CMB-PEG-DBCO;

[0028] Figure 3 Schematic diagram of the synthesis route of CMB-PEG-β-CD;

[0029] Figure 4 Schematic diagram of the synthesis route of SMB-PEG-DBCO;

[0030] Figure 5 Schematic diagram of the synthesis route of SMB-PEG-β-CD;

[0031] Figure 6 The fluorescence spectra of CMB and SMB after hybridization with the corresponding complementary chains C and S and then incubated with α-CD, β-CD and γ-CD, respectively, where a is the CMB-PEG-β-CD probe and b is the SMB-PEG-β-CD probe;

[0032] Figure 7The polyacrylamide gel electrophoresis characterization diagram of CMB-PEG-β-CD probe and SMB-PEG-β-CD probe, wherein (1) Marker, (2) CMB, (3) CMB-PEG-β-CD, (4) CMB + complementary chain C, (5) CMB-PEG-β-CD + complementary chain C, (6) SMB, (7) SMB-PEG-β-CD, (8) SMB + complementary chain S, (9) SMB-PEG-β-CD + complementary chain S;

[0033] Figure 8 The fluorescence intensity diagrams of CMB-PEG-β-CD probe, SMB-PEG-β-CD probe and corresponding complementary chain C and complementary chain S incubated for different time periods, where a is the CMB-PEG-β-CD probe and b is the SMB-PEG-β-CD probe;

[0034] Fig. 9 The CMB-PEG-β-CD probe and SMB-PEG-β-CD probe were exposed to different concentrations of Mg 2+ Fluorescence intensity diagram of hybridization with the corresponding complementary chain C and complementary chain S in a buffer solution, where a is the CMB-PEG-β-CD probe and b is the SMB-PEG-β-CD probe;

[0035] Fig.10 In vitro fluorescence spectra of CMB-PEG-β-CD probe and SMB-PEG-β-CD probe, where a is CMB-PEG-β-CD probe and b is SMB-PEG-β-CD probe;

[0036] Fig.11 This is a Gram staining microscopic examination of Staphylococcus aureus;

[0037] Fig.12 This is the laser confocal imaging of Staphylococcus aureus in MCF-7 cells using the activated supramolecular fluorescent nanoprobe system;

[0038] Fig.13 This is a laser confocal imaging image of intracellular Staphylococcus aureus in the transplanted tumor tissue of MCF-7 breast cancer mice using an activated supramolecular fluorescent nanoprobe system;

[0039] Fig.14 are bacterial morphology diagrams, where a is the bacterial morphology diagram of Staphylococcus aureus, and b is the result diagram of Staphylococcus aureus culture and identification on Columbia blood agar plate;

[0040] Fig.15 These are laser confocal microscopy images of bacteria, where a is Staphylococcus aureus and b is Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0041] The structure of DBCO-PEG-NHS is shown in formula (1):

[0042]

[0043] The concentrations of the universal activated supramolecular fluorescent nanoprobe and the specific activated supramolecular fluorescent nanoprobe were both 200 nmol / L, and the concentration ratio of the universal activated supramolecular fluorescent nanoprobe and the specific activated supramolecular fluorescent nanoprobe in the activated supramolecular fluorescent nanoprobe system was 1: 1. Azide-modified β-cyclodextrin (N3-β-CD) was purchased from Tokyo Chemical Industry Co., Ltd.

[0044] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0045] The nucleic acids used in the present invention were purchased from Shanghai Shenggong Biological Co., Ltd. The sequence table of the nucleic acids used in the present invention is shown in Table 1.

[0046] Table 1 Nucleic acid names and base sequences

[0047]

[0048] Example 1

[0049] The preparation method of the activated supramolecular fluorescent nanoprobe system of this embodiment comprises the following steps:

[0050] 1. Solution preparation

[0051] 1) Preparation of 1× PBS buffer solution (0.01 mol / L, pH 7.4): Take 10× PBS buffer solution (pH about 7.2-7.4), dilute it 10 times with ultrapure water (Milli-Q water), make up to volume with a 1000 mL volumetric flask, invert it upside down to mix it thoroughly, and store it in a 4°C refrigerator after high-pressure sterilization for later use.

[0052] 2) Preparation of hybridization buffer solution 1 (pH 7.4): 2.4228 g of tris (hydroxymethyl) aminomethane (Tris), 5.8440 g of sodium chloride (NaCl) and 0.4066 g of magnesium chloride hexahydrate (MgCl2·6H2O) were fully dissolved in a beaker containing 950 mL of Milli-Q water, and the pH was adjusted to 7.4 using a pH meter. The solution was then transferred to a 1000 mL volumetric flask and mixed thoroughly by inverting. After high pressure sterilization, the solution was sealed and placed in a refrigerator at 4°C for later use. The final concentration of Tris was 20 mmol / L, the final concentration of NaCl was 0.1 mol / L, and the final concentration of MgCl2·6H2O was 2 mmol / L.

[0053] 3) Preparation of hybridization buffer solution 2: Dissolve 1.0000g of bovine serum albumin (BSA), 5.5000g of glucose, and 0.4066g of MgCl2·6H2O in the prepared 1×PBS buffer solution. After fully dissolved, transfer to a 1000mL volumetric flask, then dilute to volume with 1×PBS buffer, mix thoroughly by inverting, and then store in a -20℃ refrigerator for later use. Take out and place in a 4℃ refrigerator to melt before use. The final concentrations of BSA, glucose, and MgCl2·6H2O are 1g / L, 25mmol / L, and 2mmol / L, respectively.

[0054] 4) Preparation of 10×TBE buffer: Dissolve the bagged 10×TBE powder in a beaker filled with Milli-Q water, stir with a magnetic stirrer until completely dissolved, then transfer to a 2000 mL volumetric flask, rinse the beaker 3 times with an appropriate amount of Milli-Q water and transfer to the volumetric flask, make up to volume with Milli-Q water, and mix thoroughly by inverting, then dispense into autoclaved glass bottles, seal and place in a 4°C refrigerator for later use.

[0055] 5) Preparation of 1×TBE buffer: Use a clean measuring cylinder to measure 50 mL of 10×TBE buffer and transfer it to a 500 mL volumetric flask. Repeat the washing of the measuring cylinder with Milli-Q water for 3 times and transfer it to the volumetric flask. Then, dilute to volume with Milli-Q water and mix thoroughly by inverting the flask. Finally, dilute to 1×TBE buffer.

[0056] 6) 0.1 mol / L sodium carbonate buffer (pH 8.2): Dissolve 5.2995 g of sodium carbonate (Na2CO3) in a beaker containing 450 mL of Milli-Q water, adjust the pH to 8.2 using a pH meter, then transfer it to a 500 mL volumetric flask and dilute to volume with Milli-Q water, invert it to mix thoroughly. After high-pressure sterilization, dispense it into 50 mL centrifuge tubes, seal and label them, and place them in a 4°C refrigerator for later use.

[0057] 7) TSB liquid culture medium: Dissolve 3.0 g of TSB powder in 100 mL of Milli-Q water, sterilize by high pressure at 121°C for 20 min, and store in a refrigerator at 4°C after sterilization.

[0058] 8) TSB solid culture medium: Dissolve 3.0 g of TSB powder and 1.5 g of agar powder in 100 mL of Milli-Q water, sterilize under high pressure at 121°C for 20 min, wait until the temperature drops to about 55°C, invert the plate in a biosafety cabinet, irradiate with UV light for 30 min, and after solidification, store it upside down in a refrigerator at 4°C.

[0059] 9) Preparation of lysozyme solution: weigh 0.1 g of lysozyme dry powder, add 1 mL of lysozyme buffer, and oscillate to mix. Lysozyme needs to be prepared before use.

[0060] 2. Principle

[0061] The experimental principle of the construction and application of activated supramolecular fluorescent nanoprobes is as follows Figure 1 As shown. The CMB in the universal activated supramolecular fluorescent nanoprobe (CMB-PEG-β-CD, pseudo color is green) is modified with a 6-FAM fluorescent group at the 5' end, a Dabcyl quenching group at the 3' end, and an amino group at the stem. The nucleic acid sequence of CMB is designed based on the non-specific sequence of 16SrRNA in bacteria, which is convenient for it to identify bacteria; the CMB nucleic acid sequence is shown in SEQ ID NO.1. The SMB in the specific activated supramolecular fluorescent nanoprobe (SMB-PEG-β-CD, pseudo color is red) is modified with a Cy3 fluorescent group at the 5' end, a BHQ-2 quenching group at the 3' end, and an amino group at the stem. The nucleic acid sequence of SMB is designed based on the specific sequence of 16S rRNA in Staphylococcus aureus, which can target and identify Staphylococcus aureus. The SMB nucleic acid sequence is shown in SEQ ID NO.2. CMB and SMB undergo dehydration condensation with DBCO-PEG-NHS to form amide bonds, respectively, to obtain intermediate products CMB-PEG-DBCO and SMB-PEG-DBCO; the intermediate products CMB-PEG-DBCO and SMB-PEG-DBCO further undergo click chemistry reaction with azide-modified β-cyclodextrin (N3-β-CD) to successfully prepare universal activated supramolecular fluorescent nanoprobes (CMB-PEG-β-CD) and specific activated supramolecular fluorescent nanoprobes (SMB-PEG-β-CD).

[0062] After CMB-PEG-β-CD and SMB-PEG-β-CD were incubated with Staphylococcus aureus, the presence of the target nucleic acid sequence in 16S rRNA activated the fluorescence signal. At the same time, due to the sensitization effect of cyclodextrin on the fluorescent group, the fluorescence signal can be further enhanced. The co-localization imaging detection of intracellular bacteria can be achieved by laser confocal imaging. Subsequently, Staphylococcus aureus was used to invade human breast cancer cells (MCF-7 cells) to construct the MCF-7 intracellular Staphylococcus aureus model and the MCF-7 breast cancer mouse transplant tumor model with intracellular Staphylococcus aureus. The activated fluorescent nanoprobe system was incubated with the intracellular bacteria model and the intracellular bacteria tissue sections obtained from the transplant tumor model. When Staphylococcus aureus was present, the non-specific sequence in 16S rRNA could activate CMB-PEG-β-CD to produce bright green fluorescence, and the specific sequence in 16S rRNA could activate SMB-PEG-β-CD to produce bright red fluorescence. Compared with monochromatic fluorescence signals, the use of colocalization signals (the superimposed color of green and red is yellow) to detect Staphylococcus aureus can improve the accuracy of detection. The reason is that colocalization signals can effectively avoid false positive signals caused by interfering factors (such as enzyme cutting, etc.) in complex environments.

[0063] 3. Preparation

[0064] 3.1 Design of CMB and SMB nucleic acid chains

[0065] The nucleic acid sequence of CMB was designed according to the non-specific sequence of 16S rRNA in bacteria, and the nucleic acid sequence of SMB was designed according to the specific sequence of 16S rRNA in Staphylococcus aureus. At the same time, the melting temperature (Tm) of the nucleic acid sequence should meet the following conditions: ① The Tm value of CMB and SMB itself should be greater than 50°C; ② The Tm value of CMB reacting with the corresponding complementary chain C should be greater than its own Tm value, and the Tm value of SMB reacting with the corresponding complementary chain S should be greater than its own Tm value. The nucleotide sequence of complementary chain C is shown in SEQ ID NO.3, and the nucleotide sequence of complementary chain S is shown in SEQ ID NO.4. In order to meet the above conditions, CMB and SMB were designed by bioinformatics software (http: / / www.unafold.org, https: / / nupack.org / ) and their hybridization with complementary chain C and complementary chain S was simulated. The Tm values ​​of the nucleic acid sequences of CMB and SMB are shown in Table 2.

[0066] Table 2 Tm values ​​of nucleic acid sequences

[0067]

[0068] 3.2 Screening of main compound cyclodextrin

[0069] In order to select the appropriate main compound cyclodextrin, the designed CMB and SMB (concentration of 1 μmol / L) were incubated with α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) in a constant temperature metal bath at 37°C, 500 r / min and light-proof conditions for 3 hours; α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin were set at two concentrations, 0.5 mmol / L and 1.0 mmol / L respectively. The fluorescence spectrometer was used for measurement, and the optimal excitation wavelength (λex) of CMB was 495 nm, and the detection band was 505 nm-560 nm; the optimal excitation wavelength (λex) of SMB was 545 nm, and the detection band was 555 nm-610 nm, and the slit was adjusted to 1.5 nm. The results were analyzed and the optimal main compound cyclodextrin was selected.

[0070] 3.3 Synthesis and purification of CMB-PEG-β-CD

[0071] Synthesis of CMB-PEG-DBCO: Dissolve 3OD of CMB in 109.5μL of 0.1mol / L sodium carbonate buffer (pH8.2) to obtain a CMB nucleic acid chain solution with a concentration of 100μmol / L. Take 3mg DBCO-PEG-NHS and dissolve it in 50μL of dimethylformamide (DMF) and 50μL of acetonitrile mixed solvent to obtain a DBCO-PEG-NHS solution with a concentration of 29.9mmol / L. Add 100μL of CMB nucleic acid chain solution to a 1.5mL ep tube sterilized by high pressure, and mix it with 100μL of DBCO-PEG-NHS ester solution in equal volumes. The resulting suspension is placed in a constant temperature metal bath in the dark at 25℃ and 700r / min, and reacted overnight to obtain the initial product of CMB-PEG-DBCO.

[0072] Separation and purification of CMB-PEG-DBCO: The CMB-PEG-DBCO initial product was diluted to 2 mL using high pressure Milli-Q water and filtered using an Amicon-3K ultrafiltration tube ( The diluted CMB-PEG-DBCO initial product was centrifuged and ultrafiltered for 30 min (6500×g, 4°C) using Ultra 4 mL centrifugal ultrafiltration tubes, and washed three times with high pressure Milli-Q water; transferred to Amicon-3K ultrafiltration tubes ( Ultra 0.5mL centrifugal ultrafiltration tube) was centrifuged for 30 min (12000×g, 4°C) for further concentration, and the centrifugal ultrafiltration washing was repeated 5 times. The CMB-PEG-DBCO volume was purified and concentrated to 100 μL for collection to obtain the CMB-PEG-DBCO solution, and the CMB-PEG-DBCO solution was placed in a -20°C refrigerator for use.

[0073] Synthesis of CMB-PEG-β-CD: Take azide-modified β-cyclodextrin (N3-β-CD) and dissolve it in anhydrous dimethyl sulfoxide (DMSO) prepared in advance to obtain a 500μmol / L N3-β-CD solution. 100μL of CMB-PEG-DBCO solution and N3-β-CD solution were mixed in equal volumes in a 1.5mL ep tube sterilized by high pressure. The obtained suspension was reacted overnight in a constant temperature metal bath at 25°C, 700r / min, and protected from light to obtain the initial product of CMB-PEG-β-CD.

[0074] Separation and purification of CMB-PEG-β-CD: The CMB-PEG-β-CD initial product was diluted to 3 mL using high pressure Milli-Q water and then filtered using an Amicon-3K ultrafiltration tube ( The diluted CMB-PEG-β-CD initial product was centrifuged and ultrafiltered for 30 min (6500×g, 4°C) using Ultra 4 mL centrifugal ultrafiltration tube, and washed 5 times by centrifugal ultrafiltration using high-pressure Milli-Q water. The CMB-PEG-β-CD was purified and concentrated to a volume of 100 μL, and the CMB-PEG-β-CD solution was collected, labeled, and placed in a -20°C refrigerator for use.

[0075] Characterization of CMB-PEG-β-CD: ① Dilute the isolated and purified CMB-PEG-β-CD solution with Milli-Q water, and measure the absorbance of CMB-PEG-β-CD at 260 nm to determine its concentration. ② Take 0.2OD and send it to Shanghai Sangon Biotechnology Co., Ltd. for matrix-assisted laser dissociation time-of-flight mass spectrometry characterization.

[0076] 3.4 Synthesis and purification of SMB-PEG-β-CD

[0077] Synthesis of SMB-PEG-DBCO: Dissolve 3OD specific molecular beacon in 114.9μL of 0.1mol / L sodium carbonate buffer (pH 8.2) to obtain a 100μmol / L SMB nucleic acid chain solution. Take 3mg of DBCO-PEG-NHS ester and dissolve it in 50μL of DMF and 50μL of acetonitrile mixed solvent to obtain a 29.9mmol / L DBCO-PEG-NHS ester solution. Add 100μL of specific molecular beacon nucleic acid chain solution to a 1.5mL ep tube after high pressure, and mix it with 100μL of DBCO-PEG-NHSester solution in equal volumes to obtain a suspension. The suspension is reacted in a constant temperature metal bath at 25℃, 700r / min, and protected from light overnight to obtain the initial product of SMB-PEG-DBCO.

[0078] Separation and purification of SMB-PEG-DBCO: The initial SMB-PEG-DBCO product was diluted to 2 mL using high pressure Milli-Q water and filtered using an Amicon-3K ultrafiltration tube ( The diluted SMB-PEG-DBCO initial product was centrifuged and ultrafiltered for 30 min (6500×g, 4°C) using Ultra 4 mL centrifugal ultrafiltration tubes, and washed three times with high pressure Milli-Q water; transferred to Amicon-3K ultrafiltration tubes ( Ultra 0.5mL centrifugal ultrafiltration tube) was centrifuged for 30 min (12000×g, 4°C) for further concentration, and the centrifugal ultrafiltration washing was repeated 5 times. The SMB-PEG-DBCO volume was purified and concentrated to 100 μL for collection to obtain the SMB-PEG-DBCO solution, and the SMB-PEG-DBCO solution was placed in a -20°C refrigerator for use.

[0079] Synthesis of SMB-PEG-β-CD: N3-β-CD was dissolved in anhydrous DMSO to obtain a 500 μmol / L N3-β-CD solution. 100 μL of SMB-PEG-DBCO solution and N3-β-CD solution were mixed in equal volumes in a 1.5 mL ep tube sterilized by high pressure to obtain a suspension, and the obtained suspension was reacted overnight in a constant temperature metal bath at 25°C, 700 r / min, and protected from light to obtain the initial product of SMB-PEG-β-CD.

[0080] Separation and purification of SMB-PEG-β-CD: The initial SMB-PEG-β-CD product was diluted to 3 mL using high pressure Milli-Q water and then filtered using an Amicon-3K ultrafiltration tube ( The diluted SMB-PEG-β-CD primary product was centrifuged and ultrafiltered for 30 min (6500×g, 4°C) using an Ultra 4 mL centrifugal ultrafiltration tube, and washed 5 times by centrifugal ultrafiltration using high-pressure Milli-Q water. The SMB-PEG-β-CD primary product was purified and concentrated to a volume of 100 μL to obtain a SMB-PEG-β-CD solution, which was labeled and placed in a -20°C refrigerator for later use.

[0081] Characterization of SMB-PEG-β-CD: ① Dilute the separated and purified product with Milli-Q water, and measure the absorbance of SMB-PEG-β-CD at 260nm to determine its concentration. ② Take 0.2OD and send it to Shanghai Sangon Biotechnology Co., Ltd. for matrix-assisted laser dissociation time-of-flight mass spectrometry characterization.

[0082] 3.5 Polyacrylamide gel electrophoresis experiment of CMB-PEG-β-CD and SMB-PEG-β-CD

[0083] Polyacrylamide gel electrophoresis is a common technique for separating and analyzing DNA. Its basic principle is to separate DNA molecules of different lengths according to the size and charge differences of DNA. Therefore, polyacrylamide gel electrophoresis was used to characterize whether the CMB-PEG-β-CD and SMB-PEG-β-CD probes were successfully synthesized and the hybridization results with the corresponding complementary chains C and S. The specific method is as follows: the samples are set as CMB, CMB-PEG-β-CD, CMB+complementary chain C, CMB-PEG-β-CD+complementary chain C, SMB, SMB-PEG-β-CD, SMB+complementary chain S, SMB-PEG-β-CD+complementary chain S, a total of 8 groups; among them, the molecular beacon, the synthesized probe and the corresponding complementary chain are mixed in the hybridization buffer solution, and the hybridization time is set to 30 minutes. Then nucleic acid non-denaturing polyacrylamide gel electrophoresis is used for characterization.

[0084] IV. Results

[0085] 4.1 Synthesis and characterization of activated supramolecular fluorescent nanoprobe system

[0086] 4.1.1 Screening and characterization of the main compound cyclodextrin

[0087] Considering that the cavity sizes of α-CD, β-CD and γ-CD are different, in order to select cyclodextrins compatible with CMB and SMB to improve the fluorescence sensitization effect, CMB and SMB were incubated with α-CD, β-CD and γ-CD, respectively. The experimental groups and results are shown in Figure 2. Figure 6 shown. Figure 6The fluorescence spectra of CMB hybridized with complementary chain C and then incubated with α-CD, β-CD and γ-CD in (a) show that the inclusion complex formed by incubation of CMB with β-CD has the best fluorescence sensitization effect. Figure 6 The fluorescence spectra of SMB hybridized with complementary chain S and then incubated with α-CD, β-CD and γ-CD in (b) show that the inclusion complex formed by incubation of SMB with β-CD has the best fluorescence sensitization effect. Therefore, the present invention selects β-CD as the main compound to construct an activated supramolecular fluorescent nanoprobe.

[0088] 4.1.2 Mass spectrometry characterization of CMB-PEG-β-CD

[0089] The amino group of the CMB stem reacts with DBCO-PEG-NHS ester to form an amide bond through dehydration condensation to obtain the intermediate product CMB-PEG-DBCO; then CMB-PEG-DBCO is covalently cross-linked with N3-β-CD through click chemistry reaction to prepare CMB-PEG-β-CD. The molecular weight of the purified final product was 11418.0Da by mass spectrometry characterization, and the theoretical molecular weight of CMB-PEG-β-CD was 11413.6Da, indicating that CMB-PEG-β-CD has been successfully synthesized.

[0090] 4.1.3 Mass spectrometry characterization of SMB-PEG-β-CD

[0091] The amino group in the stem of SMB and DBCO-PEG-NHS ester form an amide bond through dehydration condensation to obtain an intermediate product (SMB-PEG-DBCO); SMB-PEG-DBCO is then covalently cross-linked with N3-β-CD through a click chemistry reaction to prepare SMB-PEG-β-CD. The molecular weight of the final product after purification was measured to be 11480.0Da by mass spectrometry characterization, and the theoretical molecular weight of the substance is 11474.7Da. This shows that SMB-PEG-β-CD has been successfully synthesized.

[0092] 4.1.4 Characterization of CMB-PEG-β-CD and SMB-PEG-β-CD by polyacrylamide gel electrophoresis

[0093] Polyacrylamide gel electrophoresis was used to investigate whether CMB-PEG-β-CD and SMB-PEG-β-CD were successfully synthesized and whether the synthesized probes bound to the target sequence. Figure 7 shown. Figure 7As shown in the figure, the band representing CMB-PEG-β-CD significantly migrated upward compared with the band representing CMB, indicating that CMB, DBCO-PEG-NHS ester and N3-β-CD were covalently linked to form a CMB-PEG-β-CD probe with a larger molecular weight. After the addition of complementary chain C, both CMB and CMB-PEG-β-CD bound to complementary chain C, and the bands significantly shifted upward, and the nucleic acid bands of the original CMB and CMB-PEG-β-CD disappeared, indicating that both CMB and CMB-PEG-β-CD could hybridize with the target nucleic acid chain through base complementary pairing. Similarly, the band representing SMB-PEG-β-CD significantly migrated upward compared with the band representing SMB, indicating that SMB, DBCO-PEG-NHS ester and N3-β-CD were covalently linked to form a SMB-PEG-β-CD probe with a larger molecular weight. After adding complementary chain S, SMB and SMB-PEG-β-CD hybridized with complementary chain S, the bands shifted up significantly, and the nucleic acid bands of the original SMB and SMB-PEG-β-CD disappeared, indicating that SMB and SMB-PEG-β-CD can hybridize with the target nucleic acid chain through base complementary pairing. The above experimental results show that CMB-PEG-β-CD and SMB-PEG-β-CD probes have been successfully prepared and have the ability to specifically recognize the target sequence. The nucleotide sequence of complementary chain C is shown in SEQ ID NO.3, and the nucleotide sequence of complementary chain S is shown in SEQ ID NO.4.

[0094] 4.2 Characterization of the performance of activated supramolecular fluorescent nanoprobes

[0095] 4.2.1 Optimization of experimental conditions

[0096] 4.2.1.1 Optimization of incubation time between probe and complementary strand

[0097] In order to obtain the optimal reaction time for the hybridization of CMB-PEG-β-CD and SMB-PEG-β-CD probes with the corresponding complementary chains C and S, and to ensure that the experimental reaction is complete and the fluorescence signal is increased within a certain range, the incubation time between the probe and the complementary chain in the activated supramolecular fluorescent nanoprobe system was optimized. The specific results are shown in Figure 2. Figure 8 As shown in the figure, within 1-3 hours, as the hybridization time increases, the fluorescence signals generated by the hybridization of the two probes CMB-PEG-β-CD and SMB-PEG-β-CD with the corresponding complementary chains C and S increase significantly. After 3-5 hours, the hybridization efficiency decreases and the fluorescence signal also decreases. The above results prove that the optimal time for the hybridization of the CMB-PEG-β-CD and SMB-PEG-β-CD probes with the corresponding complementary chains is 3 hours.

[0098] 4.2.1.2 Mg in the probe and complementary strand hybridization buffer solution 2+ Concentration optimization

[0099] Mg 2+ Concentration is an important factor affecting hybridization efficiency. 2+ The optimal Mg concentration in the hybridization buffer solution is used to obtain the optimal Mg concentration in the hybridization buffer solution when the CMB-PEG-β-CD and SMB-PEG-β-CD probes are hybridized with the corresponding complementary chains C and S. 2+ Concentration of Mg in hybridization buffer 2+ The specific results are as follows Fig. 9 As shown in (a), CMB-PEG-β-CD was in the hybridization buffer solution Mg 2+ When the concentration is 0-2.00mmol / L, the probe hybridizes with the corresponding complementary chain in its solution system, and the generated fluorescence signal increases significantly; in the hybridization buffer solution Mg 2+ When the concentration is 2.00-10.00mmol / L, as Mg 2+ As the concentration increases, the fluorescence signal remains stable. Fig. 9 As shown in (b), SMB-PEG-β-CD was in the hybridization buffer solution Mg 2+ When the concentration is 0-0.66mmol / L, the probe hybridizes with the corresponding complementary chain in its solution system, and the generated fluorescence signal increases significantly; in the hybridization buffer solution Mg 2+ When the concentration is 0.66-10.00mmol / L, as Mg 2+ As the concentration increases, the fluorescence signal remains stable. Therefore, 2.00mmol / L Mg 2+ The above results show that CMB-PEG-β-CD and SMB-PEG-β-CD probes can be used in low Mg 2+ The hybridization buffer solution with a concentration of 0.66mmol / L MgCl2+ produced a strong fluorescence signal when hybridized with the corresponding complementary strand. 2+ The hybridization can be stably performed even at a high concentration, which is beneficial for the subsequent detection of the target sequence in Staphylococcus aureus.

[0100] 4.2.2 Characterization of spectral properties of probes

[0101] Since the β-CD cavity is a structure with high electron cloud density, when the dye is embedded in the β-CD cavity, its molecular mobility is reduced, maintaining a suitable microenvironment or protecting the excited state from contact with water molecules and quenchers, thereby causing the spectrum of the dye to shift or the fluorescence intensity to increase. Fig.10As shown in (a), when excited by 495nm excitation light, the 6-FAM fluorescent group modified by CMB and CMB-PEG-β-CD has the best emission at 520nm; when the complementary chain C is present, the fluorescence signal generated by CMB-PEG-β-CD is enhanced by about 1.32 times compared with CMB. Fig.10 (b) shows that when excited by 545nm excitation light, the Cy3 fluorescent groups modified by SMB and SMB-PEG-β-CD have the best emission at 565nm; when the complementary chain S is present, the fluorescence signal generated by SMB-PEG-β-CD is enhanced by about 1.71 times compared with SMB. The above results show that CMB-PEG-β-CD and SMB-PEG-β-CD probes have fluorescence sensitization properties. The possible reason is that cyclodextrin and CMB-PEG-β-CD, SMB-PEG-β-CD modified 6-FAM, Cy3 fluorescent groups undergo host-guest envelopment when the target is present.

[0102] 4.2.3 Characterization of the photostability of the probe

[0103] In order to demonstrate the photostability of CMB-PEG-β-CD and SMB-PEG-β-CD probes in complex systems, the fluorescence intensity changes of CMB-PEG-β-CD and SMB-PEG-β-CD probes were investigated after hybridization with the corresponding complementary chains C and S in PBS buffer solution and cell culture medium containing 10% FBS, respectively, and irradiated with UV light for different times. The results showed that in PBS buffer and cell culture medium containing 10% FBS, CMB-PEG-β-CD and SMB-PEG-β-CD hybridized with complementary chains C and S, respectively, and their fluorescence intensity could be maintained at more than 80% of the initial fluorescence intensity after continuous irradiation with UV light for 10 hours, indicating that CMB-PEG-β-CD and SMB-PEG-β-CD have good photostability, which lays a foundation for fluorescence imaging.

[0104] 4.2.4 Characterization of the probe's resistance to enzyme cleavage

[0105] In order to demonstrate the physical stability of CMB-PEG-β-CD and SMB-PEG-β-CD probes in complex systems, the anti-enzymatic properties of the synthesized CMB-PEG-β-CD and SMB-PEG-β-CD probes were studied. The results showed that the designed CMB and the constructed CMB-PEG-β-CD were incubated with 0.25U / μL Exo I, and the nucleic acid bands did not change significantly with the increase of incubation time; even after incubation with 0.25U / μL Exo I for 10h, the two could still maintain the integrity of the nucleic acid sequence. The above experimental results prove that the designed and constructed CMB and CMB-PEG-β-CD can maintain the integrity of the nucleic acid sequence in a complex biological environment and have good anti-enzymatic properties.

[0106] The designed SMB and constructed SMB-PEG-β-CD were incubated with 0.25U / μL Exo I. As the incubation time increased, the nucleic acid bands did not change significantly; even after incubation with 0.25U / μL Exo I for 10h, the two could still maintain the integrity of the nucleic acid sequence. The above experiments proved that the designed and constructed SMB and SMB-PEG-β-CD can maintain physical stability in complex biological environments and have good anti-enzymatic properties.

[0107] 4.3 Activated supramolecular fluorescent nanoprobe system for differentiating different bacteria

[0108] 4.3.1 Bacterial morphology and identification by Columbia blood agar plate culture

[0109] After Staphylococcus aureus was grown on TSB plates for 12 hours, the morphological characteristics of the colonies were observed. The colonies of Staphylococcus aureus were round in appearance, with a smooth, convex, and moist surface, and were light yellow on TSB solid culture medium plates.

[0110] Since Staphylococcus aureus can produce hemolysin to completely dissolve red blood cells, a β-hemolytic ring will appear after culturing on Columbia blood agar for 12-18 hours. Fig.14 It can be seen that Staphylococcus aureus is completely hemolyzed on the Columbia blood agar plate, and there is a clear-cut, colorless and transparent hemolysis ring around the colony, namely the β-hemolysis ring. This further verifies that the target bacteria preserved in the laboratory are Staphylococcus aureus with complete hemolysis ability.

[0111] 4.3.2 Gram staining of Staphylococcus aureus

[0112] Staphylococcus aureus is a Gram-positive bacterium. To further verify that the target bacterium preserved in the laboratory used in the experiment is Staphylococcus aureus, it was examined by Gram staining and its morphology was observed under a microscope. Fig.11As shown, the bacteria are blue-purple, Gram-positive bacteria, and round cocci in shape, mostly single or in pairs, and mainly arranged in grape-like shapes.

[0113] 4.3.3 Confocal laser imaging of bacteria

[0114] Staphylococcus aureus was used as the target bacteria and Pseudomonas aeruginosa was used as the control bacteria to study the effect of CMB-PEG-β-CD and SMB-PEG-β-CD on different bacteria. The activated supramolecular fluorescent nanoprobe system was incubated with Staphylococcus aureus and Pseudomonas aeruginosa after cell wall permeabilization for 1 hour, and the images were taken using a laser confocal microscope. The results are shown in Fig.15 As shown, both CMB-PEG-β-CD and SMB-PEG-β-CD can identify Staphylococcus aureus, showing bright yellow fluorescence (superimposed colors of green and red), and the co-localized yellow fluorescence spots representing Staphylococcus aureus overlap with the blue fluorescence of the bacteria after DAPI staining. The above experimental results show that CMB-PEG-β-CD and SMB-PEG-β-CD can target Staphylococcus aureus and achieve co-localized imaging detection of target bacteria.

[0115] The confocal imaging results of Pseudomonas aeruginosa showed that the bacteria showed bright green fluorescence, and the green fluorescence spots representing Pseudomonas aeruginosa overlapped with the blue fluorescence of the bacteria after DAPI staining. The above experimental results show that in the activated supramolecular fluorescent nanoprobe system, only CMB-PEG-β-CD can identify Pseudomonas aeruginosa.

[0116] The confocal imaging results of Staphylococcus aureus and Pseudomonas aeruginosa showed that CMB-PEG-β-CD as a universal probe can identify Staphylococcus aureus and Pseudomonas aeruginosa, while SMB-PEG-β-CD as a Staphylococcus aureus-specific probe can only identify Staphylococcus aureus. This proves that the combined use of CMB-PEG-β-CD and SMB-PEG-β-CD can be used to effectively distinguish different bacteria and achieve highly sensitive and specific co-localization imaging detection of Staphylococcus aureus.

[0117] 4.4 Activated supramolecular fluorescent nanoprobe system for detection of Staphylococcus aureus in MCF-7 cells

[0118] 4.4.1 Characterization of the effect of probes on cell viability

[0119] In order to further apply the probe to tissues and cells, it is necessary to use the MTT experiment to evaluate the effect of CMB-PEG-β-CD and SMB-PEG-β-CD probes on cell viability. The MTT experiment shows that after different concentrations of CMB and CMB-PEG-β-CD were incubated with MCF-7 cells for 2 hours, the cell survival rate could reach more than 85% after being treated with the MTT kit; similarly, after incubation with different concentrations of SMB and SMB-PEG-β-CD and MTT kit treatment, the survival rate of MCF-7 cells could also reach more than 85%. The above results show that the CMB and SMB constituting the activated supramolecular nanoprobes and the constructed CMB-PEG-β-CD and SMB-PEG-β-CD probes have little effect on cell viability, which is conducive to the subsequent application of CMB-PEG-β-CD and SMB-PEG-β-CD to the fluorescence imaging of intracellular bacteria in cells and tissues. MCF-7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0120] 4.4.2 Confocal laser imaging of Staphylococcus aureus in MCF-7 cells

[0121] In order to study the effect of the activated supramolecular fluorescent nanoprobe system on the detection of Staphylococcus aureus in MCF-7 cells, the present invention uses MCF-7 cells as target cells and Staphylococcus aureus as target bacteria, and uses Staphylococcus aureus to invade MCF-7 cells to construct an intracellular bacteria model. The activated supramolecular fluorescent nanoprobe system was incubated with the Staphylococcus aureus model in MCF-7 cells after cell wall permeabilization for 1 hour, and its fluorescence imaging was observed by laser confocal microscopy. The results are shown in Figure 2. Fig.12 As shown, Staphylococcus aureus exhibits bright yellow fluorescence (superimposed colors of green and red), and some co-localized yellow fluorescent spots representing Staphylococcus aureus exist in MCF-7 cells. The above experimental results show that the constructed activated supramolecular fluorescent nanoprobe system can be successfully used for co-localized imaging detection of Staphylococcus aureus in MCF-7 cells. At the same time, it can be seen that the activated supramolecular fluorescent nanoprobe system has high resolution in complex cellular environments and has a very obvious recognition effect on target intracellular bacteria.

[0122] 4.5 Activated supramolecular fluorescent nanoprobe system for detection of intracellular Staphylococcus aureus in transplanted tumor tissues of MCF-7 breast cancer mice

[0123] H&E staining was performed on frozen tissue sections from the transplanted tumor models of the control group and the experimental group. The results showed that in the tissue sections of the control group, the tumor tissue showed dense and disordered growth, the cell nuclei were deeply stained blue-purple, and the cytoplasm of the tumor cells was eosinophilic and pink. In the tissue sections of the experimental group, the tumor tissue also showed dense and disordered growth; and due to the injection of Staphylococcus aureus, necrotic cells appeared around the tissue; the nuclei of the tumor cells were stained lighter than those in the control group, purple-red; the cytoplasm was the same as the control group, pink. The above experimental results show that the MCF-7 breast cancer mouse transplant tumor model was successfully constructed, and Staphylococcus aureus was successfully injected into the model, which was used for the subsequent detection of tumor-resident intracellular bacteria in tissue sections by CMB-PEG-β-CD and SMB-PEG-β-CD. The experimental group is an MCF-7 breast cancer mouse transplant tumor model with intracellular Staphylococcus aureus, and the control group is an MCF-7 breast cancer mouse transplant tumor model without injection of Staphylococcus aureus.

[0124] Furthermore, IHC staining was performed on frozen tissue sections from the control group and experimental group transplant tumor models. The results showed that the MCF-7 breast cancer mouse transplant tumors in the control group were not injected with Staphylococcus aureus, and the IHC staining results showed that there were fewer Gram-positive bacterial cell wall (LTA) positive staining areas (brown-yellow). The MCF-7 breast cancer mouse transplant tumors in the experimental group were injected with Staphylococcus aureus, and the IHC staining results showed that the tumor tissue was invaded by Staphylococcus aureus and necrotic cells appeared, and brown-brown LTA positive staining spots representing Gram-positive bacteria appeared, and there were brown-yellow nonspecific staining areas. The pathological H&E staining results were consistent with the IHC staining results.

[0125] Laser confocal imaging of tissue sections using CMB-PEG-β-CD and SMB-PEG-β-CD

[0126] The frozen tissue sections used in the experiment were from an MCF-7 breast cancer mouse xenograft model with intracellular Staphylococcus aureus. The mixed solution of CMB-PEG-β-CD, SMB-PEG-β-CD and DAPI dye was incubated with the frozen sections and imaged using a laser confocal microscope. Fig.13 As shown in the figure, the cell nuclei and bacteria in the transplanted tumor tissue are stained with DAPI and show blue fluorescence; the activated supramolecular fluorescent nanoprobe system can effectively identify intracellular bacteria in the tissue, showing bright yellow fluorescence; and the co-localized yellow fluorescent spots representing Staphylococcus aureus overlap with the blue fluorescence of the bacteria after DAPI staining, and some of them exist within the cell outline and close to the cell nucleus. The above experimental results show that the constructed activated supramolecular fluorescent nanoprobe system is not easily disturbed by the tissue microenvironment and can be successfully used for co-localized imaging detection of intracellular bacteria resident in tumor tissue.

[0127] Fig.13 In the figure, the DAPI pseudocolor is set to blue, Merge 1 is the overlay of CMB-PEG-β-CD and SMB-PEG-β-CD channel images; Merge 2 is the overlay of CMB-PEG-β-CD, SMB-PEG-β-CD, DAPI and bright field (Light) channel images. Fig.15 The DAPI pseudocolor was set to blue, Merge 1 was the overlay of CMB-PEG-β-CD and SMB-PEG-β-CD channel images; Merge 2 was the overlay of CMB-PEG-β-CD, SMB-PEG-β-CD, and DAPI channel images.

Claims

1. An activated supramolecular fluorescent nanoprobe system, characterized in that: The activated supramolecular fluorescent nanoprobe comprises a universal activated supramolecular fluorescent nanoprobe and a specific activated supramolecular fluorescent nanoprobe. The universal activated supramolecular fluorescent nanoprobe comprises a universal molecular beacon and covalently modified β-cyclodextrin. The nucleotide sequence of the universal molecular beacon is designed according to the non-specific sequence of 16S rRNA in bacteria, and the universal molecular beacon is modified with a fluorescent group 1; the specific activated supramolecular fluorescent nanoprobe comprises a specific molecular beacon and covalently modified β-cyclodextrin. The nucleotide sequence of the specific molecular beacon is designed according to the specific sequence of 16S rRNA of Staphylococcus aureus, and the specific molecular beacon is modified with a fluorescent group 2. The fluorescent colors of the fluorescent group 1 and the fluorescent group 2 are different.

2. The activated supramolecular fluorescent nanoprobe system according to claim 1, characterized in that: The nucleotide sequence of the universal molecular beacon is shown in SEQ ID NO.1, and the nucleotide sequence of the specific molecular beacon is shown in SEQ ID NO.

2.

3. The activated supramolecular fluorescent nanoprobe system according to claim 1, characterized in that: The first fluorescent group is a 6-FAM fluorescent group, and the second fluorescent group is a Cy3 fluorescent group.

4. A method for preparing the activated supramolecular fluorescent nanoprobe system as claimed in claim 1, characterized in that: The following steps are involved: S1: A universal molecular beacon is designed based on the non-specific sequence of 16S rRNA in bacteria. The universal molecular beacon undergoes a dehydration condensation reaction with DBCO-PEG-NHS to form an intermediate product 1. The intermediate product 1 undergoes a click chemistry reaction with azide-modified β-cyclodextrin to obtain a universal activated supramolecular fluorescent nanoprobe. S2: Design a specific molecular beacon based on the specific sequence of 16S rRNA in Staphylococcus aureus, and react the specific molecular beacon with DBCO-PEG-NHS to form an intermediate product 2, and the intermediate product 2 reacts with azide-modified β-cyclodextrin to produce a specific activated supramolecular fluorescent nanoprobe by click chemistry; S3: Mixing the universal activated supramolecular fluorescent nanoprobe with the specific activated supramolecular fluorescent nanoprobe to obtain.

5. The method for preparing the activated supramolecular fluorescent nanoprobe system according to claim 4, characterized in that: The preparation method of the universal activated supramolecular fluorescent nanoprobe comprises the following steps: mixing a universal molecular beacon nucleic acid chain solution with a DBCO-PEG-NHS solution for overnight reaction, purifying to obtain a CMB-PEG-DBCO solution, mixing the CMB-PEG-DBCO solution with an azide-modified β-cyclodextrin solution in equal volumes, reacting in the dark overnight, and purifying to obtain the solution; the preparation method of the specific activated supramolecular fluorescent nanoprobe comprises the following steps: mixing a specific molecular beacon nucleic acid chain solution with a DBCO-PEG-NHS solution for overnight reaction, purifying to obtain a SMB-PEG-DBCO solution; mixing the SMB-PEG-DBCO solution with an azide-modified β-cyclodextrin solution in equal volumes, reacting in the dark overnight, and purifying to obtain the solution; the DBCO-PEG-NHS solution is obtained by dissolving DBCO-PEG-NHS in a mixed solvent, and the azide-modified β-cyclodextrin solution is obtained by dissolving azide-modified β-cyclodextrin in dimethyl sulfoxide.

6. The method for preparing the activated supramolecular fluorescent nanoprobe system according to claim 5, characterized in that: The concentration of the universal molecular beacon nucleic acid chain solution is 50-200 μmol / L, the concentration of the specific molecular beacon nucleic acid chain solution is 50-200 μmol / L, the concentration of the DBCO-PEG-NHS solution is 20-50 mmol / L, and the concentration of the azide-modified β-cyclodextrin solution is 400-600 μmol / L.

7. The method for preparing the activated supramolecular fluorescent nanoprobe system according to claim 5, characterized in that: The mixed solvent is a mixed solution of dimethylformamide and acetonitrile, and the volume ratio of the dimethylformamide to the acetonitrile is 1:

1.

8. The method for preparing the activated supramolecular fluorescent nanoprobe system according to claim 5, characterized in that: The volume ratio of the universal molecular beacon nucleic acid chain solution to the DBCO-PEG-NHS solution is 1:1, and the volume ratio of the specific molecular beacon nucleic acid chain solution to the DBCO-PEG-NHS solution is 1:

1.

9. The method for preparing the activated supramolecular fluorescent nanoprobe system according to claim 5, characterized in that: The preparation method of the universal molecular beacon nucleic acid chain solution is to dissolve the universal molecular beacon in a sodium carbonate buffer solution; The preparation method of the specific molecular beacon nucleic acid chain solution is to dissolve the specific molecular beacon in a sodium carbonate buffer solution; the concentration of the sodium carbonate buffer solution is 0.1-0.2 mol / L, and the pH of the sodium carbonate buffer solution is 8.0-9.

0.

10. Use of the activated supramolecular fluorescent nanoprobe system as claimed in claim 1 for co-localization imaging of intracellular Staphylococcus aureus.

Images