Enrichment enhanced luminous biological probe based on silicon dioxide-rare earth fluoride as well as preparation method and application of enrichment enhanced luminous biological probe

Abstract

The invention discloses an enrichment enhanced luminous biological probe based on silicon dioxide-rare earth fluoride as well as a preparation method and application of the enrichment enhanced luminous biological probe, and belongs to the technical field of nano materials. According to the preparation method, dendritic mesoporous silicon dioxide is used as a carrier for limited-range growth of rare earth fluoride, and a large number of rare earth fluoride nanoparticles grow in holes in an in-situ limited-range mode through the enrichment effect of ultra-large mesopores of the dendritic mesoporous silicon dioxide; the silicon dioxide-rare earth fluoride which is clear and uniform in form, strong in single particle luminescence and easy in surface functionalization is formed, and then the surface of the silicon dioxide-rare earth fluoride is modified with targeting molecules, so that the enrichment-enhanced luminescent biological probe based on the silicon dioxide-rare earth fluoride is prepared. The enrichment enhanced luminous biological probe has the characteristics of long fluorescence lifetime, strong single particle luminescence and good specific targeting, and has wide application prospects in the fields of ultra-sensitive detection of cancer markers, cancer cell imaging and in-vitro detection of cancer cells.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and in particular relates to an enrichment-enhanced luminescent biological probe based on silicon dioxide-rare earth fluoride, and a preparation method and application thereof. Background Art

[0002] Cancer is a disease caused by the unlimited proliferation and invasion of tissues and organs by abnormal cells, which seriously threatens human life. During the growth, invasion and metastasis of malignant tumors, certain cancer markers or cancer cells are released into body fluids such as blood, pleural effusion, ascites and urine. These cancer markers and detached cancer cells contain rich biological information and have specific biological significance in the occurrence, development and treatment of cancer. Early diagnosis and timely treatment of cancer can significantly improve the survival rate of patients. However, the scarcity of cancer markers and cancer cells in body fluids and the complexity of cell types have brought great difficulties to the early diagnosis of cancer.

[0003] Fluorescence bioanalysis technology has been widely used in scientific research and medical institutions. However, traditional fluorescent biological probes, such as organic fluorescent dyes and quantum dots, are easily interfered by stray light and the fluorescence of biological tissues themselves, resulting in relatively low sensitivity of biological detection. For example, a Chinese patent with publication number CN115141219A and application date June 29, 2022 discloses a fluorescent probe, synthesis method and application for ovarian cancer cell imaging. The probe functionalizes a triaryl boron structure with a pyridinium cation, and its pyridinium cation can not only bind to DNA and LPA, but also act as an electron-withdrawing group to regulate its photophysical properties. It can selectively respond to DNA and LPA (lysophosphatidic acid), but not to ATP, ADP and AMP, and has different fluorescence signal outputs for DNA and LPA, but the probe cannot be applied to other cancer cell imaging or the identification of cancer markers, and cannot meet the requirements of ultra-sensitive biological detection when the ovarian cancer cell content is low. The Chinese patent with publication number CN103513025A and application date June 21, 2012 provides gastric cancer targeted quantum dots and its preparation method and application. The quantum dots have good biocompatibility in the body and can run with the circulatory system. The targeted quantum dots are prepared to perform immunofluorescence imaging on gastric cancer cells, and further detect the tumor metastasis site in the gastric cancer animal model and combine with the tumor cells. After excitation, the tumor cells emit fluorescence, so as to image the tumor cells, thereby achieving the purpose of tumor detection. However, the immunofluorescence probe is easily interfered by non-specific light during application, thereby affecting the detection of fluorescence signals. At the same time, the fluorescent substances in the biological tissue itself will also emit fluorescence and superimpose the probe signal, reducing the signal specificity.

[0004] Rare earth ions have rich energy level structures. Rare earth-doped nanoluminescent materials have stood out from many luminescent materials in recent years due to their excellent physical and chemical properties. They have attracted widespread attention and have great application value in color display, biomedical fluorescent labeling, inorganic liquid lasers, etc. The long fluorescence lifetime of rare earth-doped inorganic nanoparticles combined with time-resolved fluorescence detection technology can effectively eliminate short-lived background fluorescence and achieve background-free ultra-sensitive detection. In particular, cerium fluoride (CeF3) nanoparticles, because of their Ce 3+ The unique 4f-5d electric dipole transition of ions shows excellent sensitization ability. 3+ Ions can enhance green luminescence and achieve a long fluorescence lifetime of milliseconds. To date, the synthesis of most CeF3 nanoparticles requires long reaction times, various organic solvents, and high temperatures. The prepared nanoparticles are usually small and non-uniform, resulting in relatively weak and non-uniform luminescence of single particles, which cannot meet the requirements of ultra-sensitive biological detection.

[0005] Therefore, it is urgent to develop highly sensitive rare earth doped fluoride luminescent bioprobes with simple synthesis strategies and strong single particle luminescence. By utilizing the enrichment of rare earth fluoride by dendritic mesoporous silica to improve the single particle luminescence intensity of nano bioprobes, a silica-rare earth fluoride luminescent nanomaterial was designed. By functionalizing its surface, it can achieve ultra-sensitive detection of cancer markers, cancer cell imaging, and in vitro detection of cancer cells. It has important reference value and practical significance for early diagnosis of cancer and evaluation of treatment effects. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides an enriched and enhanced luminescent bioprobe based on silica-rare earth fluoride, and a preparation method and application thereof. The enriched and enhanced luminescent bioprobe has the characteristics of long fluorescence lifetime, strong single particle luminescence, and good specific targeting, and has broad application prospects in the fields of ultrasensitive detection of cancer markers, cancer cell imaging and in vitro detection.

[0007] The technical solution of the present invention is as follows:

[0008] One of the purposes of the present invention is to provide a method for preparing an enriched and enhanced luminescent biological probe based on silica-rare earth fluoride, using dendritic mesoporous silica as a carrier for the confined growth of rare earth fluoride, utilizing the enrichment effect of the super-large mesopores of the dendritic mesoporous silica, so that a large number of rare earth fluoride nanoparticles are in situ confined to grow in the pores to obtain silica-rare earth fluoride nanomaterials, and then modifying the surface of the silica-rare earth fluoride nanomaterials with targeting molecules to obtain an enriched and enhanced luminescent biological probe based on silica-rare earth fluoride.

[0009] Further, the following steps are included:

[0010] S1. preparing dendritic mesoporous silica nanoparticles by anion-assisted synthesis, and modifying the surface with carboxyl groups to obtain the dendritic mesoporous silica with carboxyl groups on the surface;

[0011] S2, adsorbing rare earth cations in the dendritic mesoporous silica pores by electrostatic action, introducing sodium bifluoride NaHF2 to allow rare earth fluoride to grow in situ in the pores to form silica-rare earth fluoride nanomaterials;

[0012] S3. Introducing a targeting agent into the silica-rare earth fluoride nanomaterial solution prepared in S2 to modify its surface, washing it with water for multiple times after ultrasonic centrifugation and removing the supernatant to obtain the enriched enhanced luminescent bioprobe based on silica-rare earth fluoride.

[0013] Furthermore, the S1 comprises the following steps:

[0014] S11, add triethanolamine to ultrapure water and stir at 60-90°C, then add hexadecyltrimethylammonium bromide and sodium salicylate to the above system and continue stirring and reacting for 1 hour, then add tetraethyl orthosilicate and continue stirring and reacting for 2 hours, centrifuge at 12000 rpm for 5 minutes and remove the supernatant;

[0015] The mass ratio of ultrapure water, triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate is 6250:17:95:42; the volume ratio of ultrapure water to tetraethyl orthosilicate is 25:4;

[0016] S12, dispersing the intermediate product A obtained in S11 in a methanol solution containing concentrated hydrochloric acid, stirring evenly at 40-90° C., centrifuging at 12000 rpm for 5 minutes, washing three times with anhydrous ethanol, and removing the supernatant;

[0017] The volume ratio of concentrated hydrochloric acid to methanol is 1:60; the mass ratio of intermediate product A to methanol is 3:350;

[0018] S13, dispersing the intermediate product B obtained in S12 in an anhydrous ethanol solution containing ammonia water and 3-aminopropyltriethoxysilane, stirring at room temperature for 12 hours, centrifuging at 12000 rpm for 5 minutes, washing three times with anhydrous ethanol, and removing the supernatant;

[0019] The volume ratio of ammonia water, 3-aminopropyltriethoxysilane and anhydrous ethanol is 3:1:150; the mass ratio of intermediate product B to anhydrous ethanol is 1:160;

[0020] S14, dispersing the intermediate product C obtained in S13 again in an N,N-dimethylformamide solution containing succinic anhydride, stirring at room temperature for 6 hours, centrifuging at 12000 rpm for 5 minutes, washing three times with anhydrous ethanol, removing the supernatant and vacuum drying to obtain the dendritic mesoporous silica with carboxyl groups on the surface;

[0021] The mass ratio of intermediate product C, succinic anhydride, and N,N-dimethylformamide is 1:5:379;

[0022] The particle size of the dendritic mesoporous silica nanoparticles is 5 to 1000 nm, and the pore size is 2 to 50 nm.

[0023] Furthermore, the particle size of the dendritic mesoporous silica nanoparticles is 100-300 nm, and the pore size is 5-30 nm.

[0024] Furthermore, the S2 is added with the dendritic mesoporous silica prepared by S1 to the aqueous solution containing rare earth nitrate, and after ultrasonic reaction for 1 to 6 minutes, sodium bifluoride NaHF2 aqueous solution is added and ultrasonication is continued for 1 to 6 minutes, and after centrifugation at 8000 rpm for 3 minutes, it is washed with water three times, and the supernatant is removed to obtain the silica-rare earth fluoride nanomaterial;

[0025] The mass ratio of the dendritic mesoporous silica, rare earth nitrate and sodium bifluoride NaHF2 is 1:35:5;

[0026] The rare earth cation is Ce 3+ , Tb 3+ 、Nd 3+ 、Eu 3+ Any one or a combination of two.

[0027] Furthermore, the targeting agent in S3 is biotin or avidin.

[0028] Furthermore, when the targeting agent is avidin, the modification method is as follows:

[0029] S31, adding the silicon dioxide-rare earth fluoride nanomaterial prepared in S2 to the aqueous solution containing sodium polyacrylate, ultrasonicating for 30 to 35 minutes, centrifuging at 8000 rpm for 5 minutes, washing with water three times, and removing the supernatant;

[0030] S32, dispersing the intermediate product D obtained in S31 in a buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and N-hydroxysuccinimide sulfonic acid sodium salt NHS, reacting at 200W ultrasonic power for 20-25min, then centrifuging at 8000rpm for 5min, washing with water three times, and removing the supernatant;

[0031] S33, dispersing the intermediate product E obtained in S32 in a 4-hydroxyethylpiperazineethanesulfonic acid HEPES buffer solution, adding avidin, vortexing at 120 rpm for 30 to 35 min, centrifuging at 8000 rpm for 5 min, washing three times with water, removing the supernatant, and obtaining the avidin-modified enrichment-enhanced luminescent bioprobe based on silica-rare earth fluoride;

[0032] The mass ratio of sodium polyacrylate, silica-rare earth fluoride nanomaterials, EDC, NHS and avidin is 8:2:2:1:0.12.

[0033] Furthermore, the avidin-modified enrichment-enhanced luminescent bioprobe based on silica-rare earth fluoride is dispersed in an aqueous solution containing 2% bovine serum albumin and stored in a refrigerator at 4° C. for later use.

[0034] Furthermore, when the targeting agent is biotin, the modification method is as follows:

[0035] Ammonia water and biotin were added to 1 mL of ultrapure aqueous solution to mix the two evenly, and then the silica-rare earth fluoride nanomaterial prepared in S2 was added, and after ultrasonication for 30 to 35 minutes, the mixture was centrifuged at 8000 rpm for 5 minutes, washed with water three times, and the supernatant was removed to obtain the biotin-modified silica-rare earth fluoride-based enrichment enhanced luminescent bioprobe;

[0036] The mass ratio of ammonia water, biotin, and silicon dioxide-rare earth fluoride nanomaterials is 9:4:1.

[0037] Furthermore, the biotin-modified enriched enhanced luminescent bioprobe based on silica-rare earth fluoride is dispersed in ultrapure water and stored in a refrigerator at 4° C. for later use.

[0038] The second object of the present invention is to provide an enrichment-enhanced luminescent bioprobe based on silica-rare earth fluoride.

[0039] The third object of the present invention is to provide an enrichment-enhanced luminescent bioprobe based on silica-rare earth fluoride for use in in vitro detection of cancer markers in combination with time-resolved fluorescence detection technology.

[0040] The fourth object of the present invention is to provide an enrichment-enhanced luminescent bioprobe based on silica-rare earth fluoride for use in specific identification of cancer cells in vitro and in fluorescence-targeted cancer cell imaging.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] 1. The present invention innovates dendritic mesoporous silica as a rare earth fluoride carrier for the first time, adopts an in-situ enrichment strategy to enrich rare earth fluoride nanoparticles in the super-large mesopores of dendritic silica, and realizes the in-situ confined growth of a large number of rare earth fluoride nanoparticles in the pores, which greatly enhances the single-particle luminescence intensity of the nanoluminescent material. The present invention overcomes the problems of irregular morphology, uneven size, and weak single-particle luminescence of traditional rare earth fluoride nanomaterials.

[0043] 2. The present invention utilizes the unique millisecond-level long-lifetime characteristics of rare earth fluorides and combines it with time-resolved fluorescence detection technology to effectively distinguish the long-lifetime fluorescence signals of rare earth fluorides from the short-lifetime background fluorescence interference, so that cancer markers can be accurately detected even at extremely low concentrations, achieving ultra-sensitive detection effects and greatly improving the accuracy and sensitivity of detection.

[0044] 3. The surface of the enriched and enhanced luminescent bioprobe based on silica-rare earth fluoride prepared by the present invention has extremely high functionalization potential. Various targeting agents can be modified on the surface of the nanomaterial according to specific detection requirements to make it have excellent targeting ability, thereby broadening the application scope of the luminescent bioprobe. At the same time, the enriched and enhanced luminescent bioprobe has the significant advantage of strong single-particle luminescence, and performs well in cancer cell targeted imaging and in vitro specific detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a transmission electron microscope image of the dendritic mesoporous silica prepared in Example 1 of the present invention;

[0046] Figure 2 This is a transmission electron microscope image of the silicon dioxide-rare earth fluoride nanomaterial DMS-CeF3:Tb prepared in Example 1 of the present invention;

[0047] Figure 3 This is a transmission electron microscope image of the rare earth fluoride nanoparticle CeF3:Tb prepared in the performance test of the present invention;

[0048] Figure 4 This is a result diagram of PSA heterogeneous detection using a nanoprobe in a time-resolved detection mode in the performance test of the present invention;

[0049] Figure 5 This is a confocal imaging image of the enriched enhanced luminescent bioprobe DMS-CeF3:Tb-avidin after co-incubation with Hela cells in the performance test of the present invention;

[0050] Figure 6 This is a picture of Hela and L-02 cells combined with different nanoprobes under 254nm handheld ultraviolet light irradiation in the performance test of the present invention;

[0051] Figure 7 This is a comparison chart of the luminescence intensity at 546 nm of Hela and L-02 cells after combining with different nanoprobes in the performance test of the present invention. DETAILED DESCRIPTION

[0052] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges disclosed in the present invention and any values ​​are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be deemed to be specifically disclosed herein.

[0053] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] Example 1

[0056] This embodiment provides an enrichment-enhanced luminescent bioprobe DMS-CeF3:Tb-avidin based on silicon dioxide-rare earth fluoride, and the preparation method thereof is as follows:

[0057] S1. Add 0.068 g of triethanolamine to 25 mL of ultrapure water and stir at 80 °C for 30 min, then add 380 mg of hexadecyltrimethylammonium bromide and 168 mg of sodium salicylate to the above system and continue stirring and reacting for 60 min, then continue to add 4 mL of tetraethyl orthosilicate and continue to react for 120 min, and then centrifuge to remove the supernatant;

[0058] S2, disperse the intermediate product A obtained in S1 in 91.5 mL of methanol solution containing 1.5 mL of concentrated hydrochloric acid, stir at 60°C for 6 h, centrifuge and wash three times with alcohol, and remove the supernatant;

[0059] S3, dispersing the intermediate product B obtained in S2 in 120 mL of an ethanol solution containing 2.4 mL of aqueous ammonia and 0.8 mL of 3-aminopropyltriethoxysilane, stirring at room temperature for 12 h, centrifuging and washing with alcohol three times, and removing the supernatant;

[0060] S4, the intermediate product C obtained in S3 was dispersed again in 80 mL of N,N-dimethylformamide solution containing 1 g of succinic anhydride, stirred at room temperature for 6 h, centrifuged and washed with alcohol three times, the supernatant was removed and vacuum dried to obtain the dendritic mesoporous silica with carboxyl groups on the surface, the transmission electron microscope image of which is as shown in Figure 1As shown, the particle size is uniform, and the particle size is about 200nm;

[0061] S5, 2.5 mg of the dendritic mesoporous silica prepared in S4 was added to an aqueous solution containing 48.9 mg of cerium nitrate and 17.3 mg of terbium nitrate, and after ultrasonic reaction for 10 min, 1 mL of an aqueous solution containing 12.4 mg of sodium bifluoride NaHF2 was added, and ultrasonication was continued for 5 min. After centrifugation, the solution was washed three times with water, and the supernatant was removed to obtain a silica-rare earth fluoride nanomaterial DMS-CeF3:Tb, the transmission electron microscope image of which is shown in FIG. Figure 2 As shown, CeF3:Tb is enriched and grown in the pores of dendritic mesoporous silica;

[0062] S6, adding 10 mg of the silica-rare earth fluoride nanomaterial DMS-CeF3:Tb prepared in S5 to 1 mL of an aqueous solution containing 40 mg of sodium polyacrylate, ultrasonicating for 30 min, centrifuging and washing with water three times, and removing the supernatant;

[0063] S7, dispersing the intermediate product D obtained in S6 in 0.5 mL of a buffer solution containing 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and 5 mg of N-hydroxysuccinimide sulfonic acid sodium salt NHS, subjecting the mixture to ultrasonic reaction for 20 min, centrifuging and washing with water three times, and removing the supernatant;

[0064] S8. Disperse the intermediate product E obtained in S7 in a HEPES buffer solution, add 0.6 mg of avidin, and perform a vortex reaction for 30 minutes. After centrifugation, wash three times with water, remove the supernatant, and obtain the avidin-modified silica-rare earth fluoride-based enriched enhanced luminescent bioprobe DMS-CeF3:Tb-avidin, disperse it in 1 mL of an aqueous solution containing 2% bovine serum albumin, and store it in a refrigerator at 4°C for future use.

[0065] Example 2

[0066] This embodiment provides an enrichment-enhanced luminescent bioprobe DMS-CeF3:Tb-biotin based on silicon dioxide-rare earth fluoride, and the preparation method thereof is as follows:

[0067] S1. Add 0.068 g of triethanolamine to 25 mL of ultrapure water and stir at 80 °C for 30 min, then add 380 mg of hexadecyltrimethylammonium bromide and 168 mg of sodium salicylate to the above system and continue stirring and reacting for 60 min, then continue to add 4 mL of tetraethyl orthosilicate and continue to react for 120 min, and then centrifuge to remove the supernatant;

[0068] S2, disperse the intermediate product A obtained in S1 in 91.5 mL of methanol solution containing 1.5 mL of concentrated hydrochloric acid, stir at 60°C for 6 h, centrifuge and wash three times with alcohol, and remove the supernatant;

[0069] S3, dispersing the intermediate product B obtained in S2 in 120 mL of an ethanol solution containing 2.4 mL of aqueous ammonia and 0.8 mL of 3-aminopropyltriethoxysilane, stirring at room temperature for 12 h, centrifuging and washing with alcohol three times, and removing the supernatant;

[0070] S4, the intermediate product C obtained in S3 was dispersed again in 80 mL of N,N-dimethylformamide solution containing 1 g of succinic anhydride, stirred at room temperature for 6 h, centrifuged and washed with alcohol three times, the supernatant was removed and vacuum dried to obtain the dendritic mesoporous silica with carboxyl groups on the surface, the transmission electron microscope image of which is as shown in Figure 1 As shown, the particle size is uniform, and the particle size is about 200nm;

[0071] S5. Add 0.05 mL of ammonia water and 20 mg of biotin to 1 mL of aqueous solution respectively and mix well, then add 5 mg of the silica-rare earth fluoride nanomaterial DMS-CeF3:Tb prepared in S4, ultrasonicate for 30 min, centrifuge and wash three times with water, remove the supernatant, and obtain the biotin-modified silica-rare earth fluoride-based enriched enhanced luminescent bioprobe DMS-CeF3:Tb-biotin, disperse it in 1 mL of ultrapure water, and store it in a refrigerator at 4°C for later use.

[0072] Comparative Example 1

[0073] This comparative example provides a luminescent biological probe of rare earth fluoride, which is different from Example 1 only in that the luminescent biological probe does not contain mesoporous silica. The specific preparation method is as follows:

[0074] (1) Preparation of rare earth fluoride luminescent probes

[0075] In an aqueous solution containing 48.9 mg of cerium nitrate and 17.3 mg of terbium nitrate, 1 mL of an aqueous solution containing 12.4 mg of sodium bifluoride was added and ultrasonication was continued for 5 minutes. After centrifugation, the solution was washed three times with water and the supernatant was removed to obtain rare earth fluoride nanoparticles CeF3:Tb. The transmission electron microscope image is shown in FIG. Figure 3 shown.

[0076] (2) Rare earth fluoride nanomaterial CeF3:Tb surface modified with targeting agent avidin

[0077] 10 mg of CeF3:Tb nanoparticles were added to 1 mL of aqueous solution containing 40 mg of sodium polyacrylate, and the mixture was ultrasonically treated for 30 minutes. The mixture was washed three times with water after centrifugation, and the supernatant was removed. The product was dispersed in 0.5 mL of a buffer solution containing 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 5 mg of N-hydroxysuccinimide sulfonic acid sodium salt (NHS), and the mixture was ultrasonically treated for 20 minutes. The mixture was washed three times with water after centrifugation, and the supernatant was removed. The product was dispersed in a HEPES buffer solution, and 0.6 mg of avidin was added. The mixture was vortexed for 30 minutes, and the mixture was washed three times with water after centrifugation, and the supernatant was removed to obtain avidin-modified rare earth fluoride luminescent bioprobe CeF3:Tb-avidin. The product was dispersed in 1 mL of an aqueous solution containing 2% bovine serum albumin and stored in a refrigerator at 4°C for future use.

[0078] (3) Rare earth fluoride nanomaterial CeF3:Tb surface modified with targeting agent biotin

[0079] Add 0.05 mL of ammonia water and 20 mg of biotin to 1 mL of aqueous solution, mix well, add 5 mg of CeF3:Tb, ultrasonicate for 30 minutes, centrifuge and wash three times with water, remove the supernatant to obtain biotin-modified rare earth fluoride luminescent bioprobe CeF3:Tb-biotin, disperse the product in 1 mL of ultrapure water and store in a refrigerator at 4°C for later use.

[0080] Performance Testing

[0081] 1. Time-resolved heterogeneous detection of prostate-specific antigen (PSA)

[0082] S1. Add 100 μL of 5 μg / mL PSA antibody solution to a 96-well polysorp plate, incubate at 4°C for 12 h, pour off the liquid in the wells, and wash three times with PBST (PBS buffer solution + 0.05% Tween 20);

[0083] S2, add 290 μL 5% bovine serum albumin (BSA) blocking solution, incubate at 37°C for 1.5 h, pour off the liquid in the wells, and wash three times with PBST washing solution;

[0084] S3, add 100 μL of PSA gradient solution (starting from 2.5 μg / mL and diluting 2 times), incubate at 37°C with shaking for 1 h, pour off the liquid in the wells, and wash three times with PBST washing solution;

[0085] S4, add 100 μL of 2 μg / mL Anti-PSA-biotin, incubate at 37°C with shaking for 1 h, pour off the liquid in the well, and wash three times with PBST washing solution;

[0086] S5, adding 100 μL of the enriched enhanced luminescent bioprobe DMS-CeF3:Tb-avidin prepared in step S8 of Example 1 at a concentration of 100 μg / mL, incubating at 37°C with shaking for 0.5 h, pouring off the liquid in the well, and washing three times with PBST washing solution;

[0087] S6. Use the time-resolved mode of the multifunctional microplate reader to read the data of the microplate.

[0088] In addition to the above experimental groups, a control group was set up in which human serum albumin (HSA) solution was used instead of PSA solution, and a control group was set up in which CeF3:Tb-avidin prepared in step (2) of Comparative Example 1 was used instead of DMS-CeF3:Tb-avidin. Except for the above experimental treatments, all experimental operations were the same.

[0089] Test results such as Figure 4 As shown in the figure, it can be seen that the time-resolved fluorescence TRPL signal of the control experimental group using HSA instead of PSA is negligible, indicating that the nonspecific signal interference in this experimental design is relatively small. The detection limit (LOD) is defined as the concentration of the detected substance corresponding to the signal measured in the blank control plus three times the standard deviation. Therefore, the LOD of the in vitro heterogeneous detection of PSA based on the DMS-CeF3:Tb-avidin nanoprobe is calculated to be 0.079 pg·mL -1 The LOD of in vitro heterogeneous detection of PSA based on CeF3:Tb nanoprobe was 10 pg·mL -1 , indicating that using DMS-CeF3:Tb-avidin as a fluorescent probe combined with time-resolved fluorescence detection technology can achieve specific and highly sensitive detection of PSA.

[0090] 2. Cervical cancer cell imaging

[0091] Biotin is a growth promoter at the cellular level, and biotin receptors are overexpressed in a variety of cancer cells, such as cervical (Hela) cancer cells. Due to the high binding affinity between biotin and biotin receptors, biotinylated nanomaterials can specifically target Hela cells after co-incubation with Hela cells.

[0092] S1. Take out the cervical cancer Hela cells from the liquid nitrogen tank, and quickly add 1 mL of DMEM medium into the cell cryopreservation tube to dissolve the frozen cells;

[0093] S2. Use a pipette to remove the dissolved cells and transfer them to a centrifuge tube. Add DMEM medium to 10 mL and centrifuge at 1000 rpm for 5 min to remove the supernatant.

[0094] S3. Add 1 mL of fresh DMEM medium again, draw 20 μL of cell solution into a cell counting plate for counting, fully dilute the cell solution to an appropriate cell concentration, and then draw the cell solution into a cell culture bottle with a pipette and place it in a 37°C cell culture incubator for culture;

[0095] S4. After 2 days, observe the growth of cells in the culture flask under a microscope. If most of the cells grow adherently and no cells are suspended, it means that there is no large-scale cell death and the cell growth is good. Then add 1 mL of trypsin to the cell culture flask and allow the enzyme to react for 3 minutes. After fully mixing the cells in culture, pipette 20 μL of cell solution into a cell counting plate for counting.

[0096] S5. Adjust the cell density to 150,000 cells / mL according to the cell counting results. After sufficient pipetting, pipette 1 mL of cell solution into the cell culture dish and place it back in the 37°C cell culture incubator for culture.

[0097] S6. After 1 day, the cell culture medium in the well was aspirated, and 1 mL of culture medium containing the enriched enhanced luminescent bioprobe DMS-CeF3:Tb-biotin prepared in Example 2 (final concentration of 6 μg / mL) was added and incubated for 4 h;

[0098] S7. Remove the culture medium containing DMS-CeF3:Tb-biotin, wash three times with PBS buffer solution, add 1 mL of culture medium again, and place the cell culture dish under a laser confocal microscope for cell imaging.

[0099] A control group was further set up, and DMS-CeF3:Tb-biotin was replaced by the luminescent bioprobe CeF3:Tb-biotin prepared in Comparative Example 1. Except for the different probes added, all experimental operations were the same.

[0100] The imaging results are as follows Figure 5 As shown in the figure, the cells in the bright field image are irregularly round or spindle-shaped, indicating that the cells are in good condition; Tb can be observed on Hela cells in the experimental group. 3+ The bright green light of Tb was observed on Hela cells in the control group under the same test conditions. 3+ The luminescence of the nanoprobe was enhanced by enriching CeF3:Tb on the mesoporous silica, achieving excellent results in targeted Hela cell imaging.

[0101] 3. Cervical cancer cell detection

[0102] S1. Take out Hela cells from the incubator, wash them twice with PBS buffer solution, and then add 1 mL of trypsin to the cell culture flask for 3 minutes;

[0103] S2, add 6 mL of culture medium and centrifuge, wash twice with culture medium to remove trypsin, and add 1.5 mL of culture medium;

[0104] S3, taking the above 0.5 mL of cell solution into a centrifuge tube, adding 1 mL of culture medium containing the enriched enhanced luminescent bioprobe DMS-CeF3:Tb-biotin prepared in Example 2 (final concentration of 180 μg / mL) and mixing for 4 hours;

[0105] S4. After centrifugation at 500 rpm for 3 minutes, remove the supernatant containing excess DMS-CeF3:Tb-biotin, wash three times with PBS buffer solution, and then add 1 mL of culture medium again. Use a portable UV lamp to directly observe the luminescence of the cell fluid and use a multifunctional microplate reader to collect data.

[0106] In addition to the above experimental groups, a control group without probe, a control group using CeF3:Tb-biotin prepared in Comparative Example 1 instead of DMS-CeF3:Tb-biotin, and a control group using normal liver cells L-02 instead of Hela cells were also set up. Except for the above experimental treatments, all experimental operations were the same.

[0107] Test results such as Figure 6 As shown in the figure, under the irradiation of a portable UV lamp, it can be clearly seen that the cervical cancer cell fluid targeted by DMS-CeF3:Tb-biotin has a strong green luminescence, while the green luminescence of the cell fluid targeted by CeF3:Tb-biotin is weak. In addition, due to the low expression of biotin receptors in normal cells, the luminescence of DMS-CeF3:Tb-biotin or CeF3:Tb-biotin is basically not visible in the L-02 cell fluid.

[0108] Further spectral analysis of the cell fluid in each group was performed, and the results were as follows: Figure 7 As shown. Since the single particle of DMS-CeF3:Tb-biotin has strong luminescence and can specifically bind to Hela cells, the Hela cell fluid targeted by DMS-CeF3:Tb-biotin has the strongest luminescence at 546nm, indicating that the enriched and enhanced luminescent bioprobe based on silica-rare earth fluoride prepared by the present invention can specifically identify cancer cells and realize the in vitro detection of cancer cells.

[0109] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing an enriched and enhanced luminescent bioprobe based on silicon dioxide-rare earth fluoride, characterized in that: Dendritic mesoporous silica is used as a carrier for the confined growth of rare earth fluorides. The enrichment effect of the ultra-large mesopores of dendritic mesoporous silica is utilized to allow a large number of rare earth fluoride nanoparticles to grow in situ in the pores to obtain silica-rare earth fluoride nanomaterials. Targeting molecules are then modified on the surface of the silica-rare earth fluoride nanomaterials to obtain enrichment-enhanced luminescent bioprobes based on silica-rare earth fluorides.

2. The method for preparing a silicon dioxide-rare earth fluoride enrichment-enhanced luminescent bioprobe according to claim 1, characterized in that: The following steps are involved: S1. preparing dendritic mesoporous silica nanoparticles by anion-assisted synthesis, and modifying the surface with carboxyl groups to obtain the dendritic mesoporous silica with carboxyl groups on the surface; S2, adsorbing rare earth cations in the dendritic mesoporous silica pores by electrostatic action, introducing sodium bifluoride NaHF2 to allow rare earth fluoride to grow in situ in the pores to form silica-rare earth fluoride nanomaterials; S3. Introducing a targeting agent into the silica-rare earth fluoride nanomaterial solution prepared in S2 to modify its surface, washing it with water for multiple times after ultrasonic centrifugation and removing the supernatant to obtain the enriched enhanced luminescent bioprobe based on silica-rare earth fluoride.

3. The method for preparing a silicon dioxide-rare earth fluoride enrichment-enhanced luminescent bioprobe according to claim 2, characterized in that: The S1 comprises the following steps: S11, add triethanolamine to ultrapure water and stir at 60-90°C, then add hexadecyltrimethylammonium bromide and sodium salicylate to the above system and continue stirring and reacting for 1 hour, then add tetraethyl orthosilicate and continue stirring and reacting for 2 hours, centrifuge at 12000 rpm for 5 minutes and remove the supernatant; The mass ratio of ultrapure water, triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate is 6250:17:95:42; the volume ratio of ultrapure water to tetraethyl orthosilicate is 25:4; S12, dispersing the intermediate product A obtained in S11 in a methanol solution containing concentrated hydrochloric acid, stirring evenly at 40-90° C., centrifuging at 12000 rpm for 5 minutes, washing three times with anhydrous ethanol, and removing the supernatant; The volume ratio of concentrated hydrochloric acid to methanol is 1:60; the mass ratio of intermediate product A to methanol is 3:350; S13, dispersing the intermediate product B obtained in S12 in an anhydrous ethanol solution containing ammonia water and 3-aminopropyltriethoxysilane, stirring at room temperature for 12 hours, centrifuging at 12000 rpm for 5 minutes, washing three times with anhydrous ethanol, and removing the supernatant; The volume ratio of ammonia water, 3-aminopropyltriethoxysilane and anhydrous ethanol is 3:1:150; the mass ratio of intermediate product B to anhydrous ethanol is 1:160; S14, dispersing the intermediate product C obtained in S13 again in an N,N-dimethylformamide solution containing succinic anhydride, stirring at room temperature for 6 hours, centrifuging at 12000 rpm for 5 minutes, washing three times with anhydrous ethanol, removing the supernatant and vacuum drying to obtain the dendritic mesoporous silica with carboxyl groups on the surface; The mass ratio of intermediate product C, succinic anhydride, and N,N-dimethylformamide is 1:5:379; The particle size of the dendritic mesoporous silica nanoparticles is 5 to 1000 nm, and the pore size is 2 to 50 nm.

4. The method for preparing a silicon dioxide-rare earth fluoride enrichment-enhanced luminescent bioprobe according to claim 2, characterized in that: The S2 is prepared by adding the dendritic mesoporous silica prepared by S1 to an aqueous solution containing rare earth nitrate, and then adding a sodium bifluoride NaHF2 aqueous solution for 1 to 6 minutes of ultrasonic reaction, and then continuing ultrasonication for 1 to 6 minutes, centrifuging at 8000 rpm for 3 minutes, and then washing three times with water, and removing the supernatant to prepare a silica-rare earth fluoride nanomaterial; The mass ratio of the dendritic mesoporous silica, rare earth nitrate and sodium bifluoride NaHF2 is 1:35:5; The rare earth cation is Ce 3+ , Tb 3+ 、Nd 3+ 、Eu 3+ Any one or a combination of two.

5. The method for preparing a silicon dioxide-rare earth fluoride enrichment-enhanced luminescent bioprobe according to claim 2, characterized in that: The targeting agent in S3 is biotin or avidin.

6. The method for preparing a silicon dioxide-rare earth fluoride enrichment-enhanced luminescent bioprobe according to claim 5, characterized in that: When the targeting agent is avidin, the modification method is as follows: S31, adding the silicon dioxide-rare earth fluoride nanomaterial prepared in S2 to the aqueous solution containing sodium polyacrylate, ultrasonicating for 30 to 35 minutes, centrifuging at 8000 rpm for 5 minutes, washing with water three times, and removing the supernatant; S32, dispersing the intermediate product D obtained in S31 in a buffer solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and N-hydroxysuccinimide sulfonic acid sodium salt NHS, reacting at 200W ultrasonic power for 20-25min, then centrifuging at 8000rpm for 5min, washing with water three times, and removing the supernatant; S33, dispersing the intermediate product E obtained in S32 in a 4-hydroxyethylpiperazineethanesulfonic acid HEPES buffer solution, adding avidin, vortexing at 120 rpm for 30 to 35 min, centrifuging at 8000 rpm for 5 min, washing three times with water, removing the supernatant, and obtaining the avidin-modified enrichment-enhanced luminescent bioprobe based on silica-rare earth fluoride; The mass ratio of sodium polyacrylate, silica-rare earth fluoride nanomaterials, EDC, NHS and avidin is 8:2:2:1:0.

12.

7. The method for preparing a silicon dioxide-rare earth fluoride enrichment-enhanced luminescent bioprobe according to claim 5, characterized in that: When the targeting agent is biotin, the modification method is as follows: Ammonia water and biotin were added to 1 mL of ultrapure aqueous solution to mix the two evenly, and then the silica-rare earth fluoride nanomaterial prepared in S2 was added, and after ultrasonication for 30 to 35 minutes, the mixture was centrifuged at 8000 rpm for 5 minutes, washed with water three times, and the supernatant was removed to obtain the biotin-modified silica-rare earth fluoride-based enrichment enhanced luminescent bioprobe; The mass ratio of ammonia water, biotin, and silicon dioxide-rare earth fluoride nanomaterials is 9:4:

1.

8. An enriched and enhanced luminescent bioprobe based on silicon dioxide-rare earth fluoride prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the enrichment-enhanced luminescent bioprobe based on silica-rare earth fluoride as claimed in claim 8 in in vitro detection of cancer markers in combination with time-resolved fluorescence detection technology.

10. Use of the enriched and enhanced luminescent bioprobe based on silica-rare earth fluoride as claimed in claim 8 in specific recognition of cancer cells in vitro and imaging of cancer cells based on fluorescence targeting.

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