Functional nanoparticles, probes and applications thereof

Abstract

The application discloses a kind of functional nanoparticles and its preparation probe and application, belong to nano fluorescent probe field.The novel and general method of the present application is coupled to the un-derivatized carbohydrate on the surface of silicon dioxide nanoparticles (SiNPs) by photochemical induction of azido naphthalimide.Under ultraviolet (UV) irradiation, it is proved that azido naphthalimide is activated, and the generated aminonaphthalimide intermediate is conjugated with various bioactive molecules.The carbohydrate attached to the surface of this nanoscale platform not only maintains strong binding capacity with the corresponding lectin, but also can realize bioimaging in complex protein environment.The present application shows broad application prospects in the interaction between various bioactive molecules and proteins.

Description

Technical Field

[0001] This invention belongs to the field of nanofluorescent probes and relates to the design and application of a multifunctional platform of azidonaphthalimide-functionalized silicon nanoparticles. Background Technology

[0002] Nanoparticles (NPs) have become powerful tools in chemistry, biology, and biomedicine due to their size, comparable to biomolecules but much smaller than human cells, making them ideal for biological applications, including mimicking cell-cell interactions, bridging physiological barriers, and exploring biological mechanisms. For example, carbohydrates play a crucial role in mediating cell recognition events, including adhesion and signal transduction, through their interactions with cell surface proteins such as lectins. Studying carbohydrate-lectin interactions is essential for understanding cellular processes and improving drug delivery in living systems; however, these interactions are often weak, posing challenges to efficient analysis. Nanomaterials offer unique advantages for studying carbohydrate-protein interactions due to their small size, high surface area, and tunable physicochemical properties. Their large specific surface area facilitates multivalent interactions, and the size and shape of nanomaterials can be tailored to control ligand density and expose multiple epitopes in three dimensions.

[0003] To date, gold, iron oxide, and silica nanoparticles have been the most commonly used scaffold materials, primarily prepared by grafting glycopolymers onto their surfaces. However, gold nanoparticles often suffer from high cost and limited chemical stability, while iron oxide nanoparticles may exhibit magnetic aggregation and relatively low surface functionalization efficiency. In contrast, silica nanoparticles (SiNPs) can be mass-produced using simple processes and possess excellent chemical stability, tunable size and porosity, and abundant surface hydroxyl groups, making them a highly suitable and versatile scaffold material for glycopolymer grafting and subsequent biomedical applications. However, the design and application of silica-based glyconanomaterials face two key challenges. One lies in designing suitable surface linking chemistry, which must be tailored to the composition and morphology of the nanomaterial. The other challenge is that protein labeling imaging and characterization often require additional fluorescent probes, which can interfere with ligand labeling and ligand-protein interactions. Summary of the Invention

[0004] Based on the multivalent copying effect of nanoparticles and the molecular properties of azidonaphthalimide, this invention designs a nanoplatform based on azidonaphthalimide for photochemical coupling of underived ligands. This platform is simple to operate, highly reproducible, emits UV-activated green fluorescence for imaging, maintains ligand recognition capability, and has a high ligand loading capacity, thus enabling bacterial detection and imaging of various cells.

[0005] The technical solution of this invention: A functional silica nanoparticle, comprising a nanoparticle matrix, a linker arm, and a photoreactive group connected sequentially, with the following general structural formula:

[0006]

[0007] The nanoparticle matrix is ​​silica nanoparticles; the photoreactive group Q is selected from:

[0008] ;

[0009] Where n is an integer from 8 to 15, and m is an integer from 0 to 5.

[0010] The structure of the nanoparticles is as follows:

[0011] .

[0012] Preferably, the structure of the nanoparticles is as follows:

[0013]

[0014] Where n is an integer from 8 to 12, and m is an integer from 1 to 5.

[0015] A method for preparing functional silica nanoparticles includes the following steps:

[0016] Step 1: The silica nanoparticles are subjected to aminated functionalization treatment to obtain aminated silica nanoparticles:

[0017] Step 2: Connecting arms are attached to the surface of the aminated silica nanoparticles to obtain silica nanoparticles with surface-grafted connecting arms.

[0018] Step 3: The photoreactant is coupled with the silica nanoparticles with surface grafted connecting arms obtained in Step 2 to obtain the coupling product, namely functional silica nanoparticles.

[0019] Furthermore, in step 1, the amination functionalization treatment is performed using a silane coupling agent, wherein the silane coupling agent is an amino-containing silane coupling agent.

[0020] In step 2, connecting arms are attached to the surface of the aminated silica nanoparticles. The structure of the connecting arms is as follows: One end of it is bound to the amino group on the surface of the aminated silica nanoparticles, and the other end is connected to the photoreactant.

[0021] In step 3, the coupling reaction is carried out under light-protected conditions, and the structure of the photoreactant is as follows:

[0022] .

[0023] A specific probe, wherein the functional silica nanoparticles and specific ligands described above are prepared;

[0024] The specific ligand is:

[0025] .

[0026] Furthermore, the structure of the specific probe is as follows:

[0027] The probe prepared from the functional silica nanoparticles and specific ligand 12 is denoted as Probe 4.

[0028] The probe prepared from the functional silica nanoparticles and specific ligand 13 is denoted as Probe 5.

[0029] The probe prepared from the functional silica nanoparticles and specific ligand 14 is denoted as Probe 6.

[0030] The probe prepared from the functional silica nanoparticles and specific ligand 15 is denoted as Probe 7.

[0031] The nitride structure of the functional silica nanoparticles and the CH, NH, and OH bonds in the ligands can all be covalently bonded. Probe 4-Probe 7 are only for illustrative purposes.

[0032] A method for preparing a specific probe includes the following steps:

[0033] Step a: Mix the functional silica nanoparticles and the specific ligand in a solution;

[0034] Step b: Specific probes are obtained by reacting with low-power ultraviolet light, washing, and centrifuging.

[0035] The power of the low-power ultraviolet light is 67 mW / cm. 2 The irradiation time is 30s-90s.

[0036] The specific probes are used as follows: Probe 4 is used for specific recognition ability testing of Escherichia coli; Probe 5 is used for fluorescence imaging of HepG2 membrane proteins; Probe 6 has specific imaging ability for MCF-7 endogenous carbonic anhydrase II; and Probe 7 is used for photoaffinity labeling of human serum albumin.

[0037] A method for coupling a light-controlled probe, azidonaphthalimide, onto silica nanoparticles (SiNPs) includes the following steps:

[0038] (1) Amination-functionalization treatment was performed on silica nanoparticles with a particle size of 50 nm to obtain amination-functionalized silica nanoparticles; the amination-functionalization treatment was performed by modifying with an amino-containing silane coupling agent to ensure that amino active sites were introduced on the surface of the silica nanoparticles, providing a basis for the subsequent connection of the connecting arms.

[0039] (2) Connecting linkers are attached to the surface of aminated silica nanoparticles to obtain silica nanoparticles with surface-grafted linkers; the linker is tridecanoic acid, which has dual reactive activity, with one end bound to the amino group on the surface of the aminated silica nanoparticles and the other end bound to the azidonaphthalimide molecule, thus acting as a bridge connecting the carrier and the photoreactive group.

[0040] (3) The azidonaphthalimide molecule is coupled with the silica nanoparticles with surface grafted connecting arms obtained in step (2) to obtain the coupling product, namely the light-controlled probe azidonaphthalimide functionalized silica nanoparticles; the coupling reaction is carried out at room temperature or low temperature, the reaction is amide condensation, and the covalent bond is used to ensure the coupling stability.

[0041] Furthermore, in the coupling product prepared by the method, azidonaphthalimide, as a photoreactive group, can be converted into aminonaphthalimide under low-power, short-duration ultraviolet light irradiation, achieving rapid photochemical conversion and exhibiting fluorescence properties for target visualization; wherein the power of the low-power ultraviolet light is 67 mW / cm². 2 The irradiation time is 30-90 seconds, the photoactivation conditions are mild, and there is no need for high-power long-term ultraviolet light irradiation.

[0042] Furthermore, the preparation process of the method is simple and does not require derivatization of carbohydrates or other small molecule ligands. The carbohydrates include galactose and mannose, and the other small molecule ligands include benzenesulfonamide and ibuprofen. The above ligands can directly bind to the coupling product and can effectively retain their original molecular recognition ability.

[0043] Furthermore, using mannose-binding protein concanavalin A as a model, mannose was bound to the coupling product by photoactivation to prepare glycosyl nanomaterials, which were used to verify the specific interaction between mannose and fluorescently labeled concanavalin A (FITC-Con A).

[0044] Furthermore, the conjugation product can be used to photocouple different ligands to prepare specific probes. Specific applications include: conjugating galactose onto the conjugation product to prepare Probe 5, which is used for fluorescence imaging experiments of HepG2 membrane proteins; conjugating benzenesulfonamide onto the conjugation product to prepare Probe 6, which has specific imaging capability for MCF-7 endogenous carbonic anhydrase II; and photochemically conjugating ibuprofen onto the conjugation product to prepare Probe 7, which is used for photoaffinity labeling of human serum albumin.

[0045] The preparation steps and reaction formulas of the silica nanofluorescent probe of the present invention are as follows:

[0046]

[0047] Aminolation modification (A→B): Using silica nanospheres (A) as raw material, whose surface is rich in silanol groups (Si-OH), a silanization reaction is carried out with 3-aminopropyltriethoxysilane (APTES) in ethanol solvent to obtain aminated silica (B), which provides active sites for subsequent coupling.

[0048] Carboxylation modification (B→C): Materials B, HBTU, and DIPEA undergo amidation reaction with dodecanoic acid in the DMF system: HBTU acts as a condensing agent to activate the ester, and the amino group on the surface of B nucleophilically attacks the activated ester carbonyl group to form an amide bond. The long-chain alkyl group can reduce steric hindrance and improve the reactivity of subsequent probes.

[0049] Probe coupling (C→Probe 1): Using carboxylated silica (C) as raw material, a second amidation reaction is carried out with N-(3-aminopropyl)-4-azido-1,8-naphthalenediamine in the same HBTU / DIPEA / DMF condensation system.

[0050] The preparation process of compound 3 is as follows:

[0051]

[0052] Compound 1 reacts with NaN3 in a DMF / water mixed solvent with an aromatic nucleophilic substitution reaction to generate 4-azido-1,8-naphthalenedicarboxylic anhydride (compound 2).

[0053] The reaction temperature was controlled at 85℃. Compound 2 reacted with excess 1,2-diaminoethane in ethanol solvent to undergo an imidization reaction to obtain compound 3.

[0054] The beneficial effects of this invention are as follows: This invention uses silica nanoparticles modified with azidonaphthalimide as the core, which can be activated under short-term low-power ultraviolet light irradiation. The azidonaphthalimide generates a highly reactive intermediate, a nitroene, exhibiting characteristics such as efficient coupling reaction, high yield, simple preparation, good applicability, and convenient target enrichment. Simultaneously, azidonaphthalimide, as a photoreactive group, can be converted into aminonaphthalimide under ultraviolet light excitation, achieving rapid photochemical conversion and possessing fluorescence properties, thus enabling target visualization. The fluorescent probe provided by this invention is applicable to specific identification in cell fluorescence labeling and bacteria, and exhibits specific labeling of HSA with good biosafety.

[0055] This invention presents a near-infrared photoactivated nanoplatform for protein labeling, enrichment, and visualization in in vivo systems. Using azidonaphthalimide as the photoreactive group, target proteins can be easily labeled and separated. The photoreactive group of azidonaphthalimide can be converted to aminonaphthalimide, a fluorescent group with long-wavelength emission (maximum emission wavelength ~540 nm), thus enabling simultaneous visualization of target proteins. Based on the excellent labeling and imaging capabilities of azidonaphthalimide, azidonaphthalimide-loaded SiNPs can be developed into a promising platform for studying carbohydrate-lectin interactions and other protein-ligand interactions.

[0056] These nanoparticles can be briefly activated under low-power UV light, exhibiting highly efficient reactive binding, high yield, simple preparation, good applicability, and convenient target enrichment properties. Simultaneously, the photoactive group, azidonaphthalimide, can be converted to aminonaphthalimide, achieving rapid photochemical transformation under UV irradiation and enabling simultaneous visualization of the target analyte. Most importantly, its applicability for fluorescent labeling in cells and bacteria has been successfully demonstrated, with good safety profiles. This nanoplatform can be developed into a novel photochemical tool for future use in the photochemical coupling of drugs for drug delivery. Attached Figure Description

[0057] Figure 1 This is a graph showing the changes in UV absorption of azidonaphthalimide before and after UV irradiation.

[0058] Figure 2 This is a graph showing the fluorescence intensity changes of azidonaphthalimide.

[0059] Figure 3 This is a graph showing the change in ultraviolet absorption of diazinon under ultraviolet light irradiation over time.

[0060] Figure 4 This is a standard curve prepared by measuring the absorbance of D-mannose at 490 nm using the phenol-sulfuric acid method.

[0061] Figure 5 The absorbance was measured at 490 nm using the phenol / sulfuric acid method after D-mannose was bound to the three probes.

[0062] Figure 6 This is a competitive analysis spectrum of FITC-Con A fluorescence.

[0063] Figure 7 TEM images after incubation with Probe 4 and E. coli: (a) group without inhibitor (b) group with inhibitor.

[0064] Figure 8 Probe 5 is used for ASGPR fluorescence imaging on the surface of HepG2 cells.

[0065] Figure 9 Probe 6 is used for fluorescence imaging of MCF-7 endogenous carbonic anhydrase II.

[0066] Figure 10 The photoaffinity labeling of Probe 7 to HSA was determined using the Coomassie brilliant blue method.

[0067] Figure 11 This is a cytotoxicity assay of Probe 5 on HepG2 cells.

[0068] Figure 12 This is a cytotoxicity assay of Probe 5 on HeLa cells.

[0069] Figure 13 This is a cytotoxicity assay of Probe 6 on MCF-7 cells.

[0070] Figure 14 This is a cytotoxicity assay of Probe 6 on HeLa cells. Detailed Implementation

[0071] A novel light-triggered nanoprobe for ligand-protein interactions and cell imaging comprises a nanoplatform, a connecting arm, and a photoreactive group, and has the following structure:

[0072]

[0073] Step 1: The 50 nm silica nanoparticles of the nanoplatform are used as the core for amination and functionalization to obtain amination-modified silica nanoparticles.

[0074] ;

[0075] Step 2: Connecting arms are attached to the surface of the aminated silica nanoparticles to obtain silica nanoparticles with surface-grafted connecting arms.

[0076] ;

[0077] Step 3: The photoreactive group azidonaphthalimide is coupled with the silica nanoparticles with surface grafted connecting arms obtained in Step 2 to obtain the coupling product, namely the photocontrolled probe azidonaphthalimide coupled silica nanoparticles.

[0078] The process of amylating and functionalizing silica nanoparticles involves modifying them with a silane coupling agent, wherein the silane coupling agent is an amino-containing silane coupling agent.

[0079] The method involves attaching connecting arms to the surface of aminated silica nanoparticles. The intermediate connecting arms are selected from tridecanoic acid with a spacing of n = 11. One end of the tridecanoic acid is bonded to an amino group on the surface of the aminated silica nanoparticles, and the other end is bonded to a naphthalene azidoimide molecule.

[0080] The coupling reaction described in step 3 should be carried out under light-protected conditions, and the coupling reaction adopts a covalent bond bonding mode.

[0081] In the coupling product prepared by the method, azidonaphthalimide acts as a photoreactive group, which can be converted into aminonaphthalimide under low-power, short-duration ultraviolet light irradiation, achieving rapid photochemical conversion and exhibiting fluorescence properties for target visualization. The low-power ultraviolet light has a power of 67 mW / cm². 2 The irradiation time is 10–60 seconds.

[0082] The preparation process described is simple and does not require derivatization of carbohydrates or other small molecule ligands.

[0083] The carbohydrate or other small molecule ligands can bind directly to the coupling product and effectively retain their original molecular recognition capabilities.

[0084] The phototriggered nanoprobes can be used to prepare specific probes by photocoupling different ligands. Specific applications include: conjugating mannose onto the nanoprobes to prepare Probe 4, which is used for specific recognition testing of E. coli; conjugating galactose onto the conjugation product to prepare Probe 5, which is used for fluorescence imaging of HepG2 membrane proteins; conjugating benzenesulfonamide onto the conjugation product to prepare Probe 6, which has specific imaging ability for MCF-7 endogenous carbonic anhydrase II; and photochemically conjugating ibuprofen onto the conjugation product to prepare Probe 7, which is used for photoaffinity labeling of human serum albumin.

[0085] Example 1

[0086] Preparation of compound 3:

[0087]

[0088] Step a: Synthesis of Compound 2: Compound 1 (2.77 g, 10 mmol) was dissolved in a 100 mL round-bottom flask, and a suspension containing 50 mL of dimethylformamide (1.30 g, 20 mmol) and 3.0 mL of water was added. The mixture was stirred at room temperature for 10 h, and then the solution was slowly poured into ice water. The precipitated solid was filtered, washed with water, and dried under vacuum to give Compound 2 as a pale yellow solid in 71% yield.

[0089] Step b: Compound 2 (1.43 g, 6 mmol) was placed in a 50 mL round-bottom flask, and 20 mL of an ethanol solution of 2-diaminoethane (1.44 g, 24 mmol) was added. The reaction mixture was heated at 85°C for 4 h. After the mixture cooled to room temperature, a yellow precipitate was filtered off. The solvent was then separated under reduced pressure. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH = 20:1) to give compound 3 as a pale yellow solid in 52% yield. The structure was identified by 1H NMR. 1 H NMR (400MHz, CDCl3) δ 8.63 (d, J = 8.1 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.43 (d, J= 8.4 Hz, 1H), 7.76 - 7.72 (m, 1H), 7.46 (d, J = 7.9 Hz, 1H), 4.28 - 4.25 (m,2H), 3.08 - 3.05 (m, 2H). 13 C NMR (100 MHz, CDCl3) δ 164.3, 163.9, 143.6,132.3, 131.9, 129.2, 128.9, 126.9, 124.3, 122.5, 118.7, 114.7, 43.1, 40.4;MS-ESI m / z [M+H] + calcd for C 14 H 12 N5O2, 282.1; found, 282.1.

[0090] Preparation of compound 8:

[0091]

[0092] Steps c and d: Compound 4 (8.19 g, 70.5 mmol) was dissolved in 7N ammonia-methanol solution (70 mL methanol), and the mixture was stirred at 0 °C for 3 h until hydroxylamine-O-sulfonic acid (9.16 g, 81.1 mmol) was dissolved in methanol (60 mL) and added. The reaction mixture was stirred further overnight at room temperature, and then placed in a fume hood for 10 min to remove excess ammonia. The mixture was removed by suction filtration, and the filtrate was concentrated to give compound 5. Compound 5 was dissolved in methanol, and the mixture was cooled to 0 °C. Triethylamine (15 mL) was added to the solution, and the mixture was stirred for 5 min. Iodine was added to the solution until the solution turned reddish-brown. The solution was diluted with ethyl acetate, and the organic layer was washed with 1 mol / L hydrochloric acid, 10% sodium thiosulfate, and saturated brine. The organic layer was concentrated under reduced pressure to give compound 6 in 25% yield.

[0093] Step e: Compound 6 (167 mg, 1.3 mmol) was dissolved in CH₂Cl₂, and then DIPEA (0.33 mL, 1.5 eq) and HATU (746 mg, 1.5 eq) were added, respectively. The reaction mixture was stirred at room temperature for 1 h, and then N-Boc-ethylenediamine (0.18 mL, 0.8 eq) was added. After stirring for another 16 h, the reaction mixture was concentrated, then extracted with ethyl acetate, washed three times with saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure. The product was then purified by silica gel column chromatography to give product 7 in 35% yield.

[0094] Step f: 0.5 mL of trifluoroacetic acid (TFA) and compound 7 (138.8 mg) were added to a CH2Cl2 solution (5 mL) at 0 °C with stirring. The reaction mixture was stirred at room temperature for 5 h. The solvent was removed under vacuum to give the product in 99% yield. The structure was identified by 1H NMR. 1 H NMR (400 MHz, CD3OD) δ 3.48 - 3.45 (m, 2H), 3.10 -3.07 (m, 2H), 2.15-2.11 (m, 2H), 1.67 - 1.63 (m, 2H), 0.97 (s, 3H); 13 C NMR (100 MHz, CD3OD): δ 174.5, 39.4, 36.8, 29.7, 24.9, 18.2; MS-ESI m / z [M+H] + calcd for C7H 15 N4O, 171.2; found, 171.2.

[0095] Preparation of compound 11:

[0096]

[0097] Step g: In a 50 mL round-bottom flask, 4-benzoylbenzoic acid (compound 9) (1.13 g, 5 mmol) and HBTU (2.28 g, 6 mmol) were placed in 10 mL of dimethylformamide (DMF), followed by the addition of DIPEA (1.24 mL, 7.5 mmol) and N-Boc-ethylenediamine (1.20 g, 7.5 mmol), and the mixture was stirred for 5 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (using CH2Cl2:MeOH = 30:1 as eluent) to give compound 10 in 70% yield.

[0098] Step h: Trifluoroacetic acid (TFA) was added to 20 mL of DCM containing 1.29 g (3.5 mmol) of compound 10 at 0 °C, and the mixture was stirred at room temperature for 5 h. The solvent was removed under vacuum to give the product in 99% yield. The structure was identified by 1H NMR. 1 H NMR (400 MHz, DMSO-d6) δ 8.85 - 8.82 (m, 1H), 8.03 (d, J = 8.1 Hz,2H), 7.89 (s, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 7.8 Hz, 2H), 7.71(d, J = 7.4 Hz, 1H), 7.61 - 7.57 (m, 2H), 3.57 - 3.52 (m, 2H), 3.04 - 3.00(m, 2H); 13 C NMR (100 MHz, DMSO-d6): δ 195.9, 166.6, 139.8, 137.8, 137.1,133.6, 130.2, 129.9, 129.2, 128.0, 39.0, 37.7; MS-ESI m / z [M+H] + calcd forC 16 H 17 N2O2, 269.2; found, 269.2.

[0099] Preparation of Probe 1:

[0100]

[0101] 120 mg of material A was suspended in 30 mL of ethanol, and APTES (1.014 mL, 4.3 mmol) was added. The solution was stirred at room temperature for 18 hours, centrifuged to obtain material B, and washed with ethanol.

[0102] 60 mg of material B was suspended in 1 mL of anhydrous dimethylformamide (DMF), followed by the addition of dicarboxylic acid (0.225 mmol), diisopropylethylamine (40 μL), and HBTU (56.45 mg). After stirring overnight, the mixture was collected by centrifugation and washed with DMF. The precipitate was collected by centrifugation after three washes to obtain material C.

[0103] 60 mg of SiO2-1 was suspended in 1 mL of anhydrous dimethylformamide (DMF), followed by the addition of compound 3 (0.225 mmol), DIPEA (40 uL) and HBTU (56.45 mg). After stirring overnight, the nanoparticles were collected by centrifugation and washed with DMF to obtain Probe 1.

[0104] Referring to the steps above, use respectively , , The three photoreactive groups can be used to prepare the following three probes: Probe 1, Probe 2, and Probe 3.

[0105]

[0106] Preparation of Probe 4:

[0107]

[0108] 5 mg of Probe 1 was suspended in 100 mmol of D-mannose (compound 12) aqueous solution and then sonicated for 20 min to ensure the homogeneity of the suspension. Subsequently, it was irradiated with a 365 nm ultraviolet lamp with a power of P = 67 mW / cm2 for 60 s, and then washed three times with pure water and centrifuged to obtain Probe 4.

[0109] Referring to the steps above, use respectively , , The three ligands were photochemically coupled to Probe 1 to prepare the following probes: Probe 5, Probe 6, and Probe 7.

[0110] The intermediate nitrobenzene structure and the CH, NH, and OH bonds in the ligand can all be covalently bonded. Probe 4-Probe 7 are only for illustrative purposes.

[0111] Example 2

[0112] Screening experiment for photoreactive groups. The prepared mother solutions of compounds 3 and 8 were diluted to 100 μmol / L and 50 mmol / L, respectively, to make the total volume of the final test system 500 μL. UV-Vis absorption spectroscopy was then performed in a quartz cuvette. Figure 1 As shown, compound 3 at a power of 67 mW / cm 2 After 30 seconds of irradiation with a 365 nm wavelength ultraviolet lamp, the absorption peak attenuated significantly, but the absorption wavelength showed a noticeable red shift. According to... Figure 2 Fluorescence spectroscopy shows rapid fluorescence emission after 5 seconds of illumination, compared to compound 8, bisacrylidine ( Figure 3 The ultraviolet-visible absorption spectrum of the light indicates that a relatively long light reaction rate is required.

[0113] like Figure 4 As shown, different concentrations of D-mannose were reacted with phenol / sulfuric acid, and the absorbance at 490 nm was measured to obtain calibration curves. The D-mannose content bound to silica nanoparticles was determined using the phenol-sulfuric acid method. Different probes were optimized and screened, with higher D-mannose binding capacity being the preferred condition. Probe 1 bound 13.6 nmol / mg of mannose, while Probe 2 and Probe 3 bound 10.5 nmol / mg and 8.9 nmol / mg of mannose, respectively. Probe 1 was the optimal choice. Figure 5 ).

[0114] Example 3

[0115] Verification experiments on the specific interaction between glycosylated nanomaterials and lectins. Concanavalin A (Con A) was analyzed as a model target protein. It is known that Con A specifically binds to D-mannose, exhibiting high specificity and low affinity. A fluorescence-based competitive assay was used to quantitatively determine the binding affinity between glycosylated nanoparticles and lectins. FITC-labeled Con A was incubated with Probe 4. After centrifugation, as shown... Figure 6 The fluorescence intensity of the Probe 4 experimental group was significantly lower than that of the group with the added D-mannose inhibitor, while the decrease was less pronounced in the group with the added D-galactose inhibitor. Two equilibria were simultaneously established in this system: the binding of FITC-Con A to free D-mannose, and the binding of FITC-Con A to D-mannose linked to the nanoparticles. Both interactions were reversible and rapidly reached a stable equilibrium.

[0116] Example 4

[0117] The experiment tested the specific recognition ability of Probe 4 against *E. coli*. A single *E. coli* colony was picked from solid culture medium and inoculated into 950 μL of liquid culture medium. Probe 4 (0.5 mg / 50 μL) was added to the bacterial culture. The culture was incubated at 37°C under aerobic conditions with shaking for 6 h. One group also included the competitive inhibitor D-(+)-mannose (300 μmol / L). The sample was then centrifuged at 4000 rpm for 3 min, the supernatant was discarded, the precipitate was washed with 1 mL of ultrapure water and centrifuged again, then resuspended in 1 mL of water. The copper mesh containing the sample was dried and used for transmission electron microscopy (TEM) imaging. Figure 7 As shown, Probe 4 aggregates were clearly observed adhering to both ends of E. coli. However, this phenomenon was significantly inhibited in the presence of a mannose competitive inhibitor, confirming that Probe 4 achieves specific bacterial recognition through the specific binding of mannose to fimbriae FimH lectins.

[0118] Example 5

[0119] Probe 5 was used for fluorescence imaging of HepG2 membrane proteins. HepG2 / HeLa cell lines were cultured in DMEM medium containing 10% serum and incubated in a 37% 5% CO2 cell culture incubator until cell adhesion was achieved. The cells were digested with trypsin the following day, and cell counts were performed. Cells were counted at a rate of 1×10⁻⁶. 5 One HepG2 / HeLa cell line was seeded into a confocal glass culture dish and cultured overnight. (The text abruptly ends here.) Figure 8 In Group a, when Probe 5 was co-incubated with HepG2 cells, strong green fluorescence was observed in these cells. In Group b, green fluorescence was still observed when D-mannose was added as an inhibitor, indicating that D-mannose is a non-competitive inhibitor. Groups c and d, where D-galactose was added as an inhibitor to HepG2 and HeLa cells respectively, showed no obvious green fluorescence. Nuclear staining using the Hoechst 33342 staining kit showed blue fluorescence. Given the high expression of the ASGPR protein receptor in the liver, and the widely accepted recognition motif of galactose as the desialylate glycoprotein receptor (ASGPR), this process is mediated through the interaction of D-galactose with ASGPR in HepG2 cells.

[0120] Example 6

[0121] Fluorescence imaging experiment of MCF7 cells. Using the same cell culture method as in Example 4, 1×10 5One MCF7 / HeLa cell was seeded into a confocal glass culture dish and cultured overnight. (The rest of the text appears to be unrelated and likely refers to a different topic.) Figure 9 As shown, in Group a, significant green fluorescence was observed in MCF7 cells using Probe 6, indicating successful endogenous imaging. In Group b, where 4-(2-aminoethyl)benzenesulfonamide was added as an inhibitor, the fluorescence signal was competitively inhibited by 4-(2-aminoethyl)benzenesulfonamide, and the fluorescence intensity was significantly reduced. In Group c, the green fluorescence in HeLa cells almost disappeared. This experiment verifies that Probe 6 has specific imaging capabilities for endogenous carbonic anhydrase II.

[0122] Example 7

[0123] Photoaffinity labeling assay of human serum albumin. For the control group, lane 1 used 5 μg of human serum albumin (HSA); lane 2 used Probe 7; and in the competitive inhibition assay, Probe 7 and inhibitors including ibuprofen (lane 3), oxaliplatin (lane 4), and camptothecin (lane 5) were added. To verify the drug delivery potential of this nanoplatform, HSA was labeled with Probe 7 using the Coomassie Brilliant Blue assay. Figure 10 As shown, probe 7 achieved a labeling efficiency of 58% for HSA, which decreased to 19.4% in a competition experiment with excess ibuprofen, indicating a strong inhibitory effect. Drugs with different binding sites (camptothecin and oxaliplatin) showed weaker inhibitory effects. This study developed a nanoplatform for protein labeling, providing a new method for ligand-protein specific recognition and also applicable to drug delivery, offering promising prospects for future tumor diagnosis.

[0124] Example 8

[0125] The MTT assay was used to evaluate probe cytotoxicity. HepG2 / MCF-7 / HeLa cell lines were cultured in DMEM medium containing 10% serum and incubated in a 37% 5% CO2 cell culture incubator until cell adhesion was achieved. The following day, after trypsin digestion, cells were counted and seeded into 96-well plates (7000 cells / well, 100 μL of medium per well containing 10% FBS and 1% penicillin and streptomycin). After incubation for 24 h, the old medium was removed, and 100 μL of fresh medium containing different concentrations of Probe 5 / Probe 6 was added. The blank group was the PBS group, and the control group contained only cells without the drugs. The plates were incubated in a 37% 5% CO2 cell culture incubator for 24 h. Afterward, 0.5 mg / mL MTT solution was prepared, and DMEM medium containing 10% MTT solution was added to each well. The plates were incubated in a 37% 5% CO2 cell culture incubator for 4 h. The medium was removed, and 150 μL of DMSO was added to each well, shaken for 5 min to dissolve the formazan crystals. The absorbance of each well at 570 nm was measured using a microplate reader. Figure 11 , 12 As can be seen from 13 and 14, the probe did not show significant cytotoxicity to the HepG2 / MCF-7 / HeLa cell lines, indicating that it has reliable safety.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A functional silica nanoparticle, characterized in that: Functional nanoparticles consist of three parts connected in sequence: a nanoparticle matrix, a linker arm, and a photoreactive group. Their general structural formula is: ； The photoreactive group Q is selected from: ； Where n is an integer from 8 to 15, and m is an integer from 0 to 5.

2. The functional silica nanoparticles according to claim 1, characterized in that, The structure of the nanoparticles is as follows: 。 3. The functional silica nanoparticles according to claim 1, characterized in that, The structure of the nanoparticles is as follows: ； Where n is an integer from 8 to 12, and m is an integer from 1 to 5.

4. A method for preparing functional silica nanoparticles according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: The silica nanoparticles are subjected to aminated functionalization treatment to obtain aminated silica nanoparticles: Step 2: Connecting arms are attached to the surface of the aminated silica nanoparticles to obtain silica nanoparticles with surface-grafted connecting arms. Step 3: The photoreactant is coupled with the silica nanoparticles with surface grafted connecting arms obtained in Step 2 to obtain the coupling product, namely functional silica nanoparticles.

5. The preparation method according to claim 3, characterized in that: In step 1, the amination functionalization treatment is performed by modifying with a silane coupling agent, wherein the silane coupling agent is an amino-containing silane coupling agent; In step 2, connecting arms are attached to the surface of the aminated silica nanoparticles. The structure of the connecting arms is as follows: One end of it is bound to the amino group on the surface of the aminated silica nanoparticles, and the other end is connected to the photoreactant. In step 3, the coupling reaction is carried out under light-protected conditions, and the structure of the photoreactant is as follows: 。 6. A specific probe, characterized in that: The probe is prepared from the functional silica nanoparticles and specific ligands described in claim 3; The specific ligand is: 。 7. A specific probe according to claim 6, characterized in that, The probe prepared from the functional silica nanoparticles of claim 3 and the specific ligand 12 is denoted as Probe 4; The probe prepared from the functional silica nanoparticles of claim 3 and the specific ligand 13 is denoted as Probe5; The probe prepared from the functional silica nanoparticles of claim 3 and the specific ligand 14 is denoted as Probe6; The probe prepared from the functional silica nanoparticles of claim 3 and the specific ligand 15 is designated as Probe7.

8. A method for preparing a specific probe according to claim 6 or 7, characterized in that, Includes the following steps: Step a: Mix the functional silica nanoparticles and the specific ligand in a solution; Step b: Specific probes are obtained by reacting with low-power ultraviolet light, washing, and centrifuging.

9. The method for preparing a specific probe according to claim 8, characterized in that, The power of the low-power ultraviolet light is 67 mW / cm. 2 The irradiation time is 30s-90s.

10. The application of the specific probe according to claim 7, characterized in that, The Probe 4 is used for specific recognition ability testing of Escherichia coli; the Probe 5 is used for fluorescence imaging of HepG2 membrane proteins; the Probe 6 has specific imaging ability for MCF-7 endogenous carbonic anhydrase II; and the Probe 7 is used for photoaffinity labeling of human serum albumin.

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