Metalized microbubbles and their preparation, construction of multifunctional composite probes, rapid capture and sterilization of bacteria

Abstract

The present application relates to a kind of metalized microbubble and its preparation, the construction of multifunctional composite probe, the rapid capture and sterilization method of bacteria, belong to functional material preparation technical field.The method aims at solving the technical problems of uneven surface metal coating, not firm and poor stability of hollow glass microbubble in prior art.The technical scheme points include: the surface-activated hollow glass microbubble is mixed with polyethyleneimine solution and is reacted, and polyethyleneimine-coated hollow glass microbubble is obtained after washing and drying;Then it is reacted with single-stranded DNA buffer solution, and the intermediate product loaded with DNA is obtained;Then silver nitrate solution is introduced, and silver ion is loaded on the surface of the composite;Finally, by sodium borohydride solution reduction, metalized microbubble is prepared.The metalized microbubble prepared by the method can be used to construct multifunctional composite probe, to realize the efficient capture and synergistic antibacterial of target such as bacteria.

Description

Technical Field

[0001] This invention belongs to the field of functional materials preparation technology, specifically relating to a metallized microbubble and its preparation, the construction of a multifunctional composite probe, and a method for rapid capture and sterilization of bacteria. Background Technology

[0002] In the fields of materials science and biotechnology, endowing the surface of hollow glass microbubbles with metallic coatings (such as silver) is an important way to expand their functions (such as sensing, catalysis, or antibacterial properties). In existing technologies, metal nanoparticles are typically deposited or grown directly on the surface of hollow glass microbubbles. However, these methods often struggle to effectively control the thickness, uniformity, and bonding strength of the metal layer to the substrate. Metal particles are prone to aggregation or uneven distribution, leading to poor stability and reproducibility of the functional coating. The reason for these problems is that the smooth and chemically inert surface of hollow glass microbubbles lacks a sufficient number of uniformly distributed active sites to guide the orderly reduction and firm anchoring of metal ions. Although attempts have been made to use various polymers or biomolecules as intermediate layers to improve bonding, designing a simple, universal, and precisely controllable interface modification strategy to achieve uniform and stable metal encapsulation on the surface of hollow glass microbubbles remains a key technical challenge in this field. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages described below.

[0004] Another objective of this invention is to provide a method for preparing metallized microbubbles, which can form a uniform and firm metallic silver coating on the surface of hollow glass microbubbles through simple and controllable steps, and the method is highly versatile and reproducible.

[0005] To achieve these objectives and other advantages of the present invention, a method for preparing metallized microbubbles is provided, comprising the following steps:

[0006] S1: Add hollow glass microbubbles to a polyethyleneimine solution and rotate and mix at room temperature for 0.5-12 h. After the reaction, let it stand until the hollow glass microbubbles float to the top of the solution. Remove the lower layer of solution and wash with ultrapure water. Then, vacuum dry to obtain hollow glass microbubbles coated with polyethyleneimine.

[0007] S2: Add hollow glass microbubbles coated with polyethyleneimine to a buffer solution containing single-stranded DNA, and mix by rotation at room temperature for 0.5-4 h. After the reaction, let it stand until the complex floats to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain the intermediate product loaded with single-stranded DNA.

[0008] S3: Add silver nitrate solution to the intermediate product loaded with single-stranded DNA, rotate and mix at room temperature for 10-120 min, let stand after reaction, wait for the complex to float to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain the complex loaded with silver ions.

[0009] S4: Add sodium borohydride solution to the complex loaded with silver ions, vortex mix for 10-120 min, let stand after reaction, wait for the product to float to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain metallized microbubbles.

[0010] The concentration of the polyethyleneimine solution was 1-8 mg / mL; the amount of single-stranded DNA was 1-1000 pmol; the concentration of the silver nitrate solution was 0.1-50 mM; and the concentration of the sodium borohydride solution was 0.1-50 mM.

[0011] This invention first utilizes surface-activated hollow glass microbubbles as a substrate, forming a positively charged active layer on their surface using polyethyleneimine (PEI). Then, negatively charged single-stranded DNA molecules are introduced through electrostatic adsorption, providing abundant metal ion binding sites. Next, leveraging the strong coordination ability of specific bases in the DNA with silver ions, the silver ions are in-situ and orderly anchored to the surface of the hollow glass microbubbles. Finally, through mild chemical reduction, the silver ions are in-situ converted into silver nanoparticles under the guidance of the DNA template, thus forming a uniform, dense, and firmly bonded silver metal coating on the surface of the hollow glass microbubbles. This method is simple, mild, and uses low-cost raw materials. The DNA-mediated in-situ growth mechanism effectively solves the problems of uneven metal coating and easy detachment, significantly improving the reproducibility and structural stability of the metallized microbubble preparation, laying the foundation for its subsequent applications in sensing, catalysis, and biomedicine.

[0012] Preferably, the pretreatment method for hollow glass microbubbles is pretreatment with piranha solution, or pretreatment using the following methods:

[0013] 1) Disperse hollow glass microbubbles in anhydrous ethanol and ultrasonically clean them. After removing the ethanol, immerse the cleaned hollow glass microbubbles in a 1-5 M sodium hydroxide aqueous solution and stir the reaction at 60-90 °C for 1-4 h. Then wash with ultrapure water until neutral.

[0014] 2) The hollow glass microbubbles, washed to neutral, are redispersed in a mixed solution of ethanol and water containing a silane coupling agent. The reaction is stirred at room temperature to 50 °C for 2-12 h. After the reaction, the hollow glass microbubbles are washed three times with anhydrous ethanol. The silane coupling agent is one of aminopropyltriethoxysilane, 3-glycidyl etheroxypropyltrimethoxysilane, and 3-(2-aminoethylamino)propyltrimethoxysilane. The volume percentage of the silane coupling agent in the mixed solution is 0.5%-5%.

[0015] 3) The washed hollow glass microbubbles are vacuum dried at 80-120 ℃ for 4-12 h to obtain surface-activated hollow glass microbubbles.

[0016] This invention first controllably increases the hydroxyl density and reactivity on the surface of hollow glass microbubbles through alkali treatment. Then, using a silanization reaction, a specific silane coupling agent (such as aminopropyltriethoxysilane) is covalently bonded to the activated surface, thereby introducing a stable interface with a well-defined structure and specific functional groups such as amino or epoxy groups at the ends. By precisely controlling the intensity of the alkali treatment and the type and concentration of the silane coupling agent, this invention constructs a uniform covalently modified layer on the surface of hollow glass microbubbles, rich in specific active groups. This provides reliable, uniform, and strongly binding anchoring points for subsequent possible chemical crosslinking. This invention fundamentally ensures the consistency and controllability of the surface properties of the hollow glass microbubble starting materials, significantly improving the orientation, uniformity, batch-to-batch repeatability, and stability of the subsequent metallization coating process and the overall composite structure.

[0017] Preferably, it also includes an aging step, which specifically includes:

[0018] 4.1) The hollow glass microbubbles dried in step 3) are redispersed in a mixed solution composed of deionized water and an organic solvent; the organic solvent is one of ethanol, isopropanol, and acetone, and the volume percentage of the organic solvent in the mixed solution is 10%-40%;

[0019] 4.2) Place the mixed solution containing hollow glass microbubbles in a sealed container and age it at 25-60 ℃ for 12-72 h. After aging, remove the mixed solution and wash the microbubbles with an organic solvent.

[0020] 4.3) The washed hollow glass microbubbles are vacuum dried at 40-60 ℃ for 1-3 h to obtain surface-activated hollow glass microbubbles.

[0021] Directly subjecting hollow glass microbubbles modified with silane coupling agents to high-temperature, long-term drying may lead to excessive condensation and cross-linking of the silane layer. This results in partial embedding or reduced accessibility of surface active functional groups (such as amino or epoxy groups), weakening their effective binding with subsequent polyethyleneimine molecules. This invention places the modified hollow glass microbubbles in a mixed solution of deionized water and an organic solvent, and allows them to age at a lower temperature for a period of time. This mild solvent environment promotes slow rearrangement and reorganization of the silane molecular layer on the surface of the hollow glass microbubbles, forming a more open, uniform, and stable interface structure with higher functional group accessibility. Simultaneously, it avoids over-hardening and pore closure that may occur with high-temperature processing. The resulting benefits are a significant improvement in the quality of the silanized surface, ensuring sufficient exposure and stable existence of surface active sites. This lays a more optimized foundation for the subsequent firm and uniform coating of polyethyleneimine, further enhancing the reliability of the entire metallization process and the performance consistency of the final product.

[0022] A metallized microbubble is prepared by the aforementioned method for preparing metallized microbubbles.

[0023] A method for constructing a multifunctional composite probe for separating targets, comprising the following steps:

[0024] The metallized microbubbles were mixed with the target probe solution and rotated at room temperature for 0.5-12 h. After the reaction, the mixture was allowed to stand until the composite floated to the top of the solution. The lower layer of solution was removed and the mixture was washed with the appropriate buffer solution to obtain a composite probe with the target probe modified on the surface.

[0025] The targeting probe is selected from any of the following:

[0026] Small molecule organic compounds, aptamers, or antibodies used to target bacteria;

[0027] Antibodies against epithelial cell adhesion molecules or epidermal growth factor receptors that target circulating tumor cells.

[0028] Antibodies against tetraspan membrane proteins or phosphatidylserine-binding proteins for targeting extracellular vesicles;

[0029] Single-stranded DNA or locked nucleic acid that is complementary to the target miRNA;

[0030] Antibodies or aptamers of the protein used to target the target protein;

[0031] Chelating molecules used to target specific metal ions.

[0032] Preferably, the small molecule organic compound used to target bacteria is a solution of 4-mercaptophenylboronic acid.

[0033] A rapid bacterial capture method involves mixing the obtained composite probe with a solution containing bacteria, rotating the mixture at room temperature for 1-40 minutes, allowing it to stand until the coupling material of the composite probe and bacteria floats to the top of the solution, removing the lower layer of solution, and washing with phosphate buffer to complete the bacterial capture.

[0034] Preferably, the bacteria are one or more of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycobacterium tuberculosis, Helicobacter pylori, Salmonella, and Listeria.

[0035] A photo-sterilization method, wherein after capturing bacteria using the aforementioned rapid bacterial capture method, the complex containing the captured bacteria is treated with a laser with an excitation wavelength of 808 nm for 3-15 min.

[0036] This invention utilizes the specific interaction between the boric acid groups on the surface of a composite probe (B@Ag@4-MPBA) modified with a targeting probe and the peptidoglycan of the bacterial cell wall to achieve rapid identification and capture of target bacteria; then, by leveraging the release of silver ions loaded on the metallized microbubbles B@Ag and their photothermal conversion properties, a synergistic antibacterial effect is achieved, thereby realizing efficient removal of bacteria while capturing them.

[0037] The present invention has at least the following beneficial effects:

[0038] First, this invention employs a DNA-mediated in-situ growth method to achieve a uniform and stable coating of silver metal on the surface of hollow glass microbubbles. This method first constructs a positively charged layer on the surface of the hollow glass microbubbles using polyethyleneimine (PEI), then electrostatically adsorbs negatively charged single-stranded DNA. The specific coordination of DNA bases with silver ions guides the anchoring of the silver ions, and finally, reduction forms a silver nanoparticle coating. The entire process is simple, rapid, and uses low-cost raw materials, and the DNA template method exhibits excellent applicability and reproducibility.

[0039] Secondly, this invention constructs a multifunctional composite probe for rapid bacterial capture by modifying the surface of metallized microbubbles with the targeting molecule 4-mercaptophenylboronic acid (4-MPBA). This probe is not only simple to prepare and has a low-cost recognition unit, but also, thanks to the buoyancy of the hollow glass microbubbles themselves, enables rapid separation of captured bacteria without the need for external centrifugation or filtration, significantly reducing operational energy consumption.

[0040] Third, the composite probe of this invention exhibits unique self-aggregation and structural reconfigurability during the capture process, effectively avoiding the problem of active site shielding. It self-assembles into an ordered cluster of hollow glass microbubbles and moves autonomously using buoyancy, significantly increasing the collision frequency with bacteria. Simultaneously, the motion-induced local microfluidic field enhances the mass transfer efficiency from bacteria to the probe surface, thereby improving the capture kinetics.

[0041] Fourth, the metallized microbubbles of this invention possess both the dual functions of slow-release antibacterial silver ions and efficient photothermal conversion. Silver ions can destroy bacterial cell structure and inhibit metabolism, while the local thermal effect generated under near-infrared laser irradiation further enhances the bactericidal effect, realizing a multi-mechanism synergistic antibacterial strategy and overcoming the limitations of traditional materials with single functions.

[0042] Fifth, the metallized microbubbles prepared by this invention can serve as a general functional platform. By grafting different recognition units (such as small molecules, antibodies, aptamers, peptides, etc.) onto the surface, they can be extended to the separation and detection of various targets such as bacteria, circulating tumor cells, extracellular vesicles, nucleic acids, proteins, and metal ions. They have broad application potential in fields such as biosensing, medical diagnosis, and environmental monitoring.

[0043] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0044] Figure 1 This is a flowchart illustrating the preparation process of the metallized microbubbles B@Ag according to the present invention.

[0045] Figure 2 These are TEM, SEM, and AFM characterization images of the metallized microbubbles B@Ag of the present invention.

[0046] Figure 3 The fluorescence imaging (a) and fluorescence intensity (b) of B@PEI after being reacted with different amounts of Cy5-labeled DNA.

[0047] Figure 4 A schematic diagram of the construction of the B@Ag@4-MPBA composite probe and its bacterial capture mechanism;

[0048] Figure 5SEM images of bacteria captured by the B@Ag and B@Ag@4-MPBA composite probes are shown. Figure a shows the SEM images of B@Ag and B@Ag@4-MPBA after interaction with Staphylococcus aureus, respectively; Figure b shows the SEM images of B@Ag and B@Ag@4-MPBA after interaction with Escherichia coli, respectively; Figure c shows the SEM images of B@Ag and B@Ag@4-MPBA after interaction with a mixed bacterial system of Staphylococcus aureus and Escherichia coli, respectively.

[0049] Figure 6 Fluorescence imaging images of bacteria captured by the B@Ag and B@Ag@4-MPBA composite probes; where, image a shows the fluorescence imaging images of B@Ag and B@Ag@4-MPBA after interaction with Staphylococcus aureus; image b shows the SEM fluorescence imaging images of B@Ag and B@Ag@4-MPBA after interaction with Escherichia coli; image c shows the fluorescence imaging images of B@Ag and B@Ag@4-MPBA after interaction with a mixed bacterial system of Staphylococcus aureus and Escherichia coli.

[0050] Figure 7 The capture efficiency of the B@Ag@4-MPBA composite probe at different time points;

[0051] Figure 8 The antibacterial activity of different materials is shown in Figure a; Figure a is a comparison of the turbidity of LB medium solutions; Figure b is a statistical chart of bacterial colony count results.

[0052] Figure 9 The photothermal temperature rise curve of B@Ag of the present invention under 808 nm near-infrared laser irradiation is shown.

[0053] Figure 10 The photothermal stability of B@Ag of the present invention under repeated on-off irradiation by 808 nm near-infrared laser;

[0054] Figure 11 The images show SEM, HRSEM, and fluorescence images of the captured bacteria under both light and dark conditions. Image a shows the SEM and HRSEM characterization images, and image b shows the fluorescence images of SYTO 9 and PI dyes. The SEM, HRSEM, and fluorescence images in rows 1 and 3 are without light treatment, while the SEM, HRSEM, and fluorescence images in rows 2 and 4 are with light treatment. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0056] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0057] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0058] Experiment 1: Preparation and Characterization of Metallized Microbubbles B@Ag

[0059] according to Figure 1 The process shown illustrates the steps for preparing metallized microbubbles B@Ag:

[0060] 1) Weigh 1 g of hollow glass microbubbles (GB) pretreated with piranha solution (H2O2 / H2SO4=1:3), add 30 mL of polyethyleneimine solution (PEI solution: dissolved in ultrapure water to a concentration of 2 mg / mL), and mix by rotation at room temperature for 2 h (rotation speed of 10 rpm). After standing, the bubbles float to the top of the solution, the lower layer of solution is removed with a syringe, and then washed 3 times with ultrapure water. Finally, vacuum dry at 37 °C to obtain PEI-encapsulated hollow glass microbubbles (B@PEI).

[0061] 2) Accurately weigh 1 mg B@PEI and add it to a 1.5 mL centrifuge tube, and add 100 μL DNA buffer (100 pmol) to disperse it; after mixing and reacting at room temperature (10 rpm) for 0.5 h, the formed B@PEI@DNA complex is washed three times with ultrapure water to remove free DNA.

[0062] 3) Add 100 μL of AgNO3 solution (10 mM) to the B@PEI@DNA complex and shake to mix for 0.5 h. After the reaction is complete, wash three times with ultrapure water to remove free Ag. + A silver-loaded complex (B@PEI@DNA@Ag) was obtained. + );

[0063] 4) Add B@PEI@DNA@Ag to 100 μL of freshly prepared ice-bathed NaBH4 solution (10 mM). + The complex was vortexed for 0.5 h. B@Ag was collected and washed three times with ultrapure water to remove residual NaBH4 and reaction byproducts. After washing, B@Ag was redispersed in ultrapure water and stored in a refrigerator at 4 °C for later use.

[0064] like Figure 2As shown, the morphology of GB, B@PEI and B@Ag was characterized by scanning electron microscopy (SEM), high-resolution scanning electron microscopy (HRSEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). It can be observed that the smooth GB surface is coated with irregular PEI nanoparticles. After further reaction, silver nanoparticles are densely coated on the GB surface, proving that B@Ag was successfully prepared.

[0065] Experiment 2: Interaction of B@PEI with different concentrations of Cy5-labeled DNA strands (Cy5-DNA):

[0066] Accurately weigh 6 1 mg B@PEI portions and add them to 1.5 mL centrifuge tubes. Add 100 μL of Cy5-DNA buffer solution (dispersed in PBS) of different concentrations to each tube and vortex at room temperature for 0.5 h. Then, allow the mixture to stand until the B@PEI@Cy5-DNA complex floats completely to the top of the solution. Use a syringe to aspirate the lower layer of solution and wash three times with PBS buffer to remove free Cy5-DNA. Finally, drop the B@PEI@Cy5-DNA complex onto PDMS. After the complex aggregates at the top of the droplet, image it under a fluorescence microscope.

[0067] like Figure 3 As shown, the fluorescence intensity increases with the increase of Cy5-DNA content, eventually reaching saturation. This indicates that B@PEI can adsorb Cy5-DNA onto its surface through electrostatic interaction, and the adsorption of B@PEI reaches saturation when the Cy5-DNA content reaches 200 pmol.

[0068] Experiment 3: Construction of the B@Ag@4-MPBA composite probe and its application in bacterial capture assay:

[0069] according to Figure 4 The procedure shown is used to prepare the B@Ag@4-MPBA composite probe and apply it to the rapid capture of bacteria.

[0070] Accurately weigh 1 mg B@Ag into a 1.5 mL centrifuge tube, then add 100 μL of 4-mercaptophenylboronic acid (4-MPBA, dissolved in ultrapure water, 100 μM) solution for dispersion; after reacting at room temperature (10 rpm) for 2 h, allow to stand for separation, collect the B@Ag@4-MPBA composite probe, and wash three times with ultrapure water to remove free 4-MPBA molecules; finally, disperse the B@Ag@4-MPBA composite probe with ultrapure water and store it in a refrigerator at 4 ℃ for later use.

[0071] Accurately dispose of three 1 mg B@Ag@4-MPBA composite probes in 1.5 mL centrifuge tubes, add 90 μL of PBS buffer to disperse, and then add 10 μL of S. aureus, E. coli, and a mixed solution of S. aureus and E. coli (concentration of each bacterium 10). 8 The reaction was carried out at room temperature by rotating the sample (10 rpm) for 10 min. After the reaction, the sample was allowed to stand until the complex floated to the top of the solution. The lower layer of solution was then aspirated with a syringe and washed three times with PBS buffer to remove any uncaptured bacteria. The final result was the bacterial-coupled complex (B@Ag@4-MPBA composite probe).

[0072] Nucleic acid recognition molecules that target bacterial specific antigens, specifically including:

[0073] SA3 aptamer (targeting its surface protein A) and apt-13a aptamer (targeting its capsular polysaccharide) are targeted at Staphylococcus aureus.

[0074] EC1 aptamer (targeting lipopolysaccharide LPS) and E. coli-apt6 aptamer (targeting outer membrane proteins) for Escherichia coli (especially O157:H7 strain).

[0075] PA1 aptamer (targeting quorum sensing signaling molecule receptor) and Pseudomonas-apt1 aptamer (targeting fimbriae protein) for Pseudomonas aeruginosa.

[0076] TBapt aptamer for Mycobacterium tuberculosis (targeting bacterial surface lipoproteins) and Hp-apt12 aptamer for Helicobacter pylori (targeting urease subunits).

[0077] Immunoglobulins targeting bacterial characteristic antigens, specifically including:

[0078] Monoclonal antibody against Staphylococcus aureus capsular polysaccharide and polyclonal antibody against Staphylococcus aureus protein A;

[0079] Anti-Escherichia coli O157:H7 lipopolysaccharide (LPS) monoclonal antibody, anti-Escherichia coli outer membrane porin OmpF antibody;

[0080] Monoclonal antibody against Pseudomonas aeruginosa exotoxin A and antibody against its quorum sensing regulatory protein LasR;

[0081] Anti-Helicobacter pylori urease monoclonal antibody and anti-Mycobacterium tuberculosis heat shock protein 65 (HSP65) antibody.

[0082] Targeting probes for circulating tumor cells include antibodies such as: epithelial cell adhesion molecule (EpCAM) antibody, epidermal growth factor receptor (EGFR) antibody, anti-human epidermal growth factor receptor 2 (HER2) antibody (targeting circulating tumor cells from breast cancer), anti-cytokeratin 19 (CK19) antibody, and anti-mesothelin (MSLN) antibody (targeting circulating tumor cells from pancreatic / ovarian cancer); and aptamers such as: EpCAM aptamer (EpCAM-apt), HER2 aptamer (HER2-apt), and aptamers targeting the metastasis-associated protein CD44v6.

[0083] Targeting probes for extracellular vesicles include antibodies such as anti-CD63 antibodies, anti-CD81 antibodies, and anti-CD9 antibodies (which are universal marker antibodies for extracellular vesicles); phosphatidylserine-binding proteins, primarily Annexin V; anti-alpha-fetoprotein (AFP) antibodies (targeting extracellular vesicles derived from liver cancer), anti-carcinoembryonic antigen (CEA) antibodies (targeting extracellular vesicles derived from gastrointestinal tumors), and anti-prostate-specific antigen (PSA) antibodies (targeting extracellular vesicles derived from prostate cancer); and aptamers such as CD63 aptamers and aptamers targeting integrin αvβ3 on the surface of tumor-derived extracellular vesicles.

[0084] Targeting probes for targeting miRNAs can be single-stranded DNA, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) (resistant to nuclease degradation and with stronger affinity for miRNAs), or 2'-O-methyl modified RNA (with better stability than natural RNA).

[0085] Targeting probes for targeting target proteins, such as antibodies, can include: anti-carcinoembryonic antigen (CEA) monoclonal antibodies (tumor markers), anti-vascular endothelial growth factor (VEGF) antibodies (targeting tumor angiogenesis proteins), anti-insulin antibodies (detection of diabetes-related proteins), and anti-amyloid β (Aβ) antibodies (Alzheimer's disease-related proteins).

[0086] The aptamers can be: CEA aptamer, VEGF aptamer, insulin aptamer, and Aβ aptamer.

[0087] Chelating molecule used to target specific metal ions, such as lead ions (Pb). 2+ The following can be: ethylenediaminetetraacetic acid (EDTA), dimercaptosuccinic acid (DMSA); targeting mercury ions (Hg). 2+ It can be: sodium dimercaptopropanesulfonate (DMPS), glutathione chelating agent; targeted copper ion (Cu) 2+ Possible forms include: triethylenetetramine (TETA), penicillamine; and targeted iron ions (Fe). 3+ This could be: deferoxamine (DFO), deferoxone; targeted gallium ions (Ga...3+ It can be: transferrin, citric acid chelating agent.

[0088] These aptamers, antibodies, and chelating agents can all be used to modify the surface of metallized microbubbles (B@Ag) to construct multifunctional composite probes with specific recognition functions, enabling efficient capture and detection of various targets such as bacteria, circulating tumor cells, extracellular vesicles, nucleic acids, proteins, and metal ions.

[0089] Experiment 4: SEM characterization of bacteria captured by the B@Ag@4-MPBA composite probe:

[0090] 100 μL of glutaraldehyde (2.5%) was added to the B@Ag@4-MPBA-coupled bacterial complex. After fixing the bacteria for 4 h, they were allowed to stand for separation and washed three times with PBS. Subsequently, the samples were subjected to gradient dehydration treatment with ethanol solutions of concentrations of 30%, 50%, 70%, 80%, 90%, and 95%, with each concentration treated for 10 min. After gradient dehydration, the samples were further dehydrated twice with 100% ethanol solution, each treatment lasting 10 min. After dehydration, the samples were dried in a vacuum environment for 2 h. The dried samples were fixed on conductive adhesive, and microscopic images of the samples were acquired using SEM. The B@Ag surface without 4-MPBA assembly served as a control group.

[0091] like Figure 5 As shown, only a few non-specifically adsorbed bacteria were observed in the B@Ag group, while a large number of bacteria were observed on the surface of the B@Ag@4-MPBA composite probe. This indicates that 4-MPBA has the ability to target and recognize bacteria, and that bacteria can be rapidly captured by the B@Ag@4-MPBA composite probe. Even in the two mixed bacterial systems, the B@Ag@4-MPBA composite probe can capture them.

[0092] Experiment 5: Fluorescent characterization of bacteria captured by the B@Ag@4-MPBA composite probe:

[0093] The B@Ag@4-MPBA-coupled bacterial complex was incubated with 1 mL of green fluorescent nucleic acid dye SYTO 9 (1 μM) at room temperature in the dark for 20 min. After the reaction, the complex was allowed to float to the top of the solution, and then the lower layer of solution was aspirated with a syringe to remove free SYTO 9. The complex was washed three times with PBS buffer. Finally, the complex was dropped onto polydimethylsiloxane (PDMS), aggregated, and imaged under a fluorescence microscope.

[0094] like Figure 6As shown, the B@Ag group showed almost no fluorescence, while the B@Ag@4-MPBA composite probe group showed obvious fluorescence, indicating that the B@Ag@4-MPBA composite probe has the ability to target and capture bacteria.

[0095] Experiment 6: Bacterial capture efficiency test of B@Ag@4-MPBA composite probe:

[0096] To verify the efficiency of the B@Ag@4-MPBA composite probe in capturing bacteria, B@Ag@4-MPBA (8×10⁻⁶) was used. 5 (1) composite probes were respectively mixed with 1 mL of S. aureus or E. coli (concentration of 10). 8 Incubate with CFU / mL for different times (1, 2, 5, 10, 20 and 40 min); after the reaction, allow the B@Ag@4-MPBA coupled bacterial complex to float to the top of the solution, take 100 μL of the lower layer solution and dilute it 10000 times, then take 100 μL and spread it evenly in a plate, incubate at 37 ℃ for 16 h and count the number of bacteria; finally, calculate the capture efficiency according to the formula (capture efficiency = (number of bacteria before capture - number of bacteria after capture) / number of bacteria before capture × 100%).

[0097] like Figure 7 As shown, the efficiency of the B@Ag@4-MPBA composite probe in capturing bacteria gradually increased with the increase of capture time, and the capture efficiency tended to stabilize after 10 min. This result indicates that the B@Ag@4-MPBA composite probe has the ability to rapidly capture both S. aureus and E. coli.

[0098] The B@Ag@4-MPBA composite probe also showed similar excellent efficacy to the S. aureus or E. coli capture assays mentioned above against Pseudomonas aeruginosa, Acinetobacter baumannii, Mycobacterium tuberculosis, Helicobacter pylori, Salmonella, and Listeria. Specific experimental details and results will not be elaborated here.

[0099] Experiment 7: Characterization of the antibacterial activity of different materials:

[0100] To test the antibacterial activity of different materials, 4 mg of GB, B@Ag, and B@Ag@4-MPBA materials were respectively mixed with 1 mL of S. aureus or E. coli (concentration of 10). 8 The bacteria (CFU / mL) were incubated in LB liquid medium (37 °C) for 24 h. After that, the material was allowed to float to the top of the liquid surface. The appearance of the solution was then photographed to determine its turbidity. 100 μL of LB medium was taken, diluted, and spread evenly on the plate. The plate was then incubated at 37 °C for 16 h. Finally, the number of bacteria was counted.

[0101] like Figure 8As shown in Figure a, the LB medium solution in the GB group was turbid, which was due to rapid bacterial proliferation, indicating that GB had almost no antibacterial activity. In contrast, the LB medium in the B@Ag and B@Ag@4-MPBA groups was clear and transparent, indicating no bacterial proliferation and good antibacterial activity. This result is mainly because the B@Ag and B@Ag@4-MPBA materials can release silver ions to kill bacteria. The results of plate counting of the lower LB medium solution are shown in Figure a. Figure 8 b) This also indicates that the metallized microbubble material has antibacterial activity.

[0102] Experiment 8: Photothermal temperature rise curve of B@Ag under 808 nm near-infrared laser irradiation:

[0103] To verify the photothermal properties of B@Ag, 1 mg of B@Ag was dispersed in 100 μL of ultrapure water. After the sample formed aggregates at the top of the solution, an excitation wavelength of 808 nm and a power density of 1 W / cm² were used. 2 and 3 W / cm 2 The laser was used to irradiate the B@Ag aggregates, and the temperature data of the B@Ag aggregates was recorded and collected in real time at 10 s intervals using a handheld thermal imager. The photothermal properties of B@Ag were then analyzed by analyzing the temperature change patterns.

[0104] like Figure 9 As shown, under 808 nm light stimulation, the temperature of B@Ag shows a sharp upward trend and can remain at a stable plateau for a long time after reaching the peak. This phenomenon indicates that the material has excellent photothermal conversion efficiency and outstanding photothermal stability, and can be used in the field of photothermal therapy.

[0105] Experiment 9: Photothermal stability test of B@Ag under repeated on / off irradiation by 808 nm near-infrared laser:

[0106] To verify the photothermal stability of B@Ag, 1 mg of B@Ag was dispersed in 100 μL of ultrapure water. After the sample formed aggregates at the top of the solution, an excitation wavelength of 808 nm and a power of 1 W / cm² were used. 2 and 3 W / cm 2 A laser was used to irradiate the B@Ag aggregates in a 200-second cycle, repeatedly switching the laser on and off to achieve intermittent irradiation. Throughout the irradiation process, a handheld thermal imager was used to monitor the temperature of the B@Ag aggregates in real time, with data acquisition intervals of 10 seconds, comprehensively recording temperature changes at different power levels and time points.

[0107] like Figure 10As shown, when the laser is on, the temperature of B@Ag rises rapidly and remains within a stable temperature range without significant fluctuations during continuous illumination. When the laser is switched off, the temperature of B@Ag drops rapidly, eventually returning to a level almost identical to the ambient temperature. During the experiment involving repeated switching between laser on and off states, the temperature change trend remained consistent throughout the entire cycle, indicating that B@Ag can stably respond to illumination under 808 nm laser irradiation, achieving a cycle of heating-holding-cooling, demonstrating good photothermal stability.

[0108] Experiment 10: SEM and fluorescence characterization of captured bacteria under light and shadow:

[0109] The complex of B@Ag@4-MPBA coupled with bacteria was used, and an excitation wavelength of 808 nm and a power density of 1 W / cm² were employed. 2 The composite was irradiated with a laser for 5 min. After irradiation, the sample was divided into two equal parts. The first part was subjected to gradient dehydration with ethanol solutions of 30%, 50%, 70%, 80%, 90%, and 95% concentrations, with each concentration treated for 10 min. After gradient dehydration, the sample was further dehydrated twice with 100% ethanol solution, each treatment lasting 10 min. After dehydration, the sample was dried in a vacuum environment for 2 h. The dried sample was fixed on conductive adhesive, and its microstructure was captured using SEM. The second part was stained with 1 mL of live / dead bacterial staining agent (composed of 1 μM SYTO 9 (specifically labeled live bacteria) and 5 μM propidium iodide (PI, specifically labeled dead bacteria)) at room temperature in the dark for 20 min. After staining, the sample was washed three times with PBS to remove free dye. Finally, the washed complex was dropped onto a PDMS substrate, and after the sample aggregated, fluorescence images were acquired under a fluorescence microscope. A control group was set up during the experiment, and the preparation and processing procedures of the control group samples were the same as the above steps, except that laser irradiation was not performed.

[0110] like Figure 11 As shown in Figure a, under no-light conditions, the bacterial wall structure of *S. aureus* remained intact without significant damage or deformation, while *E. coli* showed localized damage, but not complete rupture. When *S. aureus* and *E. coli* were subjected to light treatment, the bacterial walls of both *S. aureus* and *E. coli* were found to be severely damaged, with their structural integrity significantly disrupted. This indicates that light treatment can destroy bacteria, thereby enhancing the bactericidal effect against *S. aureus* and *E. coli*. Figure 11As shown in b, when using the SYTO 9 / PI dual-fluorescence staining method to detect bacterial viability, the SYTO 9 channel showed a significant green fluorescence signal in the unlit control group, while the PI channel showed almost no red fluorescence signal, indicating that the bacteria in the control group were predominantly viable. However, in the light-treated group, the fluorescence signals showed the opposite characteristics: the green fluorescence signal in the SYTO 9 channel was significantly weakened, while the red fluorescence signal in the PI channel was significantly enhanced. This fluorescence imaging result further confirms that light treatment can be correlated with Ag... + The antibacterial system produces a synergistic effect, effectively enhancing its antibacterial efficiency against S. aureus and E. coli, and... Figure 11 The observation results of bacterial wall morphology in a corroborate each other.

[0111] Example 1: Preparation of metallized microbubbles and their application in bacterial capture and synergistic sterilization:

[0112] 1. Pretreatment of hollow glass microbubbles (GB):

[0113] 1 g of hollow glass microbubbles (average particle size of about 16 μm) were placed in a piranha solution (H2O2 / H2SO4=1:3), stirred for 1 h at 1000 rpm, and after standing, the lower layer of fragments was removed by separating with a separatory funnel. The microbubbles were washed with ultrapure water until neutral and dried at 80 °C to obtain hollow glass microbubbles pretreated with piranha solution.

[0114] 2. Preparation of metallized microbubbles (B@Ag):

[0115] 2.1 Polyethyleneimine (PEI) Encapsulation: 1 g of the prepared hollow glass microspheres GB was added to 30 mL of a 2 mg / mL aqueous solution of polyethyleneimine (PEI, molecular weight approximately 25,000). The mixture was stirred at 10 rpm for 2 h at room temperature (approximately 25 °C). After standing, the hollow glass microbubbles floated to the top of the solution. The lower supernatant was aspirated with a syringe, and the microbubbles were washed three times with ultrapure water. Subsequently, the microbubbles were vacuum dried at 37 °C to obtain PEI-encapsulated hollow glass microbubbles (B@PEI).

[0116] 2.2 DNA Adsorption and Silver Ion Loading: Accurately weigh 1.0 mg of B@PEI and place it in a 1.5 mL centrifuge tube. Add 100 μL of phosphate-buffered saline (PBS, 10 mM, pH 7.4) to disperse it. Then, add 100 μL of buffer containing a 200 pmol cytosine (C)-rich single-stranded DNA sequence (ssDNA, sequence: ccccccccccccccccccccccccccccccccccccccccccc, synthesized by Shanghai Sangon Biotech Co., Ltd.), and mix at 10 rpm for 0.5 h at room temperature. After standing separation, remove the lower layer solution and wash three times with ultrapure water to obtain the DNA-loaded intermediate product (B@PEI@DNA). Next, add 100 μL of 10 mM silver nitrate (AgNO3) aqueous solution to the intermediate product and shake to mix for 0.5 h. After the reaction, the mixture was allowed to stand, the lower layer was removed, and the solution was washed three times with ultrapure water to obtain the silver ion-loaded complex (B@PEI@DNA@Ag). + ).

[0117] 2.3 In-situ reduction of silver nanoparticles: 100 μL of freshly prepared 10 mM sodium borohydride (NaBH4) aqueous solution, placed in an ice bath, was rapidly added to the above-mentioned B@PEI@DNA@Ag. + The complex was vortexed for 0.5 h. After the reaction, the mixture was allowed to stand to allow the product to float to the top, the lower layer of solution was removed, and the mixture was washed three times with ultrapure water to remove residual reactants and byproducts. Metallized microbubbles (B@Ag) were finally obtained, redispersed in 1 mL of ultrapure water, and stored at 4 °C for later use.

[0118] 3. Construction of a multifunctional composite probe targeting bacteria (B@Ag@4-MPBA):

[0119] Take 1 mg of B@Ag prepared above (approximately 8 × 10⁻⁶ mg / kg) 5 The sample was placed in a 1.5 mL centrifuge tube. 100 μL of a 100 μM solution of 4-mercaptophenylboronic acid (4-MPBA, dissolved in ultrapure water) was added. The reaction was carried out at 10 rpm for 2 h at room temperature. After the reaction, the mixture was allowed to stand until the complex floated to the top. The lower layer was aspirated, and the sample was washed three times with ultrapure water to remove unbound 4-MPBA molecules. Finally, the mixture was redispersed with 100 μL of PBS buffer (10 mM, pH 7.4) to obtain the 4-MPBA-modified composite probe (B@Ag@4-MPBA), which was stored at 4 °C.

[0120] 4. Rapid capture of bacteria:

[0121] The B@Ag@4-MPBA composite probe (1 mg) prepared above was mixed with 1 mL of PBS suspension containing Staphylococcus aureus (S. aureus, strain ATCC 6538) (bacterial concentration approximately 1×10⁻⁶). 8 Mix (CFU / mL). Rotate the mixture at 10 rpm for 10 min at room temperature. After the reaction, let it stand for 5 min; the coupled probe and bacteria will float completely to the top of the solution. Carefully aspirate the clear solution at the bottom using a syringe, and wash the floating complex twice with 1 mL PBS buffer to remove any uncaptured bacteria. This completes the rapid capture of Staphylococcus aureus. Scanning electron microscopy (SEM) and fluorescence staining confirmed that a large number of bacteria were specifically captured on the surface of the B@Ag@4-MPBA probe, with a capture efficiency of 90% calculated by plate counting.

[0122] 5. Photothermal synergistic sterilization:

[0123] Take the bacterial-captured complex and place it in 200 μL of PBS buffer. Use a near-infrared laser emitting at 808 nm with a wavelength of 1 W / cm². 2 The sample was vertically irradiated with an 808 nm laser at a power density of 1 W / cm² for 5 minutes. Real-time monitoring with an infrared thermal imager showed that the local temperature of the sample rapidly increased and stabilized above 50 °C during irradiation. After irradiation, the samples were subjected to live / dead bacterial fluorescence staining (SYTO 9 / PI double staining). Statistical results showed that the bacterial mortality rate in the un-illuminated control group was only (2.1±0.5)%. 2 After 5 minutes of irradiation, the mortality rate of bacteria captured by the B@Ag@4-MPBA composite probe significantly increased to (95.3±1.8)%. Compared with the baseline mortality rate of (2.1±0.5)% in the un-illuminated control group, the net bactericidal efficiency exceeded 93%, confirming a significant synergistic antibacterial effect.

[0124] Example 2: Preparation of metallized microbubbles and their application in bacterial capture and synergistic sterilization:

[0125] The difference from Example 1 lies in the pretreatment of the hollow glass microbubbles as follows: 1 g of hollow glass microbubbles (average particle size approximately 16 μm) were dispersed in 30 mL of anhydrous ethanol and ultrasonically cleaned for 20 min. After removing the ethanol, the hollow glass microbubbles were immersed in 40 mL of a 3 M sodium hydroxide aqueous solution and stirred at 75 °C for 2.5 h. After the reaction, the solution was repeatedly washed with ultrapure water until it was neutral (pH≈7.0). Subsequently, the washed hollow glass microbubbles were redispersed in a mixed solution of 95 mL of ethanol and 5 mL of deionized water, and 1.5 mL (approximately 1.5% by volume) of aminopropyltriethoxysilane was added. After stirring at 35 °C for 6 h, the hollow glass microbubbles were washed three times with anhydrous ethanol. Finally, the hollow glass microbubbles were vacuum dried at 100 °C for 8 h to obtain pretreated hollow glass microbubbles (GB) for later use.

[0126] The results showed that the capture efficiency of Staphylococcus aureus in this embodiment reached over (94.5±1.5)%; after being exposed to 808nm near-infrared laser (1 W / cm²), the capture efficiency was achieved. 2 After 5 minutes of irradiation, the mortality rate of the captured bacteria increased to (96.8±1.2)%; compared with the background mortality rate of (2.1±0.5)% in the unirradiated control group, the net sterilization efficiency was calculated to be over 94%, indicating that under the surface activation process conditions, the composite probe still has a highly efficient ability to capture bacteria and perform photothermal synergistic sterilization.

[0127] Example 3: Preparation of metallized microbubbles and their application in bacterial capture and synergistic sterilization:

[0128] The difference from Example 2 is that, after the hollow glass microbubbles are vacuum dried at 100 °C for 8 h, an aging step is also included. The aging step specifically includes:

[0129] 4.1) The dried hollow glass microbubbles are redispersed in a mixed solution composed of deionized water and an organic solvent; the organic solvent is one of ethanol, isopropanol, and acetone, and the volume percentage of the organic solvent in the mixed solution is 25%.

[0130] 4.2) Place the mixed solution containing hollow glass microbubbles in a sealed container and age it at 45 °C for 40 h. After aging, remove the mixed solution and wash the hollow glass microbubbles with an organic solvent.

[0131] 4.3) The hollow glass microbubbles were vacuum dried at 50 °C for 2 h to obtain surface-activated hollow glass microbubbles.

[0132] The results showed that the capture efficiency of Staphylococcus aureus in this embodiment was further improved to (96.5±1.2)%; after being exposed to 808 nm near-infrared laser (1 W / cm²), the capture efficiency was further improved to (96.5±1.2)%. 2 After 5 minutes of irradiation, the mortality rate of the captured bacteria reached (97.5±1.0)%; compared with the baseline mortality rate of (2.1±0.5)% in the un-illuminated control group, the net sterilization efficiency was calculated to be over 95%.

[0133] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing metallized microbubbles, characterized in that, Includes the following steps: S1: Add the pretreated hollow glass microbubbles to the polyethyleneimine solution and rotate and mix at room temperature for 0.5-12 h. After the reaction, let it stand until the hollow glass microbubbles float to the top of the solution. Remove the lower layer of solution and wash with ultrapure water. Then, vacuum dry to obtain hollow glass microbubbles coated with polyethyleneimine. S2: Add hollow glass microbubbles coated with polyethyleneimine to a buffer solution containing single-stranded DNA rich in cytosine bases, and mix by rotation at room temperature for 0.5-4 h. After the reaction, let it stand until the complex floats to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain the intermediate product loaded with single-stranded DNA. S3: Add silver nitrate solution to the intermediate product loaded with single-stranded DNA, rotate and mix at room temperature for 10-120 min, let stand after reaction, wait for the complex to float to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain the complex loaded with silver ions. S4: Add sodium borohydride solution to the complex loaded with silver ions, vortex mix for 10-120 min, let stand after reaction, wait for the product to float to the top of the solution, remove the lower layer of solution and wash with ultrapure water to obtain metallized microbubbles. The concentration of the polyethyleneimine solution was 1-8 mg / mL; the amount of single-stranded DNA was 1-1000 pmol; the concentration of the silver nitrate solution was 0.1-50 mM; and the concentration of the sodium borohydride solution was 0.1-50 mM.

2. The method for preparing metallized microbubbles according to claim 1, characterized in that, The pretreatment method for hollow glass microbubbles is to pretreat them with a piranha solution, or by the following methods: 1) Disperse hollow glass microbubbles in anhydrous ethanol and ultrasonically clean them. After removing the ethanol, immerse the cleaned microbubbles in a 1-5 M sodium hydroxide aqueous solution and stir the reaction at 60-90 °C for 1-4 h. Then wash with ultrapure water until neutral. 2) The hollow glass microbubbles, washed to neutral, are redispersed in a mixed solution of ethanol and water containing silane coupling agent. The reaction is stirred at room temperature to 50 °C for 2-12 h. After the reaction is completed, the microbubbles are washed three times with anhydrous ethanol. The silane coupling agent is one of aminopropyltriethoxysilane, 3-glycidyl etheroxypropyltrimethoxysilane, and 3-(2-aminoethylamino)propyltrimethoxysilane. The volume percentage of the silane coupling agent in the mixed solution is 0.5%-5%; 3) The washed hollow glass microbubbles are vacuum dried at 80-120 ℃ for 4-12 h to obtain surface-activated hollow glass microbubbles.

3. The method for preparing metallized microbubbles according to claim 2, characterized in that, It also includes an aging process, which specifically includes: 4.1) The hollow glass microbubbles dried in step 3) are redispersed in a mixed solution composed of deionized water and an organic solvent; the organic solvent is one of ethanol, isopropanol, and acetone, and the volume percentage of the organic solvent in the mixed solution is 10%-40%; 4.2) Place the mixed solution containing hollow glass microbubbles in a sealed container and age it at 25-60 ℃ for 12-72 h. After aging, remove the mixed solution and wash the hollow glass microbubbles with an organic solvent. 4.3) The washed hollow glass microbubbles are vacuum dried at 40-60 ℃ for 1-3 h to obtain surface-activated hollow glass microbubbles.

4. A metallized microbubble, characterized in that, It is prepared by the method for preparing metallized microbubbles according to any one of claims 1-3.

5. A method for constructing a multifunctional composite probe for separating targets, characterized in that, Includes the following steps: The metallized microbubbles described in claim 4 are mixed with the targeted probe solution and rotated at room temperature for 0.5-12 h. After the reaction, the mixture is allowed to stand until the composite floats to the top of the solution. The lower layer of solution is removed and the mixture is washed with the appropriate buffer solution to obtain a composite probe with the targeted probe modified on its surface. The targeting probe is selected from any of the following: Small molecule organic compounds, aptamers, or antibodies used to target bacteria; Antibodies against epithelial cell adhesion molecules or epidermal growth factor receptors that target circulating tumor cells. Antibodies against tetraspan membrane proteins or phosphatidylserine-binding proteins for targeting extracellular vesicles; Single-stranded DNA or locked nucleic acid that is complementary to the target miRNA; Antibodies or aptamers of the protein used to target the target protein; Chelating molecules used to target specific metal ions.

6. The method for constructing a multifunctional composite probe for separating targets according to claim 5, characterized in that, The small molecule organic compound used to target bacteria is 4-mercaptophenylboronic acid.

7. A method for rapid capture of bacteria, characterized in that, The composite probe obtained in claim 6 is mixed with a solution containing bacteria and rotated at room temperature for 1-40 min. After the reaction, the mixture is allowed to stand until the coupling material of the composite probe and bacteria floats to the top of the solution. The lower layer of solution is removed and washed with phosphate buffer to complete the bacterial capture.

8. The rapid capture method according to claim 7, characterized in that, The bacteria are one or more of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycobacterium tuberculosis, Helicobacter pylori, Salmonella, and Listeria.

9. A method for sterilization by light, characterized in that, After capturing bacteria using the rapid bacterial capture method described in claim 7, the complex containing the captured bacteria is irradiated with a laser with an excitation wavelength of 808 nm for 3-15 min.

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