A method for preparing, using, and applying DNA-modified colloidal macrophages

Abstract

This invention relates to the field of bacterial detection technology and discloses a method for preparing DNA-modified colloidal macrophages, specifically as follows: After macrophages are cultured on plates to a density of 70%–85%, a solution of magnetic iron oxide nanoparticles is added and incubated for a period of time. Once the cells have phagocytosed the magnetic particles, they are digested to obtain suspended magnetized cells. A photoinitiator, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropanone, is mixed with polyethylene glycol diacrylate to prepare a gel. Then, a certain concentration of the gel is added to the cell suspension, and after incubation, the cells are collected by low-speed centrifugation and resuspended in phosphate buffer. The cells are then irradiated with a 365 nm ultraviolet lamp to initiate a polymerization reaction, obtaining colloidal macrophages. In this invention, the colloidal macrophages can not only recognize various bacteria through cell membrane surface receptors but also generate detectable fluorescent signals through changes in DNA ribozymes, specifically reporting the presence of a particular bacterium.

Description

Technical Field

[0001] This invention belongs to the field of bacterial detection technology, specifically relating to the preparation, use, and application of DNA-modified colloidal macrophages. Background Technology

[0002] Bacterial infections seriously impact human health, and rapid, accurate, and highly sensitive bacterial isolation and detection methods can provide strong support for the early diagnosis of bacterial infectious diseases. Currently, bacterial analysis mainly relies on traditional isolation and culture experiments, conventional PCR, and enzyme-linked immunosorbent assays (ELISA). Isolation and culture methods involve numerous steps, are time-consuming, and require specific operating and culture conditions, making them unsuitable for rapid identification of pathogenic bacteria. Conventional PCR detection technology utilizes nucleic acid primers for nucleic acid detection (such as polymerase chain reaction), offering good sensitivity; however, the nucleic acid detection process is complex, involving procedures such as cell lysis, nucleic acid extraction, magnetic separation, washing, and amplification, and requires additional temperature-controlled equipment. Immunological detection methods, such as immunochromatographic gold strips, are well-suited for rapid identification and analysis of pathogens due to their simplicity, low cost, and rapid signal generation. However, immunochromatographic detection methods typically require significant effort to screen and optimize a pair of highly specific biological receptors (antibodies). Furthermore, the use of immunochromatographic strips requires prior knowledge of whether the sample contains the target bacteria; when the sample contains unknown pathogens, deployment becomes difficult. In addition, bacteria secrete various exotoxins during human infection to attack the host. Currently, no technology has been reported, either domestically or internationally, that can simultaneously detect bacterial cells and related exotoxins.

[0003] Immune cells in the human body, such as macrophages Dendritic cells and neutrophils, through millions of years of evolution, possess exceptional pathogen recognition capabilities, enabling them to identify a wide range of bacteria, fungi, and their secreted virulence factors. According to current research both domestically and internationally, these immune cells primarily mediate microbial recognition and binding through protein receptors on their cell membrane surfaces, such as Toll-like receptors (TLRs), mannose receptors, scavenger receptors, and complement receptors. Notably, this receptor-ligand binding recognition model prevents immune cells from accurately identifying specific bacteria and from generating detectable signals. Furthermore, living cells rapidly die and rupture once removed from their culture environment, and their fragile cell bodies are easily affected by changes in the external environment and mechanical forces. These factors severely limit their applicability in in vitro bacterial analysis. Summary of the Invention

[0004] To address the technical problems mentioned in the background section, this invention provides a method for preparing gelled cell particles and their application in the detection of pathogenic microorganisms.

[0005] This invention is achieved using the following technical solution:

[0006] A method for preparing DNA-modified colloidal macrophages is as follows:

[0007] After macrophages reached a density of 70%–85% in a plate culture, a solution of magnetic iron oxide nanoparticles was added and incubated for a period of time. After the cells phagocytosed the magnetic particles, they were digested to obtain suspended magnetized cells.

[0008] A gel solution was prepared by mixing the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropanone with polyethylene glycol diacrylate.

[0009] Then, a certain concentration of gel solution was added to the cell suspension, and after incubation, the cells were collected by low-speed centrifugation and resuspended with phosphate buffer. The cells were then irradiated with a UV lamp with a wavelength of 365 nm to induce a polymerization reaction and obtain colloidal macrophages.

[0010] DNA ribozyme strands were incubated with colloidal macrophages, and excess DNA was removed by magnetic separation. The resulting DNA was then... Store at 4°C for subsequent use; the final concentration of the DNA ribozyme / substrate chain is 50–250 nM; incubate with colloidal macrophages for 15–20 minutes; the reaction buffer consists of 1×PBS.

[0011] Preferably, the concentration of the magnetic iron oxide nanoparticles used is 40–70 μg / mL, and they are incubated with macrophages for 8–24 hours.

[0012] Preferably, the concentration of polyethylene glycol diacrylate in the gel solution is 5-10 wt%, and the molecular weight ranges from 500 to 1000 Da.

[0013] Preferably, the final concentration of the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropanone is 1.2-1.5 g / mL, and the incubation time of the gel solution with the magnetized cells is 5-10 minutes.

[0014] Preferably, the cell suspension is irradiated with a 365nm ultraviolet lamp for 5 to 10 minutes, and the ultraviolet lamp power is 10 to 50W.

[0015] This invention proposes using the DNA-modified colloidal macrophages prepared by the above scheme for rapid identification, capture, enrichment and detection of specific pathogenic microorganisms.

[0016] This invention proposes a method for using the aforementioned DNA-modified colloidal macrophages in bacterial analysis, comprising the following steps:

[0017] The colloidal macrophage solution was incubated with a bacterial sample solution at room temperature with shaking for a period of time. Then, the DNA-modified colloidal macrophages were separated using a magnetic rack. Wash with PBS solution;

[0018] Then, take 3–5 μL of the solution for microscopic observation and analysis. The morphology and number of bacteria captured.

[0019] In this invention, because bacteria activate DNA ribozymes on the surface of colloidal macrophages, the ribozymes generate fluorescent signals after self-cleavage. The fluorescence signals on the surface of colloidal macrophages can be analyzed by fluorescence microscopy or flow cytometry to determine whether specific bacteria are present in the solution or to quantitatively analyze a certain type of bacteria.

[0020] Preferably, the The number of cell granules in the suspension ranges from 1×10³ to 1×10⁵ cells.

[0021] Preferably, the volume ratio of the solution to the sample solution is 1:1, the incubation time is 15 to 30 minutes, and the shaking speed of the mixture is 200 to 450 rpm.

[0022] Preferably, the reaction buffer solution is composed of 10 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 2 mM CaCl2, and pH 7.4.

[0023] The principle behind the preparation and use of colloidal macrophages in this invention is as follows: Macrophages are natural immune cells in mammals, possessing an extremely strong ability to recognize pathogenic microorganisms. First, utilizing the phagocytic properties of macrophages, magnetic nanoparticles are introduced into the culture medium. Macrophages can engulf these particles in large quantities, thus obtaining magnetized cells capable of responding to external magnets. Second, the monomers and initiator of the gel freely permeate through the cell membrane into the cell interior. After irradiation with ultraviolet light, the monomers undergo a polymerization reaction and cross-link with each other, forming a robust three-dimensional network structure inside the cell, greatly enhancing the cell's mechanical strength, thereby obtaining colloidal macrophages. Finally, by modifying the surface of the colloidal macrophages with DNA ribozymes, the cell particles can generate responsive fluorescent signals to specific bacteria. This is because, in the presence of specific bacteria, their surface antigens or secretions can activate DNA ribozymes, allowing the DNA ribozyme molecules to cleave themselves and generate signals. In the absence of the target bacteria, the fluorescent groups and quenching groups on the DNA ribozyme approach each other, quenching the fluorescence. However, when specific bacteria are present, DNA ribozymes are activated and cleave their own nucleic acid chains, causing quenching groups to detach from the surface of cell particles and restoring the fluorescence of fluorescent groups, thereby achieving the purpose of reporting the presence of specific bacteria.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. Colloidal macrophages, due to their colloidal core, exhibit significantly enhanced stability, enabling them to resist various adverse external conditions. Simultaneously, colloidal macrophages retain their intact cell membrane structure.

[0026] 2. Magnetized macrophages can be easily manipulated by an external magnet, facilitating in vitro magnetic sorting and collection of bacteria from samples and various routine procedures;

[0027] 3. Colloidal macrophages can not only recognize various bacteria through receptors on their cell membrane surface, but also generate detectable fluorescent signals through changes in DNA ribozymes, specifically reporting the presence of a particular bacterium. Attached Figure Description

[0028] 1. Figure 1 yes The preparation and usage principle diagram of it.

[0029] 2. Figure 2 yes The characterization results are shown in the figure.

[0030] 3. Figure 3 yes Results of the capture and detection of Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0031] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0032] Example 1:

[0033] In implementing this protocol, RAW264.7 cells (mouse macrophages) were used for the experiments.

[0034] First, RAW264.7 cells (mouse macrophages) were cultured on plates until the cell density reached 70%–85%. Then, magnetic iron oxide nanoparticles were added. After the cells fully engulfed the magnetic particles, the cells were washed three times to remove any unengulfed magnetic particles. The magnetized cells were then pipetted off to form a cell suspension. The results are as follows: Figure 2As shown in Figure A. Simultaneously, the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropanone was mixed with the gel monomer polyethylene glycol diacrylate to prepare a gel solution. Then, a certain concentration of the gel solution was added to the cell suspension. After incubation, cells were collected by magnetic sorting and resuspended in phosphate buffer. Cells were irradiated with a 365 nm UV lamp to initiate a polymerization reaction, obtaining colloidal macrophages. Finally, the stability of the obtained colloidal cell particles was verified using a pure water treatment experiment, with ordinary macrophages as a control. Since ordinary macrophages rupture rapidly in pure water, while colloidal macrophages can withstand this hypotonic environment and maintain the integrity of their cell morphology and structure, the results are as follows. Figure 2 As shown in B. The concentration of the magnetic iron oxide nanoparticles used was 40–70 μg / mL, and they were incubated with macrophages for 8–24 hours; the concentration of polyethylene glycol diacrylate in the gel solution was 5–10 wt%, and the molecular weight range was 500–1000 Da; the final concentration of the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropanone was 1.2–1.5 g / mL, and the incubation time of the gel solution with the magnetized cells was 5–10 minutes; the cell suspension was irradiated with a 365 nm ultraviolet lamp for 5–10 minutes, and the ultraviolet lamp power was 10–50 W.

[0035] Then, DNA ribozyme-modified colloidal macrophages were prepared;

[0036] Using *E. coli* as the target bacterium, a DNA ribozyme targeting *E. coli* was anchored onto the colloidal macrophage membrane via cholesterol insertion. The DNA ribozyme sequence was: 5'-FITC (fluorescent group)-TGTCGAGACCTGCGACAGGAAGACTACACACAGTTGTGTG-cholesterol-3', and the substrate sequence was: 5'-cholesterol-ACTCTTCCTAGCTrATGGTTCGATCAAGA-BHQ1 (quencher group)-3'. Equimolar concentrations of the DNA ribozyme and substrate were mixed and heated at 90°C for 3 minutes, then gradually cooled to room temperature to form a DNA ribozyme sensing element. Colloidal macrophage particles were added, and after incubation, excess nucleic acid was removed by magnetic separation. Store at 4°C for subsequent use. The final concentration of the DNA ribozyme / substrate is 50–250 nM, and the incubation time with colloidal macrophages is 15–20 minutes; the buffer solution is 1×PBS.

[0037] Example 2:

[0038] use The specific procedures for capturing and detecting bacteria in samples are as follows:

[0039] The colloidal macrophage solution and bacterial suspension were incubated with continuous shaking at room temperature for a period of time. Then, cell particles were separated using a magnet and washed twice with PBS. Finally, 3–5 μL of the colloidal macrophage solution was analyzed under an optical microscope. Gram-negative *Escherichia coli* and Gram-positive *Staphylococcus aureus* were used as examples. Figure 3 A shows the presence of large numbers of Escherichia coli and Staphylococcus aureus. Particles. Meanwhile, because bacteria activate DNA ribozymes on the surface of colloidal macrophages, the ribozymes self-cleave, generating fluorescent signals. These fluorescent signals can be analyzed using fluorescence microscopy or flow cytometry to determine the presence of specific bacteria in the solution or to quantitatively analyze a particular bacterium. Taking the detection of *E. coli* as an example, flow cytometry results show... The fluorescence increased with the increase of the number of E. coli in the sample. Figure 3 (B and 3C), proof It can be used for quantitative analysis of the number of bacteria in a sample. The number of colloidal cell particles in the suspension is 1×10³ to 1×10⁵ cells; the volume ratio of the colloidal cell particle solution to the sample solution is 1:1; the mixing solution is shaken at 200–450 rpm and incubated for 15–30 minutes; the reaction buffer consists of 10 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl₂, 2 mM CaCl₂, and pH 7.4.

[0040] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing DNA-modified colloidal macrophages, characterized by, The details are as follows: After the macrophages reach 70%~85% of the plate culture density, magnetic iron oxide nanoparticles solution is added for further incubation for a period of time, and after the cells phagocytose the magnetic particles, the suspension magnetized cells are obtained by digestion; The photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylprophenone is mixed with polyethylene glycol diacrylate to prepare a gel solution; Then, a certain concentration of the gel solution is added to the cell suspension, and after incubation, the cells are collected by low-speed centrifugation, and the cells are resuspended with phosphate buffer, and the cells are irradiated with a wavelength of 365 nm ultraviolet lamp to initiate polymerization, and obtain the gelatinized macrophages; After the DNA ribozyme chain and the substrate chain are mixed and heated and cooled to room temperature, the gelatinized macrophages are added for incubation, the excess DNA is removed by magnetic separation, and the obtained DNA-GMø is stored at 4 ℃ for subsequent use, the final concentration of the DNA ribozyme chain / substrate chain is 50~250 nM, and the incubation time with the gelatinized macrophages is 15~20 minutes. The reaction buffer component is 1×PBS.

2. The method for preparing DNA-modified colloidal macrophages as described in claim 1, characterized in that, The concentration of the magnetic iron oxide nanoparticles used is 40~70 μg / mL, and the incubation time with the macrophages is 8~24 hours.

3. The method for preparing DNA-modified colloidal macrophages as described in claim 1, characterized in that, The concentration of polyethylene glycol diacrylate in the gel solution is 5~10 wt%, and the molecular weight range is 500~1000 Da.

4. The method for preparing DNA-modified colloidal macrophages as described in claim 1, characterized in that, The final concentration of the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylprophenone is 1.2~1.5 g / mL, and the incubation time of the gel solution with the magnetized cells is 5~10 minutes.

5. The method for preparing DNA-modified colloidal macrophages as described in claim 1, characterized in that, The irradiation time of the cell suspension with a 365 nm ultraviolet lamp is 5~10 minutes, and the power of the ultraviolet lamp is 10~50 W.

6. DNA-modified colloidal macrophages according to any one of claims 1 to 5, characterized in that: It is used for rapid identification, capture, enrichment and detection of specific pathogenic microorganisms.

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