A multi-modal bioaerosol simulation material based on high-stability fluorescent protein labeling and a preparation method thereof

Abstract

The application discloses a kind of multi-modal biological aerosol simulation materials based on high stability fluorescent protein label and construction method thereof, belong to biological aerosol simulation technical field.The simulation material is made of the microorganism expressing StayGold fluorescent protein by genetic engineering modification, and is compounded with the hydrophobic silicon dioxide particles of specific particle size.The core of the present application is: first use the StayGold protein with excellent light stability as biomarker, overcome the defect that traditional fluorescent protein is easily quenched in simulation environment;Innovatively design the "microorganism-dust" composite particle structure, by optimizing the concentration of bacterial suspension, particle ratio and adding isopropyl alcohol additive, generate aerosol with particle size distribution of 1-10 μm by atomization technology, highly reproduce the physical and chemical state of biological aerosol in real environment.The simulation material maintains high survival rate and fluorescent signal stability in wide temperature range and wide humidity range.

Description

Technical Field

[0001] This invention relates to the fields of biosafety, environmental science and experimental modeling, specifically to a standardized material for high-fidelity simulation of real-world bioaerosols and its preparation method, particularly a multimodal bioaerosol simulation material constructed using highly stable fluorescent protein (StayGold) labeling technology and its preparation method. Background Technology

[0002] Bioaerosols are airborne particles composed of living microorganisms (such as bacteria, fungi, and viruses) or fragments thereof. Research on their transmission patterns is crucial for public health, biosafety, and environmental monitoring. However, directly studying pathogenic bioaerosols in real-world environments carries extremely high and uncontrollable risks; therefore, developing safe and reliable laboratory simulation techniques is essential.

[0003] Currently, laboratory simulation of bioaerosols faces the following major technical bottlenecks: 1) Poor simulation realism: Most simulation systems use single microorganisms or simple particles, which cannot reproduce the complex state in the real atmosphere where microorganisms often coexist with non-biological particles such as dust and droplets. This results in a large deviation between the simulated physicochemical properties, aerodynamic behavior and environmental durability and the actual situation.

[0004] 2) Difficulties in tracing and tracking: Traditional analogs lack effective and stable in vivo markers. Commonly used fluorescent proteins (such as GFP) are prone to photobleaching under light, especially ultraviolet light, and the fluorescence signal decays rapidly, which cannot meet the needs of long-term, outdoor or strong light conditions for simulated tracking.

[0005] 3) Single model organism: Existing simulation systems are mostly based on a single type of microorganism (such as bacteria only), lacking a multimodal simulation platform that can simultaneously cover prokaryotes (bacteria), eukaryotic microorganisms (yeast) and viruses (lentivirus models), which limits the universality of research conclusions.

[0006] 4) Low standardization: The lack of unified standards for key parameters such as the preparation process, particle size, and concentration of the simulated materials makes it difficult to compare and replicate the results of different studies.

[0007] Therefore, there is an urgent need and significant application value in developing a bioaerosol simulation material that can highly simulate the real environment, is label-stable, applicable to multiple biological models, and has standardized preparation. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a multimodal bioaerosol simulation material based on highly stable fluorescent protein labeling and its construction method. This material can safely and faithfully simulate bioaerosols in real environments, and possesses excellent stability and traceability. The specific solution is as follows: A multimodal bioaerosol mimicry material based on highly stable fluorescent protein labeling is characterized by being composed of engineered microorganisms expressing StayGold fluorescent protein and hydrophobic silica particles, wherein the engineered microorganisms are selected from at least one of Escherichia coli, yeast, or lentivirus.

[0009] The photostability of the StayGold fluorescent protein is more than 50% higher than that of the traditional GFP protein, and its fluorescence intensity retention rate is not less than 90% after continuous irradiation with 254nm wavelength ultraviolet light for 30 minutes.

[0010] The particle size of the hydrophobic silica particles is selected from one or more of 10 nm, 100 nm, or 1000 nm.

[0011] The mass mixing ratio of the engineered microorganisms to the hydrophobic silica particles is 9-11:1.

[0012] The mass mixing ratio of the engineered microorganisms to the hydrophobic silica particles is 10:1.

[0013] The final form of the material is aerosol particles (with a particle size distribution of 1-10 μm) generated by an atomization device.

[0014] A method for preparing a multimodal bioaerosol mimic material based on a highly stable fluorescent protein label includes the following steps: (1) Construction of engineered strains: The codon-optimized StayGold fluorescent protein gene was integrated into the genome of the target microorganism using CRISPR-Cas9 gene editing technology, and its expression was driven by a species-specific promoter. (2) Microbial culture and expression: The engineered strain was inoculated into an optimized culture medium for fermentation culture to induce the expression of StayGold fluorescent protein; (3) Preparation of composite suspension: Adjust the concentration of the bacterial suspension obtained in step (2) to 10. 9 CFU / mL was mixed with hydrophobic silica particles with a particle size of 10 nm, 100 nm or 1000 nm, and isopropanol was added to the mixture to disperse it evenly. (4) Aerosolization: The composite suspension prepared in step (3) is placed in an atomizing device to generate biological-dust composite aerosol particles.

[0015] In step (1), the species-specific promoter is: the J23110 promoter for Escherichia coli, the GAL1 promoter for yeast, or the CMV promoter for lentivirus.

[0016] In step (3), the concentration of isopropanol is 20%.

[0017] The present invention provides a multimodal bioaerosol simulation material based on highly stable fluorescent protein labeling that can be applied to the following scenarios: (1) laboratory simulation and research on the transmission routes of infectious disease aerosols; (2) simulation and safety assessment of the diffusion of microbial pollution in ambient air; (3) simulation research on the migration behavior of bioaerosols in building ventilation systems or public spaces; and (4) training and evaluation simulation of aerosol diffusion and decontamination effects in biological defense.

[0018] The beneficial effects and outstanding advantages of this invention are as follows: 1) Revolutionary improvement in simulation fidelity: The first-ever bio-dust composite particle design, by introducing hydrophobic silica and isopropanol of different particle sizes, controls the particle size, density and surface properties of aerosols, making their aerodynamic behavior highly consistent with that of bioaerosols in the real environment (such as microorganisms attached to dust particles).

[0019] 2) Breakthrough in tracking stability: For the first time, StayGold, an ultra-stable fluorescent protein, has been applied to the field of aerosol simulation. Compared with traditional GFP, StayGold has more than 50% improved resistance to photobleaching and can still maintain more than 90% fluorescence after 30 minutes of UV irradiation, achieving long-term, stable, and high signal-to-noise ratio fluorescence tracking of aerosols.

[0020] 3) Constructing a unified multimodal platform: By designing different gene manipulation strategies (prokaryotic expression, eukaryotic expression, and viral transduction), the labeling and simulation of three representative biological models—bacteria, yeast, and mammalian cells—were successfully achieved within the same technical system, greatly expanding the scope and representativeness of simulation research.

[0021] 4) High standardization and repeatability of the process: The concentration of the bacterial suspension (10) was clearly defined. 9 Key parameters such as CFU / mL, particle mixing ratio (10:1), additive dosage (20% isopropanol), and atomization process were established, creating a standardized and repeatable preparation process that provides a reliable basis for comparative studies in different laboratories.

[0022] 5) Good environmental adaptability and safety: The prepared simulation materials exhibit stable performance within a wide temperature range of 4-50℃ and a relative humidity range of 30-90%, with stable fluorescence signal (coefficient of variation <5%) and high survival rate (>90%). Furthermore, non-pathogenic engineered bacteria or cell models can be used to simulate high-risk scenarios under completely safe conditions. Attached Figure Description

[0023] Figure 1 : This is a fluorescence characterization verification of StayGold. (A) is the SDS detection image of high-purity StayGold protein; (B) is the StayGold fluorescent protein in the excited state.

[0024] Figure 2 Image of Escherichia coli, yeast and lentivirus transduced cells expressing StayGold under a fluorescence microscope.

[0025] Figure 3 Part A focuses on fluorescence linear correlation analysis (fluorescence linear correlation analysis) and functional excitation testing (functional excitation testing) for fluorescence characterization techniques. Figure 4 : This section describes the preparation and characterization of multimodal bioaerosols. (A) is a scanning electron microscope image of the bio-silica composite particles; (B) is a fluorescence linear correlation analysis of the bio-silica composite particles.

[0026] Figure 5 : This is an example of the application of composite bioaerosols. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0028] Example 1: In vitro purification and stability analysis of StayGold fluorescent protein The StayGold gene sequence, optimized with E. coli preferred codons, was synthesized into the expression vector pet28a and transformed into strain BL21(DE3). Protein expression was induced by IPTG at 20°C for 20 hours, followed by protein purification by affinity chromatography. Using conventional GFP as a control, fluorescence variation coefficients were analyzed after 30 minutes of UV irradiation. Figure 1 ).

[0029] Example 2: Construction of an engineered strain expressing StayGold fluorescent protein Engineered *E. coli* strain: The *StayGold* gene sequence, optimized with *E. coli* preferred codons, was synthesized and cloned into a CRISPR editing plasmid containing the constitutive strong promoter J23110. The sequence was introduced into *E. coli* DH5α competent cells via electroporation, and positive clones were screened on plates containing appropriate antibiotics. Correct gene integration was verified by sequencing.

[0030] Engineered Saccharomyces cerevisiae: The StayGold gene, with codon-optimized synthetic yeast preference, was cloned into a yeast integration vector containing a GAL1 inducible promoter. It was introduced into Saccharomyces cerevisiae strain W303 via lithium acetate conversion and induced to express on galactose medium. Strongly fluorescent colonies were screened.

[0031] Lentiviral packaging and cell transduction: The StayGold gene was cloned into a lentiviral transfer plasmid containing the CMV promoter, and co-transfected with the packaging plasmid into HEK293T cells. The viral supernatant was collected. This viral supernatant was used to infect HEK293 cells, thereby amplifying lentiviruses carrying the StayGold gene.

[0032] At the same time, the morphology of the three types of cells was observed under a microscope. Figure 2 ).

[0033] Example 3: Preparation of bio-dust composite aerosol simulation material Microbial culture: The three engineered microorganisms mentioned above were cultured on a large scale. Bacteria and yeast were cultured in LB or YPD medium, and cells were cultured in DMEM complete medium. Microorganisms in the logarithmic growth phase were harvested, washed with PBS buffer, resuspended, and their concentration was adjusted to 10⁻⁶ using a densitometer or cell counter. 9 CFU / mL.

[0034] Preparation of the composite suspension: Take 10 mL of the above bacterial suspension and add 1.0 g of hydrophobic silica particles (particle sizes of 10 nm, 100 nm, and 1000 nm, divided into three groups for the experiment). Then add 2 mL of isopropanol (to make the final concentration 20% v / v). Place the mixture on a vortex shaker and shake vigorously for 2 minutes, then sonicate (50 W power, 30 seconds, ice bath) to ensure sufficient particle dispersion and uniform adhesion of microorganisms.

[0035] Aerosolization: The prepared composite suspension is added to the reservoir of a commercial nebulizer (e.g., the Collison nebulizer). Nebulization is performed at a constant pressure (e.g., 20 psi), and the resulting aerosol is introduced into a drying mixing chamber to evaporate most of the droplet moisture, ultimately obtaining dried "microorganism-silica" composite aerosol particles. The correlation between fluorescence intensity and bacterial density is determined by a dilution-coating method coupled with an ELISA reader. Figure 3 ).

[0036] Example 4: Performance Characterization of Simulated Materials Particle size and morphology characterization: Aerosol particles were observed using scanning electron microscopy, such as... Figure 4 As shown, microbial cells (bacilli or spherical) are partially coated or tightly attached to smaller silica nanoparticles, forming a stable composite structure.

[0037] Fluorescence analysis of materials and biological complexes: Correlation tests were performed on biological complex solutions of different dry weights using an enzyme-linked immunosorbent assay (ELISA) reader. Figure 4 B), the fluorescence signal of the composite particles maintained a good linear relationship with the microbial concentration. Figure 4 B), which laid the foundation for quantitative analysis.

[0038] Example 5: The simulated material prepared in this invention (taking an engineered yeast expressing StayGold combined with 100 nm silica as an example) was applied to an experiment on bioaerosol diffusion under simulated office ventilation conditions. A fixed amount of the simulated aerosol was released at a fixed location in a sealed experimental chamber (volume 1 m³). The ventilation system inside the chamber was turned on, and different air exchange rates were set. Because the StayGold fluorescence signal is extremely stable, the sampled filter membrane can be directly observed and counted under a fluorescence microscope, or quantitatively analyzed using a fluorescence microplate reader, thus clearly and quantitatively plotting the spatiotemporal distribution of aerosols under different ventilation conditions. This application demonstrates the effectiveness and convenience of this simulated material in studying airborne propagation patterns. Figure 5 ).

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. All equivalent changes and modifications made in accordance with the technical solutions of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A multimodal bioaerosol mimicry material based on highly stable fluorescent protein labeling, characterized in that: It is composed of engineered microorganisms expressing StayGold fluorescent protein and hydrophobic silica particles, wherein the engineered microorganisms are selected from at least one of Escherichia coli, yeast or lentivirus.

2. The multimodal bioaerosol mimicry material based on highly stable fluorescent protein labeling as described in claim 1, characterized in that: The particle size of the hydrophobic silica particles is selected from one or more of 10 nm, 100 nm, or 1000 nm.

3. The multimodal bioaerosol mimicry material based on highly stable fluorescent protein labeling as described in claim 1, characterized in that: The mass mixing ratio of the engineered microorganisms to the hydrophobic silica particles is 9-11:

1.

4. The multimodal bioaerosol mimicry material based on highly stable fluorescent protein labeling as described in claim 3, characterized in that: The mass mixing ratio of the engineered microorganisms to the hydrophobic silica particles is 10:

1.

5. The multimodal bioaerosol mimicry material based on highly stable fluorescent protein labeling as described in claim 1, characterized in that: The final form of the material is aerosol particles generated by an atomizing device.

6. A method for preparing a multimodal bioaerosol mimicry material based on a highly stable fluorescent protein label as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Construction of engineered strains: The codon-optimized StayGold fluorescent protein gene was integrated into the genome of the target microorganism using CRISPR-Cas9 gene editing technology, and its expression was driven by a species-specific promoter. (2) Microbial culture and expression: The engineered strain was inoculated into an optimized culture medium for fermentation culture to induce the expression of StayGold fluorescent protein; (3) Preparation of composite suspension: Adjust the concentration of the bacterial suspension obtained in step (2) to 10. 9 CFU / mL was mixed with hydrophobic silica particles with a particle size of 10 nm, 100 nm or 1000 nm, and isopropanol was added to the mixture to disperse it evenly. (4) Aerosolization: The composite suspension prepared in step (3) is placed in an atomizing device to generate biological-dust composite aerosol particles.

7. The method for preparing a multimodal bioaerosol mimic material based on a highly stable fluorescent protein label as described in claim 6, characterized in that, In step (1), the species-specific promoter is: the J23110 promoter for Escherichia coli, the GAL1 promoter for yeast, or the CMV promoter for lentivirus.

8. The method for preparing a multimodal bioaerosol mimic material based on a highly stable fluorescent protein label as described in claim 6, characterized in that: In step (3), the concentration of isopropanol is 20%.

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