A Staphylococcus aureus simulant based on polystyrene microspheres and preparation method thereof

Abstract

The present invention discloses a Staphylococcus aureus simulant based on polystyrene microspheres and a preparation method thereof. The present invention adopts a dispersion polymerization method and a series of screening technologies to obtain highly monodisperse polystyrene fluorescent microspheres with a diameter similar to that of Staphylococcus aureus, and conducts a comparative test with Staphylococcus aureus, confirming that the simulant of the present invention can effectively simulate Staphylococcus aureus in terms of particle size, fluorescent characteristic signals and aerosol simulation, and can be widely used in bioaerosol monitoring and simulation testing of protective equipment.

Description

Technical Field

[0001] The invention relates to the technical field of bioaerosol protection, and in particular to a Staphylococcus aureus simulant based on polystyrene microspheres and a preparation method thereof. Background Art

[0002] Bioaerosols are aerosols composed of any kind of biological particles with an aerodynamic diameter of 100 μm or less. The properties of bioaerosols vary depending on environmental influences, such as humidity, airflow, and temperature. Aerosols facilitate the airborne transmission of microorganisms in the air and are composed of small particles called droplet nuclei (1-100 μm) or droplets (>100 μm); droplet nuclei can remain in the air for several hours, be transmitted over long distances, and contaminate environmental surfaces by falling. Droplets have been shown to contaminate surfaces within a range of 1 meter. Bioaerosols have been shown to contain influenza or rhinoviruses, Mycobacterium tuberculosis, Staphylococcus aureus, varicella zoster virus, Streptococcus spp., or Aspergillus spp. The spread and transmission of bioaerosols in the air has the potential to cause epidemic transmission between humans and between humans and animals.

[0003] In order to effectively provide timely warning and protection for bioaerosols in the atmosphere, it is necessary to identify the types of bioaerosols. However, in the performance testing of bioaerosol monitoring and protective equipment, there are safety risks in using infectious microbial aerosols to conduct experiments. Therefore, finding substances that can effectively replace infectious microbial aerosols has become a technical problem that needs to be solved urgently at this stage. Staphylococcus aureus is one of the most common causes of hospital-acquired infections and foodborne diseases, and is also an important component of bioaerosols. The toxins and highly toxic proteases of Staphylococcus aureus usually circulate in the host's blood vessels, causing life-threatening diseases. Typical Staphylococcus aureus is spherical with a diameter of about 0.8μm. How to construct a bioaerosol with typical Staphylococcus aureus characteristics has become a technical problem that needs to be solved urgently. Summary of the invention

[0004] Based on the above technical problems to be solved by the present invention, the present invention adopts dispersion polymerization method and series screening technology to obtain highly monodisperse polystyrene fluorescent microspheres with a diameter similar to that of Staphylococcus aureus, and proposes a Staphylococcus aureus simulator based on polystyrene microspheres and a preparation method thereof. The simulator of the present invention can effectively simulate Staphylococcus aureus in terms of particle size, fluorescent characteristic signals and aerosol simulation.

[0005] One of the purposes of the present invention is to provide a Staphylococcus aureus simulator based on polystyrene microspheres, wherein the simulator is a highly monodisperse micron-submicron polymer microsphere; the simulator comprises chloromethylated polystyrene microspheres.

[0006] Furthermore, the simulation agent has a diameter of 0.7 μm to 0.9 μm.

[0007] Furthermore, the CV value of the simulation agent is 3.0% to 7.0%.

[0008] The second object of the present invention is to provide a method for preparing a Staphylococcus aureus simulant based on polystyrene microspheres, comprising:

[0009] S1. Add polyvinyl pyrrolidone, azobisisobutyronitrile and styrene to ethanol and aqueous solution, dissolve the prepared ethanol solution and put it into a heated magnetic stirrer, introduce nitrogen to remove oxygen in the system, turn on the heated magnetic stirrer for polymerization reaction, obtain polystyrene submicron microsphere dispersion, collect by centrifugation; suspend with anhydrous ethanol, add aminosilane, stir, wash with anhydrous ethanol, and collect;

[0010] S2, swelling polystyrene microspheres in chloromethyl methyl ether, adding aluminum chloride to the mixture; stirring at 40°C to 60°C, collecting the microspheres by centrifugation, and washing them multiple times with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water; collecting by centrifugation, suspending in anhydrous ethanol solution after centrifugation, adding aminosilane, stirring at room temperature, washing with anhydrous ethanol and collecting, to obtain stable fluorescent microspheres;

[0011] S3, prepare a glycerol / water solution with a gradient mass ratio, and calculate its density and viscosity; add the prepared polystyrene microsphere sample to the glycerol / water solution, determine the glycerol / water mass ratio when the microspheres can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microspheres; replace the glycerol / water mass ratio solution, calculate the time required for the target microspheres to settle to the bottom according to the formula, settle the sample, and after reaching the calculated time, remove the upper small microspheres, repeat several times until the upper layer is nearly clear; settle the sample again, collect the upper liquid microspheres before the calculated time, remove the substrate microspheres, repeat several times until there is no obvious microsphere precipitation on the substrate, and obtain microspheres of the target particle size;

[0012] The calculation formula of the sinking time includes:

[0013] Among them, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the acceleration of gravity, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density;

[0014] When the density of the ρ liquid and the ρ ball is not equal, the microspheres will float or sink in the solution; when the density of the microspheres is less than the density of the aqueous solution, the polymer microspheres will float in the aqueous phase; when the density of the microspheres is exactly equal to the density of the aqueous solution, the microspheres can be stably suspended in the solution; when the density of the microspheres is greater than the density of the aqueous solution, the microspheres will sink to the bottom of the bottle;

[0015] S4, further screening the polystyrene microspheres using an ultrafiltration centrifuge tube, adding the microspheres of the target particle size obtained in S3 to the ultrafiltration centrifuge tube, centrifuging, and resuspending the microspheres in the upper filter with ethanol to obtain microspheres of the target particle size;

[0016] S5. Atomize the monodisperse polystyrene microspheres to form an aerosol environment with uniform and stable concentration, and obtain a Staphylococcus aureus simulant based on polystyrene microspheres.

[0017] Furthermore, in S1, the content of polyvinyl pyrrolidone is 1wt% to 5wt%, the content of azobisisobutyronitrile is 0.6wt% to 1.4wt%, the content of styrene is 6wt% to 14wt%, the ratio of water to ethanol is 0 to 1:4, and the reaction temperature is 60°C to 80°C.

[0018] Furthermore, in S1, the content of polyvinyl pyrrolidone is 2wt% to 4wt%, the content of azobisisobutyronitrile is 0.8wt% to 1.2wt%, the content of styrene is 8wt% to 12wt%, the ratio of water to ethanol is 0 to 1:4, and the reaction temperature is 65°C to 75°C.

[0019] Furthermore, the average particle size of the simulation agent is 711nm to 755nm.

[0020] Furthermore, the simulation agent produces a broad fluorescence emission peak in the range of 400nm to 500nm, and the maximum fluorescence emission peaks are stabilized at 435nm to 443nm and 458nm to 466nm, respectively.

[0021] Furthermore, the fluorescence lifetime of the mimetic agent remains stable for at least 180 days.

[0022] Furthermore, the simulation agent can simulate Staphylococcus aureus in bioaerosol.

[0023] Compared with the prior art, the present invention proposes a Staphylococcus aureus simulant based on polystyrene microspheres and a preparation method thereof, which has the following beneficial effects:

[0024] 1. The polystyrene microsphere-based Staphylococcus aureus simulant proposed in the present invention was subjected to comparative tests with real Staphylococcus aureus, which proved that the microspheres prepared in the present invention can effectively simulate Staphylococcus aureus in terms of particle size, fluorescent characteristic signals and aerosol simulation, and can be widely used in bioaerosol monitoring and simulation testing of protective equipment.

[0025] 2. The present invention adopts dispersion polymerization method and series screening technology to obtain highly monodisperse polystyrene fluorescent microspheres with a diameter of 0.7 μm to 0.9 μm, which are highly monodisperse microspheres with a CV value of 3.0% to 7.0%.

[0026] 3. The Staphylococcus aureus simulant based on polystyrene microspheres prepared by the present invention has the characteristics of good controllability, high stability, low toxicity and easy operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows the influence of different St concentrations on the average particle size and coefficient of variation of polystyrene microspheres according to the embodiment of the present invention; A: a statistical diagram of the average particle size and coefficient of variation of polystyrene microspheres; B: a SEM image of polystyrene microspheres when the St concentration is 6wt%; C: a SEM image of polystyrene microspheres when the St concentration is 10wt%; D: a SEM image of polystyrene microspheres when the St concentration is 14wt%;

[0028] Figure 2 The effect of different PVP concentrations on the particle size and coefficient of variation of polystyrene microspheres according to an embodiment of the present invention is shown; A: a statistical diagram of the average particle size and coefficient of variation of polystyrene microspheres; B: a SEM image of polystyrene microspheres when the PVP concentration is 3wt%;

[0029] Figure 3 The effects of different screening methods on the particle size dispersibility of microspheres in the embodiments of the present invention are shown; A: crude polystyrene microspheres; B: microspheres screened by gradient sedimentation method; C: microspheres screened by gradient sedimentation method and 650nm ultrafiltration centrifuge tube;

[0030] Figure 4 A comparison diagram of the fluorescence emission spectra of Staphylococcus aureus and polystyrene microspheres according to an embodiment of the present invention is shown;

[0031] Figure 5 A comparison diagram of the fluorescence lifetime of a polystyrene microsphere at 0 day (left) and 180 days (right) of an embodiment of the present invention is shown;

[0032] Figure 6 A comparison diagram of particle size distribution of Staphylococcus aureus and polystyrene microspheres in aerosols according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0033] The exemplary embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0034] In the following examples, unless otherwise specified, the technical means adopted are conventional means well known to those skilled in the art, and the reagents and materials in the present invention are obtained from the market or other public channels.

[0035] The experimental materials and equipment involved in the present invention mainly include but are not limited to:

[0036] Reagents: 2,2-azobisisobutyronitrile (A104255, Aladdin), 2,2-azobisisobutyronitrile (A104256, Aladdin), styrene (S817904, Macklin), polyvinylpyrrolidone (P274371, Aladdin), anhydrous ethanol (AR, Kermel), chloromethyl methyl ether (107-30-2, Zhengzhou Wanxiang Fine Chemical Co., Ltd.), aluminum chloride (206911, Sigma-Aldrich), hydrochloric acid solution (W530574, Sigma-Aldrich), 0.65 μm low-binding Durapore PVDF membrane (UFC40DV25, Merck), acetone (48358, Supelco);

[0037] Instruments: heat-collecting constant-temperature heating magnetic stirrer (DF-101S, Gongyi Yuhua Instrument Co., Ltd.), low-temperature constant-temperature circulating water tank (N1-2RC, Bioer), high-speed large-capacity centrifuge (CR22G, Hitachi).

[0038] The fluorescent dyeing of the present invention mainly prepares chloromethylated polystyrene microspheres by Friedel-Crafts alkylation; 5 g of polystyrene microspheres are swollen in 50 mL of chloromethyl methyl ether for 60 minutes, and then 1.2 wt % of aluminum chloride is added to the mixture; after stirring at 50° C. for 12 hours, the microspheres are collected by centrifugation and washed multiple times with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water.

[0039] The technical principles of the present invention mainly include:

[0040] Highly monodisperse polystyrene fluorescent microspheres with a diameter similar to that of Staphylococcus aureus were obtained by using a dispersion polymerization method and a series of screening technologies. The CV value of the simulator constructed by the present invention was 3.0% to 7.0%, achieving high monodispersity and a highly consistent simulation of Staphylococcus aureus. By conducting comparative tests with Staphylococcus aureus, it was confirmed that the simulator described in the present invention can effectively simulate Staphylococcus aureus in terms of particle size, fluorescent characteristic signals and aerosol simulation, and can be widely used in bioaerosol monitoring and simulation testing of protective equipment.

[0041] Based on the above principle, the present invention proposes a method for preparing a Staphylococcus aureus simulant based on polystyrene microspheres, comprising:

[0042] S1. Add polyvinyl pyrrolidone, azobisisobutyronitrile and styrene to ethanol and aqueous solution, dissolve the prepared ethanol solution and put it into a heated magnetic stirrer, introduce nitrogen to remove oxygen in the system, turn on the heated magnetic stirrer for polymerization reaction, obtain polystyrene submicron microsphere dispersion, collect by centrifugation; suspend with anhydrous ethanol, add aminosilane, stir, wash with anhydrous ethanol, and collect;

[0043] S2, 5g of polystyrene microspheres were swelled in 50mL of chloromethyl methyl ether for 60 minutes, and 1.2wt% of aluminum chloride was added to the mixture; after stirring at 50°C for 12 hours, the microspheres were collected by centrifugation, and washed with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water for multiple times; collected by centrifugation, suspended in anhydrous ethanol solution after centrifugation, aminosilane was added, stirred at room temperature, washed with anhydrous ethanol and collected to obtain stable fluorescent microspheres;

[0044] S3, prepare a glycerol / water solution with a certain gradient mass ratio, and calculate its density and viscosity; add the prepared polystyrene microsphere sample to the glycerol / water solution, determine the glycerol / water mass ratio when the microspheres can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microspheres; replace the glycerol / water mass ratio solution, calculate the time required for the target microspheres to settle to the bottom according to the formula, settle the sample, and after reaching the calculated time, remove the upper small microspheres, repeat several times until the upper layer is nearly clear; settle the sample again, collect the upper liquid microspheres before the calculated time, remove the substrate microspheres, repeat several times until there is no obvious microsphere precipitation on the substrate, and obtain microspheres of the target particle size;

[0045] The calculation formula of the sinking time includes:

[0046] Among them, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the acceleration of gravity, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density;

[0047] S4, further screening the polystyrene microspheres using an ultrafiltration centrifuge tube, adding the microspheres of the target particle size obtained in S3 to the ultrafiltration centrifuge tube, centrifuging, and resuspending the microspheres in the upper filter with ethanol to obtain microspheres of the target particle size;

[0048] S5. Atomize the monodisperse polystyrene microspheres to form an aerosol environment with uniform and stable concentration, and obtain a polystyrene-based Staphylococcus aureus simulant.

[0049] Example 1

[0050] The invention provides a preparation and screening method of high monodispersity polystyrene fluorescent microspheres simulating Staphylococcus aureus with 10 wt% of styrene.

[0051] Mainly include:

[0052] S1. Add 3wt% polyvinyl pyrrolidone, 1wt% azobisisobutyronitrile and 10wt% styrene to every 100mL ethanol and water solution, the volume ratio of ethanol to water in the ethanol solution is 1:4, the reaction temperature is 70°C, dissolve every 100mL of the prepared ethanol solution and put it into an ultrasonic reactor, introduce nitrogen to remove oxygen in the system, turn on the ultrasonic generator for polymerization reaction, obtain a polystyrene submicron microsphere dispersion, and collect it by centrifugation; suspend it with 5mL anhydrous ethanol, add 5μL aminosilane, stir for 1 hour, wash it with an equal volume of anhydrous ethanol, and collect it;

[0053] S2, 5g of polystyrene microspheres were swelled in 50mL of chloromethyl methyl ether for 60 minutes, and 1.2wt% of aluminum chloride was added to the mixture; after stirring at 50°C for 12 hours, the microspheres were collected by centrifugation, and washed and collected with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water for multiple times; collected by centrifugation, suspended in anhydrous ethanol solution after centrifugation, aminosilane was added, stirred at room temperature, washed and collected with anhydrous ethanol, and stable fluorescent microspheres were obtained;

[0054] S3, prepare glycerol / water solution with a certain gradient mass ratio of 10%, 25%, 50%, etc., and calculate its density and viscosity; add the prepared polystyrene microsphere sample to the glycerol / water solution, determine the glycerol / water mass ratio when the microsphere can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microsphere; replace the glycerol / water mass ratio solution, calculate the time required for the target microsphere to settle to the bottom according to the formula, settle the sample, and after reaching the calculated time, remove the small microspheres on the upper layer, repeat several times until the upper layer is almost clear; settle the sample again, collect the upper liquid microspheres before the calculated time, remove the substrate microspheres, repeat several times until there is no obvious microsphere precipitation on the substrate, and finally obtain microspheres of the target particle size;

[0055] The calculation formula of the sinking time includes:

[0056] Among them, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the acceleration of gravity, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density;

[0057] S4, further screening the polystyrene microspheres using a 650nm ultrafiltration centrifuge tube, adding the microspheres of the target particle size obtained in S3 to a 650nm ultrafiltration centrifuge tube, centrifuging at 5000rpm for 10min, resuspending the microspheres in the upper filter with ethanol, repeating three times to obtain microspheres of the target particle size;

[0058] S5. Atomize monodisperse polystyrene microspheres and Staphylococcus aureus particles separately to form an aerosol environment with uniform and stable concentration; place the sampler in a measurement room and use an aerodynamic particle size analyzer to measure the aerosol particle concentration upstream and downstream of the sampler.

[0059] Results: See Figure 1 A and Figure 1 C, the particle size of the microspheres prepared by using 10wt% styrene in the present invention is 722nm, and the particle size distribution of the microspheres increases with the increase of the amount of styrene used. Monodispersed polystyrene microspheres cannot be obtained at too low a styrene concentration. Since it takes a long time to form primary particles at a lower styrene concentration, the primary particles formed cannot grow due to insufficient absorption of monomers, so the particle size distribution becomes wider; while at a higher styrene concentration, more primary nuclei are formed, the probability of collision between nuclei to form larger nuclei increases, the suspension ability of the dispersant becomes worse, and the balling process is accelerated, so the distribution becomes wider.

[0060] Example 2

[0061] The invention provides a preparation and screening method of high monodispersity polystyrene fluorescent microspheres simulating Staphylococcus aureus with 6wt% of styrene.

[0062] Mainly include:

[0063] S1. Add 3wt% polyvinyl pyrrolidone, 1wt% azobisisobutyronitrile and 6wt% styrene to every 100mL ethanol and water solution, the volume ratio of ethanol to water in the ethanol solution is 1:4, the reaction temperature is 70°C, dissolve every 100mL of the prepared ethanol solution and put it into an ultrasonic reactor, introduce nitrogen to remove oxygen in the system, turn on the ultrasonic generator for polymerization, obtain a polystyrene submicron microsphere dispersion, and collect it by centrifugation; suspend it with 5mL anhydrous ethanol, add 5μL aminosilane, stir for 1 hour, wash it with an equal volume of anhydrous ethanol, and collect it;

[0064] S2, 5g of polystyrene microspheres were swelled in 50mL of chloromethyl methyl ether for 60 minutes, and 1.2wt% of aluminum chloride was added to the mixture; after stirring at 50°C for 12 hours, the microspheres were collected by centrifugation, and washed and collected with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water for multiple times; collected by centrifugation, suspended in anhydrous ethanol solution after centrifugation, aminosilane was added, stirred at room temperature, washed and collected with anhydrous ethanol, and stable fluorescent microspheres were obtained;

[0065] S3. Prepare glycerol / water solutions with a certain gradient mass ratio of 10%, 25%, 50%, etc., and calculate their density and viscosity. Add the prepared polystyrene microsphere sample to the glycerol / water solution, determine the glycerol / water mass ratio when the microspheres can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microspheres; replace the glycerol / water mass ratio solution, calculate the time required for the target microspheres to settle to the bottom according to the formula, settle the sample, and after the calculated time is reached, remove the small microspheres on the upper layer, and repeat several times until the upper layer is almost clear. Sediment the sample again, and before the calculated time, collect the upper liquid microspheres, remove the substrate microspheres, and repeat several times until there is no obvious microsphere precipitation on the substrate, and finally obtain microspheres of the target particle size;

[0066] The calculation formula of the sinking time includes:

[0067] Among them, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the acceleration of gravity, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density.

[0068] Results: See Figure 1 A and Figure 1B. The particle size of the microspheres prepared by using 6wt% styrene in the present invention is 560nm. The particle size distribution of the microspheres increases with the increase of the amount of styrene used. Monodispersed polystyrene microspheres cannot be obtained with too low a styrene concentration. Since it takes a long time to form primary particles at a lower styrene concentration, the primary particles formed cannot grow due to insufficient absorption of monomers, so the particle size distribution becomes wider; while at a higher styrene concentration, more primary nuclei are formed, the probability of collision between nuclei to form larger nuclei increases, the suspension ability of the dispersant becomes worse, and the balling process is accelerated, so the distribution becomes wider.

[0069] Example 3

[0070] The invention provides a preparation and screening method of high monodispersity polystyrene fluorescent microspheres simulating Staphylococcus aureus with 14 wt% of styrene.

[0071] Mainly include:

[0072] S1. Add 3wt% polyvinyl pyrrolidone, 1wt% azobisisobutyronitrile and 14wt% styrene to every 100mL ethanol and water solution, the volume of ethanol to water in the ethanol solution is 1:4, the reaction temperature is 70°C, and each 100mL of the prepared ethanol solution is dissolved and placed in an ultrasonic reactor, nitrogen is introduced to remove oxygen in the system, and the ultrasonic generator is turned on for polymerization to obtain a polystyrene submicron microsphere dispersion, which is collected by centrifugation; suspend with 5mL anhydrous ethanol, add 5μL aminosilane, stir for 1 hour, wash with an equal volume of anhydrous ethanol, and collect.

[0073] S2, 5g of polystyrene microspheres were swelled in 50mL of chloromethyl methyl ether for 60 minutes, and 1.2wt% of aluminum chloride was added to the mixture; after stirring at 50°C for 12 hours, the microspheres were collected by centrifugation, and washed and collected with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water for multiple times; collected by centrifugation, suspended in anhydrous ethanol solution after centrifugation, aminosilane was added, stirred at room temperature, washed and collected with anhydrous ethanol, and stable fluorescent microspheres were obtained;

[0074] S3, prepare glycerol / water solution with a certain gradient mass ratio of 10%, 25%, 50%, etc., and calculate its density and viscosity; add the prepared polystyrene microsphere sample to the glycerol / water solution, determine the glycerol / water mass ratio when the microsphere can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microsphere; replace the glycerol / water mass ratio solution, calculate the time required for the target microsphere to settle to the bottom according to the formula, settle the sample, and after reaching the calculated time, remove the small microspheres on the upper layer, repeat several times until the upper layer is almost clear; settle the sample again, collect the upper liquid microspheres before the calculated time, remove the substrate microspheres, repeat several times until there is no obvious microsphere precipitation on the substrate, and finally obtain microspheres of the target particle size;

[0075] The calculation formula of the sinking time includes:

[0076] Among them, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the acceleration of gravity, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density.

[0077] Results: See Figure 1 A and Figure 1 D, the particle size of the microspheres prepared by using 14wt% styrene in the present invention is 779nm, and the particle size distribution of the microspheres increases with the increase of the amount of styrene used. Monodispersed polystyrene microspheres cannot be obtained at too low a styrene concentration. Since it takes a long time to form primary particles at a lower styrene concentration, the primary particles formed cannot grow due to insufficient absorption of monomers, so the particle size distribution becomes wider; while at a higher styrene concentration, more primary nuclei are formed, the probability of collision between nuclei to form larger nuclei increases, the suspension ability of the dispersant becomes worse, and the balling process is accelerated, so the distribution becomes wider.

[0078] Example 4

[0079] The invention provides a preparation and screening method of high-monodispersity polystyrene fluorescent microspheres containing 5wt% of polyvinyl pyrrolidone and simulating Staphylococcus aureus.

[0080] Mainly include:

[0081] S1. Add 5wt% polyvinyl pyrrolidone, 1wt% azobisisobutyronitrile and 10wt% styrene to every 100mL ethanol and water solution, the volume ratio of ethanol to water in the ethanol solution is 1:4, the reaction temperature is 70°C, dissolve every 100mL of the prepared ethanol solution and put it into an ultrasonic reactor, introduce nitrogen to remove oxygen in the system, turn on the ultrasonic generator for polymerization, obtain a polystyrene submicron microsphere dispersion, and collect it by centrifugation; suspend it with 5mL anhydrous ethanol, add 5μL aminosilane, stir for 1 hour, wash it with an equal volume of anhydrous ethanol, and collect it;

[0082] S2, 5g of polystyrene microspheres were swelled in 50mL of chloromethyl methyl ether for 60 minutes, and 1.2wt% of aluminum chloride was added to the mixture; after stirring at 50°C for 12 hours, the microspheres were collected by centrifugation, and washed and collected with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water for multiple times; collected by centrifugation, suspended in anhydrous ethanol solution after centrifugation, aminosilane was added, stirred at room temperature, washed and collected with anhydrous ethanol, and stable fluorescent microspheres were obtained;

[0083] S3. Prepare glycerol / water solutions with a certain gradient mass ratio of 10%, 25%, 50%, etc., and calculate their density and viscosity; add the prepared polystyrene microsphere sample to the glycerol / water solution, determine the glycerol / water mass ratio when the microspheres can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microspheres; replace the glycerol / water mass ratio solution, calculate the time required for the target microspheres to settle to the bottom according to the formula, settle the sample, and after the calculated time is reached, remove the small microspheres on the upper layer, and repeat several times until the upper layer is almost clear. Sediment the sample again, collect the upper liquid microspheres before the calculated time, remove the substrate microspheres, and repeat several times until there is no obvious microsphere precipitation on the substrate, and finally obtain microspheres of the target particle size;

[0084] The calculation formula of the sinking time includes:

[0085] Among them, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the acceleration of gravity, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density.

[0086] Results: Figure 2 A and Figure 2 As shown in Figure B, in a system with a low PVP concentration (1wt% to 5wt%), the system viscosity is small, the surface tension is small, and the diameter of the formed pronuclei is large; but the suspension and dispersion capabilities of the system are weak, and the probability of collision and fusion of the formed pronuclei is high. The primary nuclei are not effectively dispersed and protected, so the size distribution of the primary particles formed at this time is relatively wide. In the present invention, a 5wt% PVP concentration is selected for the experiment.

[0087] Example 5

[0088] The present invention proposes an experiment for selecting a screening method based on the influence of the screening method on the dispersibility of microsphere particle size.

[0089] Prepare glycerol / water solutions with a certain gradient mass ratio of 10%, 25%, 50%, etc., and calculate their density and viscosity; add the prepared polystyrene microsphere sample to the glycerol / water solution to determine the glycerol / water mass ratio when the microspheres can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microspheres; replace the glycerol / water mass ratio solution, calculate the time required for the target microspheres to settle to the bottom according to the formula, settle the sample, and after the calculated time is reached, remove the small microspheres in the upper layer, and repeat several times until the upper layer is almost clear; settle the sample again, collect the upper liquid microspheres before the calculated time, remove the substrate microspheres, repeat several times until there is no obvious microsphere precipitation on the substrate, and finally obtain microspheres of the target particle size; further use a 650nm ultrafiltration centrifuge tube to further screen the polystyrene microspheres, add the microspheres of the target particle size obtained in S3 to a 650nm ultrafiltration centrifuge tube, centrifuge at 5000rpm for 10 minutes, resuspend the microspheres in the upper filter with ethanol, and repeat three times to obtain microspheres of the target particle size.

[0090] The calculation formula of the sinking time includes:

[0091] Among them, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the acceleration of gravity, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density.

[0092] Results: See Figure 3 A, the average particle size of the polystyrene microspheres after washing and centrifugation is 752nm, and the CV value is 17.4%; please refer to Figure 3 B, after gradient sedimentation treatment, the average particle size of polystyrene microspheres is 722nm, and the CV value is 9.8%; please refer to Figure 3 C, after screening by 650nm ultrafiltration centrifuge tube, the average particle size of polystyrene microspheres is 733nm, and the CV value is 3.0%. This indicates that the dispersed polymerization method and series screening technology adopted by the present invention have obtained highly monodisperse polystyrene fluorescent microspheres with a diameter similar to that of Staphylococcus aureus, and the CV value of the simulator constructed by the present invention is 3.0% to 7.0%, achieving high monodispersity and high consistency simulation of Staphylococcus aureus.

[0093] Example 6

[0094] The invention proposes a fluorescence spectrum comparison experiment of polystyrene fluorescent microspheres and Staphylococcus aureus.

[0095] The frozen standard strain was streaked on nutrient agar to revive, cultured at 37℃ for 18-24 hours, single Staphylococcus aureus colony was picked from the plate, inoculated into nutrient broth, cultured at 37℃ for 18-24 hours, the bacterial solution was inoculated into Baird-Parker medium, cultured at 37℃ until the colonies grew, then 10 colonies were randomly picked for Gram staining, and observed under a microscope. The bacteria were arranged in a staphylococcal shape, without spores or capsules, and with a diameter of about 0.5 micron to 1.0 micron. The colony containing 10 per milliliter was selected. 8 The fluorescence spectra of Staphylococcus aureus and fluorescent polystyrene microspheres were observed using a steady-state / transient fluorescence spectrometer; the average particle size and particle size distribution of Staphylococcus aureus and microspheres were determined by a nanoparticle size and zeta potential analyzer.

[0096] Results: See Figure 4 , polystyrene microspheres can produce a wide fluorescence emission peak in the range of 400-500nm, and the maximum fluorescence emission peaks are stabilized at 439nm and 462nm, respectively, which coincide with the maximum fluorescence emission peaks of Staphylococcus aureus, and can better simulate the fluorescence signal of Staphylococcus aureus.

[0097] Example 7

[0098] The invention proposes a fluorescence lifetime experiment of polystyrene microspheres to verify the stability of fluorescent polystyrene microspheres.

[0099] Use a steady-state / transient fluorescence spectrometer to detect the fluorescence lifetime of polystyrene microspheres after storage at 4°C for 180 days; add the solution into a cuvette, put it into the instrument, select the corresponding light source, adjust the parameters, test the spectrum with a step size of 1nm and an integration time of 0.2s, select channel 1024 for fluorescence lifetime, and select channel 2000 for phosphorescence lifetime.

[0100] Results: See Figure 5 The fluorescence lifetime of the polystyrene microspheres is substantially consistent with that of the polystyrene microspheres 180 days ago. Therefore, the polystyrene microspheres prepared by the present invention have good stability in simulating the fluorescence signal of Staphylococcus aureus.

[0101] Example 8

[0102] The invention proposes an experiment of using polystyrene microspheres to simulate Staphylococcus aureus to produce aerosols, and at the same time is used to verify the accuracy of simulation results of fluorescent polystyrene microspheres.

[0103] Polystyrene fluorescent microspheres with a particle size of 800 nm were used to simulate Staphylococcus aureus to produce aerosols. Staphylococcus aureus with a similar particle size was selected, cultured overnight, and the bacteria were collected by centrifugation, washed 5-10 times, mixed with ultrapure water, and ultrasonically treated for 30 minutes. Bioaerosols were generated by atomization to form an aerosol environment with uniform and stable concentration. The sampler was placed in a measuring room, and the aerosol particle concentration upstream and downstream of the sampler was measured with an aerodynamic particle size analyzer, and its particle size distribution was analyzed.

[0104] Results: See Figure 6 The particle sizes of polystyrene fluorescent microspheres and Staphylococcus aureus are basically concentrated in the range of 500nm-1000nm. The particle size distribution of aerosol simulation test is relatively consistent, and the simulation results are more accurate.

[0105] Comparative Example

[0106] A comparison between the present invention and commercially available and conventional submicron functional microspheres is proposed.

[0107] 3 wt% of polyvinyl pyrrolidone, 1 wt% of azobisisobutyronitrile and 10 wt% of styrene were added to every 100 mL of ethanol and water solution, the volume of ethanol to water in the ethanol solution was 1:4, the reaction temperature was 70°C, every 100 mL of the prepared ethanol solution was dissolved and put into an ultrasonic reactor, nitrogen was introduced to remove oxygen in the system, and the ultrasonic generator was turned on for polymerization to obtain a polystyrene submicron microsphere dispersion, which was collected by centrifugation; it was suspended with 5 mL of anhydrous ethanol, 5 μL of aminosilane was added, stirred for 1 hour, and then suspended with an equal volume of anhydrous The method comprises the following steps: washing with ethanol and collecting; swelling 5 g of polystyrene microspheres in 50 mL of chloromethyl methyl ether for 60 minutes, and adding 1.2 wt % of aluminum chloride to the mixture; stirring at 50° C. for 12 hours, collecting the microspheres by centrifugation, and washing and collecting the microspheres with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water for multiple times to obtain stable fluorescent microspheres; observing images of different polystyrene microspheres by scanning electron microscopy, determining the average particle size and particle size distribution of the microspheres by laser diffraction particle size analyzer, and evaluating the particle size distribution by the coefficient of variation. The larger the coefficient of variation, the wider the particle size distribution.

[0108] Results: For ideal monodisperse microspheres, the uniformity is zero, while for most commercially available and conventionally prepared monodisperse microspheres, this value is usually between 10% and 20%. If the coefficient of variation is higher than 20%, the particle size distribution is too wide to achieve the effectiveness of the Staphylococcus aureus simulant of the present invention.

[0109] It should be noted that the term "comprises" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only these elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprises..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0110] The above are only embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included within the scope of the claims of the present application.

Claims

1. A method for preparing a Staphylococcus aureus simulant based on polystyrene microspheres, characterized in that: include: S1. Add polyvinyl pyrrolidone, azobisisobutyronitrile and styrene to ethanol and aqueous solution, dissolve the prepared ethanol solution and put it into a heated magnetic stirrer, introduce nitrogen to remove oxygen in the system, turn on the heated magnetic stirrer to carry out polymerization reaction, obtain polystyrene submicron microsphere dispersion, and collect it by centrifugation; Suspend with anhydrous ethanol, add aminosilane, stir, wash with anhydrous ethanol, and collect; S2, swelling the polystyrene microspheres in chloromethyl methyl ether, adding aluminum chloride to the mixture; stirring at 40°C to 60°C, collecting the microspheres by centrifugation, and washing them multiple times with acetone, hydrochloric acid solution, anhydrous ethanol and deionized water; Collect by centrifugation, suspend in anhydrous ethanol solution after centrifugation, add aminosilane, stir at room temperature, wash and collect with anhydrous ethanol, and obtain stable fluorescent microspheres; S3, prepare a glycerol / water solution with a gradient mass ratio, and calculate its density and viscosity; add the prepared polystyrene microsphere sample to the glycerol / water solution, determine the glycerol / water mass ratio when the microspheres can be suspended in the solution for a long time, and the density of the aqueous solution is the same as the density of the microspheres; replace the glycerol / water mass ratio solution, calculate the sinking time according to the formula, the sinking time is the time required for the target microspheres to settle to the bottom, settle the sample, and after reaching the calculated time, remove the upper small microspheres, and repeat several times until the upper layer is close to clear; The sample is precipitated again. Before the calculation time, the upper liquid microspheres are collected and the substrate microspheres are removed. This process is repeated several times until there is no obvious microsphere precipitation on the substrate, and the microspheres of the target particle size are obtained. The calculation formula of the sinking time includes: , Among them, t is the sinking time, d is the distance the microsphere falls, v is the speed of the microsphere, μ is the dynamic viscosity of the water phase, R is the radius of a microsphere, g is the gravitational acceleration, ρ 液 is the density of aqueous solution, ρ 球 is the microsphere density; S4, further screening the polystyrene microspheres using an ultrafiltration centrifuge tube, adding the microspheres of the target particle size obtained in S3 to the ultrafiltration centrifuge tube, centrifuging, and resuspending the microspheres in the upper filter with ethanol to obtain microspheres of the target particle size; S5, atomizing the monodisperse polystyrene microspheres to form an aerosol environment with uniform and stable concentration, and obtaining a Staphylococcus aureus simulant based on polystyrene microspheres; The Staphylococcus aureus simulant based on polystyrene microspheres is chloromethylated polystyrene microspheres; the diameter of the simulant is 0.7 μm to 0.9 μm; and the CV value of the simulant is 3.0% to 7.0%.

2. The preparation method according to claim 1, characterized in that: In S1, the content of polyvinyl pyrrolidone is 1 wt% to 5 wt%, the content of azobisisobutyronitrile is 0.6 wt% to 1.4 wt%, the content of styrene is 6 wt% to 14 wt%, the water:ethanol=0~1:4 and the water content is not zero, and the reaction temperature is 60°C to 80°C.

3. The preparation method according to claim 1, characterized in that: In S1, the content of polyvinyl pyrrolidone is 2 wt%~4 wt%, the content of azobisisobutyronitrile is 0.8 wt%~1.2 wt%, the content of styrene is 8 wt%~12 wt%, the water:ethanol=0~1:4 and the water content is not zero, and the reaction temperature is 65°C~75°C.

4. The preparation method according to claim 1, characterized in that: The average particle size of the simulant is 711nm~755nm.

5. The preparation method according to claim 1, characterized in that: The simulant produces a broad fluorescence emission peak in the range of 400nm to 500nm, and the maximum fluorescence emission peaks are stabilized at 435nm to 443nm and 458nm to 466nm, respectively.

6. The preparation method according to claim 1, characterized in that: The fluorescence lifetime of the mimetic agent remained stable for at least 180 days.

7. The preparation method according to claim 1, characterized in that: The simulant can simulate Staphylococcus aureus in bioaerosol.

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