A "step" automatic device suitable for rapid separation and purification of bacterial spores, preparation method and application

Abstract

The application belongs to the field of spore separation, and relates to a 'ladder type' magnetic flow separation device suitable for rapid separation and purification of bacterial spores, a preparation method and application. The 'ladder type' automatic device comprises a 'ladder type' magnetic flow cavity and a 'ladder type' magnet mounting groove, the 'ladder type' magnetic flow cavity and the 'ladder type' magnet mounting groove are arranged in cooperation, and the 'ladder type' magnetic flow cavity is provided with a sample inlet and a sample outlet. The 'ladder type' automatic device combines Fe3O4-Van antibiotic magnetic bead targeted capture of bacterial bodies with specific recognition function for gram-positive bacterial bodies, shortens the separation time of bacterial bodies and spores, can realize operation automation, and thus realizes the purpose of rapid purification of spores in a large-volume sample.

Description

Technical Field

[0001] This invention belongs to the field of spore isolation, and relates to a "step-like" automated device, preparation method and application suitable for rapid isolation and purification of bacterial spores. Background Technology

[0002] Bacillus is typically a Gram-positive bacterium. In its later stages of growth, under nutrient deprivation or adverse environmental conditions, it forms a highly resilient dormant body with a unique structure characterized by thick walls, high refractive index, and resistance to harsh environments (high temperature, high pressure, radiation, etc.), known as a spore. Under normal conditions, spores can be preserved for several years to decades, germinating and multiplying when nutrients and conditions are suitable. Due to their high stability, long storage time, and minimal damage during processing, Bacillus spores are widely used in various fields. Probiotic Bacillus spores (Bacillus cereus and Bacillus subtilis) are often used as microbial preparations in feed processing and pesticide additives. Simultaneously, because spores are among the most resistant organisms in nature, their elimination has become an important criterion for various disinfection and sterilization methods; for example, Bacillus stearothermophilus spores are used to evaluate the bactericidal ability of pressure steam sterilization. To achieve more efficient utilization of spores, high purity is required.

[0003] Achieving rapid, convenient, and high-purity purification of bacterial spores is an indispensable step in elucidating spore structure and germination mechanisms, as well as in the production and application of microbial preparations. Currently, spore purification methods primarily employ filtration and centrifugation, which rely on the differences in physicochemical properties such as size and density between spores and other microorganisms to achieve separation and purification. However, traditional filtration and centrifugation methods are cumbersome, time-consuming, and labor-intensive, and the iohexol solution used for centrifugation is expensive, making them unsuitable for large-scale purification and production of probiotic Bacillus and for further mechanistic studies of single spores.

[0004] Magnetic separation technology is a technique that captures and separates target analytes by combining them with magnetic nanomaterials. It has been widely used for the separation and enrichment of target analytes in complex samples. For example, patent CN115058359A discloses a method for the magnetic separation and enrichment of Bacillus cereus. This method includes the coupling of magnetic nanoparticles with streptavidin, the coupling of carboxylic acid-polyethylene glycol-biotin with cefepime, and the binding of cefepime-carboxylic acid-polyethylene glycol-biotin to Bacillus cereus in the sample solution. Under the action of an external magnetic field, the captured Bacillus cereus is separated from the sample solution and resuspended. However, this method uses a conventional magnetic separation rack, which is a simple magnetic separation device, but its limited centrifuge tube volume makes it unsuitable for the separation and purification of large-volume samples and small-volume, low-concentration samples. Therefore, developing an automated magnetic flow device is of great significance for the rapid separation, purification, and production applications of bacterial spores. Summary of the Invention

[0005] To address the technical problem that conventional magnetic separation racks are unsuitable for separating and purifying large-volume samples and small-volume, low-concentration samples, this invention proposes a "step-like" automated device, its preparation method, and its application suitable for the rapid separation and purification of bacterial spores. Vancomycin (which has a specific recognition function for Gram-positive bacterial vegetative cells) is modified onto the surface of magnetic nanoparticles to target and capture bacterial cells. The "step-like" structure increases the contact area and collision probability between the sample and the magnetic components, shortening the separation time between bacterial cells and spores, thereby achieving rapid spore purification.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] The first aspect of the present invention provides a "step-type" automatic device suitable for rapid separation and purification of bacterial spores. The "step-type" magnetic flow separation device includes a "step-type" magnetic flow cavity and a "step-type" magnet mounting groove, which are configured to cooperate with each other. The "step-type" magnetic flow cavity is provided with an inlet and an outlet.

[0008] Furthermore, the "stepped" magnetic flow cavity has an n+1 level "stepped" configuration, and the "stepped" magnet mounting slot has an n level "stepped" configuration, where n≥3.

[0009] Furthermore, the inlet and outlet are located on the left and right sides of the "stepped" magnetic flow cavity, respectively; the inlet and outlet are respectively provided with connecting tube I and connecting tube II, which are truncated rings. The inner diameter of the upper ring of the truncated ring is 0.8 cm and the outer diameter is 1.0 cm. The inner diameter of the lower ring is 0.4 cm and the outer diameter is 0.6 cm. The inlet is connected to the upper ring of connecting tube I, and the outlet is connected to the upper ring of connecting tube II.

[0010] Furthermore, the "stepped" magnetic flow cavity also includes a "stepped" magnetic flow cavity cover plate, which cooperates with the "stepped" magnetic flow cavity. The height of the "stepped" magnetic flow cavity cover plate is 0.6~0.8cm.

[0011] Furthermore, a "stepped" gap is provided between the "stepped" magnetic flow cavity and the "stepped" magnet mounting groove, and the thickness of the "stepped" gap is 0.3~0.5 cm.

[0012] Furthermore, the wall thickness of the "stepped" magnetic flow cavity and the "stepped" magnet mounting groove is 0.6~0.8cm, and the height is 2.6~2.8cm.

[0013] A method for preparing a "step-by-step" automated device suitable for rapid isolation and purification of bacterial spores includes the following five steps:

[0014] Step 1: Pretreatment of polydimethylsiloxane (PDMS): Mix the PDMS precursor and its curing agent in a beaker at a ratio of 10:1 and stir thoroughly with a glass rod. Vacuum for 60-120 minutes to remove air bubbles generated during stirring. After treatment, seal and set aside for later use.

[0015] Step 2: Preparation of the main unit (including the "stepped" magnetic flow cavity, the "stepped" magnet mounting groove, the inlet, and the outlet). The pretreated PDMS colloid from Step 1 is slowly poured into the main unit mold. The mold is placed in an oven and dried at 70 °C for 48 h until the PDMS colloid is completely solidified. The mold is then demolded to obtain the main unit of the "stepped" automatic device, including the "stepped" magnetic flow cavity, the "stepped" magnet mounting groove, the inlet, and the outlet. The cavity wall thickness of the main unit is maintained at 0.6–0.8 cm, and the height of the main unit is 2.6–2.8 cm. The thickness of the "stepped" gap between the "stepped" magnetic flow cavity and the "stepped" magnet mounting groove is maintained at 0.3–0.5 cm. The inlet and outlet are circular holes of the same size, with a diameter of 0.8–1.0 cm.

[0016] Step 3: Preparation of the "stepped" magnetic flow cavity cover plate. The pretreated PDMS colloid from Step 1 is slowly poured into the "stepped" magnetic flow cavity cover plate mold of the device. The drying and demolding operations are the same as in Step 2, resulting in the PDMS cover plate used for sealing the "stepped" magnetic flow cavity. The shape of the "stepped" magnetic flow cavity cover plate mold is consistent with the "stepped" magnetic flow cavity in the main unit, but its corresponding length and width are 0.4~0.6 cm larger than the cross-section of the "stepped" magnetic flow cavity. The thickness of the "stepped" magnetic flow cavity cover plate is 0.6~0.8 cm.

[0017] Step 4: Preparation of Connecting Tube I and Connecting Tube II. The pretreated PDMS colloid from Step 1 is slowly poured into the connecting tube I mold and connecting tube II mold of the device, respectively. The drying and demolding operations are the same as in Step 1, resulting in connecting tube I and connecting tube II that connect to the "stepped" magnetic flow cavity. Connecting tube I and connecting tube II are identical annular frustums. The inner diameter of the upper ring of the annular frustum is 0.8 cm, and the outer diameter is 1.0 cm; the inner diameter of the lower ring is 0.4 cm, and the outer diameter is 0.6 cm. The sample inlet is connected to the upper ring of connecting tube I, and the sample outlet is connected to the upper ring of connecting tube II.

[0018] Step 5: Assembly of the "stepped" automatic device. The "stepped" magnetic flow cavity, "stepped" magnet mounting slot, "stepped" magnetic flow cavity cover, connecting pipe I, and connecting pipe II obtained in steps 2, 3, and 4 are sealed together using silicone sealant, and then dried in an oven at 40 ℃ for 1-2 hours. Finally, a permanent magnet is installed in the magnet mounting slot, and the "stepped" magnetic flow separation device is complete.

[0019] According to a second aspect of the present invention, a method for rapid isolation and purification of bacterial spores based on a "step-like" automated device is also provided, comprising:

[0020] (1) Preparation of Fe3O4-Van antibiotic magnetic beads. Fe3O4 magnetic beads and 11-mercaptoundecanoic acid (MUA) ethanol solution were added to a round-bottom flask and sonicated for 2 h. The mixture was then washed three times with anhydrous ethanol and ultrapure water, respectively. The obtained Fe3O4-MUA particles were dissolved in 2-(N-morpholine)ethanesulfonic acid (MES) buffer and sonicated for 15 min to ensure complete dispersion. Carbodiimide (EDC) and vancomycin solution (Van) were added sequentially, and the reaction was continued with sonication for 2 h. The mixture was washed three times with ultrapure water to obtain Fe3O4-Van antibiotic magnetic beads. Finally, the beads were resuspended in PBS solution to obtain Fe3O4-Van antibiotic magnetic bead solution, which was stored at 4 °C for later use.

[0021] (2) Add Fe3O4-Van antibiotic magnetic beads to the solution containing bacterial cells and spores and mix by shaking (200 r / min, 5 min) to ensure that the Fe3O4-Van antibiotic magnetic beads in the mixture are fully combined with the bacterial cells. Then, put the mixture into a sample bottle and use a peristaltic pump to input the above mixture into the "stepped" magnetic flow chamber from the "stepped" automatic device inlet. This allows the bacterial cells combined with the Fe3O4-Van antibiotic magnetic beads to be fully adsorbed on the "stepped" inner wall of the magnetic flow chamber. The "stepped" structure of the magnetic flow chamber increases the contact area and collision probability between the sample and the magnetic part. The sloping structure shortens the distance between the sample and the magnetic part and also facilitates the flow of sample liquid. The purified spores are collected into the collection bottle at the outlet.

[0022] Furthermore, in step (1), the concentration of MUA ethanol is 40 μM, each mL of MUA ethanol solution contains 1 mg of Fe3O4 magnetic beads, the concentration of MES buffer is 0.1 M, and the pH is 5.5; the mass ratio of Fe3O4 magnetic beads to EDC and Van is 1:(0.6~0.7):(0.4~0.5); the concentration of PBS solution is 0.1 M, and the pH is 7.4; the concentration of Fe3O4-Van antibiotic magnetic bead solution is 0.8~1.2 mg / mL.

[0023] Furthermore, in step (2), the volume ratio of the bacterial cell and spore mixture to the Fe3O4-Van antibiotic magnetic beads is 9:1.

[0024] The above-mentioned "step-like" automated device suitable for the rapid isolation and purification of bacterial spores is applied in the rapid isolation and purification of bacterial spores.

[0025] The present invention has the following beneficial effects:

[0026] This invention provides a "stepped" automated device suitable for the rapid separation and purification of bacterial spores. The "stepped" structure of the magnetic flow chamber increases the contact area and collision probability between the sample and the magnetic parts, while the sloping structure shortens the distance between the sample and the magnetic parts and facilitates the flow of sample liquid. Combined with Fe3O4-Van antibiotic magnetic beads that specifically recognize Gram-positive bacterial cells, the separation time between bacterial cells and spores is shortened, and the operation can be automated, thereby achieving the goal of rapid purification of spores from large-volume samples. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the overall structure of the "step-type" automatic device in this invention.

[0029] Figure 2 This is a schematic diagram of the preparation of the main unit mold of the "stepped" automatic device in this invention, including a "stepped" magnetic flow cavity, a "stepped" magnet mounting groove, an inlet, and an outlet.

[0030] Figure 3 This is a schematic diagram of the cover preparation of the "step-type" automatic device in this invention.

[0031] Figure 4 This is a schematic diagram of the mold for the connecting pipe I and connecting pipe II of the "step-type" automatic device in this invention.

[0032] Figure 5 This is a schematic diagram of the assembly process of the "step-type" automatic device in this invention.

[0033] Figure 6 This is a schematic diagram of a method for rapid isolation and purification of bacterial spores based on a "step-by-step" automated device.

[0034] Figure 7 Characterization of the Fe3O4-Van antibiotic magnetic beads prepared in this invention: (A) is a scanning electron microscope image of the Fe3O4-Van antibiotic magnetic beads, (B) is a layered EDS image of the Fe3O4-Van antibiotic magnetic beads, (C) is a Fe elemental mapping map (green) from Fe3O4, and (D) is a N elemental mapping map (red) from Van.

[0035] Figure 8 To compare phase-contrast microscopy images before and after rapid isolation and purification of bacterial spores using a "step-like" automated device: (A) Phase-contrast microscopy image of a mixture of Clostridium perfringens and its spores; (B) Phase-contrast microscopy image of purified spores from a mixture of Clostridium perfringens and its spores; (C) Phase-contrast microscopy image of a mixture of Bacillus subtilis and its spores; (D) Phase-contrast microscopy image of purified spores from a mixture of Bacillus subtilis and its spores; (E) Phase-contrast microscopy image of a mixture of Bacillus cereus and its spores; (F) Phase-contrast microscopy image of purified spores from a mixture of Bacillus cereus and its spores.

[0036] Reference numerals in the attached drawings: 1. "Stepped" magnetic flow cavity; 2. "Stepped" magnet mounting groove; 3. Sample inlet; 4. Sample outlet; 5. "Stepped" magnetic flow cavity cover plate; 6. Connecting pipe I; 7. Connecting pipe II. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In this invention, both the ECD solution and the NHS solution are solvents of ultrapure water.

[0039] Example 1

[0040] A method for preparing a "step-by-step" automated device suitable for rapid isolation and purification of bacterial spores includes the following five steps:

[0041] Step 1: Pretreatment of polydimethylsiloxane (PDMS): Mix the PDMS precursor and its curing agent in a beaker at a ratio of 10:1 and stir thoroughly with a glass rod. Vacuum for 60-120 minutes to remove air bubbles generated during stirring. After treatment, seal and set aside for later use.

[0042] Step 2: Preparation of the main unit (including the "stepped" magnetic flow cavity, the "stepped" magnet mounting groove, the inlet, and the outlet). The pretreated PDMS colloid from Step 1 is slowly poured into the main unit mold. The mold is placed in an oven and dried at 70 °C for 48 h until the PDMS colloid is completely solidified. The mold is then demolded to obtain the main unit of the "stepped" magnetic flow separation device, including the "stepped" magnetic flow cavity, the "stepped" magnet mounting groove, the inlet, and the outlet. The cavity wall thickness of the main unit is maintained at 0.6–0.8 cm, and the height of the main unit is 2.6–2.8 cm. The thickness of the "stepped" gap between the "stepped" magnetic flow cavity and the "stepped" magnet mounting groove is maintained at 0.3–0.5 cm. The inlet and outlet are circular holes of the same size, with a diameter of 0.8–1.0 cm.

[0043] Step 3: Fabrication of the "stepped" magnetic flow cavity cover plate. (For example...) Figure 3As shown, the pretreated PDMS colloid from step 1 is slowly poured into the "stepped" magnetic flow cavity cover plate mold of the device. The drying and demolding operation is the same as shown in step 2, resulting in a PDMS cover plate for sealing the "stepped" magnetic flow cavity. The shape of the "stepped" magnetic flow cavity cover plate mold is consistent with that of the "stepped" magnetic flow cavity in the main unit, but its length and width are 0.4~0.6 cm larger than the cross-section of the "stepped" magnetic flow cavity. The thickness of the "stepped" magnetic flow cavity cover plate is 0.6~0.8 cm.

[0044] Step 4: Preparation of connecting tube I and connecting tube II. (e.g.) Figure 4 As shown, the pretreated PDMS colloid from step 1 is slowly poured into molds I and II of the device, respectively. The drying and demolding operations are the same as in step 1, resulting in connecting tubes I and II that connect to the "stepped" magnetic flow cavity. Connecting tubes I and II are identical annular frustums, with the upper ring having an inner diameter of 0.8 cm and an outer diameter of 1.0 cm; the lower ring has an inner diameter of 0.4 cm and an outer diameter of 0.6 cm. The inlet is connected to the upper ring of connecting tube I, and the outlet is connected to the upper ring of connecting tube II.

[0045] Step 5: Assembly of the "step-by-step" automated device. (For example...) Figure 5 As shown, the "stepped" magnetic flow cavity, the "stepped" magnet mounting groove, the "stepped" magnetic flow cavity cover plate, connecting pipe I, and connecting pipe II obtained in steps 2, 3, and 4 are bonded and sealed with silicone sealant, and then dried in an oven at 40 ℃ for 1-2 h. Finally, a permanent magnet is installed in the magnet mounting groove, and the "stepped" magnetic flow separation device is completed.

[0046] Example 2

[0047] like Figure 1 and 2As shown, a "stepped" automated device suitable for the rapid separation and purification of bacterial spores is disclosed. The "stepped" magnetic flux separation device mainly includes a "stepped" magnetic flux chamber 1 and a "stepped" magnet mounting groove 2, which are configured in conjunction. The "stepped" magnetic flux chamber 1 is equipped with an inlet 3 and an outlet 4. The "stepped" magnetic flux chamber 1 provides an environment for the rapid separation and purification of spores. The stepped structure increases the contact area between the mixed solution to be separated and the steps after entering the "stepped" magnetic flux chamber 1, and facilitates the adsorption of bacterial cells bound to Fe3O4-Van antibiotic magnetic beads by the permanent magnet installed in the "stepped" magnet mounting groove 2. Furthermore, the stepped structure avoids the poor performance and relatively easy detachment of bacteria that occurs with non-stepped structures when the magnetic beads adsorb too many bacterial cells. Furthermore, its sloping structure (inclination angle 30~45°) allows residual liquid to flow out quickly along the slope, facilitating collection and accelerating the separation and purification rate.

[0048] Example 3

[0049] like Figure 2 As shown, the main unit of the device includes a stepped magnetic flow cavity 1, a stepped magnet mounting slot 2, connecting pipe I 6, and connecting pipe II 7. The stepped magnetic flow cavity 1 has an n+1-stage stepped configuration, and the stepped magnet mounting slot 2 has an n-stage stepped configuration, where n ≥ 3. The inlet 3 and outlet 4 are located on the left and right sides of the stepped magnetic flow cavity 1, respectively. Connecting pipe I 6 and connecting pipe II 7 are respectively installed inside the inlet 3 and outlet 4. Connecting pipe I 6 and connecting pipe II 7 are truncated rings. The inner diameter of the upper ring of the truncated ring is 0.8 cm, and the outer diameter is 1.0 cm. The inner diameter of the lower ring is 0.4 cm, and the outer diameter is 0.6 cm. Figure 4As shown; the inlet 3 is connected to the upper ring of connecting tube I 6, and the outlet 4 is connected to the upper ring of connecting tube II 7. Both the "stepped" magnetic flow chamber 1 and the "stepped" magnet mounting groove 2 are "stepped". When the mixture of Fe3O4-Van antibiotic magnetic beads and bacterial cells is injected into the "stepped" magnetic flow chamber 1 through the inlet 3, the permanent magnet in the "stepped" magnet mounting groove 2 can magnetically adsorb the bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads in the solution to be separated onto the "stepped" inner wall of the "stepped" magnetic flow chamber, thereby separating them from other liquids. Finally, the remaining liquid... The spores flow out through the sample outlet 4 and are collected, thus obtaining purified spores. The "stepped" magnetic flow chamber 1 and the "stepped" magnet mounting groove 2, due to their stepped structure, increase the contact area and collision probability between the bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads in the solution to be separated and the magnetic parts. Simultaneously, the sloping structure shortens the distance between the sample and the magnetic parts while facilitating rapid flow of the liquid sample. The purified spores are collected in a collection bottle at the sample outlet.

[0050] The other structures of the “step-type” automatic device are the same as those in Embodiment 2.

[0051] Example 4

[0052] like Figure 3 As shown, the "stepped" magnetic flow cavity 1 also includes a "stepped" magnetic flow cavity cover plate 5, which cooperates with the "stepped" magnetic flow cavity 1. The corresponding length and width are both 0.4~0.6 cm larger than the cross-section of the "stepped" magnetic flow cavity, so that the "stepped" magnetic flow cavity cover plate can completely adhere and seal the "stepped" magnetic flow cavity during the assembly of the "stepped" magnetic flow separation device. The height of the "stepped" magnetic flow cavity cover plate 5 is 0.6~0.8 cm. The "stepped" magnetic flow cavity cover plate 5 provides a sealed environment for the "stepped" magnetic flow cavity 1, ensuring the cleanliness and hygiene of the surrounding environment during the magnetic separation process. It also prevents liquid splashing during the rapid separation and purification of the solution to be separated.

[0053] The other structures of the “step-type” automatic device are the same as those in Embodiment 2.

[0054] Example 5

[0055] A method for rapidly separating and purifying bacterial spores using the "step-by-step" automated device prepared in Example 1, suitable for the rapid separation and purification of bacterial spores, is as follows: Figure 6 As shown, the steps are as follows:

[0056] (1) Preparation of Fe3O4-Van antibiotic magnetic beads. 500 mg of Fe3O4 magnetic beads were added to 500 mL of 11-mercaptoundecanoic acid (MUA) ethanol solution (40 μM) in a round-bottom flask. After sonication for 2 h, the beads were washed three times with anhydrous ethanol and ultrapure water, respectively. The obtained Fe3O4-MUA particles were dissolved in 200 mL of 2-(N-morpholine) ethanesulfonic acid (MES) buffer (0.1 M, pH 5.5) and sonicated for 15 min to ensure complete dispersion. 0.3 g of carbodiimide (EDC) and 0.2 g of vancomycin solution (Van) were added sequentially, and the reaction was continued with sonication for 2 h. The beads were washed three times with ultrapure water to obtain Fe3O4-Van antibiotic magnetic beads with a concentration of 1 mg / mL. Finally, the beads were resuspended in 500 mL of PBS (0.01 M, pH 7.4) and stored at 4 °C for later use.

[0057] The Fe3O4-Van antibiotic magnetic beads prepared above were characterized, and the characterization results are as follows: Figure 7 As shown. Figure 7 Characterization of Fe3O4-Van antibiotic magnetic beads; (A) Scanning electron microscope image of Fe3O4-Van antibiotic magnetic beads, (B) Layered EDS image of Fe3O4-Van antibiotic magnetic beads, (C) Fe elemental mapping map from Fe3O4 (green), (D) N elemental mapping map from Van (red). Figure 7 (A) is the scanning electron microscopy characterization result of Fe3O4-Van antibiotic magnetic beads. The result shows that Fe3O4-Van antibiotic magnetic beads have uniform particle size and good dispersion. Rough particles can be clearly observed on the surface of Fe3O4 magnetic beads, indicating that Van has been successfully adsorbed on the surface of Fe3O4 magnetic beads. Figure 7 (B)~(D) show the EDS elemental mapping analysis results of Fe3O4-Van antibiotic magnetic beads. Dense N elements (red) are observed to be uniformly distributed on the surface of Fe elements (green). N is a characteristic element of Van, indicating that Van is tightly bonded to the surface of the Fe3O4 magnetic beads. These characterization results demonstrate the successful preparation of Fe3O4-Van antibiotic magnetic beads.

[0058] (2) Add 50 mL of Fe3O4-Van antibiotic magnetic bead solution obtained in step (1) to 450 mL of the mixed solution containing bacterial cells and spores to be separated. After thorough shaking and mixing (200 r / min, 5 min), pump the solution through the inlet 3 into the "stepped" magnetic flow chamber 1 using a peristaltic pump. The permanent magnet in the "stepped" magnet mounting groove 2 adsorbs the bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads onto the inner wall of the "stepped" magnetic flow chamber 1. The remaining solution flows out through the outlet 4 and the connecting tube II 7, and the purified spores are collected. The bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads are fully adsorbed on the "stepped" inner wall of the magnetic flow chamber. The "stepped" structure of the magnetic flow chamber increases the contact area and collision probability between the sample and the magnetic part. The sloping structure shortens the distance between the sample and the magnetic part and also facilitates the flow of sample liquid. The purified spores are collected into the collection bottle at the outlet.

[0059] Example 6

[0060] A method for rapidly separating and purifying bacterial spores using the "step-by-step" automated device prepared in Example 1, suitable for the rapid separation and purification of bacterial spores, is as follows: Figure 6 As shown, the steps are as follows:

[0061] (1) Preparation of Fe3O4-Van antibiotic magnetic beads. 500 mg of Fe3O4 magnetic beads were added to 500 mL of 11-mercaptoundecanoic acid (MUA) ethanol solution (40 μM) in a round-bottom flask. After sonication for 2 h, the beads were washed three times with anhydrous ethanol and ultrapure water, respectively. The obtained Fe3O4-MUA particles were dissolved in 200 mL of 2-(N-morpholine) ethanesulfonic acid (MES) buffer (0.1 M, pH 5.5) and sonicated for 15 min to ensure complete dispersion. 0.32 g of carbodiimide (EDC) and 0.22 g of vancomycin solution (Van) were added sequentially, and the reaction was continued with sonication for 2 h. The beads were washed three times with ultrapure water to obtain Fe3O4-Van antibiotic magnetic beads. Finally, the beads were resuspended in 500 mL of PBS (0.01 M, pH 7.4) and stored at 4 °C for later use.

[0062] (2) Add 50 mL of Fe3O4-Van antibiotic magnetic bead solution obtained in step (1) to 450 mL of mixed solution containing bacterial cells and spores to be separated. After thorough shaking and mixing (200 r / min, 5 min), pump the solution from the connecting tube I 6 through the sample inlet 3 into the “stepped” magnetic flow chamber 1 using a peristaltic pump. The permanent magnet in the “stepped” magnet mounting groove 2 adsorbs the bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads onto the inner wall of the “stepped” magnetic flow chamber 1. The remaining solution flows out through the sample outlet 4 via the connecting tube II 7 and the sample outlet 4, and the purified spores are collected. The bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads are fully adsorbed onto the "stepped" inner wall of the magnetic flow chamber. The "stepped" structure of the magnetic flow chamber increases the contact area and collision probability between the sample and the magnetic part. The sloping structure shortens the distance between the sample and the magnetic part while also facilitating the flow of sample liquid. The purified spores are collected into the collection bottle at the sample outlet.

[0063] Example 7

[0064] A method for rapidly separating and purifying bacterial spores using the "step-by-step" automated device prepared in Example 1, suitable for the rapid separation and purification of bacterial spores, is as follows: Figure 6 As shown, the steps are as follows:

[0065] (1) Preparation of Fe3O4-Van antibiotic magnetic beads. 500 mg of Fe3O4 magnetic beads were added to 500 mL of 11-mercaptoundecanoic acid (MUA) ethanol solution (40 μM) in a round-bottom flask. After sonication for 2 h, the beads were washed three times with anhydrous ethanol and ultrapure water, respectively. The obtained Fe3O4-MUA particles were dissolved in 200 mL of 2-(N-morpholine) ethanesulfonic acid (MES) buffer (0.1 M, pH 5.5) and sonicated for 15 min to ensure complete dispersion. 0.35 g of carbodiimide (EDC) and 0.25 g of vancomycin solution (Van) were added sequentially, and the reaction was continued with sonication for 2 h. The beads were washed three times with ultrapure water to obtain Fe3O4-Van antibiotic magnetic beads. Finally, the beads were resuspended in 500 mL of PBS (0.01 M, pH 7.4) and stored at 4 °C for later use.

[0066] (2) Add 50 mL of Fe3O4-Van antibiotic magnetic bead solution obtained in step (1) to 450 mL of mixed solution containing bacterial cells and spores to be separated. After thorough shaking and mixing (200 r / min, 5 min), pump the solution from the connecting tube I 6 through the sample inlet 3 into the “stepped” magnetic flow chamber 1 using a peristaltic pump. The permanent magnet in the “stepped” magnet mounting groove 2 adsorbs the bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads onto the inner wall of the “stepped” magnetic flow chamber 1. The remaining solution flows out through the sample outlet 4 via the connecting tube II 7 and the sample outlet 4, and the purified spores are collected. The bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads are fully adsorbed onto the "stepped" inner wall of the magnetic flow chamber. The "stepped" structure of the magnetic flow chamber increases the contact area and collision probability between the sample and the magnetic part. The sloping structure shortens the distance between the sample and the magnetic part while also facilitating the flow of sample liquid. The purified spores are collected into the collection bottle at the sample outlet.

[0067] Application Example 1

[0068] This application example uses the corresponding bacterial cells and spores of Clostridium perfringens, Bacillus subtilis, and Bacillus cereus as examples to verify the feasibility and practicality of the method for rapid spore isolation and purification.

[0069] Bacterial cell and spore culture: Clostridium perfringens, Bacillus subtilis, and Bacillus cereus stored at -80 °C were activated on nutrient agar medium and then transferred to nutrient broth for enrichment culture on a shaker at 180 r / min for 22 h. The cells were washed three times with sterile deionized water, and the cell concentration was adjusted to 10⁻⁶ cells / mL by plate counting. 4 CFU / mL was prepared for use. The activated *Clostridium perfringens* from the above steps was transferred to 20 mL of liquid mercaptoacetate broth (FTG) and cultured for 7 h. Then, it was transferred at a 1:100 ratio to spore-producing broth and cultured at 37°C for 16–24 h to collect *Clostridium perfringens* spores. The activated *Bacillus subtilis* from the above steps was transferred to DSM spore-promoting medium and cultured at 37°C for 36 h to collect *Bacillus subtilis* spores. The *Bacillus cereus* from the above steps was transferred to LB agar medium supplemented with 50 mg / L MnSO4 and cultured at 37°C for 6 days to collect *Bacillus cereus* spores. Finally, the collected spores were heat-treated at 80°C for 15 min, and the spore concentration was adjusted to 10⁻⁶ using plate counting. 4 CFU / mL available for use.

[0070] The concentration is 10. 4After CFU / mL of Clostridium perfringens and its spores, Bacillus subtilis and its spores, and Bacillus cereus and its spores were mixed in a single system at a volume ratio of 1:1, the spores in the bacterial cell and spore mixture were rapidly purified according to the operating steps in Examples 5 and 6.

[0071] The isolation and purification effect of spores was determined by comparing the microscopic examination results before and after purification using phase contrast microscopy. Figure 8 To compare phase-contrast microscopy images before and after rapid separation and purification of bacterial spores using a "stepped" magnetic flux separation device; (A) is a phase-contrast microscopic image of a mixture of *Clostridium perfringens* and its spores; (B) is a phase-contrast microscopic image of purified spores from the mixture of *Clostridium perfringens* and its spores; (C) is a phase-contrast microscopic image of a mixture of *Bacillus subtilis* and its spores; (D) is a phase-contrast microscopic image of purified spores from the mixture of *Bacillus subtilis* and its spores; (E) is a phase-contrast microscopic image of a mixture of *Bacillus cereus* and its spores; (F) is a phase-contrast microscopic image of purified spores from the mixture of *Bacillus cereus* and its spores. Figure 8 It can be clearly observed that after purification using this method, the three bacterial spores were essentially sterile under a phase-contrast microscope, indicating good spore isolation and purification effects. Heat treatment at 80 ℃ for 15 min can kill bacterial cells but not spores, and to some extent stimulates spore germination and cell formation. Based on this, the three spore purification solutions in the collection bottle were subjected to heat treatment at 80 ℃ for 15 min, and the concentration of bacterial spores in the collection solution was calculated by plate counting to determine the purification efficiency. Table 1 shows the purification efficiency of spores in the mixed solutions of the three bacterial cells and spores using this method.

[0072] Spore purification efficiency = Spore concentration after purification / Spore purification concentration

[0073] Table 1. Purification efficiency of spores in a single mixed system of three bacterial cells and spores based on this method.

[0074]

[0075] As shown in Table 1, the purification efficiency of all three spores reached over 98%. Therefore, the method for rapid separation and purification of bacterial spores based on a "step-by-step" automated device is effective for spore purification in a single mixed system of bacterial cells and spores.

[0076] Application Example 2

[0077] This application example uses a cross-mixing system of corresponding cells and spores of Bacillus subtilis and Bacillus cereus to verify the feasibility and practicality of the method for rapid spore isolation and purification.

[0078] Bacterial cell and spore culture: Bacillus subtilis and Bacillus cereus stored at -80 °C were activated on nutrient agar medium and then transferred to nutrient broth liquid culture medium for enrichment culture on a shaker at 180 r / min for 18 h. The cells were washed three times with sterile deionized water, and the cell concentration was adjusted to 10⁻⁶ cells / mL by plate counting. 4 CFU / mL was prepared for use. The activated Bacillus subtilis from the above steps was transferred to DSM (dimethylformamide) medium and cultured at 37°C for 24 h, after which Bacillus subtilis spores were collected. Similarly, Bacillus cereus from the above steps was transferred to LB agar medium supplemented with 50 mg / L MnSO4 and cultured at 37°C for 5 days, after which Bacillus cereus spores were collected. Finally, the collected spores were heat-treated at 80°C for 15 min, and the spore concentration was adjusted to 10⁻⁶ using plate counting. 4 CFU / mL available for use.

[0079] The concentration is 10. 4 CFU / mL of Bacillus subtilis and Bacillus cereus cells and spores were mixed separately at a 1:1 volume ratio. Experimental group 1 was a cross-mixture of Bacillus subtilis, Bacillus cereus, and Bacillus subtilis spores (equal volume ratio); Experimental group 2 was a cross-mixture of Bacillus subtilis, Bacillus cereus, and Bacillus cereus spores (equal volume ratio); and Experimental group 3 was a cross-mixture of Bacillus subtilis and Bacillus cereus cells and spores (equal volume ratio). Rapid purification of spores from the bacterial cell and spore mixture was performed following the procedures described in Application Example 1.

[0080] Spore purification efficiency = Spore concentration after purification / Spore purification concentration

[0081] Table 3. Purification efficiency of spores in the bacterial cell and spore cross-mixing system based on this method.

[0082]

[0083] As shown in Table 2, the spore purification efficiency in all three experimental groups reached over 96%. Therefore, the method for rapid separation and purification of bacterial spores based on a "step-by-step" automated device is effective for spore purification in systems with cross-mixing of bacterial cells and spores.

[0084] Application Example 3

[0085] This application example uses a complex mixture of Staphylococcus aureus, Salmonella, and Bacillus subtilis and Bacillus cereus cells and spores as an example to verify the feasibility and practicality of this method for rapid spore isolation and purification.

[0086] Culture of bacterial cells and spores: Staphylococcus aureus and Salmonella stored at -80 °C were activated by culturing on LB agar at 37 °C for 18–24 h, and then transferred to LB liquid culture medium for enrichment culture on a shaker at 180 rpm for 18–24 h. The concentration was adjusted to 10⁻⁶ using plate counting. 4 CFU / mL was prepared for use. Corresponding cells and spores of Bacillus subtilis and Bacillus cereus were cultured according to Application Example 1 and adjusted to a concentration of 10⁻⁶ by plate counting. 4 CFU / mL available for use.

[0087] The concentration is 10. 4 CFU / mL of Staphylococcus aureus, Salmonella, and Bacillus subtilis and Bacillus cereus cells and spores were mixed separately at a 1:1 volume ratio. Experimental group 1 was a complex mixture of Staphylococcus aureus, Salmonella, and Bacillus subtilis cells and spores (equal volume ratio); Experimental group 2 was a complex mixture of Bacillus subtilis, Bacillus cereus, and Bacillus cereus spores (equal volume ratio); and Experimental group 3 was a complex mixture of Staphylococcus aureus, Salmonella, and Bacillus subtilis and Bacillus cereus cells and spores (equal volume ratio). Rapid purification of spores from the bacterial cell and spore mixture was performed following the procedures described in Application Example 1.

[0088] Spore purification efficiency = Spore concentration after purification / Spore purification concentration

[0089] Table 3. Purification efficiency of spores in the bacterial cell and spore cross-mixing system based on this method.

[0090]

[0091] As shown in Table 3, the spore purification efficiency in all three experimental groups reached over 95%. Therefore, the method for rapid separation and purification of bacterial spores based on a "step-by-step" automated device is effective for spore purification in complex mixtures of bacterial cells and spores.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A "stepwise" automatic device suitable for rapid isolation and purification of bacterial spores, characterized in that: The "stepped" automatic device includes a "stepped" magnetic flow cavity and a "stepped" magnet mounting slot, which are configured to work together; the "stepped" magnetic flow cavity is provided with an inlet and an outlet. The inlet and outlet are located on the left and right sides of the "stepped" magnetic flow cavity, respectively. Connecting tube I and connecting tube II are respectively provided inside the inlet and outlet. Connecting tube I and connecting tube II are truncated rings. The inner diameter of the upper ring of the truncated ring is 0.8 cm and the outer diameter is 1.0 cm. The inner diameter of the lower ring is 0.4 cm and the outer diameter is 0.6 cm. The inlet is connected to the upper ring of connecting tube I, and the outlet is connected to the upper ring of connecting tube II. The "stepped" magnetic flow cavity also includes a "stepped" magnetic flow cavity cover plate, which cooperates with the "stepped" magnetic flow cavity. A stepped gap is provided between the stepped magnetic flow cavity and the stepped magnet mounting slot, and the thickness of the stepped gap is 0.3~0.5 cm. The wall thickness of the "stepped" magnetic flow cavity and the "stepped" magnet mounting groove is 0.6~0.8 cm, and the height is 2.6~2.8 cm.

2. The "step-type" automated device for rapid isolation and purification of bacterial spores according to claim 1, characterized in that: The "stepped" magnetic flow cavity has an n+1 level "stepped" configuration, and the "stepped" magnet mounting slot has an n level "stepped" configuration, where n≥3.

3. The method for rapid separation and purification of bacterial spores using the "staged" robot of claim 1 or 2, characterized by, The steps are as follows: (1) Preparation of Fe3O4-Van antibiotic magnetic beads: Fe3O4 magnetic beads were mixed with MUA ethanol solution, sonicated, washed and dissolved in MES buffer, sonicated and dispersed, EDC solution and Van solution were added in sequence and sonicated again, washed to obtain Fe3O4-Van antibiotic magnetic beads. Fe3O4-Van antibiotic magnetic beads were resuspended in PBS solution to obtain Fe3O4-Van antibiotic magnetic bead solution. (2) Add the Fe3O4-Van antibiotic magnetic bead solution obtained in step (1) to the solution to be separated containing bacterial cells and spores. After thorough shaking and mixing, pump it from the inlet to the "step-type" magnetic flow chamber. The permanent magnet in the "step-type" magnet mounting slot adsorbs the bacterial cells bound to the Fe3O4-Van antibiotic magnetic beads onto the inner wall of the "step-type" magnetic flow chamber. The remaining solution flows out through the outlet and the purified spores are collected.

4. The method for rapid separation and purification of bacterial spores by "staged" robotic device according to claim 3, characterized by the fact that: In step (1), the concentration of MUA ethanol is 40 μM, each milliliter of MUA ethanol solution contains 1 mg of Fe3O4 magnetic beads, the concentration of MES buffer is 0.1 M, and the pH is 5.5; the mass ratio of Fe3O4 magnetic beads to EDC and Van is 1:(0.6~0.7):(0.4~0.5); the concentration of PBS solution is 0.1 M, and the pH is 7.

4.

5. The method for rapid separation and purification of bacterial spores by "staged" robotic device according to claim 3, characterized by: In step (1), the concentration of the Fe3O4-Van antibiotic magnetic bead solution is 0.8~1.2 mg / mL; in step (2), the volume ratio of the mixture of bacterial cells and spores to the Fe3O4-Van antibiotic magnetic beads is 9:1.

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