CN113817602B - Simple microbial separation device and separation method - Google Patents

Abstract

The invention provides a simple microorganism separation device and a simple microorganism separation method. The device comprises a centrifuge tube and a high gradient magnetic separator fixed in the middle of the centrifuge tube. When the device is used for separating microorganisms, magnetic materials modified with biological recognition molecules are firstly added into a centrifuge tube, the magnetic materials are captured in a channel hole by using an array high-gradient magnetic field, a magnetic material chain is formed in the hole, then a sample containing the target microorganisms is added, and the centrifuge tube is repeatedly reversed, so that the target microorganisms are captured by the biological recognition molecules on the magnetic material chain when flowing through the channel hole, and the on-site and rapid separation of the target microorganisms is realized. The novel device for separating the target microorganisms in the large system sample has the characteristics of simplicity and convenience in operation, field separation, wide application range and the like.

Description

Simple microbial separation device and separation method

Technical Field

The present invention relates to microorganism separation technology, in particular to a simple microorganism separation device and a separation method.

Background technique

In recent years, there are many types of foodborne diseases caused by edible microorganisms contaminating food, and the incidence rate has shown a significant growth trend, which has seriously hindered the development of social economy. Since the concentration of pathogenic microorganisms in food is usually very low, conventional analytical methods cannot directly detect pathogenic microorganisms, and the background of food samples is complex, which will seriously interfere with the detection of target microorganisms and easily cause false positives. Therefore, it is very necessary to separate and enrich the target microorganisms from complex samples, which is also a key problem that needs to be solved urgently.

At present, common microbial separation methods mainly include conventional biological separation technology based on physical mechanisms and selective immunoseparation technology. Conventional biological separation methods based on physical mechanisms mainly include centrifugation and filtration, which are based on the physical and chemical properties of the target microorganisms such as mass and size to achieve the separation of target microorganisms, and have no specificity. Selective immunoseparation methods usually use biological recognition elements (antibodies, aptamers, etc.) to specifically recognize target microorganisms to achieve selective separation of target microorganisms.

Immunomagnetic separation technology is a new biological separation technology based on antigen-antibody immune binding, which has been widely used for the separation and enrichment of targets in complex samples. Conventional immunomagnetic separation can usually only handle small-volume samples and has high requirements on the professionalism of operators and equipment. At present, many foodborne pathogenic microorganisms are required not to be detected in ready-to-eat foods. However, at low concentrations, even if all target microorganisms in small-volume samples are separated, it is difficult to reach the detection limit of subsequent detection methods. Therefore, it is of great significance to develop a new method for on-site specific separation and efficient enrichment of target microorganisms in large-volume samples using a simple microbial separation device based on a centrifuge tube and a magnetic material chain.

Summary of the invention

The purpose of the present invention is to provide a simple microorganism separation device and a separation method.

In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a simple microorganism separation device, comprising a centrifuge tube and a high gradient magnetic separator fixed in the middle of the centrifuge tube;

The high gradient magnetic separator is composed of a closed cylindrical support, n cylindrical magnet groups and n-1 capillary glass tubes;

The cylindrical bracket is composed of a cover and a body, the body is used to load the cylindrical magnet group, and the cover and the body are connected by a slot structure to form a closed cylindrical bracket;

A cylindrical magnet hole is provided in the middle of the body, and cylindrical magnet holes of the same size are radially arranged at equal intervals along the direction of the cross line with the cylindrical magnet hole as the center, and the cylindrical magnet group is embedded in each magnet hole; each cylindrical magnet group is composed of two cylindrical magnets that repel each other, that is, the N poles of the two magnets are bonded together, or the S poles of the two magnets are bonded together; and the magnetic fields of two adjacent cylindrical magnet groups are in opposite directions;

In the body, a channel hole is provided between two adjacent cylindrical magnet holes, and the channel hole penetrates the cover of the cylindrical bracket; a capillary glass tube is inserted into the channel hole of the cover in the direction of the centrifuge tube cover, and it should be ensured that the centrifuge tube cover can be covered; a capillary glass tube is inserted into the channel hole of the body in the direction of the bottom of the centrifuge tube;

The body is also provided with two symmetrical air holes and two symmetrical screw holes, and both the air holes and the screw holes penetrate the cover of the cylindrical bracket; the air holes are used to balance the air pressure in the centrifuge tube; the screw holes are used to facilitate the removal of the cylindrical bracket from the centrifuge tube (during the experiment, it is necessary to take out the cleaned and sealed magnetic separator, and then use a pipette to inject magnetic material into the capillary glass tube channel, and then put it into the centrifuge tube and get it stuck near the middle of the centrifuge tube);

The cover has a protruding plug that matches the cylindrical magnet hole on the body and is used to seal the cylindrical magnet group in the cylindrical magnet hole.

In the aforementioned separation device, the outer diameter of the cylindrical support is consistent with the inner diameter of the centrifuge tube.

In the aforementioned separation device, the upper and lower surfaces of the cylindrical support are conical concave surfaces. The conical concave surface is a conical concave surface similar to an oil leak, and its function is to make the solution flow down completely without any residue. If it is a flat design, the solution will not flow out completely, and there will be residue, which will affect the capture detection.

Preferably, the height of the cylindrical support is 13% to 14% of the height of the centrifuge tube; more preferably, the height of the cylindrical support (15.5 mm) is 13.2% of the height of the centrifuge tube (117.1 mm).

Preferably, the diameter of the cylindrical magnet hole provided on the cylindrical bracket is 19% to 20% of the bracket diameter; more preferably, the diameter (5mm) of the cylindrical magnet hole provided on the cylindrical bracket is 19.2% of the bracket diameter (26.04mm).

Preferably, the aperture of the channel hole provided on the cylindrical support is 4% to 5% of the diameter of the support; more preferably, the aperture (1.3 mm) of the channel hole provided on the cylindrical support is 4.5% of the diameter of the support (26.04 mm).

Preferably, the diameter of the pores provided on the cylindrical support is 4% to 5% of the diameter of the support; more preferably, the diameter of the pores provided on the cylindrical support (1.3 mm) is 4.5% of the diameter of the support (26.04 mm).

Preferably, the diameter of the screw hole provided on the cylindrical bracket is 9% to 10% of the diameter of the bracket; more preferably, the diameter of the screw hole provided on the cylindrical bracket (2.5 mm) is 9.6% of the diameter of the bracket (26.04 mm).

Preferably, the cylindrical bracket is a 3D printed cylindrical bracket, and the bracket is made of resin material (preferably photosensitive resin).

Preferably, the material of the cylindrical magnet is N40 neodymium iron boron permanent magnet.

In the aforementioned separation device, the cover and the body of the cylindrical bracket are further tightly connected by a slot and an adhesive (such as glue).

In a second aspect, the present invention provides use of the separation device in separation of microorganisms.

In a third aspect, the present invention provides a method for separating microorganisms, which uses the separation device to separate microorganisms in a sample. The method includes: first adding a magnetic material (such as an immune nickel wire) modified with a biological recognition molecule into a centrifuge tube, and using a magnetic field to capture the magnetic material in the channel hole; then adding a sample solution containing a target microorganism into the centrifuge tube, repeatedly inverting the centrifuge tube, so that the target microorganism is captured by the magnetic material when flowing through the channel hole, thereby achieving separation of the target microorganism.

The magnetic material includes but is not limited to nickel wire and magnetic beads.

In a specific embodiment of the present invention, the method for preparing the immunonickel wire solution comprises:

(1) Mix 75 μL of 1 M nickel chloride hexahydrate solution (purity 99.9%) and 15 mL of ethylene glycol (99.8%) in a beaker and heat to 100°C;

(2) Add 0.5 mL of hydrazine monohydrate dropwise to the above mixture, keeping the temperature at 100°C;

(3) Continue heating for 30 min until a dark gray product is formed and floats on the surface of the solution;

(4) collecting the generated dark gray product, namely the nickel nanowire, and washing it three times with deionized water combined with magnetic separation, and washing it twice with anhydrous ethanol combined with magnetic separation;

(5) dispersing the washed nickel nanowires in ethanol containing 0.03% w/v polyvinyl pyrrolidone to obtain a 1 mg/mL nickel nanowire solution;

(6) Ultrasonic mixing of the nickel nanowire solution to form a uniform dispersion system; 500 μL of the nickel nanowire solution was added to a reaction bottle, and washed twice with ultrapure water (to remove the protective agent on the surface of the nickel wire, ethanol);

(7) Add 100 μL H ₂ O ₂ , 100 μL NH ₄ OH and 500 μL deionized water to the washed nano-nickel wires (for hydroxylation modification of the nano-nickel wires), place the mixed solution in a water bath at 80° C. for 30 min to obtain nano-nickel wires modified with hydroxyls (the reaction bottle is not covered);

(8) Recovering the hydroxyl-modified nickel nanowires with a magnetic rack, washing them once with ultrapure water and once with anhydrous ethanol, and then drying them in an oven (drying at 65°C for about 8 minutes);

(9) 1% [3-(methoxysilyl)propyl]succinic anhydride solution was prepared with anhydrous ethanol, and the dried hydroxyl-modified nickel nanowires were resuspended with 1% [3-( _{methoxysilyl)propyl]succinic anhydride (molecular formula C10H18O6Si ) solution} (10 μL [3-(methoxysilyl)propyl]succinic anhydride + 990 μL anhydrous ethanol) (carboxylation of the nickel nanowires) and then placed on a mixer to react overnight at room temperature (15 h) to obtain carboxyl-modified nickel nanowires;

The structure of [3-(methoxysilyl)propyl]succinic anhydride is shown in Formula I):

(10) The next day, the nickel nanowires modified with carboxyl groups were recovered using a magnetic rack, washed three times with anhydrous ethanol (to remove the residual carboxylation reagent on the surface), and then washed twice with ultrapure water (to remove the residual ethanol), and then 500 μL of 1 M NaOH solution (pH 9, pH adjusted with HCl) was added and reacted for more than 5 h (it can be observed with the naked eye that the water solubility of the nickel wire solution has improved, that is, the carboxyl groups are fully opened);

(11) The nickel nanowires modified with carboxyl groups resuspended in alkaline solution were washed three times with ultrapure water (to remove residual NaOH), and then washed once with PB solution at pH 7.4;

(12) Add 500 μg EDC (10 mg/mL, dissolved in PB solution, pH 7.4) and NHSS (10 mg/mL, dissolved in PB solution, pH 7.4) and react for 1 h to activate the carboxyl group (final system 500 μL, PB solution, pH 7.4);

(13) Wash the nickel nanowires modified with activated carboxyl groups twice with a PB solution at pH 7.4 (to remove residual EDC and NHSS);

(14) The nickel nanowires modified with activated carboxyl groups were re-dissolved with 420 μL of PB solution (pH 7.4), and then 41.67 μL of 1.2 mg/mL streptavidin solution and 200 μg of skim milk (for binding of streptavidin to nickel wires and stabilizing the reaction system) were added and reacted at room temperature for 1 h.

(15) The nickel nanowire modified with activated carboxyl groups was washed twice with a PB solution at pH 7.4 (to remove unbound streptavidin), and then 500 μL of 1% skim milk (dissolved in a PB solution at pH 7.4) was added for blocking for 1 h to obtain streptavidin-labeled nickel wires;

(16) Add 60 μg of streptavidin-conjugated nickel wire to 500 μL PBS solution, wash twice by magnetic separation, and remove the skim milk solution;

(17) Then, the nano-nickel wires were re-dissolved with 500 μL PBS solution and 4 μg of capture antibody was added, and the mixture was incubated at room temperature for 45 min; wherein the capture antibody can specifically recognize and bind to the target microorganism (when the target microorganism is Salmonella typhimurium, the capture antibody can be an anti-Salmonella typhimurium biotinylated polyclonal antibody with a concentration of 2.25 mg/mL);

(18) Magnetic separation and washing to remove excess unbound antibodies.

In the present invention, the solution circulation principle is: when the liquid above the bracket flows down to the bottom of the bracket through the channel hole, the air below the bracket is squeezed to the top of the bracket by the liquid through the pores, and the more air above the bracket, the more liquid will be pushed to flow to the bottom of the bracket.

By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

The simple microorganism separation device based on centrifuge tube and magnetic material provided by the present invention, when the separation device is used for microorganism separation, after adding the magnetic material modified with biological recognition molecules into the centrifuge tube, due to the presence of the high gradient magnetic field of the array, the magnetic material will be captured in the channel hole, and a magnetic material chain will be formed in the hole. After adding the sample solution containing the target microorganism, by repeatedly inverting (standing upside down) the centrifuge tube, the target microorganism is captured by the biological recognition molecules on the magnetic material chain when flowing through the channel hole, thereby improving the capture efficiency, thereby improving the separation efficiency of the target microorganism from the sample, simplifying the operation steps, and realizing the on-site and rapid separation of the target microorganism. Therefore, the separation device can be used for the separation of target microorganisms in large system samples, effectively improving the utilization efficiency of magnetic materials, reducing costs, being easy to operate, not relying on professional operators and complex instrumentation facilities, and meeting the on-site detection needs with limited resources.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overall structural diagram of a simple microorganism separation device in a preferred embodiment of the present invention.

Figure 2 is a structural diagram of a 3D printed cylindrical bracket in a preferred embodiment of the present invention, which is divided into an upper part and a lower part (i.e., a cover and a body). The body is mainly provided with cylindrical holes for loading magnets, and a channel hole for placing a capillary glass tube is provided between every two cylindrical magnet holes, and two symmetrical screw holes and two symmetrical air holes are provided in the remaining blank space.

FIG. 3 is a three-view diagram of the upper and lower parts of the 3D printed cylindrical bracket in a preferred embodiment of the present invention.

FIG. 4 is a schematic diagram of the overall structure of an assembled high gradient magnetic separator (loaded with magnets and inserted into a capillary glass tube) in a preferred embodiment of the present invention.

FIG. 5 is a picture of a high gradient magnetic separator loaded with a cylindrical magnet group in a preferred embodiment of the present invention.

FIG. 6 is a picture of a simplified microorganism separation device in a preferred embodiment of the present invention.

FIG. 7 is a picture of a 3D printed cylindrical bracket in a preferred embodiment of the present invention.

FIG. 8 is a picture of the upper portion (lid) of the 3D printed cylindrical bracket in a preferred embodiment of the present invention.

FIG. 9 is a picture of the lower portion (body) of the 3D printed cylindrical bracket in a preferred embodiment of the present invention.

FIG. 10 is a cross-sectional view of the upper and lower parts of a 3D printed cylindrical bracket in a preferred embodiment of the present invention.

FIG. 11 is a cross-sectional view of a closed 3D printed cylindrical bracket in a preferred embodiment of the present invention.

FIG. 12 shows the separation of target microorganisms using a simple microorganism separation device in a preferred embodiment of the present invention.

FIG. 13 is a magnetic field simulation diagram of a high gradient magnetic field array in an embodiment of the present invention.

FIG. 14 is a graph showing the optimization result of the number of cycles in an embodiment of the present invention.

FIG. 15 is a result of optimizing the amount of nickel wire in an embodiment of the present invention.

In Figures 1, 2, 10 and 11, 1: centrifuge tube; 2: 3D printed cylindrical bracket; 3: air hole; 4: capillary glass tube; 5: screw hole; 6: cylindrical magnet hole; 7: channel hole; 8: cover; 9: body; 10: convex structure of the slot; 11: concave structure of the slot; 12: concave concave surface; 13: plug; 14: slot structure.

Detailed ways

The present invention aims to provide a simple microorganism separation device and separation method based on a centrifuge tube and a magnetic material chain, so as to solve the problems of small sample volume, complicated operation process and dependence on instruments and equipment in the conventional magnetic separation technology.

The present invention adopts the following technical solution:

On the one hand, the present invention provides a simple microorganism separation device based on a centrifuge tube and a magnetic material chain, comprising a high gradient magnetic separator and a conventional centrifuge tube.

The high gradient magnetic separator consists of a closed cylindrical support, n cylindrical magnet groups and n-1 capillary glass tubes.

The cylindrical bracket is composed of a cover and a body, the body is used to load the cylindrical magnet group, and the cover and the body are connected through a slot structure to form a closed cylindrical bracket.

A cylindrical magnet hole is provided in the middle of the body, and cylindrical magnet holes of the same size are arranged radially at equal intervals along the direction of the cross line with the cylindrical magnet hole as the center, and the cylindrical magnet group is embedded in each magnet hole; each cylindrical magnet group is composed of two cylindrical magnets that repel each other, that is, the N poles of the two magnets are bonded together, or the S poles of the two magnets are bonded together; and the magnetic field directions of two adjacent cylindrical magnet groups are opposite. That is, first place a group of magnets (two) in the cylindrical magnet hole at the center position, and the two magnets have the same poles, such as two N poles bonded together, then place a magnet group with two S poles bonded together at each of the four positions 7mm above, below, left and right of the center magnet group, and then set a channel hole (diameter 1.3mm) between every two N poles and S poles, that is, at the middle position of 2mm. Then expand to the surroundings in this way.

In the body, a channel hole is provided between two adjacent cylindrical magnet holes, and the channel hole passes through the cover of the cylindrical bracket; a capillary glass tube is inserted into the channel hole of the cover in the direction of the centrifuge tube cover, and it should be ensured that the centrifuge tube cover can be covered; a capillary glass tube is inserted into the channel hole of the body in the direction of the bottom of the centrifuge tube.

The main body is also provided with two symmetrical air holes and two symmetrical screw holes, and both the air holes and the screw holes penetrate the cover of the cylindrical bracket; the air holes are used to balance the air pressure in the centrifuge tube; and the screw holes are used to facilitate the removal of the cylindrical bracket from the centrifuge tube.

The cover has a protruding plug that matches the cylindrical magnet hole on the body and is used to seal the cylindrical magnet group in the cylindrical magnet hole.

The high gradient magnetic separator is composed of an array high gradient magnetic field and magnetic materials that modify the biorecognition element. The array high gradient magnetic field is composed of a series of cylindrical magnet groups that are vertically crossed and equally spaced. The magnet group is composed of two cylindrical magnets that repel each other. The adjacent vertical intersection points of any magnet group are magnet groups with opposite magnetic field directions. The magnet group is installed in a closed 3D printed cylindrical bracket. A channel hole is provided between each two adjacent magnet groups, and each channel hole has a high gradient magnetic field. Two channel holes are randomly selected from the channel holes, and a capillary glass tube higher than the liquid column is inserted in the upper and lower parts respectively. The circular bracket is installed near the middle of the centrifuge tube. When there is liquid in the centrifuge tube and it is placed upright, the liquid will flow down from the top of the bracket through the channel hole to the bottom of the bracket, and vice versa.

The conventional centrifuge tube is designed to match the size of the 3D printed cylindrical bracket and is combined with the high gradient magnetic separator to add a magnetic material modified with a biorecognition molecule into the centrifuge tube, capture the magnetic material in the channel hole using the array high gradient magnetic field, and form a magnetic material chain in the hole, then add a sample solution containing target microorganisms, and repeatedly invert the centrifuge tube so that the target microorganisms are captured by the biorecognition molecules on the magnetic material chain when flowing through the channel hole, thereby achieving on-site and rapid separation of the target microorganisms.

Furthermore, the high-gradient magnetic separator includes the closed 3D printed cylindrical bracket, on which cylindrical magnet holes are arranged in a vertically crossed and equidistant manner from the center to the surroundings, a channel hole is provided between each two adjacent cylindrical magnet holes, a group of slots with concave-convex structures, two symmetrical air holes and two symmetrical screw holes; the high-gradient magnetic separator includes a plurality of magnet groups, each of which is respectively embedded in the cylindrical magnet hole, and the magnet group is composed of two mutually repelling cylindrical magnets, and the adjacent vertical intersection points of any magnet group are magnet groups with opposite magnetic field directions. The high-gradient magnetic separator also includes a capillary glass tube, the length of which is higher than the liquid column and is inserted into the channel hole.

The slot is designed around the outer periphery of the cylindrical bracket, and its function is to tightly connect the cover and the body of the circular bracket together to prevent the solution from penetrating into the magnet. The function of the pores is to circulate air and balance the atmospheric pressure. When the liquid above the 3D printed circular bracket flows from the capillary glass tube to the bottom of the bracket, it will occupy the position of the air, so the air below will pass from the pores to the top of the bracket, and the air pressure above will increase, pressing the liquid to continue to flow to the bottom of the bracket through the capillary glass tube. The function of the screw hole is to facilitate the removal of the 3D printed circular bracket from the centrifuge tube. Since the 3D printed bracket is stuck in the middle of the centrifuge tube, there is a certain friction. If the screw hole is not designed, the bracket cannot be taken out. If there is a screw hole, a copper column of a certain length can be screwed into the screw hole to take out the bracket.

Furthermore, the outer circumference of the 3D printed cylindrical support is consistent with the inner diameter of the conventional centrifuge tube.

Furthermore, the upper and lower surfaces of the 3D printed cylindrical bracket are conical concave surfaces.

Furthermore, the material of the 3D printed cylindrical bracket is photosensitive resin.

Furthermore, the cover and the body of the cylindrical bracket can be further tightly connected by using a slot and an adhesive.

On the other hand, the present invention also provides an array high-gradient magnetic field separation method for a simple microorganism separation device based on a centrifuge tube and a magnetic material chain, comprising: when using the separation device to separate microorganisms, first add a magnetic material modified with a biological recognition molecule into the centrifuge tube, use the array high-gradient magnetic field to capture the magnetic material in the channel hole, and form a magnetic material chain in the hole; then inject a sample solution containing the target microorganism into the centrifuge tube, and by repeatedly inverting the centrifuge tube, the target microorganism is captured by the biological recognition molecules on the magnetic material chain when flowing through the channel hole, thereby achieving on-site and rapid separation of the target microorganism in a large system sample.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

In the description of the present invention, unless otherwise specified, "multiple" means two or more than two. The directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention.

The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

Example 1 Simple microbial separation device and separation method

1 to 11 , this embodiment provides a simple microorganism separation device and method based on a centrifuge tube and a magnetic material chain, including: a high gradient magnetic separator and a conventional centrifuge tube 1 .

The high gradient magnetic separator is composed of a closed cylindrical support, n cylindrical magnet groups and n-1 capillary glass tubes;

The 3D printed cylindrical bracket is composed of a cover 8 and a body 9, the body is used to load the cylindrical magnet group, and the cover (a convex structure 10 with a slot) and the body (a concave structure 11 with a slot) are connected through a slot structure 14 to form a closed 3D printed cylindrical bracket;

A cylindrical magnet hole is provided in the middle of the body, and cylindrical magnet holes 6 of the same size are radially arranged at equal intervals along the direction of the cross line with the cylindrical magnet hole as the center, and the cylindrical magnet group is embedded in each magnet hole; each cylindrical magnet group is composed of two cylindrical magnets that repel each other, that is, the N poles of the two magnets are bonded together, or the S poles of the two magnets are bonded together; and the magnetic fields of two adjacent cylindrical magnet groups are in opposite directions;

In the body, a channel hole 7 is provided between two adjacent cylindrical magnet holes, and the channel hole passes through the cover of the 3D printed cylindrical bracket; a capillary glass tube is inserted into the channel hole of the cover in the direction of the centrifuge tube cover, and it should be ensured that the centrifuge tube cover can be covered; a capillary glass tube 4 is inserted into the channel hole of the body in the direction of the bottom of the centrifuge tube;

The body is also provided with two symmetrical air holes 3 and two symmetrical screw holes 5, both of which penetrate the cover of the 3D printed cylindrical bracket; the air holes are used to balance the air pressure in the centrifuge tube; the screw holes are used to facilitate the removal of the 3D printed cylindrical bracket from the centrifuge tube;

The cover has a protruding plug 13 matching the cylindrical magnet hole on the body, and is used to seal the cylindrical magnet group in the cylindrical magnet hole.

The outer diameter of the 3D printed cylindrical support is consistent with the inner diameter of the centrifuge tube.

The upper and lower surfaces of the 3D printed cylindrical bracket are conical concave surfaces 12. The conical concave surface is a conical concave surface similar to an oil leak, and its function is to make the solution flow down completely without any residue. If it is a flat design, the solution will not flow out completely, and there will be residue, which will affect the capture detection.

Wherein, the high gradient magnetic separator is composed of an array high gradient magnetic field and a magnetic material for modifying a biorecognition element. The array high gradient magnetic field is composed of a series of cylindrical magnet groups that are vertically crossed and equally spaced, and the magnet group is composed of two mutually repelling cylindrical magnets. The adjacent vertical intersections of any magnet group are magnet groups with opposite magnetic field directions. The magnet group is installed in a magnet hole 6 of a closed 3D printed cylindrical bracket 2, and a channel hole 7 is provided between each two adjacent magnet groups. Preferably, the cylindrical magnet is an N40 neodymium iron boron permanent magnet with a diameter of 5 mm and a thickness of 2 mm, and the magnetization direction is shown in Figure 13. The magnetic field simulation of the array high gradient magnetic field is shown in Figure 13. It can be seen from the simulation results that each channel hole has a high gradient magnetic field, and the magnetic lines of force are evenly distributed, which can induce the magnetic material in it to be distributed in a chain. Two channel holes are randomly selected from the channel holes, and a capillary glass tube 4 higher than the liquid column is inserted at the top and bottom, respectively. Preferably, the inner diameter of the capillary glass tube is 0.9-1.1 mm, the length is about 45 mm, and the circular bracket is installed near the middle of the centrifuge tube. When the centrifuge tube contains liquid and is placed upright, the liquid will flow from the top of the bracket through the channel hole to the bottom of the bracket, and vice versa. A group of grooves with a concave-convex structure, two symmetrical air holes 3 and two symmetrical screw holes 5 are provided between every two adjacent holes on the 3D printed cylindrical bracket.

The height of the 3D printed cylindrical bracket is 15.5 mm, which is the height of the centrifuge tube 117.1 mm.

The cylindrical magnet hole provided on the 3D printed cylindrical bracket has a hole diameter of 5 mm, and the bracket diameter is 26.04 mm.

The diameter of the channel hole provided on the 3D printed cylindrical bracket is 1.3 mm.

The diameter of the pores provided on the 3D printed cylindrical bracket is 1.3 mm.

The diameter of the screw hole provided on the 3D printed cylindrical bracket is 2.5 mm.

Furthermore, the cover and the body of the 3D printed cylindrical bracket can be further tightly connected by using a slot and an adhesive.

Furthermore, the material of the 3D printed cylindrical bracket is photosensitive resin.

The solution flow principle of the device is: when the liquid above the bracket flows down to the bottom of the bracket through the channel hole, the air below the bracket is squeezed to the top of the bracket by the liquid through the pores, and the more air above the bracket, the more it will push the liquid to flow below the bracket.

As shown in FIG1 , the figure schematically shows that the simple microorganism separation device based on a centrifuge tube and a magnetic material chain includes: a high gradient magnetic separator and a conventional centrifuge tube.

The high gradient magnetic separator can effectively separate the target microorganisms in the solution. When the separation device is combined with a magnetic material for microbial separation, a magnetic material modified with a biorecognition molecule is first added to the centrifuge tube, and the array high gradient magnetic field is used to capture the magnetic material in the channel hole, and a magnetic material chain is formed in the hole, and then a sample containing the target microorganism is added. By repeatedly inverting the centrifuge tube, the target microorganism is captured by the biorecognition molecules on the magnetic material chain when it flows through the channel hole, thereby achieving on-site and rapid separation of the target microorganism. The simple microbial separation device based on a centrifuge tube and a magnetic material chain of the present invention can inject 10mL of a sample solution containing the target microorganism at one time, and therefore can be used for the separation of target objects in large system samples, effectively improving the utilization efficiency of magnetic materials, reducing costs, and being easy to operate, and can be used for on-site separation.

It should be noted that the magnetic material modified by the biorecognition element is fixed in the channel hole by the magnetic field to form a chain, and can capture the target microorganism in the sample solution.

The present invention also provides a simple microorganism separation method based on a centrifuge tube and a magnetic material chain, comprising:

Step 1. Open the clean bench, wipe the table with absorbent paper and alcohol, prepare the following items: 1.5mL centrifuge tube box, filter box, centrifuge tube rack, syringe, 75% alcohol, ultrapure water, sterilized PBS, simple microbial separation device, screw stick, gun tip, 15mL centrifuge tube, 50mL centrifuge tube, etc., place them on the clean bench, and then turn on the ultraviolet light for sterilization for 10 minutes.

Step 2. In the clean bench, sterilize the device with 75% alcohol, repeatedly inverting it for 2 cycles; pour out the alcohol, wash it with ultrapure water for 2 cycles, and then rinse it with sterile PBS for 2 cycles until it is clean.

Step 3, weigh the required BSA in a 50mL centrifuge tube in advance, dissolve it in sterile PBS on a clean bench to make about 40mL 1% BSA, and then filter it into sterile BSA in a clean bench. After the above disinfection and cleaning steps are completed, add 1% sterile BSA to the device, and then place the device on a mixer and seal it for 45 minutes.

Step 4: After sealing, wash the device with sterile PBS until it is clean and free of bubbles. During this period, the immunonickel wire can be cleaned.

Step 5, when cleaning the immune nickel wire, take out the immune nickel wire bottle and the sealed sterile glass bottle from the refrigerator, take 200 μL of immune nickel wire (concentration of 1 μg/μL) and add it to the sealed glass bottle, wash it with 500 μL of sterilized PBS each time, wash it once (too many times will wash off the antibody), magnetic separation for 3 minutes each time, and finally dissolve it with 200 μL of sterile PBS after magnetic separation to a concentration of 1 μg/μL.

Step 6, after the device is sealed and cleaned, the immune nickel wire is injected, and 100 μg (100 μL) is injected into each channel (the channel refers to the capillary glass tube channel formed by inserting the capillary glass tube into the channel hole of the 3D printed circular bracket), and then 10 mL of Salmonella solution with a concentration of 1.0×10 ⁴ CFU/mL is added, and the reaction is started for 20 cycles (the cycle refers to adding the sample solution, first allowing it to flow to the bottom of the bracket, and then turning it upside down to allow the solution originally under the bracket to flow to the top of the original bracket, which is one cycle).

Step 7: After the reaction is completed, the supernatant is taken out for magnetic separation for 10 minutes, and then the supernatant is taken out and plated, and the capture rate is verified the next day.

The separation of target microorganisms using a simple microorganism separation device is shown in Figure 12.

According to the above experimental scheme, two parameters were optimized for this device - nickel wire dosage and cycle number. The experimental optimization results are as follows:

The optimization result of the number of cycles is shown in Figure 14, which shows that the usage of the immune nickel wire directly affects the separation efficiency of bacteria. Therefore, the optimal usage of the immune nickel wire is determined by the separation efficiency of capturing Salmonella typhimurium with different amounts of immune nickel wire. Different amounts of (100μg, 150μg, 200μg, 250μg and 300μg) of immune nickel wire (1mg/mL) are injected into the capillary glass channel to capture 10mL of Salmonella typhimurium with a concentration of 4.8×10 ⁶ CFU/mL, respectively. The number of bacteria is obtained by plate counting method, and the capture efficiency of different amounts of immune nickel wire is calculated. When the usage of the immune nickel wire is increased from 100μg to 300μg, the capture efficiency increases from 26.4% to 58.4%. However, when the usage of the immune nickel wire is increased to 250μg and 300μg, the capture efficiency does not change significantly, which indicates that 200μg of immune nickel wire is sufficient for capturing target bacteria. Therefore, the present invention selects the best usage of immune nickel wire as 200μg.

The results of the optimization of the nickel wire dosage are shown in Figure 15, which shows that the number of cycles also directly affects the contact time between the immune nickel wire and the bacteria, thereby affecting the separation efficiency. 200 μg of immune nickel wire was reacted with 10 mL of 7.2×10 ⁶ CFU/mL of Salmonella typhimurium using different number of cycles (5 times, 10 times, 15 times, 20 times and 25 times). When the number of cycles was 5 times, the separation efficiency was 24.2%, when the number of cycles was increased to 10 times, the separation efficiency was 36.1%, and when the number of cycles was further increased to 15 times, the separation efficiency increased to 42.1%, and when the number of cycles was increased to 20 times, the separation efficiency was 52.5%, and when the number of cycles was 25 times, the separation efficiency was 54.8%, which remained basically unchanged, so the optimal number of cycles of the present invention is 20 times.

Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (7)

1. A simple microorganism separation device, characterized in that it comprises a centrifuge tube and a high gradient magnetic separator fixed in the middle of the centrifuge tube; The high gradient magnetic separator is composed of a closed cylindrical support, n cylindrical magnet groups and n-1 capillary glass tubes; The cylindrical bracket is composed of a cover and a body, the body is used to load the cylindrical magnet group, and the cover and the body are connected by a slot structure to form a closed cylindrical bracket; A cylindrical magnet hole is provided in the middle of the body, and cylindrical magnet holes of the same size are radially arranged at equal intervals along the direction of the cross line with the cylindrical magnet hole as the center, and the cylindrical magnet group is embedded in each magnet hole; each cylindrical magnet group is composed of two cylindrical magnets that repel each other, that is, the N poles of the two magnets are bonded together, or the S poles of the two magnets are bonded together; and the magnetic fields of two adjacent cylindrical magnet groups are in opposite directions; In the body, a channel hole is provided between two adjacent cylindrical magnet holes, and the channel hole penetrates the cover of the cylindrical bracket; a capillary glass tube is inserted into the channel hole of the cover in the direction of the centrifuge tube cover, and it should be ensured that the centrifuge tube cover can be covered; a capillary glass tube is inserted into the channel hole of the body in the direction of the bottom of the centrifuge tube; The body is also provided with two symmetrical air holes and two symmetrical screw holes, and both the air holes and the screw holes penetrate the cover of the cylindrical bracket; the air holes are used to balance the air pressure in the centrifuge tube; the screw holes are used to facilitate the removal of the cylindrical bracket from the centrifuge tube; The cover has a protruding plug that matches the cylindrical magnet hole on the body and is used to seal the cylindrical magnet group in the cylindrical magnet hole; The outer diameter of the cylindrical support is consistent with the inner diameter of the centrifuge tube; The height of the cylindrical support is 13% to 14% of the height of the centrifuge tube; The diameter of the cylindrical magnet hole provided on the cylindrical bracket is 19% to 20% of the diameter of the bracket; The diameter of the channel hole provided on the cylindrical support is 4% to 5% of the diameter of the support; The diameter of the pores provided on the cylindrical support is 4% to 5% of the diameter of the support; The diameter of the screw hole provided on the cylindrical bracket is 9% to 10% of the diameter of the bracket. 2. The separation device according to claim 1 is characterized in that the upper and lower surfaces of the cylindrical bracket are conical concave surfaces. 3. The separation device according to claim 1 is characterized in that the cylindrical bracket is a 3D printed cylindrical bracket, and the material of the bracket is resin material. 4. The separation device according to any one of claims 1 to 3 is characterized in that the cover and the body of the cylindrical bracket are further tightly connected by a slot and an adhesive. 5. Use of the separation device according to any one of claims 1 to 4 in separation of microorganisms. 6. A method for separating microorganisms, characterized in that the separation device according to any one of claims 1 to 4 is used to separate microorganisms in a sample, the method comprising: first adding a magnetic material modified with a biorecognition molecule into a centrifuge tube, and capturing the magnetic material in the channel hole using a magnetic field; then adding a sample solution containing a target microorganism into the centrifuge tube, and repeatedly inverting the centrifuge tube so that the target microorganism is captured by the magnetic material when flowing through the channel hole, thereby separating the target microorganism. 7. The method according to claim 6, characterized in that the magnetic material comprises nickel wire and magnetic beads.