Magnetic nano-particles, immobilized enzyme microspheres and preparation devices of magnetic nano-particles and immobilized enzyme microspheres

Abstract

The utility model belongs to the technical field of immobilized enzyme, particularly relates to magnetic nanoparticles, immobilized enzyme microspheres and a preparation device thereof, and solves the problem that enzyme is difficult to fix. The magnetic nanoparticles are formed by self-assembly of chitosan long chains in water, and metal nanoparticles or metal oxide nanoparticles are electrostatically adsorbed on the chitosan long chains. The immobilized enzyme microsphere comprises a magnetic inner core, a cross-linking layer and an enzyme layer which are sequentially arranged from inside to outside, wherein the magnetic inner core is the magnetic nanoparticle disclosed by the invention. The effect that the magnetic immobilized enzyme microspheres are convenient to separate is achieved.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme technology, specifically relating to a magnetic nanoparticle, an immobilized enzyme microsphere, and a device for preparing the same. Background Technology

[0002] Alzheimer's disease (AD) is a chronic, progressive dementia caused by central nervous system degeneration. Existing research suggests that the pathogenesis of AD includes excessive accumulation of amyloid-β protein, neurofibrillary tangles formed by hyperphosphorylation of tau protein, and a deficiency of the neurotransmitter acetylcholine. Therefore, acetylcholinesterase inhibitors have become one of the most studied anti-Alzheimer's drugs.

[0003] Aconite (Aconitilateralis Radix Praeparata) plays an important role in the treatment of Alzheimer's disease (AD). For example, the treatment of Alzheimer's disease with ephedra preparations is mainly attributed to the good antioxidant and anti-acetylcholinesterase activities of aconite. This indicates that aconite contains abundant acetylcholinesterase inhibitors. If the acetylcholinesterase inhibitors in aconite can be isolated and purified, the aconite resource can be utilized more fully.

[0004] Immobilized acetylcholinesterase is an effective method for isolating acetylcholinesterase inhibitors from Aconitum carmichaelii extract. The amino groups in chitosan have a high affinity for various proteins, and its three-dimensional multi-spatial structure makes it an excellent carrier for immobilizing enzymes, antibodies, and other bioactive substances. Therefore, chitosan can immobilize enzymes under mild conditions with high efficiency and good activity. Furthermore, chitosan is non-toxic, biodegradable, and its excellent physical adsorption and biocompatibility provide a new and highly efficient carrier for enzyme immobilization.

[0005] However, due to the small size of chitosan, separating the enzyme immobilized in chitosan microspheres from the immobilization solution and enzyme reaction medium requires centrifugation or filtration, a cumbersome and laborious process. Therefore, overcoming this obstacle is of great significance for enzyme immobilization. Summary of the Invention

[0006] The first objective of this invention is to provide magnetic nanoparticles and immobilized enzyme microspheres, which not only serve as a good carrier for immobilized enzymes but also facilitate the rapid and effective separation of immobilized enzyme products.

[0007] To achieve this innovative objective of this utility model, the following technical solutions can be used:

[0008] A magnetic nanoparticle is formed by the self-assembly of chitosan long chains in water, wherein metal nanoparticles or metal oxide nanoparticles are electrostatically adsorbed on the chitosan long chains.

[0009] The magnetic nanoparticles of this invention are formed in water. In water, metal nanoparticles first adsorb onto chitosan long chains. Then, the chitosan long chains that have adsorbed metal nanoparticles or metal oxide nanoparticles curl up and intertwine with other chitosan long chains to form spherical magnetic nanoparticles. The interior of the magnetic nanoparticles consists of chitosan and metal nanoparticles, and the ends of the chitosan long chains extend to the outside of the magnetic nanoparticles, providing support for the immobilized enzymes.

[0010] In the magnetic nanoparticles of this invention, not only can chitosan serve as a good carrier for immobilized enzymes, but metal nanoparticles or metal oxide nanoparticles can also facilitate the separation of immobilized enzymes, achieving two goals at once.

[0011] In the aforementioned magnetic nanoparticles, the metal oxide nanoparticles are iron oxide nanoparticles.

[0012] Preferably, the radius of the magnetic nanoparticles of this invention is between 1 and 5 nm.

[0013] The second objective of this invention is to address the aforementioned problems in the prior art by proposing an immobilized enzyme microsphere.

[0014] To achieve this innovative objective of this utility model, the following technical solutions can be used:

[0015] An immobilized enzyme microsphere comprises a magnetic core, a cross-linking layer, and an enzyme layer arranged sequentially from the inside out; wherein the magnetic core is the aforementioned magnetic nanoparticle.

[0016] Preferably, in the above-mentioned immobilized enzyme microspheres, the cross-linking layer is a glutaraldehyde layer.

[0017] In the above-mentioned immobilized enzyme microspheres, the enzyme layer may be an acetylcholinesterase layer or other enzyme layers.

[0018] Preferably, the radius of the immobilized enzyme microspheres is between 50 and 100 nm.

[0019] The third objective of this invention is to address the aforementioned problems in the prior art by proposing an immobilized enzyme microsphere.

[0020] To achieve this innovative objective of this utility model, the following technical solutions can be used:

[0021] An apparatus for preparing immobilized enzyme microspheres, comprising: a first mixer, wherein a magnetic stirrer assembly is provided below the first mixer for preparing a first mixture containing metal oxides; a centrifuge assembly for centrifuging the first mixture to separate metal oxide nanoparticles or metal nanoparticles; an ultrasonic oscillator assembly for agitating and mixing nanoparticles and chitosan solution to obtain a second mixture; a second mixer for mixing the second mixture with an auxiliary agent to prepare a third mixture containing magnetic nanoparticles; a third mixer for mixing the magnetic nanoparticles obtained by filtering the third mixture with acetylcholinesterase to carry out a cross-linking reaction, and removing the supernatant after the reaction is completed and precipitation occurs; and a drying assembly for drying the immobilized enzyme microsphere precipitate left in the third mixer.

[0022] The preparation apparatus of this invention is used to prepare magnetic nanoparticles and immobilized enzyme microspheres based on these magnetic nanoparticles. The first mixer and centrifuge assembly are used to prepare metal oxide nanoparticles, such as iron oxide. A mixed solvent, such as ferric chloride and ferrous sulfate, is added to the first mixer in a specific molar ratio. An agent such as ammonia is added to adjust the pH to alkaline. The reaction is carried out under stirring and heating conditions, and after aging at this temperature, a corresponding iron oxide mixture, i.e., mixture number one, is obtained. The centrifuge assembly separates the iron oxide particles in this mixture. As an optimization, the separated iron oxide particles need to be rinsed multiple times with deionized water until the pH is neutral. Chitosan solution is prepared by dissolving solid chitosan in acetic acid solution. An ultrasonic oscillator assembly is used for the preliminary mixing of chitosan solution and metal oxide to obtain mixture number two. Mixture number two, along with appropriate auxiliary agents, is added to the second mixer to achieve adsorption of chitosan solution and metal oxide, thus realizing the preparation of magnetic nanoparticles. The third mixer is used for the final formation of immobilized enzyme microspheres. Magnetic nanoparticles, buffer solution, acetylcholinesterase, and glutaraldehyde solution are added sequentially, and the cross-linking reaction is completed under the corresponding temperature conditions. After the reaction, the precipitate is separated to obtain the immobilized enzyme microspheres. Drying the components can improve the purity of the immobilized enzyme microspheres. This preparation device can achieve efficient and high-quality preparation of immobilized enzyme microspheres.

[0023] In the above-mentioned apparatus for preparing immobilized enzyme microspheres, the first mixer, the second mixer, and the third mixer all include a cylindrical body with an open top. The upper opening of the cylindrical body is detachably closed by a cap, and a feeding port is provided at the top. A stirring assembly is provided on the cap. The cylindrical body is connected to a temperature control mechanism. A magnet is provided at the bottom of the third mixer, and a supernatant removal mechanism is also provided on the cap of the third mixer.

[0024] The cylindrical body contains a mixing chamber. A cap seals the opening to prevent impurities from entering, and multiple feeding ports can be installed for adding the appropriate raw materials. A stirring assembly is mounted on the cap and can be removed along with the removable cap for easy maintenance. A temperature control mechanism regulates the temperature within the mixing chamber, providing the necessary temperature conditions for each reaction step. The third mixer ultimately yields magnetically immobilized enzyme microspheres. Magnets help settle the immobilized enzyme microspheres at the bottom, aiding in separation. A supernatant removal mechanism is used to remove the supernatant after stratification, making the process more convenient.

[0025] In the aforementioned apparatus for preparing immobilized enzyme microspheres, the temperature control mechanism includes at least an annular interlayer cavity disposed on the side wall of the cylindrical body, and a thermometer assembly with its temperature measuring end extending into the cylindrical body. The interlayer cavity is connected to a hot water input assembly, a cold water input assembly, and a circulating water output assembly, respectively. The stirring assembly includes at least one vertically arranged stirring rod, which is eccentrically rotatably connected to the bottom of the cap via an L-shaped connecting rod. The vertical section of the L-shaped connecting rod passes through the cap and its upper end is connected to a rotary actuator. The supernatant removal mechanism includes a suction tube that is vertically telescopically moved through the cap of the third mixer. The lower end of the suction tube is located outside the rotation path of the stirring rod, and its upper end is connected to a negative pressure suction assembly for absorbing the supernatant after precipitation. A limiting structure for maintaining the height of the suction tube is provided between the suction tube and the cap.

[0026] The jacketed cavity is located between the inner and outer walls of the cylindrical body and can accommodate the liquid used for heat exchange, such as water. Furthermore, it can also be set between the upper and lower bottom surfaces to improve temperature control efficiency. The hot water input component and the cold water input component are used to input hot or cold water. By controlling the ratio of the input hot and cold water, the temperature of the liquid in the jacketed cavity is controlled, thereby achieving temperature control of the mixing chamber of the cylindrical body. This temperature control is equivalent to a water bath, with uniform temperature control and no contact with the specific solution. The thermometer component is used to specifically detect the internal temperature as a basis for adjusting the input ratio of hot and cold water. The specific control logic is common knowledge and will not be elaborated further. The stirring assembly is achieved through vertically extending and circumferentially distributed stirring rods on the underside of the cap. These stirring rods can rotate circumferentially around the central axis of the cap in a horizontal plane. The lower end of the stirring rod is almost in contact with but not in contact with the inner bottom surface of the mixing chamber, serving for stirring. Multiple stirring rods can be used to improve stirring efficiency. An L-shaped connecting rod is used to install the stirring rods. Its horizontal inner end is connected to the vertical section, and its outer end is connected to the stirring rod. The vertical section is rotatably connected to the center position of the cap. The output shaft of the rotary actuator is connected to the upper end of the vertical section to provide the driving force required for rotation. Moreover, the rotary actuator is located at the upper end of the cap, which facilitates maintenance and extends service life. The supernatant suction mechanism mainly uses a suction tube to suck out the supernatant. This suction tube can be vertically raised and lowered to adapt to the height of the corresponding sediment layer in the mixing chamber, and is fixed by a limiting structure after adjustment. The negative pressure suction assembly is used to provide the negative pressure power required for supernatant suction.

[0027] Specifically, the limiting structure includes a limiting clamp located at the upper end of the cap. The limiting clamp corresponds to the suction tube through-hole on the cap, through which the suction tube passes and is clamped and limited by the limiting clamp. The limiting clamp has a retractable clamping hole located directly above the suction tube through-hole, ensuring that the suction tube can pass through simultaneously. When vertical movement is required, the limiting clamp is released from clamping the suction tube; once in place, it is clamped again, making operation convenient.

[0028] Compared with the prior art, the technical effects of this utility model are reflected in:

[0029] 1. The magnetic nanoparticles of this invention are formed in water. In water, metal nanoparticles or metal oxide nanoparticles are first adsorbed onto positively charged chitosan chains through electrostatic adhesion. Then, the chitosan chains adsorbed with metal nanoparticles or metal oxide nanoparticles curl up and intertwine with other chitosan chains to form spherical magnetic nanoparticles. The interior of these magnetic nanoparticles consists of chitosan and metal nanoparticles, with the ends of the chitosan chains extending to the outside of the magnetic nanoparticles to provide support for immobilized enzymes. Therefore, these magnetic nanoparticles provide chitosan as a good carrier for immobilized enzymes on the one hand, and metal nanoparticles or metal oxide nanoparticles on the other hand, facilitating the separation of immobilized enzymes, achieving two benefits at once.

[0030] 2. In the preparation apparatus of this invention, the jacketed cavity is located between the inner and outer walls of the cylindrical body, capable of accommodating the liquid used for heat exchange. This temperature control is equivalent to a water bath, providing uniform temperature control without contact with the specific solution. The stirring assembly can be implemented using vertically extending and circumferentially distributed stirring rods on the lower side of the cap. The rotary actuator is located at the upper end of the cap, facilitating maintenance and extending service life. The suction tube of the supernatant removal mechanism can be vertically raised and lowered to adapt to the height of the corresponding sediment layer within the mixing chamber. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of the immobilized enzyme microspheres provided by this utility model;

[0032] Figure 2 This is a schematic diagram and a measured diagram of the formation process of the magnetic nanoparticles provided by this utility model;

[0033] Figure 3 This is a measured image of the immobilized enzyme microspheres provided by this utility model;

[0034] Figure 4 This is a simplified schematic diagram of the apparatus for preparing immobilized enzyme microspheres provided by this utility model.

[0035] In the figure, the components are: magnetic core 1, chitosan long chain 11, nanoparticles 12, cross-linking layer 2, enzyme layer 3, first mixer 41, second mixer 42, third mixer 43, cylindrical body 44, cap 45, feeding port 46, stirring assembly 47, temperature control mechanism 48, magnet 49, jacketed cavity 50, hot water input assembly 51, cold water input assembly 52, circulating water output assembly 53, stirring rod 54, L-shaped connecting rod 55, rotary actuator 56, suction tube 57, negative pressure suction assembly 58, limiting structure 59, limiting clamp 60, supernatant suction mechanism 61, thermometer assembly 62, and magnetic stirrer assembly 71. Detailed Implementation

[0036] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0037] Specific implementation examples Figure 1 As shown, this immobilized enzyme microsphere includes a magnetic core 1, a cross-linking layer 2, and an enzyme layer 3 arranged sequentially from the inside out.

[0038] The magnetic core 1 is a magnetic nanoparticle with a radius of 1-5 nm. The magnetic nanoparticle is self-assembled in water by chitosan long chains 11, and metal oxide nanoparticles 12 are electrostatically adsorbed on the chitosan long chains 11. In this embodiment, the metal oxide nanoparticles 12 are iron oxide nanoparticles.

[0039] like Figure 2 As shown, the magnetic nanoparticles are formed in water. In water, iron ions are first adsorbed onto the chitosan long chains 11 through electrostatic adhesion and hydrogen bonding. Then, under high temperature and pressure, on the one hand, the iron ions are oxidized to form iron oxide; on the other hand, the chitosan long chains 11 with adsorbed iron oxide nanoparticles curl up and intertwine with other chitosan long chains 11 to form spherical magnetic nanoparticles. The interior of the magnetic nanoparticles consists of chitosan and iron oxide nanoparticles, and the chain ends of the chitosan long chains 11 extend to the outside of the magnetic nanoparticles, providing support for the immobilized enzyme.

[0040] In this embodiment, the crosslinking layer 2 is a glutaraldehyde layer, which serves to link the magnetic core 1 and the immobilized enzyme layer 3.

[0041] In this embodiment, enzyme layer 3 is an acetylcholinesterase layer, and the immobilized enzyme microspheres thus prepared can be used to screen for acetylcholinesterase inhibitors. Clearly, to achieve other screening functions of the immobilized enzyme microspheres, simply changing the type of enzyme in enzyme layer 3 is sufficient.

[0042] like Figure 3 As shown, the radius of the immobilized enzyme microspheres in this embodiment is between 50 and 100 nm.

[0043] Examples of apparatuses for preparing immobilized enzyme microspheres, such as Figure 4As shown, the device includes: a first mixer 41, with a magnetic stirrer assembly 71 below the first mixer 41 for preparing a first mixture containing metal oxides; a centrifuge assembly for centrifuging the first mixture to separate metal oxide nanoparticles; an ultrasonic oscillator assembly for agitating and mixing the metal oxide nanoparticles and chitosan solution to obtain a second mixture; a second mixer 42 for mixing the second mixture with an auxiliary agent to prepare a third mixture containing magnetic nanoparticles; a third mixer 43 for mixing the magnetic nanoparticles obtained by filtering the third mixture with acetylcholinesterase to carry out a cross-linking reaction, and removing the supernatant after the reaction is completed and precipitation occurs; and a drying assembly for drying the immobilized enzyme microsphere precipitate left by the third mixer 43.

[0044] Specifically, this preparation apparatus is used to prepare magnetic nanoparticles and immobilized enzyme microspheres based on these magnetic nanoparticles. The first mixer 41 and centrifuge assembly are used to prepare metal oxide nanoparticles. A mixture of ferric chloride and ferrous sulfate is added to the first mixer 41 in a specific molar ratio, and ammonia or other adjusting agents are added to adjust the pH to alkaline. The reaction is carried out under stirring and heating conditions, and after aging at this temperature, a corresponding iron oxide mixture, i.e., mixture number one, is obtained. The centrifuge assembly separates the iron oxide particles, which are then washed multiple times with deionized water until the pH is neutral. Chitosan solution is prepared by dissolving solid chitosan in acetic acid solution. The ultrasonic oscillator assembly is used for the preliminary mixing of the chitosan solution and metal oxides to obtain mixture number two. Mixture number two, along with appropriate auxiliary agents, is added to the second mixer 42 to achieve adsorption of the chitosan solution and metal oxides, thus preparing magnetic nanoparticles. The separated magnetic nanoparticles also need to be washed multiple times with deionized water. The third mixer 43 is used for the final formation of immobilized enzyme microspheres. Magnetic nanoparticles, buffer solution, acetylcholinesterase, and glutaraldehyde solution are added sequentially, and the cross-linking reaction is completed under the corresponding temperature conditions. After the reaction, the precipitate is separated to obtain the immobilized enzyme microspheres. Drying the components can improve the purity of the immobilized enzyme microspheres. This preparation device can achieve efficient and high-quality preparation of immobilized enzyme microspheres.

[0045] In this embodiment, the first mixer 41, the second mixer 42, and the third mixer 43 all include a cylindrical body 44 with an open upper end. The upper opening of the cylindrical body 44 is detachably closed by a cap 45, and a feeding port 46 is provided at the top. A stirring assembly 47 is provided on the cap 45. The cylindrical body 44 is connected to a temperature control mechanism 48. A magnet 49 is provided at the bottom of the third mixer 43, and a supernatant suction mechanism 61 is also provided on the cap 45 of the third mixer 43.

[0046] Specifically, a mixing chamber is formed inside the cylindrical body 44. A cap 45 is used to seal the opening to prevent impurities from falling in. A feeding port 46 is used to feed the corresponding raw materials. The first mixer 41 and the second mixer 42 have three feeding ports 46, while the third mixer 43 has one. A stirring assembly 47 is mounted on the cap 45 and can be removed along with the removable cap 45 for easy maintenance. A temperature control mechanism 48 is used to control the temperature inside the mixing chamber and provide the temperature conditions required for each reaction process. The third mixer 43 ultimately yields magnetically immobilized enzyme microspheres. A magnet 49 is provided to precipitate the immobilized enzyme microspheres at the bottom, aiding in separation. A supernatant removal mechanism 61 is used to remove the supernatant after stratification, making the process more convenient.

[0047] As a specific optimization of this embodiment, the temperature control mechanism 48 includes an annular interlayer cavity 50 disposed on the side wall of the cylindrical body 44, and a thermometer assembly 62 with its temperature measuring end extending into the cylindrical body 44. The interlayer cavity 50 is connected to the hot water input assembly 51, the cold water input assembly 52, and the circulating water output assembly 53, respectively. The stirring assembly includes two vertically arranged stirring rods 54. The stirring rods 54 are eccentrically rotatably connected to the bottom of the cover 45 via an L-shaped connecting rod 55. The vertical section of the L-shaped connecting rod 55 passes through the cover 45 and its upper end is connected to the rotary driver 56. The supernatant suction mechanism 61 includes a suction tube 57 that is vertically telescopically movable and passes through the cover 45 of the third mixer 43. The lower end of the suction tube 57 is located outside the rotation path of the stirring rod 54, and its upper end is connected to the negative pressure suction assembly 58 for suctioning the supernatant after sedimentation. A limiting structure 59 for maintaining the height of the suction tube 57 is provided between the suction tube 57 and the cover 45.

[0048] Specifically, the interlayer cavity 50 is located between the inner and outer walls of the cylindrical body 44. The hot water input component 51 and the cold water input component 52 are used to input hot or cold water. By controlling the ratio of the input hot and cold water, the temperature of the liquid in the interlayer cavity 50 is controlled, thereby achieving temperature control of the mixing chamber of the cylindrical body 44. This temperature control is equivalent to a water bath, with uniform temperature control and no contact with the specific solution. The thermometer component 62 is located near the lower part to detect the temperature of the internal liquid. The stirring assembly 47 can be achieved by stirring rods 54 extending vertically and distributed circumferentially on the lower side of the cover 45. The stirring rods 54 can rotate circumferentially on the horizontal plane around the central axis of the cover 45. The lower end of the stirring rods 54 is almost in contact with but not in contact with the inner bottom surface of the mixing chamber for stirring. Multiple stirring rods 54 can be provided to improve stirring efficiency. The L-shaped connecting rod 55 is used to install the stirring rods 54. Its horizontal inner end is connected to the vertical section, and its outer end is connected to the stirring rods 54. The vertical section is rotatably connected to the center position of the cover 45. The output shaft of the rotary driver 56 is connected to the upper end of the vertical section to provide the driving force required for rotation. Moreover, the rotary driver 56 is located at the upper end of the cover 45, which helps with maintenance and extends service life. The supernatant suction mechanism 61 mainly uses the suction tube 57 to suction out the supernatant. The suction tube 57 can be raised and lowered vertically to adapt to the height of the corresponding sediment layer in the mixing chamber, and is fixed by the limiting structure 59 after adjustment. The negative pressure suction assembly 58 is used to provide the negative pressure power required for supernatant suction.

[0049] Specifically, the limiting structure 59 includes a limiting clamp 60 disposed on the upper end of the cap 45. The limiting clamp 60 corresponds to the suction tube through hole on the cap 45. The suction tube 57 passes through the suction tube through hole and is clamped and limited by the limiting clamp 60. The limiting clamp 60 has a retractable clamping hole located directly above the suction tube through hole, ensuring that the suction tube 57 can pass through simultaneously. When vertical movement is required, the limiting clamp 60 can be operated to release the clamping of the suction tube 57. After moving into position, it can be clamped again, making the operation convenient.

[0050] Brief working principle: First, a mixture of ferric chloride and ferrous sulfate in a molar ratio of 9:5 is weighed into the first mixer 41. Then, ammonia is added, and simultaneously, stirring rod 54 is rotated to adjust the pH to 10⁻¹². Next, the magnetic stirrer assembly 71 is activated. After stirring for a period of time, the first mixer 41 continues to heat and maintain the temperature for a certain period to obtain mixture number one. Mixture number one is then transferred to a centrifuge assembly for centrifugation to separate the precipitate. The precipitate is washed multiple times with deionized water to obtain iron oxide nanoparticles. Chitosan solution and the iron oxide nanoparticles are added and mixed by shaking. Then, the shaken mixture is added to the second mixer 42, and auxiliary agents such as paraffin oil are added. After heating and stirring for a certain period, sodium hydroxide is added to adjust the pH to 10⁻¹², and the reaction continues for a certain period. After filtration and washing multiple times with deionized water, magnetic nanoparticles are obtained. The magnetic nanoparticles were then added to the third mixer 43, and phosphate buffer solution was added to fully swell them. Acetylcholinesterase solution was then added, and the temperature was adjusted to a low temperature to allow for full adsorption. Glutaraldehyde solution was then added, and the temperature was increased and maintained to allow for cross-linking. The immobilized enzyme microspheres generated after the reaction precipitated at the bottom of the third mixer 43. The height of the suction tube 57 was adjusted, and the supernatant was removed. The immobilized enzyme microsphere precipitate was taken out and rinsed several times with phosphate buffer solution before being placed in the drying assembly for drying. Finally, high-purity immobilized enzyme microspheres were obtained.

[0051] Specific details such as the feeding ratio and reaction temperature are not the focus of this patent, and therefore are not described in detail.

[0052] The specific embodiments described herein are merely illustrative examples illustrating the spirit of this utility model. Those skilled in the art to which this utility model pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of this utility model or exceeding the scope defined by the appended claims.

Claims

1. A magnetic nanoparticle, characterized in that, It is formed by the self-assembly of chitosan long chains (11) in water, and metal nanoparticles or metal oxide nanoparticles (12) are adsorbed on the chitosan long chains (11).

2. The magnetic nanoparticles according to claim 1, characterized in that, The metal oxide nanoparticles (12) are iron oxide nanoparticles.

3. The magnetic nanoparticles according to claim 1, characterized in that, The radius is between 1 and 5 nm.

4. An immobilized enzyme microsphere, characterized in that, It includes a magnetic core (1), a cross-linking layer (2), and an enzyme layer (3) arranged sequentially from the inside out. The magnetic core (1) is the magnetic nanoparticle described in any one of claims 1-3.

5. The immobilized enzyme microspheres according to claim 4, characterized in that, The cross-linking layer (2) is a glutaraldehyde layer.

6. The immobilized enzyme microspheres according to claim 4, characterized in that, The enzyme layer (3) is an acetylcholinesterase layer.

7. The immobilized enzyme microspheres according to claim 4, characterized in that, The radius is between 50 and 100 nm.

8. An apparatus for preparing immobilized enzyme microspheres, used to prepare the immobilized enzyme microspheres according to claim 4, characterized in that, include: A first mixer (41) is provided below the first mixer (41) for preparing a first mixture containing metal oxides; Centrifuge assembly, used to centrifuge the No. 1 mixture to separate metal nanoparticles or metal oxide nanoparticles; An ultrasonic oscillator assembly is used to vibrate and mix nanoparticles and chitosan solution to obtain mixture No.

2. The second mixer (42) is used to mix the second mixture with the auxiliary agent to prepare the third mixture containing magnetic nanoparticles; The third mixer (43) is used to mix the magnetic nanoparticles obtained by filtering the third mixture with acetylcholinesterase for cross-linking reaction, and remove the supernatant after the reaction is completed and precipitation occurs. A drying component is used to dry the immobilized enzyme microsphere precipitate left by the third mixer (43).

9. The apparatus for preparing immobilized enzyme microspheres according to claim 8, characterized in that, The first mixer (41), the second mixer (42) and the third mixer (43) all include a cylindrical body (44) with an open top. The upper opening of the cylindrical body (44) is detachably closed by a cap (45), and a feeding port (46) is provided at the top. The cap (45) is provided with a stirring assembly (47); The cylindrical body (44) is connected to the temperature control mechanism (48); The bottom of the third mixer (43) is provided with a magnet (49), and the cover (45) of the third mixer (43) is also provided with a supernatant suction mechanism (61).

10. The apparatus for preparing immobilized enzyme microspheres according to claim 9, characterized in that, The temperature control mechanism (48) includes an annular interlayer cavity (50) at least disposed on the side wall of the cylindrical body (44), and a thermometer assembly (62) with its temperature measuring end extending into the cylindrical body (44). The interlayer cavity (50) is connected to the hot water input assembly (51), the cold water input assembly (52), and the circulating water output assembly (53), respectively. The stirring assembly (47) includes at least one vertically arranged stirring rod (54), which is eccentrically rotatably connected to the bottom of the cover (45) via an L-shaped connecting rod (55). The vertical section of the L-shaped connecting rod (55) passes through the cover (45) and its upper end is connected to the rotary driver (56). The supernatant suction mechanism (61) includes a suction tube (57) that is vertically telescopically inserted through the cap (45) of the third mixer (43). The lower end of the suction tube (57) is located outside the rotation path of the stirring rod (54), and the upper end is connected to the negative pressure suction assembly for suctioning the supernatant after sedimentation. A limiting structure (59) for maintaining the height of the suction tube (57) is provided between the suction tube (57) and the cap (45).

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