Anti-adhesion spiral micro-nano robot based on biological enzyme modification and preparation method thereof

Abstract

This invention provides an anti-adhesive helical micro / nanorobot based on bio-enzyme modification and its preparation method. The preparation method includes the following steps: Step S1, preparing a helical micro / nanorobot by sequentially depositing a layer of Fe and Ti on the surface of the helical micro / nanorobot to obtain a magnetically controlled helical micro / nanorobot; Step S2, depositing a gold film on the surface of the magnetically controlled helical micro / nanorobot, then immersing it in a β-mercaptoethylamine solution for reaction, modifying the helical surface with an amino group through a gold-sulfur bond reaction, and then using glutaraldehyde to link hyaluronidase to the helical surface of the helical micro / nanorobot to obtain an anti-adhesive helical micro / nanorobot based on bio-enzyme modification. Using the technical solution of this invention, the HAase on the surface of the obtained helical micro / nanorobot can catalyze the decomposition of HA, enabling drag-reduced movement of the magnetically driven helical micro / nanorobot in viscous solutions.

Description

Technical Field

[0001] This invention relates to the field of surface biomodification technology for micro and nanostructures, and in particular to an anti-adhesive helical micro / nano robot based on bioenzyme modification and its preparation method. Background Technology

[0002] Helical micro-nano robots (H-MNMs) are characterized by their small size, light weight, and strong driving force, and have broad application prospects in the biomedical field, such as achieving directional movement in complex physiological environments, improving tissue permeability, promoting cellular uptake, carrying and releasing drugs, etc.

[0003] However, in highly complex biofluid environments, factors such as high viscosity, adhesion of biological protein molecules, the blocking effect of blood cells, the flow effect of liquids, and the phagocytic activity of the immune system can significantly affect the controllability and stability of microrobot motion. Therefore, how to achieve efficient actuation of micro- and nanorobots in viscous and complex biofluids is an important problem in the field. Summary of the Invention

[0004] To address the above technical problems, this invention discloses an anti-viscous helical micro / nano robot based on bio-enzyme modification and its preparation method. It has significant anti-viscous drag reduction effects in viscous biological fluids and can solve the problems of difficulty in driving and poor controllability of traditional micro / nano robots in viscous and complex biological fluids.

[0005] The technical solution adopted by this invention is as follows:

[0006] A method for fabricating an anti-adhesive helical micro / nano robot based on bio-enzyme modification includes the following steps:

[0007] Step S1: Prepare a spiral micro / nano robot by sequentially depositing a layer of Fe and Ti on the surface of the spiral micro / nano robot to obtain a magnetron-controlled spiral micro / nano robot.

[0008] Step S2: A gold film is deposited on the surface of the magnetically controlled helical micro / nano robot, and then it is immersed in a β-mercaptoethylamine solution for reaction. An amino layer is modified on the helical surface through a gold-sulfur bond reaction. Then, hyaluronidase is linked to the helical surface of the helical micro / nano robot with glutaraldehyde to obtain an anti-adhesive helical micro / nano robot based on bio-enzyme modification.

[0009] The helical micro-nano robots using this technical solution are biocompatible. By modifying the surface of the helical micro-nano robots with biological enzymes and attaching hyaluronidase to the surface, the hyaluronidase can catalyze the decomposition of HA, thereby achieving drag-reduced motion of the helical micro-nano robots in viscous solutions. This is of great significance for realizing the efficient driving role of micro-nano robots in the actual biomedical field.

[0010] As a further improvement of the present invention, in step S1, a helical micro / nano robot is printed on a glass slide using a 3D printer, and then a layer of Fe and Ti is sequentially deposited on the glass slide using a high-vacuum electron beam evaporation coating machine. Further, the diameter of the helical micro / nano robot is 4.5-6 μm, and its length is 15-20 μm.

[0011] As a further improvement of the present invention, step S1 includes: using IP-S photoresist to print a double helix array on a glass slide, setting the laser power to 40mW and the scanning speed to 100mm / s, and developing and drying with isopropanol after printing.

[0012] As a further improvement of the present invention, step S1 includes: depositing an 80nm Fe layer using a high-vacuum electron beam evaporation coating machine, followed by depositing a 10nm Ti layer; the vacuum level is controlled at 10 during the evaporation process. -4 The sample stage rotation speed is 6-10 revolutions per minute. Further, the sample stage rotation speed is 8 revolutions per minute.

[0013] As a further improvement of the present invention, in step S2, a gold film is deposited on the glass slide treated in step S1, and then the glass slide is immersed in a 5-15 μM β-mercaptoethylamine solution for at least 3 hours to modify the spiral surface with amino groups, resulting in an amino-modified glass slide. The amino-modified glass slide is then placed in a glutaraldehyde PBS solution for at least 3 hours, rinsed, and then reacted overnight with a hyaluronidase (HAase) PBS solution. Further, the material slide is rinsed 3-5 times with purified water. Further, the HAase is selected from a concentrated, highly active (3000 U / mg) sample produced by Shanghai Yuanye.

[0014] As a further improvement of the present invention, the glutaraldehyde PBS solution is prepared by mixing glutaraldehyde and PBS solution at a volume ratio of 1:60-80, wherein the concentration of the PBS solution is 2-3 mg / mL.

[0015] As a further improvement of the present invention, step S2 further includes: cutting off the processed spiral micro-nano robots with a glass cutter and placing them in a container, adding water, and ultrasonically dispersing them to obtain a spiral dispersion.

[0016] This invention discloses an anti-adhesive helical micro / nano robot based on bio-enzyme modification, which is prepared by any of the above-described methods for preparing anti-adhesive helical micro / nano robots based on bio-enzyme modification.

[0017] This invention discloses the application of the enzyme-modified anti-adhesive helical micro / nanorobot described above, used to prepare helical micro / nanorobots that achieve efficient and controllable motion in viscous biofluids containing HA, enabling targeted drug delivery. The enzyme-modified anti-adhesive helical micro / nanorobot can be configured according to the concentration of HA in the viscous biofluid. Further, the helical micro / nanorobot has a diameter of 4.5 μm and a length of 15 μm.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The technical solution of this invention imparts magnetism and biocompatibility to a helical micro / nanorobot by electron beam evaporation of a layer of Fe and Ti. Then, an amino group is modified onto the helical surface using a gold-sulfur bond reaction. Finally, hyaluronidase (HAase) is linked to the helical surface using glutaraldehyde. The resulting helical micro / nanorobot with HAase on its surface can catalyze the decomposition of HA, enabling drag-reduced movement of the magnetically driven helical micro / nanorobot in viscous solutions. This invention provides a valuable reference for the efficient and controllable actuation of micro / nanorobots in complex biological fluids (such as cerebrospinal fluid, vitreous humor, synovial fluid, and intestinal mucosa). Attached Figure Description

[0020] Figure 1 This is a flowchart of a method for preparing a bio-enzyme-modified helical micro / nano robot according to the present invention.

[0021] Figure 2 This is a schematic diagram of the drag reduction method for bioenzyme-modified helical micro / nano robots in viscous solutions according to the present invention.

[0022] Figure 3 This is a bar chart showing the change in magnetically driven motion speed of the spiral micro / nano robot with and without HAase modification under an 8mT, 1Hz magnetic field as a function of HA concentration in Embodiment 1 of the present invention.

[0023] Figure 4 It is the distance traveled by the spiral microrobot in 6 mg / mL sodium hyaluronate solution for 10 seconds in Example 1 of the present invention, where (a) is the unmodified hyaluronidase; and (b) is the modified hyaluronidase.

[0024] Figure 5 This is a bar chart showing the change in magnetically driven motion speed of helical micro-nano robots with and without HAase modification under an 8mT, 1Hz magnetic field as a function of HA concentration in Embodiment 2 of the present invention; wherein, "with enzyme" refers to helical microrobots with HAase modification, and "without enzyme" refers to helical microrobots without HAase modification.

[0025] Figure 6This is a bar chart showing the movement speed of helical microrobots with and without HAase modification in a 6 mg / mL sodium hyaluronate solution in Example 3 of the present invention; wherein, "with enzyme" refers to helical microrobots with HAase modification, and "without enzyme" refers to helical microrobots without HAase modification. Detailed Implementation

[0026] The preferred embodiments of the present invention will be described in further detail below.

[0027] A bio-enzyme-modified anti-adhesive helical micro / nano robot, such as Figure 1 As shown, its preparation method includes the following steps:

[0028] Step S1, Fabrication of magneto-controlled helical micro / nano robots: A 22*22mm model was fabricated using a two-photon 3D printer. 2 A spiral array was printed on a glass slide, and then a layer of Fe and Ti was deposited using a high-vacuum electron beam evaporation coating machine to give the spiral micro-nano robot magnetic and biocompatible properties.

[0029] The specific materials and manufacturing process are as follows: A PPGT2 two-photon 3D printer manufactured by Nanoscribe (Germany) was selected, and IP-S photoresist was used to create a 0.16mm thick, 22*22mm substrate. 2 A double-helix array with a diameter of 6 μm and a length of 20 μm was printed on a glass slide. The laser power was set to 40 mW, and the scanning speed was 100 mm / s. After printing, it was developed and dried with 99.5% isopropanol. Then, Fe and Ti were deposited using a high-vacuum electron beam evaporation coating machine. First, an 80 nm Fe layer was deposited to impart magnetism to the helical micro / nano robot, and then a 10 nm Ti layer was deposited to impart biocompatibility. The vacuum level was controlled at 10°C during the evaporation process. -4 The sample stage rotation speed is 8 revolutions per minute to ensure the uniformity of the coating.

[0030] Step S2, surface enzyme modification of the helical micro / nano robot: A gold film is deposited on the glass slide treated in S1, and then the glass slide is immersed in β-mercaptoethylamine solution for reaction. An amino layer is modified on the helical surface through gold-sulfur bond reaction, and then hyaluronidase (HAase) is linked to the helical surface with glutaraldehyde.

[0031] The specific experimental preparation process is as follows: A gold film was deposited on the spiral surface using an SBC12 miniature ion sputtering instrument, with the sputtering current controlled at 5 mA and the sputtering time at 330 s; then, a 10 μM β-mercaptoethylamine solution was prepared, and the gold-plated glass slide was placed in the prepared β-mercaptoethylamine solution and reacted with shaking for 4 h to modify the spiral surface with amino groups; then, the material was rinsed with pure water 3-5 times, and then the glass slide was placed in a glutaraldehyde PBS solution and reacted for 4 hours (V PBS / V戊二醛 =70 / 1); Rinse the material sheet 3-5 times with purified water, then add 2.5 mg / mL hyaluronidase (HAase) in PBS solution and react overnight. The HAase used was a concentrated high-activity (3000 U / mg) sample produced by Shanghai Yuanye, resulting in a hyaluronidase (HAase) modified helical array. Rinse the helical surface 3-5 times with purified water to thoroughly wash away any unconjugated hyaluronidase. Then, cut the helical array with a glass cutter and place it in a 1.5 mL transparent centrifuge tube. Add 0.5 mL of water and sonicate the tube for 1 min to form a uniform dispersion of the helices. The control group used unmodified HAase helices to prepare the same volume of helical dispersion.

[0032] The aforementioned bio-enzyme-modified anti-adhesive helical micro / nano robots possess HAase enzymes on their surface, which can catalyze the decomposition of HA, thereby achieving drag reduction performance of the magnetically driven helical micro / nano robots in HA solutions. Figure 2 As shown. The specific research experiment includes the following steps:

[0033] Prepare a sodium hyaluronate (HA) solution of appropriate concentration. Add the HA and spiral solution sequentially into the groove of a 10 mm × 10 mm acrylic plate. Then place it under a triaxial Helmholtz coil for magnetic drive experiment. Adjust the relevant parameters of the magnetic field and observe and record the magnetically controlled motion video under a 10x eyepiece × 20x objective microscope. Observe and compare the difference in motion speed between the spiral micro-nano robots with and without enzyme modification, calculate the motion speed and summarize the data.

[0034] The specific process flow is as follows: Prepare a 6 mg / mL HA (①H107141 sodium hyaluronate, derived from Streptococcus equi, 95%) solution, heat it in a 90℃ water bath for 30 min while stirring to ensure the HA powder is fully dissolved. First, add 100 μL of 6 mg / mL HA to a 10×10 mm groove, then add 40 μL of helical dispersion, and finally add another 100 μL of 6 mg / mL HA. Cover with a glass slide to ensure the helical solution is completely dispersed in the HA solution. Then, attach a 30*40 mm polyimide heating film (5V 1W) to the bottom of the groove and connect it to a DC power supply. Set the voltage to 4.5V and the current to 0.2A, and heat for 5 min to achieve uniform mixing of the helical solution and HA, maintaining the temperature at 37℃. Next, place the groove within the working range of a triaxial Helmholtz coil, apply a rotating magnetic field, and establish the motion velocity distribution map of the helical microrobot by adjusting the magnetic field strength and rotation frequency. Through preliminary exploratory experiments, magnetically driven motion was carried out at a magnetic field parameter of 8mT and 1Hz. Videos of more than 5 spiral movements for 20s were recorded for both the experimental and control groups. The difference in motion speed between the spiral micro-nano robots with and without enzyme modification was observed and compared. The motion speed was calculated and the data was summarized to plot the speed curve.

[0035] The following description uses specific examples to illustrate the point.

[0036] Example 1

[0037] A bio-enzyme-modified helical micro / nano robot was prepared using the above method, and its drag-reduction performance in viscous HA solution was investigated. Specifically, this includes:

[0038] (1) Using a two-photon 3D printer and IP-S photoresist, a 0.16mm thick, 22*22mm thick lithography process was carried out. 2 A double-helix array with a diameter of 6 μm and a length of 20 μm was printed on a glass slide. The laser power was set to 40 mW and the scanning speed to 100 mm / s. After printing, it was developed and dried with 99.5% isopropanol. Then, Fe and Ti were deposited using a high-vacuum electron beam evaporation coating machine. An 80 nm Fe layer was deposited to impart magnetism to the helical micro / nano robot, followed by a 10 nm Ti layer to impart biocompatibility. The vacuum level was controlled at 10°C during the evaporation process. -4 The sample stage rotation speed is 8 rpm to ensure the uniformity of the coating.

[0039] (2) A gold film was deposited on the spiral surface using an SBC12 miniature ion sputtering instrument, with the sputtering current controlled at 5 mA and the sputtering time at 330 s; then a 10 μM β-mercaptoethylamine solution was prepared, and the gold-plated glass slide was placed in the prepared β-mercaptoethylamine solution and shaken for 4 h to modify the spiral surface with amino groups; then the material was rinsed with pure water 3-5 times, and then the glass slide was placed in a glutaraldehyde PBS solution and reacted for 4 hours (V PBS / V 戊二醛 =70 / 1); Rinse the material sheet 3-5 times with purified water, then add 2.5 mg / mL PBS solution of hyaluronidase and react overnight. The hyaluronidase (HAase) used was a concentrated high-activity (3000 U / mg) sample produced by Shanghai Yuanye, resulting in a hyaluronidase (HAase) modified helical array. Then rinse the helical surface 3-5 times with purified water to thoroughly wash away unconnected hyaluronidase. Then cut the helical array with a glass cutter and place it in a 1.5 mL transparent centrifuge tube, add 0.5 mL of water, and sonicate the centrifuge tube for 1 min to form a uniform dispersion of the helices. The control group used unmodified HAase helices to prepare the same volume of helical dispersion.

[0040] (3) Prepare HA (①H107141 sodium hyaluronate, derived from Streptococcus equi, 95%) solutions with concentrations of 9, 6, and 3 mg / mL, respectively. Heat in a 90℃ water bath for 30 min with stirring to ensure complete dissolution of the HA powder. First, add HA (100 μL) to a 10×10 mm groove, then add the spiral dispersion (40 μL), and finally add HA (100 μL). Cover with a glass slide to ensure complete dispersion of the spiral solution in the HA solution. Then, attach a 30*40 mm polyimide heating film (5V 1W) to the bottom of the groove and connect it to a DC power supply. Set the voltage to 4.5V and the current to 0.2A, and heat for 5 min to achieve uniform mixing of the spiral solution and HA, maintaining the temperature at 37℃. Next, place the groove under a triaxial Helmholtz coil rotating magnetic field. By adjusting the magnetic field rotation frequency and magnetic field strength, establish a cluster formation phase diagram. Through preliminary exploratory experiments, magnetically driven motion was carried out at a magnetic field parameter of 8mT and 1Hz. Videos of more than 5 spiral movements for 20s were recorded for the three experimental groups (9, 6 and 3 mg / mL HA) and the control group. The difference in motion speed between the spiral micro-nano robots with and without enzyme modification was observed and compared. The motion speed was calculated and the data were summarized to draw speed curves.

[0041] The movement velocities of helices with and without HAase modification in HA solutions of different concentrations are as follows: Figure 3 and Figure 4As shown in the figure. The experimental results indicate that the motility of the hyaluronidase-modified helices in 3 mg / mL and 9 mg / mL HA solutions was not significantly different, and was higher than that of the unmodified helices; however, in a 6 mg / mL HA solution, the motility... Figure 4 Video screenshots taken before and after helical modification at 6 mg / mL within 10 seconds also show that the helical movement speed of the modified hyaluronidase is significantly faster than that of the unmodified helix. This indicates that hyaluronidase can decompose hyaluronidase at 37℃, reducing the concentration of surrounding HA and thus increasing the helical movement speed.

[0042] Example 2

[0043] This embodiment includes the following steps:

[0044] (1) Select the PPGT2 two-photon 3D printer and use IP-S photoresist with a thickness of 0.16mm and a size of 22*22mm. 2 A double-helix array with a diameter of 4.5 μm and a length of 15 μm was printed on a glass slide. The laser power was set to 40 mW and the scanning speed to 100 mm / s. After printing, it was developed and dried with 99.5% isopropanol. Then, Fe and Ti were deposited using a high-vacuum electron beam evaporation coating machine. An 80 nm Fe layer was deposited to impart magnetism to the helical micro / nano robot, followed by a 10 nm Ti layer to impart biocompatibility. The vacuum level was controlled at 10°C during the evaporation process. -4 The sample stage rotation speed is 8 rpm to ensure the uniformity of the coating.

[0045] (2) A gold film was deposited on the spiral surface using an SBC12 miniature ion sputtering instrument, with the sputtering current controlled at 5 mA and the sputtering time at 300 s; then a 20 μM β-mercaptoethylamine solution was prepared, and the gold-plated glass slide was placed in the prepared β-mercaptoethylamine solution and reacted with shaking for 4 h to modify the spiral surface with amino groups; then the material was rinsed with pure water 3-5 times, and then the glass slide was placed in a glutaraldehyde PBS solution and reacted for 4 h (V PBS / V 戊二醛 =70 / 1); Rinse the material sheet 3-5 times with purified water, then add 5mg / mL PBS solution of hyaluronidase and react overnight. The hyaluronidase (HAase) used was a concentrated high-activity (3000U / mg) sample produced by Shanghai Yuanye, resulting in a modified hyaluronidase (HAase) helical array. Rinse the helical surface 3-5 times with purified water to thoroughly wash away any unconjugated hyaluronidase. Then, cut the helical array with a glass cutter and place it in a 1.5mL transparent centrifuge tube. Add 0.5mL of water and sonicate the tube for 1 minute to form a uniform dispersion of the helices. The control group used unmodified HAase helices to prepare the same volume of helical dispersion.

[0046] (3) This experiment was refined based on Example 1, specifically including: preparing HA (①H107141 sodium hyaluronate, derived from Streptococcus equi, 95%) solutions with concentrations of 9, 7.5, 6, 4.5 and 3 mg / mL respectively, heating in a 90℃ water bath for 30 min with stirring to ensure complete dissolution of the HA powder. First, add HA (100 μL) to a 10×10 mm groove, then add a spiral dispersion (40 μL), and finally add another HA (100 μL). Cover with a glass slide to ensure complete dispersion of the spiral solution in the HA solution. Then, attach a 30*40 mm polyimide heating film (5V 1W) to the bottom of the groove and connect it to a DC power supply, setting the voltage to 4.5V and the current to 0.2A, heating for 5 min to achieve uniform mixing of the spiral solution and HA, and maintaining the temperature at 37℃. Next, place the groove under a triaxial Helmholtz coil rotating magnetic field, and establish a cluster formation phase diagram by adjusting the magnetic field rotation frequency and magnetic field strength. Through preliminary exploratory experiments, magnetically driven motion was achieved by setting the magnetic field parameters to 8 mT and 1 Hz. Five experimental groups (9, 7.5, 6, 4.5 and 3 mg / mL HA) and a control group were each filmed with more than 5 spiral motions for 20 seconds. The difference in motion speed between the spiral micro-nano robots with and without enzyme modification was observed and compared. The motion speed was calculated and the data was summarized to plot the speed curve.

[0047] Experimental results are as follows Figure 5 As shown, it can be observed that a decrease in helical size leads to a decrease in speed. The helical speed gradually decreases with increasing HA concentration. Particularly noteworthy is that in HA solutions with concentrations of 6 and 7.5 mg / mL, the helical structure modified with hyaluronidase moved significantly faster than the unmodified helical structure. The helical speed of the hyaluronidase-modified helix in HA solutions with concentrations of 3 mg / mL and 9 mg / mL showed little difference but was still higher than that of the unmodified helix. This indicates that hyaluronidase can decompose HA at 37°C, reducing the concentration of surrounding HA and thus increasing the helical speed.

[0048] Example 3

[0049] This embodiment includes the following steps:

[0050] (1) A PPGT2 two-photon 3D printer was selected. Using IP-S photoresist, a double helical array with a diameter of 6μm and a length of 20μm was printed on a glass slide with a thickness of 0.16mm and a size of 22*22mm². The laser power was set to 40mW and the scanning speed to 100mm / s. After printing, the image was developed and dried with 99.5% isopropanol. Then, Fe and Ti were deposited using a high-vacuum electron beam evaporation coating machine. A 50nm Fe layer was deposited to impart magnetism to the helical micro / nano robot, and a 10nm Ti layer was deposited to impart biocompatibility. The vacuum level was controlled at 10°C during the evaporation process. -4 The sample stage rotation speed is 8 rpm to ensure the uniformity of the coating.

[0051] (2) This embodiment extends the reaction time of β-mercaptoethylamine with the gold film (from 4h to 24h) based on Example 1: A gold film is deposited on the spiral surface using an SBC12 miniature ion sputtering instrument, with the sputtering current controlled at 5mA and the sputtering time at 200s; then a 20μM β-mercaptoethylamine solution is prepared, and the gold-plated glass slide is placed in the prepared β-mercaptoethylamine solution and reacted with shaking for 24h to modify the spiral surface with amino groups; then the material is rinsed with pure water 3-5 times, and then the glass slide is placed in a glutaraldehyde PBS solution and reacted for 4 hours (V PBS / V 戊二醛 =70 / 1); Rinse the material sheet 3-5 times with purified water, then add 5mg / mL hyaluronidase PBS solution and react overnight. The hyaluronidase (HAase) used is Aladdin's product (≥300U / mg), resulting in a hyaluronidase (HAase) modified helical array. Then rinse the helical surface 3-5 times with purified water to thoroughly wash away unconnected hyaluronidase. Then cut the helical array with a glass cutter and place it in a 1.5mL transparent centrifuge tube, add 0.5mL of water, and sonicate the centrifuge tube for 1min to form a uniform dispersion of the helices. The control group uses unmodified HAase helices to prepare the same volume of helical dispersion.

[0052] (3) Prepare a 6 mg / mL HA (①H107141 sodium hyaluronate, derived from Streptococcus equi, 95%) solution, heat it in a 90℃ water bath for 30 min while stirring to ensure the HA powder is fully dissolved. Add 100 μL of HA to a 10×10 mm groove, then add 40 μL of spiral dispersion, and finally add another 100 μL of HA. Cover with a glass slide to ensure the spiral solution is completely dispersed in the HA solution. Then, attach a 30*40 mm polyimide heating film (5V 1W) to the bottom of the groove and connect it to a DC power supply. Set the voltage to 4.5V and the current to 0.2A, and heat for 5 min to achieve uniform mixing of the spiral solution and HA, maintaining the temperature at 37℃. Next, place the groove under a triaxial Helmholtz coil rotating magnetic field. By adjusting the magnetic field rotation frequency and magnetic field strength, establish a cluster formation phase diagram. Through preliminary exploratory experiments, magnetically driven motion was carried out at a magnetic field parameter of 8mT and 1Hz. Videos of more than 5 spiral movements for 20s were recorded for both the experimental and control groups. The difference in motion speed between the spiral micro-nano robots with and without enzyme modification was observed and compared. The motion speed was calculated and the data was summarized to plot the speed curve.

[0053] Experimental results are as follows Figure 6 As shown, it can be observed that changing the type of hyaluronidase still achieves the drag reduction effect of the helical micro / nanorobot in HA, although the enzyme activity decreases and the drag reduction effect is also reduced. In a 6 mg / mL HA solution, the speed of the helical structure modified with hyaluronidase is approximately twice that of the unmodified helical structure, indicating that the anti-adhesion helical micro / nanorobot based on bio-enzyme modification in this embodiment still has a drag reduction effect.

[0054] Based on the above experimental procedures and results, it is clear that the hyaluronidase (HAase) modified helical micro-nano robots exhibit significant drag reduction performance in sodium hyaluronate solution at 37℃.

[0055] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for fabricating an anti-adhesive helical micro / nano robot based on bio-enzyme modification, characterized in that, Includes the following steps: Step S1: Prepare a spiral micro / nano robot by sequentially depositing a layer of Fe and Ti on the surface of the spiral micro / nano robot to obtain a magnetron-controlled spiral micro / nano robot. Step S2: A gold film is deposited on the surface of the magnetically controlled helical micro / nano robot, and then it is immersed in a β-mercaptoethylamine solution for reaction. An amino layer is modified on the helical surface through a gold-sulfur bond reaction. Then, hyaluronidase is linked to the helical surface of the helical micro / nano robot with glutaraldehyde to obtain an anti-adhesive helical micro / nano robot based on bio-enzyme modification.

2. The method for preparing the anti-adhesive helical micro / nano robot based on bio-enzyme modification according to claim 1, characterized in that: In step S1, a spiral micro-nano robot is printed on a glass slide using a 3D printer, and then a layer of Fe and Ti is sequentially deposited on the glass slide using a high-vacuum electron beam evaporation coating machine.

3. The method for preparing the anti-adhesive helical micro / nano robot based on bio-enzyme modification according to claim 2, characterized in that: Step S1 includes: printing a double helix array on a glass slide using IP-S photoresist, setting the laser power to 40mW and the scanning speed to 100 mm / s, and developing and drying with isopropanol after printing.

4. The method for preparing the anti-adhesive helical micro / nano robot based on bio-enzyme modification according to claim 3, characterized in that: Step S1 includes: depositing an 80 nm Fe layer using a high vacuum electron beam evaporation coating machine, followed by depositing a 10 nm Ti layer; the sample stage rotation speed during the evaporation process is 6-10 rpm.

5. The method for preparing the anti-adhesive helical micro / nano robot based on bio-enzyme modification according to claim 2, characterized in that: In step S2, a gold film is deposited on the glass slide treated in step S1, and then the glass slide is immersed in a 5-15 μM β-mercaptoethylamine solution for more than 3 hours to modify the helical surface with amino groups, thus obtaining an amino-modified glass slide; then the amino-modified glass slide is placed in a glutaraldehyde PBS solution for more than 3 hours, rinsed, and then a hyaluronidase PBS solution is added and reacted overnight.

6. The method for preparing the anti-adhesive helical micro / nano robot based on bio-enzyme modification according to claim 5, characterized in that: The glutaraldehyde PBS solution is prepared by mixing glutaraldehyde and PBS solution at a volume ratio of 1:60-80, and the concentration of the PBS solution is 2-3 mg / mL.

7. The method for preparing the anti-adhesive helical micro / nano robot based on bio-enzyme modification according to claim 6, characterized in that: Step S2 also includes: cutting the processed helical micro-nano robots with a glass cutter and placing them in a container, adding water, and ultrasonically dispersing them to obtain a helical dispersion.

8. A bio-enzyme-modified anti-adhesive helical micro / nano robot, characterized in that: The anti-adhesive helical micro / nano robot based on bio-enzyme modification was prepared using any one of claims 1 to 7.

9. Application of the anti-viscous helical micro / nano robot based on bio-enzyme modification as described in claim 8: controllable movement in viscous biofluids containing HA for targeted drug delivery.

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