Magnetically-controlled spiral structure micro-nano machine and preparation method and application thereof

Abstract

The invention discloses a micro-nano machine with a magnetic control spiral structure and a preparation method and application of the micro-nano machine with the magnetic control spiral structure, the micro-nano machine with the magnetic control spiral structure takes a spirulina biological template as a matrix, and the surface of the matrix is coated with a magnetic metal coating. According to the magnetic control micro-nano machine disclosed by the invention, the sperm carrying magnetic control micro-nano machine is prepared by a chemical plating method based on a biological template and a mechanical cutting method based on magnetic field arrangement, and the obtained magnetic control micro-nano machine has good manufacturing cost performance, size uniformity and biocompatibility; the magnetic control micro-nano machine is applied to sperm carrying, a spirulina biological template matched in size is selected, and the magnetic control spiral structure micro-nano machine can penetrate into the sperm from the tail of the sperm to realize sperm carrying by changing the direction of a rotating magnetic field. In the experimental test of the invention, the magnetic control spiral structure micro-nano machine realizes complex path movement under the rotation control; and at the movement speed of 15 mu m / s, the precise and quick-response movement capability is realized.

Description

Technical Field

[0001] This invention belongs to the field of micro-nano materials technology, specifically relating to a magnetically controlled helical structure micro-nano machine, its preparation method, and its application. Background Technology

[0002] Micro- and nanomachines refer to entities at the micrometer or nanometer scale that can convert various forms of energy into mechanical motion and perform specific functions. Due to their extremely small size, they can function within the human body, especially in narrow and complex areas, and hold great promise in the biomedical field. After years of development, micro- and nanomachines are currently mainly categorized into soft machines, smart material machines, helical micro- and nanomachines, tubular / rod-shaped micro- and nanomachines, and spherical micro- and nanomachines, playing important roles in precision surgery, drug delivery, disease diagnosis, biosensing, and targeted delivery. Based on the energy source driving micro- and nanomachines, they can generally be divided into chemical energy-driven and physical field-driven (magnetic, electric, optical, and acoustic fields). Compared to chemical energy-driven systems, which require special solution media, physical field-driven systems typically do not have significant requirements for solution media and exhibit good biocompatibility. Furthermore, physical field-driven systems not only continuously provide the energy required for the movement of micro- and nanomachines but also allow for control of the direction of movement by adjusting physical field parameters. Different physical field-driven methods have different driving principles and their own advantages and disadvantages. Among them, magnetic field manipulation technology for micro- and nano-machines can achieve precise control of micro- and nano-machines through an external magnetic field, with its main advantage being the selective manipulation of ferromagnetic entities. Furthermore, low-intensity, low-frequency magnetic fields have been proven to have good biocompatibility and strong penetrability, allowing them to propagate freely in water without interference, and can be applied both in vivo and in vitro. Currently, magnetic field-controlled micro- and nano-machines, with their micro- and nano-scale size and high-precision motion, are widely used in the capture, transport, and targeted release of single cells, especially in sperm delivery and assisted reproductive applications.

[0003] In the fields of sperm transport and assisted reproduction, magnetically controlled micro / nanomachines can capture sperm through specialized structures, control their movement direction using an external magnetic field, and release sperm directionally at any location, thus achieving sperm capture, transport, and directional release. Sanchez et al. used 3D laser direct writing and electron beam evaporation technology to fabricate a magnetic helical structure micro / nanomachine suitable for sperm-scale applications. Xu et al. proposed a sperm hybridization micro / nanomachine targeted drug delivery system, with the micro / nanomachine fabricated by 3D printing. It is evident that the current fabrication of sperm-scale micro / nanomachines heavily relies on two-photon 3D printing technology (i.e., two-photon polymerization lithography). Although this technology allows for flexible control of the structural dimensions of micro / nanomachines (such as pitch and diameter), its industrial application faces significant economic barriers. Mainstream commercial equipment (such as the Nanoscribe Photonic Professional GT2) costs between 800,000 and 5 million yuan per unit, and equipment maintenance is also expensive. Furthermore, specialized photoresists are costly, and printing time increases exponentially with volume. Taking the helical micro / nanomachines required for sperm transport as an example, the service cost for fabricating 400 micro / nanomachines in a single batch can reach as high as 20,000 yuan. Because two-photon technology uses a point-by-point scanning mode, the yield per batch is extremely low, making it difficult to meet the high-throughput demands of medical scenarios. This has resulted in the limited widespread adoption of such equipment and a scarcity of commercial services. Furthermore, the photocurable resins commonly used in two-photon 3D printing contain acrylate monomers, oligomers, and photoinitiators. After printing, certain carcinogenic volatiles such as 1,4-dioxane and methanol remain. Even after secondary curing with ultraviolet light and cleaning with ethanol, unreacted functional groups still exist on the surface of the resin-based micro / nanomachines, potentially inducing inflammation or apoptosis, significantly affecting sperm cell activity and survival rate. In addition, the original structure printed by two-photon is not magnetic and requires the addition of metal layers such as nickel or iron through electron beam evaporation or physical vapor deposition to achieve magnetic responsiveness. This process requires a vacuum environment and precision coating equipment, increasing the cost per processing run. To address these shortcomings, there is an urgent need to develop a magnetically controlled micro / nanomachine that does not require two-photon 3D printing. Summary of the Invention

[0004] To overcome the problems existing in the prior art, one objective of this invention is to provide a magnetically controlled helical structure micro / nanomachine. A second objective is to provide a method for fabricating the aforementioned magnetically controlled helical structure micro / nanomachine. A third objective is to provide applications of the aforementioned magnetically controlled helical structure micro / nanomachine.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a magnetically controlled spiral structure micro / nanomachine, wherein the magnetically controlled spiral structure micro / nanomachine uses a spirulina biotemplate as a substrate, and the surface of the substrate is coated with a magnetic metal coating.

[0007] Preferably, the spirulina biotemplate is Spirulina major.

[0008] Preferably, the magnetic metal coating is a micron-sized nickel layer.

[0009] Preferably, the inner diameter of the magnetron-controlled spiral micro / nanomachine is 0.5-2 μm.

[0010] More preferably, the inner diameter of the magnetron-controlled spiral micro / nanomachine is 0.8-1.2 μm.

[0011] Preferably, the outer diameter of the magnetron-controlled spiral micro / nanomachine is 3-5 μm.

[0012] More preferably, the outer diameter of the magnetron-controlled spiral micro / nanomachine is 3.2-4 μm.

[0013] Preferably, the wire diameter of the magnetron-controlled spiral micro / nanomachine is 1.2-1.4 μm.

[0014] Preferably, the length of the magnetron-controlled spiral micro / nanomachine is 10-40 μm.

[0015] Among them, the filament diameter refers to the diameter of the spirulina; the inner diameter and outer diameter refer to the inner and outer diameters of the phycoel that bends in a regular spiral shape to form a hollow spiral loop (i.e., a complete spiral shape).

[0016] Preferably, the thickness of the metal coating is 100-400 nm.

[0017] Preferably, the thickness of the metal coating is 150-250 nm.

[0018] The second aspect of this invention provides a method for fabricating the magnetically controlled helical structure micro / nano machine described in the first aspect, comprising the following steps: S1. Select a Spirulina template and fix it; perform surface activation treatment on Spirulina in a palladium activation solution; S2. The activated spirulina is placed in a metal plating solution for chemical metal plating; the metal-plated spirulina is then mechanically cut to obtain a magnetron-controlled spiral structure micro / nano machine.

[0019] Preferably, step S1 further includes conditioned culture and size control of the Spirulina template.

[0020] More preferably, the cultivation method includes: cultivating in a light incubator at 20-30°C and 1000-3000 lux.

[0021] Preferably, in step S1, the specific steps of fixing include: soaking spirulina in a glutaraldehyde solution.

[0022] Preferably, the method further includes the following step: resuspending the spirulina in water after fixation to obtain a spirulina concentration of 1x10⁻⁶. 6 -1x10 4 Spirulina suspension with 1 / mL of spirulina.

[0023] Preferably, the palladium activation solution comprises stannous chloride, palladium chloride, concentrated hydrochloric acid, sodium stannate, and water.

[0024] More preferably, the method for preparing the palladium activation solution includes the following steps: preparing solution A by mixing stannous chloride, palladium chloride, concentrated hydrochloric acid, and water; preparing solution B by mixing stannous chloride, concentrated hydrochloric acid, and sodium stannate; and mixing solution A and solution B to obtain the palladium activation solution.

[0025] More preferably, in solution A, the mass ratio of stannous chloride to palladium chloride is 1:(0.1-0.8).

[0026] More preferably, the mass ratio of stannous chloride in solution A to stannous chloride in solution B is 1:(15-30).

[0027] More preferably, the mass ratio of stannous chloride in solution A to sodium stannate in solution B is 1:(1-3).

[0028] Preferably, the method further includes the following step: after surface activation treatment, spirulina is placed in an aqueous solution of sodium hypophosphite for desquamation treatment.

[0029] Preferably, the surface activation treatment specifically includes immersing in a palladium activation solution at 25-40°C for 10-30 minutes.

[0030] Preferably, the components of the metal plating solution include nickel sulfate, sodium hypophosphite, sodium citrate, ammonia, and water.

[0031] More preferably, in terms of concentration, the components of the metal plating solution include 20-40 g / L nickel sulfate, 20-40 g / L sodium hypophosphite, 20-40 g / L sodium citrate, 20-40 mL / L ammonia and water.

[0032] Preferably, the electroless metal plating is performed under oscillating conditions.

[0033] More preferably, the process specifically includes the following steps: placing the activated spirulina in a metal plating solution and reacting it at 50-70°C with shaking for 2-10 minutes.

[0034] The metal plating method of the present invention uses oscillation instead of the traditional mechanical stirring method, which can solve the problem that the slightly metallized spirulina sinks to the bottom and spirulina agglomerates because the mechanical stirring head cannot reach the bottom to stir. Compared with mechanical stirring, oscillation can more effectively and evenly mix all the spirulina in the liquid, increase the yield and reduce agglomeration.

[0035] Preferably, the mechanical cutting specifically includes the following steps: adding the metal-plated spirulina into a mold, arranging the metal-plated spirulina neatly and parallel by a uniform magnetic field, then adding paraffin wax to solidify it into a wax block; finally, embedding the wax block into an embedding box, slicing it, washing away the paraffin wax from the slices, and obtaining the magnetically controlled spiral structure micro-nano machine.

[0036] More preferably, the mold is made of polydimethylsiloxane (PDMS).

[0037] More preferably, the method further includes the following steps: arranging nickel-plated spirulina in ethanol, and then adding paraffin wax after the ethanol evaporates to solidify it into a wax block.

[0038] The spirulina of this invention is first coated with a ferromagnetic material (such as iron, cobalt, nickel) layer on its surface. Therefore, under the action of a uniform magnetic field, multiple spirulina can gradually be arranged in the same direction from random directions. Further mechanical cutting can yield a magnetically controlled spiral structure micro-nano machine with uniform length.

[0039] More preferably, the curing is carried out at -30 to -10°C.

[0040] The third aspect of the present invention provides the application of the magnetically controlled helical micro / nanomachinery described in the first aspect in carrying cells or preparing products for carrying cells.

[0041] Preferably, the cell is a sperm.

[0042] The beneficial effects of this invention are: This invention provides a magnetically controlled helical micro / nanomachine, which uses a spirulina biotemplate as a substrate. The surface of the substrate is coated with a magnetic metal layer. Specific advantages include: (i) The magnetically controlled micro / nanomachine of this invention does not require two-photon 3D printing technology. It uses a chemical coating method based on a biotemplate and a mechanical cutting method based on magnetic field alignment to fabricate the sperm-carrying magnetically controlled micro / nanomachine. The resulting magnetically controlled micro / nanomachine has good manufacturing cost-effectiveness, size uniformity, and biocompatibility; (ii) The magnetically controlled micro / nanomachine of this invention is designed for sperm transport applications. By selecting a size-matched spirulina biotemplate, the direction of the rotating magnetic field can be changed to control the magnetically controlled helical micro / nanomachine to penetrate from the sperm tail to achieve sperm transport. In experimental tests of this invention, the magnetically controlled helical micro / nanomachine achieved complex path movements under rotational control, achieving precise and rapid response movement capabilities at a speed of 15.72 μm / s.

[0043] This invention also provides a method for fabricating a magnetically controlled spiral structure micro / nano machine. By introducing magnetic field alignment and embedding slicing technology, the magnetic field alignment enables all spirulina in the spirulina suspension to be aligned in the same direction, thus successfully achieving length control of the micro spiral structure micro / nano machine. Attached Figure Description

[0044] Figure 1 A flowchart provided for an embodiment; Figure 2 A schematic diagram of the micro / nano machine embedding process for a magnetized helical structure provided in this embodiment; Figure 3 Optical images and scanning electron microscope images of the Spirulina template provided for the embodiments; wherein (A) and (B) are at different magnifications; Figure 4 Optical images and scanning electron microscope images of the magnetically controlled spiral structure micro / nano machine provided in the embodiment; where (A) and (B) are at different magnifications; Figure 5 Cross-sectional scanning electron microscope image and elemental analysis results of the magnetically controlled spiral structure micro / nanomachine provided for the embodiment; Figure 6 The magnetic field strength-magnetic moment response curve of the magnetically controlled helical structure micro / nanomachine provided in the embodiment; Figure 7 A schematic diagram of the embedded wax block of the magnetically controlled spiral structure micro / nano machine provided in the embodiment; where (AC) represents the size measurement of the embedded wax block; and (D) is a physical image of the embedded wax block. Figure 8 Size distribution diagrams of spiral structure micro / nano machines after being cut with different set slice thicknesses for the embodiments; Figure 9The following are the results of micro / nanomachines with helical structures of different lengths provided for the embodiments; Figure 10 The diagram shows the results of sperm cell survival rate using the spiral structure micro / nanomachinery provided in the embodiment. Figure 11 The diagram shows the effect of the helical micro / nanomachinery provided in the example on sperm DNA integrity. Figure 12 A motion path diagram of a spiral structure micro / nanomachine provided for an embodiment under the control of a rotating magnetic field; Figure 13 A schematic diagram (A) and a result diagram (B) of the spiral structure micro / nano machine provided in the embodiment realizing sperm transport under the control of a rotating magnetic field; Figure 14 This diagram illustrates the process of sperm transport using a spiral-structured micro / nanomachine provided in this embodiment, controlled by a rotating magnetic field. Detailed Implementation

[0045] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0046] Example 1 This embodiment provides a magnetically controlled helical micro / nanomachine based on a biotemplate method, the fabrication process of which is as follows: Figure 1 As shown, the specific implementation process is as follows: 1. Culture of Spirulina template 1) Initial Culture: Spirulina Major was purchased from the UTEX Algal Culture Bank in the United States. The conical flasks used for culture were boiled for sterilization. After cooling, the resulting algal blocks were transferred into sterilized conical flasks. The original culture medium was discarded, and 8 mL of fresh Enriched Seawater Medium (ES) was added. The flasks were sealed with sealing film and cultured at 25°C, 3000 lx, with a photoperiod L:D = 12h:12h. The flasks were shaken 2-3 times daily. A small amount of culture medium was taken weekly to observe for the formation of free algal filaments. If no flocculent matter or bacterial growth was observed in the culture medium, the medium was changed monthly, retaining the algal blocks and discarding the old medium each time. When multiple algal blocks developed multiple green spots, a second expansion culture was conducted. A small amount of culture medium was taken weekly to observe for the formation of free algal filaments. If free algal filaments were formed, a second expansion culture was performed. 2) Scale-up Culture: When multiple algal masses show multiple green spots, place each algal mass into a separate sterile centrifuge tube for scale-up culture. For the first scale-up culture, use 2 ml of ES Medium at 25℃, 3000 lx, and a photoperiod of L:D = 12h:12h, shaking the flask 2-3 times daily. Take a small amount of culture medium weekly to observe for the formation of free algal filaments. Following the above steps, if the algal masses do not turn yellow and the OD value is <1, scale-up culture is performed every 2 weeks. However, from the second run onwards, use a mixture of Erdschreiber's Medium (ERD) and ES Medium in sterile medium, with mixing ratios of 1:3, 2:2, and 3:1 respectively. For the fifth run, use only ERD Medium. Each time, retain 1 ml of the old medium, and increase the amount of fresh medium by 1 ml compared to the previous run. After 5 rounds of scale-up culture, the algal solution in the centrifuge tubes can be transferred to Erlenmeyer flasks for routine culture.

[0047] 3) Routine Cultivation: Sterilize the conical flasks used for cultivation by boiling. After cooling, transfer all the expanded algal culture (including algal clumps) to sterilized conical flasks. Add sterile ERD Medium at an algal culture to medium ratio of 1:2. Seal the flasks with sealing film and incubate at 25℃, 3000 lx, with a photoperiod L:D = 12h:12h. Shake the flasks 2-3 times daily. If the algal clumps have not turned yellow and the OD value of the algal culture is <1, change the medium every 2 weeks at an algal culture to medium ratio of 1:3. When Spirulina is in its growth phase, algae can be harvested for use (this can be accurately determined by the OD value or the concentration of Spirulina Major, approximately within 5-11 days after adding fresh medium).

[0048] 4) Spirulina Template Size Control and Screening: After routine culture and culture medium replacement, the Spirulina solution was transferred to sterile Erlenmeyer flasks, and the flasks were sealed with sealing film. The flasks were then placed at three temperatures (15℃, 25℃, and 35℃) and three light intensities (2000 lx, 3000 lx, and 4000 lx), respectively. The photoperiod was consistently set to L:D = 12 h:12 h, and the flasks were shaken 2-3 times daily. Samples were taken from each culture medium every 4 days, and the morphological characteristics of the Spirulina were observed under a microscope. Dimensions (such as filament length, spiral diameter, or spiral pitch) were precisely measured. Higher light intensities and temperatures caused the Spirulina spirals to contract, producing a tight spiral. This process was continued until the Spirulina size measurements stabilized, indicating that its morphology had reached a relative equilibrium under different culture conditions. Finally, the sizes of the Spirulina under different culture conditions were compared, and the culture conditions corresponding to a Spirulina template inner diameter of 1.5 μm were selected for routine culture.

[0049] 2. Cleaning and fixing of the spirulina template 1) Spirulina washing: Take 10 mL Spirulina major Centrifuge the algal solution at 6000 rpm for 5 minutes and discard the supernatant; add 1 mL of deionized water to resuspend the spirulina, centrifuge again and discard the supernatant, repeat 5 times.

[0050] 2) Spirulina fixation: Add 2 mL of 2.5% glutaraldehyde aqueous solution and fix at 4 ℃ for 6 hours. After fixation, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, add 1 mL of deionized water to resuspend the spirulina, centrifuge again and discard the supernatant, repeat 5 times.

[0051] 3) Concentration adjustment: Resuspend the spirulina in deionized water, observe the number of algal filaments under a microscope, and adjust the spirulina concentration to 1 x 10⁻⁶. 5 cells / mL 3. Palladium activation of spirulina template 1) Preparation of palladium activation solution: The palladium activation solution is prepared by mixing solution A and solution B. Solution A consists of 0.075 g SnCl2·2H2O, 0.03 g PdCl2, 10 mL 12 M HCl and 20 mL deionized water. Solution B consists of 0.14 g Na2SnO3·3H2O, 1.5 g SnCl2·2H2O and 20 mL 12 M HCl. Solutions A and B are mixed in a 30℃ water bath with magnetic stirring at 500 rpm for 10 minutes, and then kept in a 60℃ water bath for 3 hours. After the incubation period, the mixture is stored at room temperature for later use.

[0052] 2) Palladium activation of Spirulina template: Add 1 mL of Spirulina suspension to 20 mL of palladium activation solution and react for 15 minutes in a water bath at 30°C.

[0053] 3) Centrifugation and washing: After the reaction is complete, centrifuge all reaction solutions at 6000 rpm for 5 minutes and discard the supernatant. Add 1 mL of deionized water to resuspend the palladium-activated spirulina, centrifuge, discard the supernatant, and repeat 5 times.

[0054] 4) Decapitation treatment: Resuspend the activated spirulina in 10 mL of 0.28 M NaH2PO2 solution, incubate at room temperature for 5 minutes to decapitate, then centrifuge at 6000 rpm for 5 minutes and discard the supernatant. Repeat 3 times. Resuspend in 1 mL of deionized water, centrifuge, discard the supernatant, and repeat 3 times.

[0055] 5) Concentration adjustment: Resuspend the de-shelled palladium-activated spirulina in deionized water, observe the number of algal filaments under a microscope, and adjust the concentration to 1x10. 4 per mL.

[0056] 4. Electroless nickel plating of spirulina templates 1) Preparation of nickel plating solution: Weigh 3 g NiSO4·6H2O, 3 g NaH2PO2·H2O, 2.5 g C6H5O7Na3·2H2O, and 31.82 mL NH3·H2O, add them to 30 mL of deionized water, transfer to a 100 mL volumetric flask and make up to volume to obtain the nickel plating solution.

[0057] 2) Spirulina nickel plating: Take 1 mL of deshelled palladium-activated spirulina suspension and add it to 10 mL of nickel plating. React in an oscillator at 60 ℃ and 800 rpm for 4 minutes. After the reaction is complete, use a magnet to separate the magnetically controlled spiral micro-nano machine and rinse it repeatedly with deionized water. Finally, disperse it in 1 mL of ethanol solution.

[0058] 5. Mechanical cutting of micro- and nano-machines 1) The embedding process of micro / nano machines is as follows: Figure 2 First, the micro / nanomachines were aligned to the same orientation using a magnetic field. Then, Spirulina was embedded into a tissue embedding cassette through a two-stage embedding process: PDMS prepolymer (solution A:solution B = 10:1) was mixed and degassed, then cured at 70°C for 2 hours, resulting in a PDMS chamber with a hollow structure of 4 mm × 10 mm × 4 mm. The PDMS chamber was placed in a 4 cm diameter culture dish, and 3 mL of liquid paraffin was added. After the paraffin solidified, 200 μL of ethanol-dispersed, magnetically controlled spiral micro / nanomachines were added to the chamber and oriented using a magnetic field. After the ethanol completely evaporated, liquid paraffin was added to fill the culture dish, and the mixture was solidified and cooled at -20°C for 10 minutes. After solidification, the rectangular wax block containing the micro-nano machines was removed using a scalpel. The wax block was then placed in the embedding mold, with the long axis of the neatly arranged micro-nano machines perpendicular to the bottom surface of the embedding mold. Liquid paraffin was poured in to embed the wax block into the tissue embedding cassette. After cooling, the tissue embedding cassette containing the micro-nano machines was removed for later use.

[0059] 2) Cutting of micro-nano machines: Fix the tissue embedding cassette containing the micro-nano machines onto the tissue slides, adjust the trimming thickness to 20 μm and trim the slides until the black cross section of the micro-nano machines is exposed in the paraffin block, and then use different slide thicknesses (15, 20, 25, 30 μm) to cut the slides and collect all the slides.

[0060] 3) Cleaning of micro / nano machine slices: Immerse the slices in 20 mL of environmentally friendly transparent dewaxing solution for 10 minutes, then wash with 5 mL of 100% ethanol for 5 min, repeat twice. Then wash with 5 mL of 95% ethanol for 5 min, 5 mL of 85% ethanol for 5 min, 5 mL of 80% ethanol for 5 min, 5 mL of 70% ethanol for 5 min, and 5 mL of deionized water for 5 min, repeating each three times. Finally, disperse in 3 mL of deionized water for later use.

[0061] biological template Spirulina major Microscopic representation Observation using an optical microscope Spirulina major And 200 spirals were measured to obtain their geometric dimensions. Its optical images and scanning electron microscope images are as follows: Figure 3 As shown, the obtained geometric dimensions are: outer diameter 3.6 ± 0.11 μm, inner diameter 1.0 ± 0.06 μm, and wire diameter 1.3 ± 0.10. This invention is based on the capture of sperm using a magnetically controlled helical micro / nanomachine; therefore, the template size of the micro / nanomachine must be related to the sperm. Research indicates that the human sperm head is 3.7–4.7 μm long and 2.5–3.2 μm wide, the midsegment is 5–7 μm long and 1–1.8 μm wide, and the tail is approximately 20–30 μm long, with a width smaller than the midsegment (another width is not mentioned). To enable the helical micro / nanomachine to capture and immobilize sperm, the inner diameter of the template for the helical micro / nanomachine must be smaller than the width of the sperm head and larger than the width of the sperm tail. Therefore, the dimensions used in this invention… Spirulina major The template size requirements are met.

[0062] Microscopic characterization of magneto-controlled helical micro / nano machines.

[0063] First, optical images of the magnetized helical structure were obtained using an optical microscope, such as... Figure 4 As shown, the helical structure of the magneto-controlled helical micro / nanomachine is clearly visible and has a certain inner diameter. Subsequently, the magneto-controlled helical micro / nanomachine was mechanically cut open, and scanning electron microscopy (SEM) images of the magneto-controlled helical micro / nanomachine were obtained, as shown below. Figure 5 As shown, the surface of the micro-nano machine is composed of nickel particles. The magnetron-controlled spiral structure micro-nano machine has a nickel layer with a thickness of 160 nm. The cross-sectional structure was observed using a scanning electron microscope and the elemental analysis of the cross-section was performed. Only a small amount of nickel element was distributed inside.

[0064] Next, the magnetic response curves of the magnetically controlled helical micro / nanomachine were tested using a comprehensive physical property measurement system, such as... Figure 6As shown, a saturation magnetization of 32.633 emu / g and a coercivity of 149.371 Gs were obtained. The higher coercivity endows the magnetized helical micro / nanomachine with better temperature stability and magnetic preservation capabilities. Figure 6 The smaller hysteresis loop region in the micro-nano machine also means that the micro-nano machine has better magnetic permeability.

[0065] Evaluation of the results of micro / nano-machine cutting of a magnetized helical structure Figure 7 The paraffin block and tissue embedding cassette containing micro / nano machines were displayed. The lengths of the micro / nano machines after cutting were then observed and counted using an optical microscope. Figure 8 and 9 As shown, mechanical cutting of H-MNMs using an embedding slicing method can effectively control the length, thereby efficiently preparing H-MNMs with relatively uniform length.

[0066] Biocompatibility characterization of magnetically controlled helical micro / nanomachines Inoculate 90 μL / well of sperm suspension (sperm concentration 1 x 10⁻⁶) into 96-well plates. 6 (0, 1000, 2000, and 3000 micro-nanomachines / mL dispersed in PBS) were added, and 10 μL of PBS-dispersed micro-nanomachine suspensions were added respectively. After incubation in a CO2 incubator at 37 ℃ for 1 and 3 h respectively, 20 μL of 2 μM Calcein-AM and 5 μM pyridine iodide staining solution were added. After incubation at 37 ℃ for 20 min, the staining results were observed under a fluorescence microscope to characterize the effect of the magnetically controlled helical micro-nanomachines on sperm cell survival. Figure 10 As shown, the magnetically controlled helical micro / nanomachinery has a low impact on cell survival rate at low concentrations and over short periods of time.

[0067] Subsequently, the effect of the magnetically controlled helical micro / nanomachinery on sperm DNA integrity was characterized using sperm staining structure analysis. 45 μL / well of sperm suspension was seeded into 48-well plates, and 5 μL of PBS (0, 1000, 2000, and 3000 sperm / mL) was added to disperse the micro / nanomachinery suspension. After incubation at 37 °C for 1 h in a CO2 incubator, H-MNMs were separated from the sperm using a magnet. Sperm under different conditions were redispersed in 45 μL of PBS, followed by 90 μL of detergent solution (0.08 MHCL, 150 mM NaCl, 0.1% Triton X-100, adjusted to pH 1.2). After 30 s, 180 μL of staining solution (6 mg / L AO, 0.1 M Citric acid, Na2HPO4, 1 mM disodium EDTA, 150 mM NaCl, adjusted to pH 6.0) was added, and staining was performed for 20 seconds. After staining, the 48-well plate was placed under an inverted laser focusing microscope to observe the staining results. Figure 11 As shown, lower concentrations of micro / nano machines cause less damage to sperm DNA.

[0068] Application Example 1 This application example provides a method for magnetic field manipulation and sperm capture of a magnetically controlled helical micro / nanomachine based on a biotemplate method, specifically comprising the following steps: 1. Magnetic field manipulation of magneto-controlled spiral micro / nano machines Sample preparation: The PDMS prepolymer (solution A:solution B = 10:1) was mixed and degassed, then cured at 70℃ for 2 hours. The resulting PDMS chamber was cut to obtain a PDMS chamber with a central control structure of 4 mm × 10 mm × 4 mm. The PDMS chamber was placed on a 2.5 cm × 3.5 cm glass slide, which was then placed on the stage of a three-dimensional magnetron coil. 20 μL of a 25 μm long micro / nano-machine suspension was added dropwise into the PDMS chamber.

[0069] Magnetic field manipulation: The rotating magnetic field frequency was set to 15 Hz and the magnetic field strength to 30 Gs. The motion video of the magnetically controlled helical micro-nano machine was observed and recorded under a microscope.

[0070] Data processing: Software was used to analyze the motion speed and path of the magnetically controlled helical micro-nano machine, and images of the motion path and posture of the magnetically controlled helical micro-nano machine were generated.

[0071] 2. Sperm transport using magnetically controlled helical micro / nano machines Sperm sample preparation: Liquefy the semen sample in a 37℃ water bath for 30 minutes; take 1 mL of liquefied sperm, centrifuge at 200g for 5 minutes, and discard the supernatant; resuspend the sperm in 1 mL of TNE solution (Tris-NaCl-EDTA buffer), centrifuge and discard the supernatant, repeat twice; resuspend the sperm in 1 mL of deionized water, centrifuge and discard the supernatant, repeat twice; resuspend the sperm in deionized water and adjust the sperm concentration to 1×10⁻⁶ using a cell counting chamber. 4 / mL; Add 10 μL of sperm suspension to the PDMS chamber. Sperm capture: The rotating magnetic field frequency was set to 15 Hz and the rotating magnetic field strength to 30 Gs. Under a microscope, the direction of the rotating magnetic field was changed to control the magnetically controlled helical micro-nano machine to penetrate the sperm tail and achieve sperm transport. Figure 13 ).

[0072] 3. Characterization of the motion capability of magnetically controlled helical micro / nanomachines under magnetic fields Figure 12 The study demonstrated that a magnetically controlled helical micro / nanomachine can achieve complex path motion under rotational manipulation, achieving precise and rapid motion response at a speed of 15.72 μm / s.

[0073] 4. Evaluation of sperm transport using magnetically controlled helical micro / nanomachinery Figure 14 This demonstrates the ability of a magnetically controlled micro / nanomachine to transport sperm. The target sperm undergoes a positional change after binding with the micro / nanomachine, achieving sperm transport via a magnetically controlled helical micro / nanomachine.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A magnetically controlled helical micro / nano machine, characterized in that, The magnetically controlled spiral structure micro-nano machine uses a spirulina biotemplate as a substrate, and the surface of the substrate is coated with a magnetic metal layer.

2. The magnetically controlled helical micro / nano machine according to claim 1, characterized in that, The inner diameter of the magnetically controlled spiral structure micro / nano machine is 0.5-2 μm, and the outer diameter is 3-5 μm; And / or, the wire diameter of the magnetically controlled spiral structure micro / nanomachine is 1-1.5 μm.

3. The magnetically controlled helical micro / nano machine according to claim 1, characterized in that, The length of the magnetically controlled spiral structure micro / nano machine is 10-40 μm; And / or, the Spirulina biotemplate is Spirulina major .

4. The magnetically controlled helical micro / nano machine according to claim 1, characterized in that, The thickness of the metal coating is 100-400 nm; And / or, the magnetic metal coating is a micron-sized nickel layer.

5. The method for fabricating the magnetically controlled helical structure micro / nano machine according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Select a Spirulina template and fix it; perform surface activation treatment on Spirulina in a palladium activation solution; S2. The activated spirulina is placed in a metal plating solution for chemical metal plating; the metal-plated spirulina is then mechanically cut to obtain a magnetron-controlled spiral structure micro / nano machine.

6. The method for fabricating a magnetically controlled helical micro / nano machine according to claim 5, characterized in that, The palladium activation solution comprises stannous chloride, palladium chloride, concentrated hydrochloric acid, sodium stannate, and water.

7. The method for fabricating a magnetically controlled helical micro / nano machine according to claim 5, characterized in that, The components of the metal plating solution include nickel sulfate, sodium hypophosphite, sodium citrate, ammonia, and water.

8. The method for fabricating a magnetically controlled helical micro / nano machine according to claim 5, characterized in that, The mechanical cutting specifically includes the following steps: adding the metal-plated spirulina into a mold, arranging the metal-plated spirulina neatly and parallel by a uniform magnetic field, then adding paraffin wax to solidify it into a wax block; finally, embedding the wax block into an embedding box, slicing it, washing away the paraffin wax from the slices, and obtaining the magnetically controlled spiral structure micro-nano machine.

9. The use of the magnetically controlled helical micro / nanomachinery of any one of claims 1-4 in carrying cells or preparing products for carrying cells.

10. The application according to claim 9, characterized in that, The cells in question are sperm.

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