CN112375560A - Functionalized biological hybrid micro-nano motor and preparation method thereof - Google Patents
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Abstract
The invention belongs to the field of material technology and micro-nano manufacturing, and relates to a functionalized biological hybrid micro-nano motor and a preparation method thereof. The functionalized biological hybrid micro-nano motor comprises: a biological entity inner core, a magnetic nanoparticle coating, and a probe layer. The invention selects fungal spores, pollen and micro/nano plant seeds as inner cores, is easy to obtain or culture in large quantity, has uniform material size and is easy to control; the inner core and the nano particles are hybridized by a step-by-step coating method, and probes are also loaded on the surface of the nano coating. The functionalized biological hybrid micro-nano motor can actively move under accurate control, is very easy to observe, and overcomes the existing defects.
Description
Technical Field
The invention belongs to the field of material technology and micro-nano manufacturing, and relates to a functionalized biological hybrid micro-nano motor and a preparation method thereof.
Background
The micro-nano motor (also called micro-nano robot) realizes autonomous continuous motion in a three-dimensional space by converting external energy (light, electricity, magnetism, heat, chemical energy and the like) into driving force of the micro-nano motor, so that a specific micro operation task is executed. Its autonomous continuous motion in different liquid environments can lead to both micro-scale eddy currents and surface dynamic chemical effects. The two effects cooperate to realize solution micromixing, accelerate solute molecule diffusion, improve chemical reaction rate and yield, enable the micro-nano motor to participate in various dynamic reactions as a dynamic active reactor, and improve the contact chance of functional molecules and target molecules. In view of these enhancement effects, micro-nano motors have been widely used in the biomedical field and have been completely open in the sensing field. The multiple and controllable motion modes also make the development of intelligent sensing devices possible, and the device has great prospect in the aspects of noninvasive diagnosis and treatment of diseases and the like.
However, the existing micro-nano motor has poor controllability of the whole size and nonuniform size distribution, for example, the length of the spirulina-based micro motor is mainly determined by the length of spirulina and can reach dozens of microns to hundreds of microns. This difference in size is not conducive to scale-up on the one hand; on the other hand, the driving speed, the drug loading amount, the target combination and the like are seriously influenced.
In addition, when oriented to biomedical applications, the biocompatibility of the micro-nano motor is a research focus. The existing Chinese patent application CN11082705A discloses a biocompatible iron-manganese dioxide system micro-nano motor. Compared with the micro-nano motors based on low-toxicity inorganic oxides, the biological hybrid micro-nano motor based on natural materials shows more excellent biocompatibility. Many biological entities such as bacteria, viruses, cells, etc. were started to be used for the preparation of micro-nano motors. However, the functions of these biological entities are not fully utilized. For example, the method of coating the micro-nano motor with the cell membrane alone cannot solve the core driving problem of the synthetic micro-nano motor. The biological hybrid micro-nano motor with easy observation and consistent controllable driving still faces the challenges of uniform preparation and controllable driving.
Disclosure of Invention
Aiming at the problems, the invention provides a functionalized biological hybrid micro-nano motor and a preparation method thereof. The invention selects fungal spores, pollen and micro/nano-scale plant seeds as inner cores, realizes the hybridization of the inner cores and nano-particles by a step-by-step coating method, and loads probes on the surface of the nano-coating. The functionalized biological hybrid micro-nano motor can actively move under the accurate control, is very easy to observe, and overcomes the defects.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a functionalized biological hybridization micro-nano motor, which comprises: a biological entity inner core, a magnetic nanoparticle coating, and a probe layer.
Preferably, the inner core of the biological entity is a natural material, including fungal spores, pollen, and micro/nano-scale plant seeds; preferably, the surface of the inner core of the biological entity is rough or porous. Natural plant seeds, fungal spores, and the like have unique and complex three-dimensional structures and can be cultured or obtained in large quantities. The rough or porous structure provides abundant active sites for the adhesion coating of the functional nanoparticles, so that the detection probes can be loaded in high capacity, and a large number of reaction sites in contact with target molecules are provided, so that the micro-nano motor has good sensitivity (equivalent to clinical immunoassay) below the level of nanogram/milliliter. The inner core thereof may also be a synthetic core, and preferably a synthetic porous inner core; for example, the inner core may comprise a mesoporous silica micro-nano structure.
Preferably, the magnetic nanoparticles in the magnetic nanoparticle coating comprise iron, nickel and/or a magnetic metal oxide, preferably the magnetic nanoparticles are Fe3O4And (3) nanoparticles. Preferably, the magnetic nanoparticle coating has a thickness of about 50 to 200 nm. The magnetic nano coating can be a micro-nano horseTo provide magnetic response capability for movement in a magnetron system. Where the term "magnetic" refers to a material property that responds to a magnetic field, but is not limited to paramagnetic and ferromagnetic.
Preferably, the magnetic nanoparticle coated surface may further comprise a self-assembled monolayer. The self-assembled monolayer includes, but is not limited to, thiolated self-assembled monolayers and other chemical and/or biological coupling agents. The self-assembled monolayer may better immobilize the detection probes or other structures.
Preferably, the probe layer comprises a carbon dot with fluorescence emission and response capability and various specific groups or ligand molecules with targeting function, and the probe generates fluorescence emission or quenching phenomenon when being combined with a target molecule; the specific group or aptamer molecule with the targeting function comprises oligosaccharide, RNA aptamer, phenylboronic acid and derivatives thereof; the probes may also include other fluorophores with the same or similar targeted detection functionality, such as agglomeration-inducing luminophores, polymer dots, silicon nanoparticles, molybdenum disulfide nanoparticles, MXene quantum dots, and combinations thereof. These fluorescent nanoparticles may emit light having the same or different emission wavelengths in response to light having the same or different excitation wavelengths of the carbon dots.
The probe may comprise two or more probes, which can bind to different target molecules and can simultaneously detect multiple types of target molecules. The probe molecules in the probe layer can bind to and form complexes with the target molecules. The formation of the complex can be monitored by the fluorescence response and intensity change of the continuously moving functionalized biological hybridization micro-nano motor, and finally the existence of the target molecule can be confirmed. Targets in the fluid to be tested may include bacterial toxins such as toxin a and toxin B of clostridium difficile toxin, endotoxins of gram-negative bacteria, mycotoxins from fungi in rotten foods (ochratoxin a) and fumonisin B1(fumonisine B1)), and phytoricin B toxin. Thus, the fluid to be tested may comprise both such media containing toxins and also media containing bacteria to which such toxins correspond.
The invention also provides a preparation method of the functionalized biological hybrid micro-nano motor, which comprises the following steps:
hybridizing magnetic nanoparticles on the surface of the inner core of the biological entity to form a magnetic nanoparticle coating; the probe is fixed on the surface of the magnetic nano-particle coating by using a biological/chemical coupling technology.
Preferably, the hybridization of the inner core of the biological entity with the magnetic nanoparticles is achieved by a step-by-step coating method.
The method also comprises the step of pretreating the inner core of the biological entity to remove surface impurities. The method further comprises coating the magnetic nanoparticles with a self-assembled monolayer prior to immobilizing the probes.
The biological/chemical coupling technology comprises oligosaccharide-compound repeated oligopeptide combination, avidin-biotin combination and weak interaction.
Compared with the prior art, the invention has the following advantages:
according to the invention, natural plant seeds, fungal spores and the like are used as inner cores, and the natural materials can be cultured or obtained in a large scale, so that the large-scale production of the micro-nano motor is facilitated; and the size is uniform, and the magnetic response capability and the magnetic motion control capability of the micro-nano motor are enhanced due to the complex three-dimensional structure and the porous hollow structure, so that the motion track of the micro-nano motor can be accurately controlled.
The micro-nano motor comprises a specific recognition probe and can selectively detect target molecules; and the probe can generate fluorescence emission or quenching phenomenon when being combined with the target molecule, and the accurate and rapid detection of the target molecule can be realized through the change of fluorescence intensity in the movement process. The micro-nano motor can move in various fluids. Therefore, the micro-nano motor can be widely applied to the fields of food, biology, chemistry, medicine and the like.
The functionalized biological hybridization micro-nano motor realizes the hybridization of the biological entity inner core and the magnetic nano particles by a step-by-step coating method in the preparation process, and compared with a micro-nano structure obtained by conventionally using a template (such as an anodic alumina template) for deposition and post-treatment, the preparation method is time-saving and low in cost.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
Fig. 1 is a flow chart of a process for preparing a functionalized bio-hybrid micro-nano motor according to an embodiment of the present invention;
fig. 2 is a microstructure diagram of a functionalized biological hybrid micro-nano motor according to an embodiment of the present invention;
fig. 3 is an optical image (a) and a fluorescence image (B) under green light excitation of the functionalized bio-hybrid micro-nano motor according to an embodiment of the present invention;
fig. 4 is a movement pattern of the functionalized biological hybrid micro-nano motor in the magnetic driving technology according to a specific embodiment of the present invention.
Fig. 5 is a diagram of a motion trajectory of the functionalized bio-hybrid micro-nano motor in various fluids according to an embodiment of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
Aiming at the problems of the background technology, the invention provides a functionalized biological hybrid micro-nano motor and a preparation method thereof.
The functionalized biological hybrid micro-nano motor comprises a biological entity inner core, a magnetic nano particle coating and a probe layer. The inner core of the biological entity can be natural plant seeds, fungal spores, pollen and the like, has a unique and complex three-dimensional structure and can be cultured or obtained in a large quantity. The surface of the biological entity inner core is provided with a magnetic nano-particle coating for realizing the driving and control of the micro-nano motor, and the magnetic nano-particle coating can be formed by self-assembling magnetic nano-particles deposited by chemical adsorption. On the surface of the inner core of the biological entity wrapped by the magnetic nano coating, probes are fixed by self-assembly functional groups, and the probes comprise carbon points with fluorescence emission and response capability and various specific groups or ligand molecules with targeting functions, such as oligosaccharide of targeting complex repeated oligopeptides, phenyl boronic acid (PAPA) of targeting endotoxin, different RNA aptamers combined with mycotoxin and ricin B toxin, and the like. The probe layer may also include other fluorescent probes having the same or similar targeted detection functionality. The probes are used to bind target molecules (e.g., toxin molecules) in the fluid to be tested, thereby producing rapid, real-time fluorescence changes for follow-up monitoring. Therefore, the functionalized biological hybridization micro-nano can be suitable for detecting the existence of target molecules, and the existence and the content of biological targets in a sample can be judged through the change of fluorescence intensity in the movement process.
The preparation process of gradual deposition on the inner core of the biological entity, such as natural spores, can be carried out to produce the functional biological hybrid micro-nano motor in a large scale. The functionalized biological hybrid micro-nano motor can be prepared by the following method: the magnetic coating is first deposited on the pretreated biological entity, then the magnetic biological entity is functionalized with a self-assembled monolayer, and finally the fluorescent probe is immobilized by a biological/chemical coupling technique. The magnetic coating can enable the biological hybrid micro-nano motor to move controllably. The self-assembled monolayer may better immobilize the detection probes or other structures. As shown in fig. 1, an exemplary manufacturing schematic diagram of a functionalized bio-hybrid micro-nano motor of the disclosed technology is given. An exemplary preparation process includes three steps, e.g., mixing Fe3O4Depositing nanoparticles on natural Ganoderma spore with size of 10 μm, and modifying Fe with thiolated self-assembled monolayer3O4Coating spore, and fixing with NHS/EDC techniqueAnd (3) spotting the oligosaccharide functionalized fluorescent carbon on the magnetic spore. Schematic 110 shows an example of magnetic nanoparticles deposited on a spore core. In embodiments, the inner core of the biological entity may also be synthetic. In embodiments, the magnetic nanoparticles may have a particle size between 10-80 nm. In some examples, magnetic Fe3O4The nanoparticles may be deposited on spores or pollen of plants or fungi. In embodiments, the deposited nanoparticles form a coating on the surface of the inner core of the biological entity. Schematic 120 illustrates an exemplary modification of magnetic particle coated spores, for example using 3-mercaptopropionic acid (MPA) to form a linker layer. Schematic 130 shows an example of immobilization of a detection probe, e.g., immobilization of an oligosaccharide functionalized carbon site, by EDC/NHS chemical coupling technique. The disclosed deposition techniques can be implemented to produce magnetically driven bio-hybrid micro-nano motors. The structure, size, and functionality of each portion of the motor can be configured to various parameters of the disclosed functionalized bio-hybrid micro-nano motors, including specific surface area, magnetic properties, optical properties, and targeted detection capabilities. Examples of functional parameters include magnetic properties (intensity), optical properties (fluorescence or phosphorescence), and targeted detectability (specific fluorescence on or off). In the embodiment, the saturation magnetization of the micro-nano motor can be between 40 and 60emu/g, which is beneficial to the smooth implementation of magnetic driving motion. In embodiments, due to differences in natural biological entities, the size may range from 5 μm to 20 μm. In embodiments, the detection probe is about 1-10nm in size. Relative to the inner core of the biological entity and the magnetic coating, the detection probe has no obvious coating thickness and can be ignored.
In embodiments, various types of naturally occurring spores can be used as the inner core of the biological entity. In embodiments, the inner core may also extend to a synthetic inner core. The disclosed deposition or encapsulation techniques can be applied to obtain micro-nano motors with various structures and sizes. For example, stone pine spores or ganoderma spores can be used. Other examples may include depositing magnetic layers such as iron or nickel or oxide nanoparticles thereof. Other exemplary designs may also include detection probes with different targeting functionalities. For example, exemplary embodiments of modifying the micro-nano motor with PAPA or related functional groups can enable detection of targeted bacteria or endotoxins. The exemplary design of the functionalized biological hybrid micro-nano motor can make the inner core of the porous biological entity have multiple functions, so that the inner core can detect target molecules in fluid.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Examples
Taking the oligosaccharide functionalized spore-based micro-nano motor as an example, a functionalized biological hybrid micro-nano motor and a preparation method thereof are described in detail.
The preparation method comprises the following steps:
And 2, ultrasonically dispersing the spores pretreated in the step 1 into 60mL of deionized water, and stirring for 5min to form a brown suspension of uniformly dispersed spores. Then adding FeSO into the suspension4After stirring for 20min, 20mL of ammonia water (25-27 wt%) is added dropwise, and the adding time is controlled within 10 min. Following sealing and further stirring for 2h, the spores covered by the black magnetic coating can be collected with a magnet. The collected black precipitate was washed several times with ethanol and deionized water and freeze-dried for use.
Step 3. the sample obtained in step 2 was dispersed into 200mL of ethanol and stirred for 10min to form a homogeneous suspension, followed by the addition of 0.2mmol of 3-mercaptopropionic acid. The mixed suspension was stirred for 10min and left at room temperature for one day. After collection using a magnet, the functionalized magnetic spores were washed several times with ethanol to remove residual functionalizing agent and lyophilized for use.
And 4, dispersing 50mg of the freeze-dried functionalized magnetic spores obtained in the step 3 into 60mL of deionized water. Then adding a chemical coupling reagent: 0.5mmol EDC and 0.5mmol NHS and stirred for 2h to activate the coupling reagent. The functionalized fluorescent carbon dots obtained by hydrothermally treating a mixture of 0.16g of aspartic acid, 0.16g of glucose and 0.16g of p-phenylenediamine for 12 hours were added to the above mixed suspension, and stirred at room temperature for 24 hours. Finally, the magnetic spore-based micro-nano motor with the oligosaccharide functionalized is obtained through EDC/NHS chemical coupling, and can be collected by a magnet, washed by ethanol and deionized water for several times and freeze-dried for later use.
An oligosaccharide-functionalized magnetic spore-based micro-nano motor scanning electron microscope image is shown in fig. 2. As shown in FIG. 2A, the microstructures of the drop spores have an average size of about 6-10 μm. Fig. 2B shows nanoparticles coated on the surface of spores, including magnetic iron oxide and carbon dots, from a few to tens of nanometers. The fractured structure in FIG. 2C shows a hollow structure and double walls of about 0.5-1 μm. This combined microscopic combination of the original porous, hollow structures and the deposited coarse nanoparticles renders them approximately 12.96m2The high specific surface area per gram can provide more active sites for adsorption and reaction. The magnetic spore-based micro-nano motor with the oligosaccharide functionalized can have uniform particle size; red fluorescence can also be exhibited under green light (excitation filter: 537-552nm) excitation, as shown in fig. 3.
The oligosaccharide-functionalized magnetic spore-based micro-nano motor can also drive movement in different fluids such as deionized water (DIW), Phosphate Buffered Saline (PBS), cell culture medium (DMEM), Fetal Bovine Serum (FBS) and gastrointestinal Mucus (Mucus). The speed of motion of the motor may increase in DIW, PBS and DMEM as the frequency increases, for example from 0 to 15 Hz; it is also possible in FBS and Mucus to increase first and then decrease with increasing frequency, presenting a maximum movement speed in a frequency range, e.g. between 7 and 9 Hz. When the motor is operated in a tumbling motion mode along the axis of rotation, the highest speed of motion and long displacement of motion can be achieved in all media, as shown in fig. 4.
The flow performance of the oligosaccharide functionalized magnetic spore-based micro-nano motor in deionized water (DIW), Phosphate Buffered Saline (PBS), cell culture medium (DMEM), Fetal Bovine Serum (FBS) and gastrointestinal Mucus (Mucus) liquid is examined. The speed of motion of the motor may increase in DIW, PBS and DMEM as the frequency increases, for example from 0 to 15 Hz; it is also possible in FBS and Mucus to increase first and then decrease with increasing frequency, presenting a maximum movement speed in a frequency range, e.g. between 7 and 9 Hz. The motor can be operated in a tumbling motion along the spinning shaft to achieve the highest speed of motion and long displacement of motion in all media, as shown in fig. 4.
The fluorescent tracing track of the oligosaccharide functionalized spore hybridization micro-nano motor in DIW, PBS, DMEM, FBS and Mucus in a controllable driving mode is shown in figure 5. Therefore, the oligosaccharide functionalized spore hybrid micro-nano motor can perform controllable motion in various fluids.
The results prove that the motor has good fluorescence performance and controllable active motion capability in different fluids. The exemplary detailed fabrication processes described herein can also be extended to and applied to the fabrication of other functionalized bio-hybrid micro-nano motors (e.g., PAPAs, RNA aptamers, etc.).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A functional biological hybridization micro-nano motor, which is characterized in that the motor comprises: a biological entity inner core, a magnetic nanoparticle coating and a probe layer.
2. The functionalized biohybrid micro-nano motor according to claim 1, wherein the inner core of the biological entity is a natural material comprising fungal spores, pollen, micro/nano plant seeds;
preferably, the surface of the inner core of the organism is rough or porous.
3. The functionalized biohybrid micro-nano motor according to claim 1, wherein the magnetic nanoparticles in the magnetic nanoparticle coating compriseIron, nickel and/or a magnetic metal oxide, preferably the magnetic nanoparticles are Fe3O4A nanoparticle;
preferably, the magnetic nanoparticle coating has a thickness of about 50 to 200 nm.
4. The functionalized biohybrid micro-nano motor according to claim 1, wherein the magnetic nanoparticle coating surface further comprises a self-assembled monolayer.
5. The functionalized bio-hybrid micro-nano motor according to claim 4, wherein the self-assembled monolayer comprises a thiolated self-assembled monolayer.
6. The functionalized biohybrid micro-nano motor according to claim 1, wherein the probe layer comprises carbon dots with fluorescence emission and response capability and various specific groups or ligand molecules with targeting function;
preferably, the specificity group or aptamer molecule with targeting function comprises oligosaccharide, RNA aptamer, phenylboronic acid and derivatives thereof;
preferably, the detection probe may comprise two or more probes, which may bind to different target molecules and may detect multiple types of target molecules simultaneously.
7. The method for preparing the functionalized biological hybrid micro-nano motor of any one of claims 1 to 6, which comprises the following steps:
hybridizing magnetic nanoparticles on the surface of the inner core of the biological entity to form a magnetic nanoparticle coating; fixing the probe on the surface of the magnetic nanoparticle coating by using a biological/chemical coupling technology;
preferably, the hybridization of the inner core of the biological entity with the magnetic nanoparticles is achieved by a step-by-step coating method.
8. The method of claim 7, further comprising pre-treating the inner core of the biological entity to remove surface impurities.
9. The method of claim 7, further comprising forming a self-assembled monolayer on the surface of the magnetic nanoparticle coating layer before immobilizing the probe.
10. The method of claim 7, wherein the bio/chemical coupling technique comprises oligosaccharide-complex repeat oligopeptide binding, avidin-biotin binding, weak interaction.
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