CN110385088B

CN110385088B - Method for preparing microparticles

Info

Publication number: CN110385088B
Application number: CN201810344015.8A
Authority: CN
Inventors: 沈峰; 屈海军; 俞梦超
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2018-04-17
Filing date: 2018-04-17
Publication date: 2021-12-21
Anticipated expiration: 2038-04-17
Also published as: CN110385088A; CN114345261A; CN114345261B

Abstract

The present invention discloses a method for preparing microparticles, comprising: injecting a solution of a substance for preparing the microparticles into a fluid channel formed by overlapping the micropores on the surface of the upper sub-chip of the microfluidic chip with the micropores on the surface of the lower sub-chip thereof; moving the upper sub-chip relative to the lower sub-chip so that the micro-holes on the surface of the upper sub-chip are no longer overlapped with the micro-holes on the surface of the lower sub-chip; and converting the solution in the micropores on the surface of the upper sub-chip and/or the lower sub-chip from a liquid state to a non-liquid state to form the microparticles. Also disclosed are methods for preparing microparticles comprising at least two layers and methods for preparing microparticles by biological or chemical reactions. These methods have the advantages of relatively simple operation and control, capability of preparing microparticles of various shapes and sizes, capability of preparing multi-layer microparticles, capability of being applied to various raw materials, and the like.

Description

Method for preparing microparticles

Technical Field

The invention relates to the fields of biological, chemical, medical analysis and the like, in particular to a method for preparing microparticles by utilizing a microfluidic chip.

Background

Micro-particles have a very wide range of potential applications in biology, medicine, engineering, etc., such as optical materials, MEMS, biomaterials, self-assembly, etc. The controllable preparation of microparticles with different sizes and shapes has important scientific research and application significance. The current methods for synthesizing microparticles include batch production (batch process) or microfluidic methods, but they have great limitations in terms of materials, shapes, composite particles and the like.

The conventional monodispersion method (mono dispersion) is used for preparing spherical microparticles of a single size, and has been available for industrial mass production. However, this method is not suitable for preparing microparticles having non-spherical shapes (e.g., square, cylindrical, triangular, elliptical, etc.). In recent years, as a new attempt, researchers have started to apply a microfluidic (microfluidic) method to the preparation of various microparticles. Microfluidics refers to a technique for manipulating micro-volumes of fluid (e.g., microliters μ L, nanoliters nL, picoliters pL, or even subpicoliters) through microfluidic channels, microwells, microchambers, and the like. It can control the surface property of chip, fluid property and external environment (such as pressure, temperature, humidity and light).

Methods for using microfluidic chips for preparing microparticles are mainly classified into the following (review articles Micromachines 2017,8, 255): 1) first, a liquid material to be solidified to form microparticles and an organic phase immiscible with the liquid material are injected into a fluid channel (e.g., a T-shaped channel or a droplet-generating cross-shaped channel) of a microfluidic chip. The liquid for making microparticles can be separated by organic spacers to generate droplets with required size, and then the droplets are irradiated by external ultraviolet light to initiate photochemical reaction of chemical substances in the droplets, so as to solidify into microparticles (angeltide Chemie-International Edition,2010,49, 87-90); 2) secondly, liquid containing reaction monomers is injected into a fluid channel of the microfluidic chip, a mask with a designed shape is placed at the bottom end of the fluid channel, and ultraviolet light is used for irradiation from the bottom end. Ultraviolet light can be cured into microparticles by initiating a chemical reaction of the liquid monomer through the transparent portions of the mask (Nature Materials,2006,5, 365-. However, the above method for preparing microparticles using a microfluidic chip still has some disadvantages: 1) are complex and generally require precise fluid control and optical equipment; 2) difficult to use for preparing microparticles of different sizes and specific shapes; 3) is only suitable for synthesizing microparticles by utilizing photo-induced reaction, and is not suitable for other materials (such as hydrogel and the like or other biochemical materials); 4) multilayer microparticle synthesis is not easily achieved.

In view of the above, those skilled in the art have made an effort to develop a novel method for preparing microparticles using a microfluidic chip, which can use a variety of materials as long as the materials can change from a liquid state to a non-liquid state (e.g., a solid state or a gel state) under certain conditions. The liquid to non-liquid change may be a physical change, a chemical change, or a liquid to non-liquid conversion caused by other biological and chemical reactions. Ideally, it can be used to prepare microparticles of various sizes and shapes (including specific shapes), can be used for materials such as hydrogels, and can enable the preparation of multi-layered microparticles without the need for sophisticated equipment and controls.

Disclosure of Invention

To achieve the above object, according to one aspect of the present invention, there is provided a method for preparing microparticles, comprising: injecting a solution of a substance for preparing the microparticles into a fluid channel formed by overlapping the micropores on the surface of the upper sub-chip of the microfluidic chip with the micropores on the surface of the lower sub-chip thereof; moving the upper sub-chip relative to the lower sub-chip so that the micro-holes on the surface of the upper sub-chip are no longer overlapped with the micro-holes on the surface of the lower sub-chip; and converting the solution in the micropores on the surface of the upper sub-chip and/or the lower sub-chip from a liquid state to a non-liquid state to form the microparticles.

Preferably, the non-liquid state includes a solid state and a gel state.

Preferably, converting the solution from a liquid state to a non-liquid state comprises converting the solution from a liquid state to a non-liquid state by physical means, chemical reaction, or biological reaction.

Preferably, the physical means comprises changing the temperature of the solution.

Preferably, the converting the solution from the liquid state to the non-liquid state by the chemical reaction includes irradiating the solution with ultraviolet light to activate the photocuring reaction.

According to another aspect of the present invention, there is provided a method for preparing microparticles comprising at least two layers, comprising: overlapping a first series of micropores on the surface of an upper sub-chip of the microfluidic chip with a first series of micropores on the surface of a lower sub-chip of the microfluidic chip to form a first fluid channel; injecting a first solution of a substance for preparing the microparticles into the first fluid channel; moving the upper sub-chip to a first position relative to the lower sub-chip such that the first series of micro-holes on the surface of the upper sub-chip no longer overlap with the first series of micro-holes on the surface of the lower sub-chip; converting the first solution in the first series of microwells on the surface of the upper or lower chiplet from a liquid state to a non-liquid state to form a first layer of the microparticles; overlapping the second series of micro-holes on the surface of the upper sub-chip with the second series of micro-holes on the surface of the lower sub-chip to form a second fluid channel; injecting a second solution of a substance for preparing the microparticles into the second fluid channel; moving the upper sub-chip to a second position relative to the lower sub-chip such that a second series of micro-wells on the surface of the lower sub-chip or the upper sub-chip into which the second solution is injected overlaps with a first series of micro-wells on the surface of the upper sub-chip or the lower sub-chip into which the first layer of micro-particles has been formed; and converting the second solution in the second series of wells on the surface of the lower or upper chiplet from a liquid state to a non-liquid state to form a second layer of microparticles.

According to still another aspect of the present invention, there is provided a method for preparing microparticles by a biological or chemical reaction, comprising: injecting a first solution into a first fluid channel formed by overlapping a first series of micropores on the surface of an upper sub-chip of the microfluidic chip with a first series of micropores on the surface of a lower sub-chip of the microfluidic chip; injecting a second solution into a second fluid channel formed by the overlapping of the second series of wells on the surface of the upper biochip and the second series of wells on the surface of the lower biochip; moving the upper sub-chip relative to the lower sub-chip such that a first series of microwells on the surface of the upper sub-chip or the lower sub-chip into which the first solution is injected and a second series of microwells on the surface of the lower sub-chip or the upper sub-chip into which the second solution is injected overlap each other; and the first solution and the second solution are biologically or chemically reacted to convert the solutions from a liquid state to a non-liquid state to form the microparticles.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic diagram illustrating a method of making microparticles according to one embodiment of the present invention;

FIG. 2 is a schematic diagram illustrating a method of making microparticles according to another embodiment of the present invention;

fig. 3 is a schematic view illustrating a method of preparing multi-layered (two or more layers) microparticles according to an embodiment of the present invention; .

FIG. 4a is a perspective view of an upper sub-chip of an exemplary microfluidic chip;

FIG. 4b is a side view of the upper sub-chip of FIG. 4 a;

FIG. 4c is a top view of the upper sub-chip of FIG. 4 a;

FIG. 4d is a perspective view of a lower sub-chip of an exemplary microfluidic chip;

FIG. 4e is a side view of the lower sub-chip of FIG. 4 d;

FIG. 4f is a top view of the lower sub-chip of FIG. 4 d;

FIG. 4g is a perspective view of the microfluidic chip obtained by assembling the upper sub-chip of FIG. 4a and the lower sub-chip of FIG. 4 d;

FIG. 4h is a side view of the microfluidic chip of FIG. 4 g;

FIG. 4i shows the basic operation of moving the upper sub-chip relative to the lower sub-chip of the microfluidic chip in FIG. 4 g;

fig. 5 is a schematic view illustrating a process of preparing microparticles using relative displacement of upper and lower sub-chips of a displacement type microchip.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

In the drawings, structurally identical elements are represented by like reference numerals, and structurally or functionally similar elements are represented by like reference numerals throughout the several views. The size and thickness of each component shown in the drawings are arbitrarily illustrated, and the present invention is not limited to the size and thickness of each component. The dimensions and thicknesses of components in the figures may be exaggerated where appropriate to improve clarity.

The present invention prepares microparticles by using one of the microfluidic chips, namely a displacement microfluidic chip. The microfluidic chip provided by the invention can be prepared from various materials and processing methods. For example, the materials of the microfluidic chip can be selected from glass, quartz glass, plastic, ceramic, metal, inorganic materials, fiber materials, polymers, and the like. There are many different methods for the fabrication of the microfluidic chip, including wet etching, dry etching, micromachining, 3D printing, thermoforming, pressure forming, injection molding, and the like.

Specifically, a glass chip is prepared by wet etching. Soda lime glass, which has been coated with a chrome layer and a photoresist layer, is commercially available from Telic Corporation (California, USA). The design of the photomask is done by AutoCAD software. The photomask is printed and prepared by Shenzhen exquisite photoelectricity shares Limited. First, a photomask was closely covered on the side of glass to which a photoresist was attached, and the glass was placed in a full-function ultraviolet exposure machine (Intelli-Ray 400W). The exposure is carried out for 10-20 seconds with a light source intensity of 50%. Subsequently, the exposed glass was immersed in a 0.1mol/L sodium hydroxide (Chinese medicine) solution for 1 minute to remove the portion of the photoresist that reacted with the ultraviolet light. The glass was then transferred to a dechroming solution for 1 minute, so that the photoresist had been removed and the bare chromium layer was removed. The dechroming solution contained 0.6mol/L perchloric acid (national medicine) and 0.365mol/L cerium ammonium nitrate (Michalin's reagent) in water. The treated glass was then rinsed thoroughly with deionized water and blown dry with nitrogen. And after the side, which does not need to be etched, of the glass is protected by a waterproof adhesive tape, the side, which needs to be etched, of the glass is upwards immersed in the glass etching liquid, and the exposed glass, from which chromium is removed, is etched. In order to better control the etching rate and achieve more uniform etching, wet etching was performed in a constant temperature water bath shaker at 40 degrees celsius. The glass etching solution contained 1mol/L hydrofluoric acid (Aladdin corporation), 0.5mol/L ammonium fluoride (Lingfeng chemical) and 0.75mol/L nitric acid (Chinese medicine) solution. The desired micro-holes may be formed on the glass chip by the wet etching method. The depth of the micro-holes on the glass chip can be controlled by the time of the wet etching.

The prepared chip needs to be subjected to corresponding surface treatment. Taking a glass chip as an example, the surface of the glass chip needs to be subjected to hydrophobic treatment, and the specific method is as follows: firstly, fully cleaning the surface of glass by using deionized water, and drying the surface by using nitrogen; next, the glass chip was placed in a plasma cleaner (Harrick, USA) for 1 minute of surface plasma cleaning and activation. Finally, the glass chip was placed in a desiccator containing 20. mu.l of dichlorodimethylsilane (Inay Kay technology) to undergo a vapor phase silylation reaction for 1 hour. And (3) washing the treated chip with chloroform (Chinese medicines), acetone (Chinese medicines) and absolute ethyl alcohol (Chinese medicines), and drying by blowing with nitrogen gas to obtain the chip assembly and the microparticle preparation.

The assembly of the chip is that the surfaces of the upper and lower sub-chips containing the micropores are opposite, organic solvent such as mineral oil is added between the surfaces, and then the micropores on the upper and lower sub-chips are staggered and overlapped according to the design to form communicated fluid channels. Subsequently, the upper and lower sub-chips may be fixed by a jig. The added organic solvent can provide corresponding lubrication and can also provide the required surface energy and surface property with the treated surface. Generally, the organic solvent needs to well wet the treated solid surface.

Hereinafter, a microfluidic chip according to the present invention and its basic operation will be described with reference to the accompanying drawings. Fig. 4a is a perspective view of an upper sub-chip of an exemplary microfluidic chip, as shown in fig. 4. Fig. 4b is a side view of the upper sub-chip of fig. 4 a. Fig. 4c is a top view of the upper sub-chip in fig. 4 a. Fig. 4d is a perspective view of the lower sub-chip of an exemplary microfluidic chip. Fig. 4e is a side view of the lower sub-chip of fig. 4 d. Fig. 4f is a top view of the lower sub-chip of fig. 4 d. Fig. 4g is a perspective view of the microfluidic chip obtained after the upper sub-chip in fig. 4a and the lower sub-chip in fig. 4d are assembled together. Fig. 4h is a side view of the microfluidic chip in fig. 4 g. Fig. 4i shows the basic operation of moving the upper sub-chip relative to the lower sub-chip of the microfluidic chip in fig. 4 g.

Next, how to prepare desired microparticles specifically according to the present invention will be described with reference to the accompanying drawings. General microparticle preparation procedure referring to fig. 1, the corresponding reaction solution can be injected into the assembled chip through the inlet port by means of a pipette, a fluid pump or other positive pressure. This injection process may also be accomplished by applying a negative pressure or vacuum at the outlet end. (FIG. 1-A and FIG. 1-B). After the liquid injection is completed (fig. 1-C), the upper sub-chip can be moved relative to the lower sub-chip manually or by an external mechanical carrier, so that the micro-holes on the corresponding surfaces of the upper sub-chip and the lower sub-chip are not overlapped with each other any more, and relatively independent reaction micro-holes are formed (fig. 1-D). Note that the reaction wells may include wells in the upper and/or lower sub-chip, and the reaction solution is in the reaction wells, thereby forming relatively independent reaction wells. The liquid in the reaction wells is solidified to form microparticles by a corresponding biological, chemical, or physical process (or combination of processes) (fig. 1-E). Finally, the upper and lower sub-chips are separated, and the microparticles are taken out from the micropores of the sub-chips by ultrasonic oscillation or physical stripping, etc. (FIG. 1-F).

The specific method for preparing the microparticles by the photocuring chemical reaction is as follows: referring to fig. 2, wherein fig. 2-a shows the upper and lower sub-chips of the assembled microfluidic chip. First, a liquid photo-curable resin material (nova smart) is injected into the assembled chip by means of a pipette or positive pressure (fig. 2-B). Specifically, the uncured resin material is injected into the chip through a fluid channel formed by overlapping the micro-holes of the upper and lower sub-chips, and fig. 2-C shows a case where the uncured resin material in a liquid state fills the fluid channel after the injection is completed. After the injection is completed, the upper sub-chip is moved to a second position relative to the lower sub-chip manually or by a carrier, so that the corresponding micropores on the upper sub-chip and the lower sub-chip are not connected with each other any more. The chips were placed in a full-function UV exposure machine (Intelli-Ray 400W). Exposure was carried out for 5-10 seconds using 50% light source intensity (FIG. 2-D). The specific exposure parameters need to be optimized according to the photo-curable resin material used. Finally, the upper and lower daughter chips were separated and the microparticles were removed with tweezers (FIG. 2-E). Note that, here, the resin material in the upper sub-chip and/or the lower sub-chip may be subjected to ultraviolet light irradiation to form desired cured microparticles.

As previously mentioned, the microparticle preparation method according to the present invention may utilize a variety of raw materials, and it may also use a variety of different methods to effect a change of material from a liquid state to a non-liquid state (e.g., solid or gel state). Previously, it has been illustrated by way of example how microparticles are prepared by irradiation with ultraviolet light. Hereinafter, how to prepare microparticles using a temperature change will be explained.

Specifically, the method for preparing gel microparticles by temperature change is as follows: first, an aqueous solution containing Agarose, Agarose (Bio-Rad, USA), was heated to dissolve the Agarose sufficiently. The concentration of Agarose in the solution may be from 0.1% to 10%, the higher the concentration the greater the hardness after solidification. The hot Agarose solution is injected into the chip through a fluid channel formed by overlapping the micropores of the upper and lower sub-chips. After the injection is completed, the upper sub-chip is moved to a second position relative to the lower sub-chip manually or by a carrier, so that the corresponding micropores on the upper and lower sub-chips are not connected/overlapped with each other any more. The chip is cooled and the Agarose solution solidifies to a gel or solid state at room temperature or below. And finally, separating the upper sub-chip from the lower sub-chip, and stripping colloidal microparticles from the sub-chips by using ultrasound or tweezers, so that the microparticles can be recovered.

Ice crystal microparticles can also be prepared by temperature variation, and the method comprises the following steps: first, an aqueous solution is injected into the chip through a fluid channel formed by overlapping micro-holes of upper and lower sub-chips at a temperature higher than the freezing point thereof. After the injection is completed, the upper sub-chip is moved to a second position relative to the lower sub-chip manually or by a carrier, so that the corresponding micropores on the upper and lower sub-chips are not connected/overlapped with each other any more. The chip was cooled below the freezing point of the aqueous solution (note that at the selected temperature, the organic phase was still in the liquid state). The aqueous solution in the micropores will coagulate into ice crystal microparticles. Finally, the upper and lower sub-chips are separated, and the ice crystal microparticles are peeled off from the sub-chips by using ultrasound or tweezers.

In addition to preparing a monolayer of microparticles as described above, the present invention may also be used to prepare microparticles comprising at least two layers. First, a first solution is injected into the chip through a fluid channel formed by overlapping a first series of wells of the upper and lower sub-chips (as shown in FIGS. 3-A to 3-B). After the injection is completed, the upper sub-chip is moved to a second position relative to the lower sub-chip, manually or by a carrier, such that the corresponding first series of micro-holes on the upper and lower sub-chips are no longer connected/overlapped with each other (as shown in fig. 3-C). The first solution is solidified in the corresponding microwells by biological, chemical, or physical change (solidification by irradiation with ultraviolet light in fig. 3-C), forming a first layer of microparticles. In a second position, the second solution is injected into the chip through the fluid channel formed by the overlapping of the second series of wells of the upper and lower sub-chips (as shown in FIG. 3-D). After the injection is completed, the upper and lower sub-chips are displaced to a third position relative to the lower sub-chip, either manually or by means of a carrier, and the second series of wells containing the second solution (e.g., the second series of wells of the lower or upper sub-chip) is overlapped with the first series of wells containing the first solution that has solidified (e.g., the first series of wells of the upper or lower sub-chip), and the second reaction solution is brought into contact with the solidified first substance. The second solution is then cured in the corresponding microwells by biological, chemical, or physical change (as shown in fig. 3-E, which illustrates curing with ultraviolet light irradiation), forming a second layer of microparticles. Finally, the upper and lower sub-chips are separated, and microparticles comprising the two layers are peeled off from the sub-chips by ultrasound or tweezers (as shown in fig. 3-F), thereby recovering the microparticles.

In addition, the microchip of the present invention can also be prepared by solidifying a liquid by causing a biological or chemical reaction through a pore-pairing reaction on a chip. Firstly, injecting a first solution into a first fluid channel formed by mutually overlapping a first series of micropores of the upper sub-chip and the lower sub-chip; a second solution is injected into a second fluid channel formed by the overlapping of the second series of wells of the upper and lower chiplets (as shown in FIGS. 5-A-5-B). The first solution and the second solution are both liquids, but they react to become solid after mixing with each other. After the injection is completed (as shown in fig. 5-C), the upper sub-chip is displaced to a second position relative to the lower sub-chip, either manually or by a carrier, such that the first series of wells of the upper or lower sub-chip infused with the first solution overlaps the second series of wells of the lower or upper sub-chip infused with the second solution (as shown in fig. 5-D), thereby initiating a biological or chemical reaction between the first and second solutions (as shown in fig. 5-E). Finally, the upper and lower sub-chips are separated, and the solidified microparticles are peeled off from the sub-chips by using ultrasound or tweezers (as shown in fig. 5-F), so that the microparticles can be recovered.

Specific examples of preparing microparticles by causing a biological or chemical reaction to solidify a liquid by a microwell pairing reaction on a chip will be given below. This example is an example of the preparation of blood coagulation microparticles. EDTA anticoagulation tube collects blood which is not coagulated because calcium ions are bound by EDTA. The preparation of blood coagulation microparticles of different sizes and shapes is useful for studies relating to blood clotting and, in particular, for studies relating to methods for lysing blood clots. Firstly, passing blood treated by EDTA through a first fluid channel formed by mutually overlapping a first series of micropores of an upper sub-chip and a lower sub-chip; passing the solution containing calcium ions through a second fluid channel formed by the overlapping of the second series of wells of the upper and lower sub-chips (as shown in FIGS. 5-A to 5-B). After the injection is completed (as shown in fig. 5-C), the upper sub-chip is displaced to a second position with respect to the lower sub-chip, manually or by means of a carrier, so that the first series of wells of the upper or lower sub-chip filled with EDTA-treated blood overlaps with the second series of wells of the lower or upper sub-chip filled with a solution containing calcium ions (as shown in fig. 5-D), and thus calcium ions are introduced into the EDTA-treated blood to initiate a blood coagulation reaction, forming a blood clot of a specific size and shape in the corresponding wells (as shown in fig. 5-E). Finally, the upper and lower sub-chips are separated, and the cured clot microparticles are peeled off from the sub-chips using ultrasound or forceps (as shown in FIG. 5-F), thereby recovering the microparticles.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. A method for preparing microparticles comprising at least two layers, characterized in that it comprises:

overlapping a first series of micropores on the surface of an upper sub-chip of the microfluidic chip with a first series of micropores on the surface of a lower sub-chip of the microfluidic chip to form a first fluid channel;

injecting a first solution of a substance for preparing the microparticles into the first fluid channel;

moving the upper sub-chip to a first position relative to the lower sub-chip such that the first series of micro-holes on the surface of the upper sub-chip no longer overlap with the first series of micro-holes on the surface of the lower sub-chip;

converting the first solution in the first series of microwells on the surface of the upper or lower chiplet from a liquid state to a non-liquid state to form a first layer of the microparticles;

overlapping the second series of micro-holes on the surface of the upper sub-chip with the second series of micro-holes on the surface of the lower sub-chip to form a second fluid channel;

injecting a second solution of a substance for preparing the microparticles into the second fluid channel;

moving the upper sub-chip to a second position relative to the lower sub-chip such that a second series of micro-wells on the surface of the lower sub-chip or the upper sub-chip into which the second solution is infused overlaps or partially overlaps with a first series of micro-wells on the surface of the upper sub-chip or the lower sub-chip into which the first layer of microparticles has been formed; and

converting the second solution in the second series of wells on the surface of the lower or upper chiplet from a liquid state to a non-liquid state to form a second layer of the microparticles.

2. The method of claim 1, wherein converting the solution from the liquid state to the non-liquid state comprises converting the solution from the liquid state to the non-liquid state by physical means, chemical reactions, or biological reactions.

3. The method of claim 2, wherein the physical means comprises changing the temperature of the solution.

4. The method of claim 2, wherein said converting the solution from the liquid state to the non-liquid state by a chemical reaction comprises irradiating the solution with ultraviolet light to activate a photocuring reaction.