KR100957200B1 - Production Method of Mono-Dispersion Bead Using Microfluidic Channel Structure, the Produced Bead by This Method, and Production Method of Microfluidic Chip - Google Patents

Abstract

The present invention relates to a method for synthesizing monodisperse beads and a method for manufacturing microfluidic chips using a microfluidic channel structure, and more particularly, simple microfluidic focusing, microfluidic focus containing a mixed channel. The microfluidic channel structure is characterized by synthesizing uniform monodisperse beads using a microfluidic chip manufactured by a method selected from the group consisting of a biased shear force driven microflow using a flow method and a shear stress. Preparing monodisperse beads, monodisperse beads prepared by the method, and microchannels; Injecting the reactants; And it relates to a method for producing a microfluidic chip for producing a monodisperse biopolymer, characterized in that it comprises the step of analyzing the generated by-products. The method for synthesizing monodisperse beads of the present invention can effectively produce monodisperse beads, such as biocompatible monodisperse hydrogel beads, which are widely used in the fields of biotechnology and medicine. It can be useful to make.

Hydrogels, monodisperse beads, microfluidics, micro reactors

Description

Production method of mono-dispersion beads using microfluidic channel structure, the produced beads by this method, and production method of Microfluidic Chip}

The present invention provides a microfluid produced by a method selected from the group consisting of a simple microflow focusing method, a microfluidic focus flow method including a mixed channel, and a biased shear force driven microflow method. A method for synthesizing monodisperse beads using a microfluidic channel structure comprising synthesizing uniform monodisperse beads using a chip, monodisperse beads prepared by the above method, and a method for manufacturing a microfluidic chip will be.

A microfluidic chip refers to a chip capable of analyzing a variety of materials present therein while flowing a small amount of analyte into a microchannel formed in the chip. The microfluidic chip is called a lab-on-a-chip (LOC: 'lab on chip'), and is a chip that can analyze analytes at a time in a small chip. Is being developed. Microfluidic chips are being used for the analysis, separation, and synthesis of materials, and their fields of use are gradually expanding. Therefore, in order to develop the lab-on-a-chip, a circuit is formed into a microchannel through which a solution can flow on a surface of plastic, glass, silicon, and the like, and then pretreatment, separation, dilution, mixing, biochemical reaction, and detection of a sample are performed. It must be able to be miniaturized and integrated in the chip. Therefore, the importance of microfluidic engineering technology in the lab-on-a-chip becomes very large. Microfluidic chips are microchips made of micromachining techniques such as photolithography, hot-embossing, and molding to cover and cover or control a small amount of fluid. It has the advantage of reducing the amount of reagent consumed and shortening the analysis time.

In particular, when DNA denaturation, annealing, and extension reactions require different temperatures, such as polymerase chain reaction, the reactions are repeated by repeated temperature cycles. The reaction volume and large area allow for rapid temperature transfer within the microchamber, which reduces the time required for the temperature cycle. There are many ways to detect PCR reactions in real time, but most of them use fluorescence detection. The method used here is a method using a dye such as SYBR Green I which binds to double stranded DNA generated by a PCR reaction to improve fluorescence, and in addition to the primer used for PCR, A tag polymerase (Taq) is used for DNA synthesis by using a DNA sequence capable of binding between primers as a probe and binding a fluorophore and a quencher to both ends of the probe. Using the exonuclease activity of the polymerase, the probe is cut, and the DNA bound between the fluorophore and the luminescent inhibitor is cut, and the fluorescence is generated as the bond between the fluorophore and the luminescent inhibitor is cleaved. Various methods have been developed, such as the TaqMan (R) method.

Microfluidics is an active field of research and development of key technologies that are the basis for the commercialization of micro-total analysis systems (μ-TAS) and lab-on-a-chip. Research and commercialization are in progress. The micro-combination analysis system is a system in which chemical and biological experiments and analyses, which undergo a plurality of experimental steps and reactions, are comprehensively implemented in a unit on a bench. The micro-combination analysis system includes a sampling area, a microfluidic circuit, a detector, and a controller for controlling the sampling area, a microfluidic circuit, and a detector. However, unlike the microarray field, which has been actively commercialized since the human genome project, the field of microfluidics has still been concrete and practical in the product scene, despite technological advances made in recent years. Products with significant influence have not been developed (D & MD Reports # 9082-Microbiotechnology, 2002).

Companies such as Caliper Technology, Cepheid, and Fluidigm, known as the leading group in microfluidics, have developed technologies based on their specific platform technologies, while maintaining the appropriate marketability and marketability. Developing products As a technique capable of controlling the flow of a solution without a separate pump or valve, an electrokinetic microfluidic technique has been proposed (Anal. Chem. 64, 1926, 1992). Caliper Technology Inc. has recently developed an analytical method for separating DNA, RNA, and proteins by size using electrophoretic separation, a kind of electrokinetics (Anal. Chem. 73: 1207-1212, 2001). Electrophoresis 21: 128-134, 2001; Lab Chip 2: 42N-47N, 2002; Anal.Biochem. 316: 92-102, 2003). However, the analysis method using the electrophoretic separation technique has a problem in that a high voltage power supply capable of providing a plurality of electrodes and several kHz when controlling the fluid is required.

In addition, techniques have been developed that use molecular diffusion (diffusion) due to laminar flow in microfluidic channels. Micronics has developed a way to analyze blood directly using this technology, and by integrating microfluidic circuits in credit card-sized plastics to separate blood cells and plasma using diffusion, enzymes, proteins, electrolytes and pharmacological substances in plasma And the like (Biomedical Microdevices 3: 267-274, 2001).

In addition, there are biofluid control methods such as Tecan and Gyros' centrifugal force method (Biotechniques 26: 758-767, 1999) and electrowetting method developed by UCLA (Journal of MEMS 12). , 70-80, 2003).

On the other hand, in general, microfluidic control methods of bio-related materials include pretreatment of samples (eg, separation and purification of DNA from living organisms, reaction and washing in immune reactions between antigens and antibodies), and serialization. It is a very important factor in controlling the analysis process. Cepheid has developed a variety of sample pretreatment devices using microfluidic technology. In particular, it has developed a polymerase chain reaction (PCR) device for DNA and a portable analysis system for DNA concentration, microbial lysis, and microbial sample preparation. (Clin. Chem. 47: 1917-1918, 2001; μ-TAS 2001: 670-672, 2001; Anal. Chem. 73: 286-289, 2001). Cepheid Inc. used an external membrane valve and diaphragm pump separately mounted to control the fluid inside the chip in the PCR device and portable assay system.

Meanwhile, Fluidigm has developed a microfluidic chip using a multi-layered channel in which a fluid flow channel and an air flow channel intersect using a plastic soft layered lithography method (Science). 288: 113-116, 2000; Science 290: 1536-1539, 2002). Fluidigm is using the microfluidic chip using the multi-layered channel as a protein crystallizer and immunoassay method (PNAS 99: 16531-16536, 2002; Anal. Chem. 75: 3581-3586, 2003). Nature Biotechnology 21: 1179-1183, 2003). In addition, Fluidigm solves the interfacing problem of connecting the microfluidic chip (Anal. Chem. 75: 4718-4723, 2003), and uses an external optical detector separately for measuring the reaction result. .

As described above, various microfluidic control schemes developed to date and products manufactured using the microfluidic control schemes are compared with existing analytical methods in fields other than the microfluidics field. In terms of efficiency, accuracy and product competitiveness, there is still much room for improvement. In particular, the use of existing microfluidic chips in processing various sample groups has been extremely limited.

The use of the microfluidic chip is extremely limited because the characteristics of each of the samples are different in inspecting, measuring, and analyzing unknown samples. That is, since a sample having a relatively high concentration exceeds the concentration measuring range of the analysis detector, there is a problem in that the sample must be diluted at an appropriate ratio to exist within the concentration measuring range of the analysis detector. Therefore, there is a need for a method for actively utilizing microfluidic chips.

Hydrogels, on the other hand, are hydrophilic polymers with three-dimensional conformation that can easily contain large amounts of water and genes, peptides, proteins and cells. In particular, spherical hydrogel beads are known to be highly valuable materials for the implantation of biosensors, contrast agents, drug delivery devices, cell carriers, and cells or tissues due to their inherent biocompatibility and hydrophilic properties. In such applications, monodisperse hydrogel beads have great advantages due to excellent signal detection, analysis, and modeling, and there is an absolute need for a technology capable of producing monodisperse particles while controlling particle size.

Method for producing conventional hydrogel beads or polymer beads (Method for producing polymer beads by emulsion polymerization (Dec. 15, 2006), Patent application No. 2000-0056980; Water-soluble chitosan porous bead capsule and method for producing same (2002.04.04), Patent Application No. 2002-0018477; Alginate bead for sustained-release drug delivery system using alginic acid and its manufacturing method (1997.07.09), Patent application No. 1997-0031780 use a syringe or emulsion polymerization method Therefore, it produced polydisperse beads with very high polydispersity, but did not produce monodisperse beads.

In addition, published publications (J. Amer. Chem. Soc, Zhang et al., 128, 12205 -12210, 2006) produce monodisperse hydrogel beads, but basically employ a fluid-focused flow method and continuously Diluting a small amount of the reaction agent (CaI) in the phase is produced by the diffusion between interfaces, the reaction proceeds depending on the diffusion has a very slow reaction rate. In addition, the existing Talu et al. Studies proposed a method of forming a hydrogel by diffusion by adding a crosslinking agent calcium to the dispersion phase, but showed a very weak productivity and mechanical properties.

 As such, micro-sized hydrogels have generally been synthesized by emulsion polymerization. However, the reality is that the size of the particles can be easily adjusted and it is very difficult to obtain monodisperse particles.

Thus, the inventors of the present invention, while trying to more effectively produce monodisperse particles, was able to easily produce and produce micron-sized monodisperse particles using a microfluidic system. More specifically, since the hydrogel particles using the microfluidic system have a very high viscosity due to the high viscosity of the polymer used, the present inventors have an optimal production process through various microchannel structures. I tried to find the effect of the structure.

As a result, the microfluidic method using an optimal microfluidic channel structure, that is, a shear stress, can be used to easily control stable mechanical properties and various particle sizes while simultaneously producing monodisperse hydrogel particles. The present invention was completed by synthesizing uniform monodisperse beads through the formation of a small and stable emulsion, and producing a robust microfluidic chip having the same properties as the slope.

An object of the present invention is to solve the problems of the conventional method for producing particles using the bulk emulsion polymerization method, to provide a method for easily synthesizing monodisperse polymer hydrogel beads in real time using microfluidic in the microfluidic channel will be.

Still another object of the present invention is to provide a monodisperse polymer hydrogel bead prepared by the above method.

Still another object of the present invention is to provide a method of manufacturing a microfluidic chip.

In order to achieve the above object, the present invention is selected from the group consisting of a simple microflow focusing method, a microfluidic focus flow method containing a mixed channel and a biased shear force driven microflow method. Provided is a method for synthesizing monodisperse beads using a microfluidic channel structure, which comprises synthesizing uniform monodisperse beads using a microfluidic chip manufactured by the method.

The present invention also provides monodisperse beads prepared by the above method.

In addition, the present invention provides a method for manufacturing a microfluidic chip, i) manufacturing a microchannel to withstand the pressure drop due to reactant injection while the channel surface has the same hydrophobic character; Ii) injecting the reactants into the inlet of the microchannels; And iii) analyzing the by-products generated from the microchannels.

Hereinafter, the present invention will be described in detail.

First, definitions and explanations of various terms used in the present invention are as follows.

First, the channel structure will be described.

14 shows a microfluidic channel structure using a simple microfluidic focal flow method. At this time, the height of the channel is 50 μm. The microfluidic focal flow method uses hexadecane (unmixed) flowing in both channels to form two reactants in aqueous phase, calcium and alginate emulsions in series. However, due to the laminar flow characteristic of the microfluidic channel, the mechanical strength of the alginate produced is not good because it is a reaction dependent on diffusion at the reactant interface.

15 shows a microfluidic channel structure using a microfluidic focal flow method containing a mixed channel. At this time, the height of the channel is 50 μm. The microfluidic focal flow method containing the mixing channel forms an additional curved channel to increase the efficiency of the mixing. Although structural changes and reaction times were increased, sufficient mixing to synthesize monodisperse alginate beads, i.e. strong turbulence could not be formed inside the emulsion, yielding similar results to the simple microfluidic focal flow method.

16 shows a microfluidic channel structure using a microfluidic method using shear stress. At this time, the height and width of the channel is 50 μm. The microfluidic method using the shear stress was used to form an emulsion using one hexadecane, unlike the previous microfluidic focusing method. The resulting emulsion passes through the structure of the bent channel, and the friction area with the wall and the wall of the channel is different. Strong turbulence was formed therein to maximize mixing efficiency and form desired alginate beads. The mixing principle is explained below.

Rapid reactions used in the present invention are as follows.

The behavior of the fluid is defined as the Reynolds number (Re). In general, the fluid flow in the microfluidic channel is less than 1, resulting in laminar flow. As shown in Figure 17 (a) below, assuming that the reactants A, B are injected side by side in the microchannels, eventually the mixing in the microchannels is very slow due to the behavior of the fluid, depending on the diffusion at the contact surface. The efficiency is also very low.

In order to overcome this, the emulsion is continuously formed by using a fluid that is not mixed with the reactants, and then turbulent flow is induced in a limited space to mix the reactants. As shown in FIG. 17 (b), the reactants A and B do not proceed until they meet the oil, but after being formed into an emulsion, a) and d) pass through the bent channel form very quickly. Will be done. The emulsion that rubs against the wall flows inwardly as opposed to the direction of movement. In the case of a) and c), both emulsions are touched and the mixing proceeds at the same time, resulting in relatively slow mixing. However, in case of b) and d), the contact surface of the other side is very low compared to the one side, which enables cross-mixing of the reactants. Repetition of such intervals allows for very fast mixing.

The concept of the biopolymer used in the present invention is as follows. Biopolymer refers to a polymer material having biocompatibility and biodegradability or that can be directly extracted from nature. For example, alginate or carrageenan can be used for drug delivery, cosmetics, and food additives.

Fabrication of specific microfluidic chips used in the present invention is as described in FIG. More specifically, first, the glass substrate is washed with an organic solvent, and then PDMS is spin coated (6000 rpm, 60 seconds) thereon. The coated surface is partially cured in the oven (50 minutes, 65 ° C.) to induce stickiness. PDMS molds containing channels through which fluid can flow (curing PDMS on a silicon master obtained through photolithography and then peeled off to obtain PDMS molds containing channels) are placed on a sticky surface and additionally in an oven The microfluidic chip can be manufactured by curing (2 hours, 65 ° C.) to form a completely adhered microfluidic channel.

The present invention provides a microfluid produced by a method selected from the group consisting of a simple microflow focusing method, a microfluidic focus flow method including a mixed channel, and a biased shear force driven microflow method. Provided is a method for synthesizing monodisperse beads using a microfluidic channel structure, which comprises synthesizing uniform monodisperse beads using a chip.

In the method for synthesizing monodisperse beads using the microfluidic channel structure of the present invention, the method is preferably a microfluidic method using shear stress.

In addition, in the method for synthesizing monodisperse beads using the microfluidic channel structure of the present invention, the monodisperse beads are preferably hydrogel beads, and the hydrogel is more preferably an alginate polymer gel reacted with calcium as a crosslinking agent. In this case, the synthesis of the hydrogel bead is first injected with hexadecane (hexadecane) in the inlet of the microfluidic chip, and then injected with alginate and calcium chloride (CaCl ₂ ), it is synthesized by their reaction Most preferred.

In addition, in the method of synthesizing monodisperse beads using the microfluidic channel structure of the present invention, the synthesis method preferably prevents adhesion of the beads in the channel by rapid reaction, and the microfluidic channel has a channel surface. It is desirable to have the same hydrophobicity and to withstand the pressure drop due to reactant injection, and the microfluidic chip preferably has the same hydrophobicity on four sides.

In addition, in the method of synthesizing monodisperse beads using the microfluidic channel structure of the present invention, the microfluidic chip may comprise the steps of: i) washing the glass substrate with an organic solvent and spin coating the PDMS thereon; Ii) curing the coated surface to induce adhesion; Iii) placing a PDMS mold including a channel through which fluid can flow on the tacky surface; And iii) further curing the mold to form a fully bonded microfluidic channel, wherein the PDMS mold including the channel is made by curing PDMS on a silicon master obtained through photolithography. It is more preferable.

The characteristic of the method of synthesizing monodisperse beads of the present invention is that the reaction rate is very slow when the reaction proceeds depending on the diffusion, so that the mixture is completely mixed in microseconds using a rapidly bent channel in a shear stress system. This is to increase the reaction rate.

The present invention provides a simple microfluidic focus flow method (microflow focusing 1: FIG. 2), a microfluidic focus flow method containing a mixed channel (microflow focusing 2: FIG. 3), and a microfluidic method using shear stress (biasedshear force driven microflow 3). : Characterization of polymer hydrogel beads synthesized using microreactors having different microfluidic structures of FIG. 4) and fusion of hydrogel beads synthesized by developing a new synthesis method for monodisperse polymer hydrogel beads The present invention provides a method for preventing bead synthesis and easily implementing bead synthesis having a very uniform physical particle size of the beads and excellent mechanical strength of the particles.

In order to produce the monodisperse micro hydrogel beads of the present invention, it is necessary to fabricate a microreactor having the same surface properties, and the present inventors have found the structure and surface properties of the optimum microchannel through the examples, By using this, it was possible to obtain beads of high efficiency through economical and time saving by increasing production speed and dispersing as much as possible.

The reaction mechanism of the two reactants in the channel for producing the hydrogel beads can be briefly shown in FIG. 12. Alginate, a hydrogel used in the present invention, reacts with calcium, which is a crosslinking agent, to form a polymer gel (FIG. 12).

The method for producing an effective hydrogel bead according to the present invention can be achieved through optimized mixing and prevention of coalescence according to the channel structure, and it is basically uniform to a desired product by injection of a solid and stable reactant by a bonding method of a micro device. And stable beads can be formed.

The present invention also provides monodisperse beads prepared by the above method.

In addition, the present invention provides a method for manufacturing a microfluidic chip, i) manufacturing a microchannel to withstand the pressure drop due to reactant injection while the channel surface has the same hydrophobic character; Ii) injecting the reactants into the inlet of the microchannels; And iii) analyzing the by-products generated from the microchannels.

The manufacturing process of the microfluidic chip of the present invention is as follows. In order to manufacture the microfluidic chip, a PDMS mold is prepared and the glass to be used as the bottom surface of the chip is cleaned. The PDMS mold can be obtained by mixing PDMS prepolymer and curing agent in a ratio of 10: 1 to the silicon master with channels formed by photolithography and separating from the silicon master after thermal curing at 65 ° C. for 2 hours. 1 and 11

The cleaned glass substrate is spincoated with PDMS prepolymer and thermally cured at 65 ° C. for 50 minutes. During that time, the PDMS will not fully cure and the surface will be tacky. The PDMS containing the previously prepared channels can be bonded to the tacky glass surface and completely cured for 2 hours to complete one microfluidic chip.

In the drawing for the chip of the present invention, the microchannel structure of the present invention uses an autocad program. 2 to 4 show the effect of microfluidic channel structure on monodisperse hydrogel beads as the structure of each microchannel used in the present invention. 2 and 3 is a microfluidic focal flow method, Figure 2 has a structure in which the reaction section is a linear structure, Figure 3 has a structure in which a straight line of the same width is transformed into a curved shape. FIG. 4 is designed to induce a very fast reaction through recirculation within a limited space by forming a spherical droplet with a continuous shear force in which two reactants flowing in a laminar flow in a microfluidic flow method using a shear stress do not mix. All microchips used in the present invention consist of a passage for continuous phase injection of hexadecane and a passage for injection of hydrophilic reactants (alginate, calcium chloride).

In the fluid injection method of the present invention, both hydrophobic and hydrophilic non-mixing fluids were respectively injected using a micro syringe pump (Harvard apparatus PHD 2000, USA). Disposable syringe of 1 ml volume contains two hydrophilic reactants, alginate, calcium chloride (CaCl ₂ ) and hydrophobic hexadecane, and each solution is micro-treated using a Tygon tube. The chip was injected. Alginate and calcium chloride are injected separately through two inlets as a dispersed phase and hydrophobic solution (hexadecane) is injected into the remaining inlets in the continuous phase (FIG. 13).

In the bead analysis method of the present invention, the observation of the formed beads was performed using a microscope (NIKON, SMZ800, Japan) equipped with a color CCD camera. Image J and Image Pro (Media cybermetic) were used to confirm the distribution of generated bead sizes. Bead size distribution was calculated as the coefficient of variation (CV), defined as the standard deviation divided by the mean size.

The present inventors manufactured the microfluidic chip using the bonding method using the adhesive used in the present invention. First, the glass substrate is cleaned with isopropyl, acetone, and ethanol, respectively. After spin-coating the PDMS prepolymer, the channel is blocked by the capillary force when combined with the PDMS including the channel. By preventing, the PDMS undergoes thermal curing but not fully cured to form a tacky thin film, and the PDMS having a fluid-fluid structure is bonded to the tacky surface to complete a microfluidic chip having hydrophobic front surface. Injecting the fluid required for the reaction while adjusting the flow rate using the syringe pump to the prepared microfluidic chip. The connection between the microfluidic chip and the syringe pump is directly connected to the formed inlet port so that the pressure of the injected fluid does not leak. By blocking the adsorption as much as possible to prevent the variables caused in the formation of hydrogel beads.

To determine the effect of the structure of the microfluidic channel used to form monodisperse hydrogel beads in a rapid and stable form, a syringe pump is used to inject the required reactants and hydrophobic fluid. When the fluid is injected and beads are formed by shearing hydrophobic fluids, a stable bead generation region is designated based on the time when the hydrophobic fluid is maintained in a stable form. Find.

As a result of investigating the influence of the fraction of the dispersed phase at various fluid flow rates to determine the optimum state for forming stable hydrogel beads, the simple microfluidic focal flow method results in the hydrogel beads being formed in a stable form only in certain regions (FIG. 5). Reference). As the flow velocity of the continuous phase increases, the area for forming stable beads increases, and when the flow velocity of the continuous phase decreases, it is difficult for the fluid of the dispersed phase to flow in laminar flow or to form droplets. The simple microfluidic focal flow method can form hydrogel beads continuously but there is not enough mixing between the two reactants of alginate and calcium chloride. The boundary between the two reactants shows the shape of a hydrogel bead clearly indicating that mixing was not done properly (see FIG. 6). Inadequate hydrogel beads are present in various sizes due to coalescence between the beads with weak mechanical strength at the exit (see FIG. 7). In the simple microfluidic focal flow channel structure, calcium chloride only reacts with alginate depending on diffusion. Diffusion-dependent reactions require longer reaction times, which require longer channels. 7 illustrates a problem of the microfluidic focal flow method through an image of non-uniform beads. In addition, it can be confirmed through the micrograph that the channel method is a reaction that depends only on the diffusion inside the channel (see FIG. 18). That is, FIG. 18 is visually proved by adding a dye to the reactants, and the two reactants have a very low reaction rate as the reaction proceeds depending only on diffusion. Thus, the emulsion formed indicates that it does not mix uniformly between the two reactants over time and remains in a separated form.

The microfluidic focal flow method with mixed channels adds a bent section for more effective mixing to the previously used simple microfluidic focal flow method. The mixing efficiency between alginate and calcium chloride is improved, but the hydrogels that are not completely reacted merge in a wide bent section (see FIG. 8). The reason why beads are adhered in the channel is, firstly, that the faster the flow rate of the beads behaving in the channel, the more randomly they behave in the channel space and the pressure drop in the channel causes the beads to accumulate. The random behavior of the beads thus produced results in coalescence in the channel. Second, fluctuations in the generated hydrogel bead movement increase the likelihood of collision in the channel structure. Third, despite the addition of the mixing channel is not enough mixing because the mechanical strength falls.

The problem found in the microfluidic focus method is that the mixing efficiency is low and adhesion occurs due to the pressure drop present in the channel depending on the diffusion at the interface. Unlike the two channel structures previously used, the microfluidic method using shear stress does not cause the problem of mixing and coalescence. The hydrophilic hydrogel is formed in the vertical direction by the shear force of the hydrophobic continuous phase (see FIG. 9A). The hydrogel formed is rapidly mixed by recycling in the channel. Compared to the microfluidic focal flow containing the previous mixing channel, the narrow channel structure not only increases the mixing efficiency but also can be sufficiently mixed to prevent coalescence. Micrograph showing the hydrogel obtained at the exit shows that no coalescence between hydrogels occurred (see FIG. 9B). As a result of measuring the size and the dispersion of the hydrogel obtained from the outlet, the dispersion was very low, monodispersed and excellent in mechanical strength (see FIG. 10).

As described above, the microfluidic focal flow method is generally used to form microbeads, but it is impossible to rely on diffusion at an interface to form hydrogel beads. Therefore, in order to solve this problem, the present invention was able to construct a channel structure that can produce monodisperse hydrogel beads by adopting a microfluidic method using shear stress and preventing adhesion through rapid mixing in a channel.

The method for synthesizing monodisperse beads using the microfluidic channel structure of the present invention can effectively produce monodisperse beads such as biocompatible monodisperse hydrogel beads which can be widely used in biotechnology and medicine. It can be usefully used to fabricate microfluidic chips, such as micro reactors.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Fabrication of Microfluidic Chips

The present inventors manufactured the microfluidic chip using the bonding method using the adhesive used in the present invention. First, the glass substrate to be used as the bottom surface is cut to length X length (2.5 cm X 5.0 cm), and each 5 minutes cleaning process with isopropyl, acetone, and ethanol is followed by PDMS prepolymer. (prepolymer) was spin-coated (spin-coating) at 6000 rpm for 1 minute. This process is to prevent the blockage of the channel by the capillary force when combined with the PDMS containing the channel. As a result, the PDMS was thermally cured at 65 ° C. for 50 minutes but was not completely cured to form a tacky thin film. PDMS having a microfluidic structure was bonded to the adhesive surface to complete a microfluidic chip having a hydrophobic front surface (FIG. 11).

More specifically, as shown in FIG. 11, first, the glass substrate was washed with an organic solvent, and then PDMS was spin coated (6000 rpm, 60 seconds) thereon. The coated surface was partially cured in the oven (50 minutes, 65 ° C.) to induce stickiness. PDMS molds containing channels through which fluid can flow (curing PDMS on a silicon master obtained through photolithography and then peeled off to obtain PDMS molds containing channels) are placed on a sticky surface and additionally in an oven Curing (2 hours, 65 ℃) to produce a microfluidic chip in which a microfluidic channel adhered completely.

Example 2 Injection of Fluid Required for Reaction with a Syringe Pump

The microfluidic chip prepared in Example 1 was placed on a microscope equipped with a color CCD camera (NIKON, SMZ800, Japan), and the fluid required for the reaction was injected while controlling the flow rate using a syringe pump. The connection of the microfluidic chip and the syringe pump was made by using a Tygon tube to connect the syringe needle directly to the formed inlet so that the pressure of the injected fluid did not leak. The injection of fluid allowed the continuous phase hexadecane to flow into the channel first to prevent the adsorption of reactants alginate and calcium chloride into the channel to prevent the variables caused in the formation of hydrogel beads.

Example 3 Optimal Channel Structure for Monodisperse Hydrogel Bead Formation

In order to investigate the effect of the structure of the microfluidic channel used to form monodisperse hydrogel beads in a rapid and stable form, a syringe pump was used to inject the required reactants and hydrophobic fluids. To determine the mechanism and shape of the beads in the channels, the microfluidic chip was placed on a microscope. When the fluid was injected and beads were formed by shearing of hydrophobic fluids, a stable bead generation region was designated based on the time when it lasted for more than 5 minutes in a stable form, and found differences and problems according to the respective channel structures. The optimal channel structure was found.

Example 4 Simple Fluid Flow Focus Flow Method

The reactant was injected into the microfluidic chip having various channel structures prepared in Example 1 using a syringe pump. The simple microfluidic focal flow method first proposed in the present invention is known to be very easy to form stable water droplets. The influence of the fraction of the dispersed phase (volume of the dispersed phase / (volume of the dispersed phase + volume of the continuous phase)) at various fluid flow rates was investigated to determine the optimal state for forming stable hydrogel beads. 5 is a graph showing that the hydrogel beads are formed in a stable form only in a specific region. As shown in FIG. 5, the present invention can be divided into two types, a stable section I and an unstable section II. For example, at high dispersion phase fractions (> 0.6) and low dispersion phase fractions (<0.2), stable hydrogel beads cannot be produced due to the imbalance, viscosity, and wettability between interfaces. However, in the range of 0.2-0.6, the fraction is the optimal value for obtaining stable hydrogel beads. It can be seen that as the flow velocity of the continuous phase increases, the zone for forming stable beads increases. On the contrary, when the flow velocity of the continuous phase decreases, it becomes difficult for the fluid in the dispersed phase to flow in laminar flow or to form water droplets.

The hydrogel beads can be formed continuously through a simple microfluidic focal flow, but there is not enough mixing between the two reactants of alginate and calcium chloride (FIG. 5). 6 and 7 illustrate this in a simple manner, and FIG. 6 shows the shape of the hydrogel beads having clear boundaries between two reactants in the channel, which shows that mixing was not performed properly. FIG. 7 shows that poorly mixed hydrogel beads exist in various sizes due to adhesion between the beads having weak mechanical strength at the outlet. As a result, in the simple microfluidic focal flow channel structure of FIG. 2, calcium chloride reacts with alginate only depending on diffusion. Diffusion-dependent reactions require longer reaction times, which require longer channels.

Example 5 Microfluidic Focal Flow Method Containing Mixed Channels

The microfluidic focal flow method containing the mixing channel adds a bent section for more effective mixing to the previously used simple microfluidic focal flow method (FIG. 3). Although the mixing efficiency between alginate and calcium chloride was improved further, the hydrogels that were not completely reacted merged over a wide bent section (FIG. 8). There are three reasons why beads can coalesce in a channel. The first is that the faster the flow rate of the beads behaving in the channel, the random behavior in the channel space. In addition, the pressure drop occurring in the channel causes the beads to accumulate. Eventually, the random behavior of the accumulated beads causes adhesion within the channel. The second generated variation in hydrogel bead movement increases the likelihood of collision in the channel structure. Thirdly, although the mixing channel is added, the mechanical strength is lowered because the mixing is not enough as the previous channel.

Example 6 Microfluidic Method Using Shear Stress

The problem found in the microfluidic focus method is that the mixing efficiency is low and adhesion occurs due to the pressure drop present in the channel depending on the diffusion at the interface. The microfluidic method using the shear stress that solves the problems seen in the two channel structures used previously solves the problems of mixing and coalescence discussed in the previous two. The hydrophilic hydrogel is formed in the vertical direction by the shear force of the hydrophobic continuous phase (FIG. 9A). The hydrogel formed is rapidly mixed by recycling in the channel. Compared to the microfluidic focal flow containing the previous mixing channel, the narrow channel structure not only increases the mixing efficiency but also can be sufficiently mixed to prevent coalescence. FIG. 9 is a micrograph showing the hydrogel obtained at the exit, which shows that no coalescence between hydrogels occurred (FIG. 9B). The hydrogel obtained at the exit was sized using an image analysis program and the dispersion degree was measured (FIG. 10). As a result, the degree of dispersion obtained a value of 2.5% or less, it can be seen that it is very monodisperse and also excellent mechanical strength.

As described above, the microfluidic focal flow method is generally used to form microbeads, but it is not possible to rely on diffusion at the interface to form hydrogel beads. Therefore, in order to solve this problem, the present invention was able to construct a channel structure that can produce monodisperse hydrogel beads by adopting a microfluidic method using shear stress and preventing adhesion through rapid mixing in a channel.

1 is an actual photograph of a microfluidic chip used in the present invention.

2 is a CAD drawing photograph of a simple microfluidic focusing method (microflow focusing 1).

FIG. 3 is a CAD drawing photograph of a microfluidic focusing method 2 containing mixed channels.

4 is a CAD drawing photograph of a microsedshear force driven microflow 3 using shear stress.

FIG. 5 is a graph showing a region where stable beads are generated when the simple microfluidic focal flow method is used.

Figure 6 is a micrograph showing the shape of the beads produced when using the simple microfluidic focal flow method.

FIG. 7 is a micrograph at the channel exit showing problems when using the simple microfluidic focal flow method. FIG.

FIG. 8 is a micrograph showing the problem seen when using a microfluidic focal flow method containing mixed channels.

Figure 9 is a micrograph showing the bead shape in the channel and the beads obtained at the exit when using the microfluidic method using the shear stress.

FIG. 10 is a graph measuring polydispersity after measuring the length of the beads obtained at the exit using an image program.

Figure 11 briefly shows the manufacturing process of the microfluidic chip produced in the present invention.

12 is a reaction mechanism of two reactants in a channel for producing a hydrogel bead used in the present invention, wherein the hydrogel alginate used in the present invention reacts with calcium as a crosslinking agent to form a polymer gel. Formula shown.

13 is a cross-sectional view showing a method of injecting a reactant into the microfluidic chip manufactured in the present invention.

14 is a microfluidic channel structure using a simple microfluidic focal flow method.

15 is a microfluidic channel structure using a microfluidic focal flow method with mixed channels.

16 is a microfluidic channel structure using a microfluidic method using shear stress.

Figure 17 is a schematic diagram illustrating the rapid reaction used in the present invention, wherein (a) is a step of injecting reactants A, B into the microchannel, (b) is continuously using a fluid that does not mix with the reactants After forming an emulsion, turbulence is induced within a defined space to indicate the degree of mixing between reactants.

FIG. 18 is a micrograph in a channel showing the problem of using a simple microfluidic focal flow method, which is a reaction that depends only on diffusion within the channel.

Claims (12)

Iii) washing the glass substrate with an organic solvent and spin coating the PDMS thereon; Ii) curing the coated surface to induce adhesion; Iii) placing a PDMS mold including a channel through which fluid can flow on the tacky surface; And Iv) additionally curing the mold to form a fully adhered microfluidic channel; a microfluidic channel structure comprising synthesizing uniform monodisperse beads using a microfluidic chip manufactured by the method comprising Synthesis method of monodisperse beads using. The method of claim 1, The monodisperse beads are hydrogel beads, the method of synthesizing monodisperse beads using a microfluidic channel structure. The method of claim 2, The hydrogel is a method of synthesizing monodisperse beads using a microfluidic channel structure, characterized in that the alginate polymer gel reacted with calcium as a crosslinking agent. The method of claim 2, Synthesis of the hydrogel beads is first injected with hexadecane (hexadecane) in the inlet of the microfluidic chip, and then injected with alginate (alginate) and calcium chloride (CaCl ₂ ), characterized in that synthesized by their reaction Synthesis method of monodisperse beads using microfluidic channel structure. The method of claim 1, The microfluidic channel has the same hydrophobic characteristics as the surface of the channel has a characteristic of withstanding the pressure drop due to the injection of reactants, characterized in that the monodisperse beads using a microfluidic channel structure. The method of claim 1, The microfluidic chip has a method of synthesizing monodisperse beads using a microfluidic channel structure, characterized in that the slope has the same hydrophobicity. The method of claim 1, The PDMS mold containing the channel is a method of synthesizing monodisperse beads using a microfluidic channel structure, characterized in that the PDMS is produced by curing the PDMS on the silicon master obtained through photolithography. delete delete delete delete delete

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