Microdroplets, methods of making and using the same

Abstract

A microdroplet, its preparation method, and its application are disclosed. The microdroplet has a core-shell structure, wherein the core is an aqueous droplet containing biomolecules, and the shell is a cross-linked polymer of a surfactant, the shell encapsulating the core. The microdroplet is capable of free movement in the oil phase in which it is prepared. The microdroplets of this disclosure have the advantages of good stability and uniformity, and the microdroplets are capable of free movement.

Description

Technical Field

[0001] This disclosure relates to, but is not limited to, the field of biotechnology, and particularly to a microdroplet, its preparation method, and its application. Background Technology

[0002] Microdroplets have important applications in drug controlled release, virus detection, particulate material synthesis, and catalysts. Microdroplets are often used as microreactors to achieve biochemical reactions, rapid reagent mixing, and microparticle synthesis. For example, gene chips, protein chips, single-cell analysis, and droplet digital polymerase chain reaction (ddPCR) technology all require the use of microdroplets. Summary of the Invention

[0003] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of protection of this application.

[0004] This disclosure provides a microdroplet having a core-shell structure, wherein the core is an aqueous droplet containing biomolecules and the shell is a cross-linked polymer of a surfactant, the shell encapsulating the core, and the microdroplet is able to move freely in the oil phase in which the microdroplet is prepared.

[0005] In an exemplary embodiment of this disclosure, the shell may include a hydrophilic first shell and an oleophilic second shell, the first shell being formed by the hydrophilic end of the crosslinked polymer of the surfactant, the second shell being formed by the oleophilic end of the crosslinked polymer of the surfactant, the first shell extending into the core, the second shell covering the core and connected together with the first shell, the second shell having a crosslinked structure.

[0006] In exemplary embodiments of this disclosure, the biomolecules may include any one or more of cells, nucleic acids, proteins, carbohydrates, and fats.

[0007] This disclosure also provides a method for preparing microdroplets, comprising:

[0008] The oil phase, surfactant, and aqueous phase containing biomolecules are mixed to form aqueous droplets containing biomolecules.

[0009] The surfactant is initiated by an initiator to undergo a crosslinking or polymerization reaction, forming a crosslinked polymer of the surfactant coating the aqueous droplet containing biomolecules on the surface of the droplet, thus obtaining the microdroplet.

[0010] In an exemplary embodiment of this disclosure, the preparation method may include:

[0011] The oil phase, the monomer used to prepare the surfactant, the initiator, and the aqueous phase containing biomolecules are mixed.

[0012] The initiator initiates the polymerization reaction of the monomers used to prepare the surfactant to form the surfactant;

[0013] In the presence of the surfactant, aqueous droplets containing biomolecules are formed;

[0014] The initiator initiates the surfactant to undergo a crosslinking or polymerization reaction, forming a crosslinked polymer of the surfactant coating the aqueous droplet containing the biomolecule on the surface of the droplet, thus obtaining the microdroplet.

[0015] In exemplary embodiments of this disclosure, the crosslinking or polymerization reaction of the surfactant can be carried out on the oil phase side of the interface between the aqueous phase and the oil phase.

[0016] In an exemplary embodiment of this disclosure, the surfactant may be of type AB or type ABA, where A is the lipophilic end of the surfactant containing unsaturated bonds and B is the hydrophilic end of the surfactant.

[0017] In exemplary embodiments of this disclosure, the unsaturated bond may include any one or more of carbon-carbon double bonds and carbon-carbon triple bonds.

[0018] In an exemplary embodiment of this disclosure, the monomer used to prepare the surfactant may be a hydrophilic monomer.

[0019] In exemplary embodiments of this disclosure, the hydrophilic monomer may include any one or more hydrophilic vinyl monomers.

[0020] In exemplary embodiments of this disclosure, the hydrophilic vinyl monomer may include any one or more of acrylamide, acrylic acid, methacrylic acid, vinylpyridine, styrene, N-vinylpyrrolidone, a tertiary ammonium salt of dimethylaminoethyl methacrylate, a quaternary ammonium salt of dimethylaminoethyl methacrylate, and hydroxyethyl acrylate.

[0021] In an exemplary embodiment of this disclosure, the initiator may be a lipophilic initiator.

[0022] In exemplary embodiments of this disclosure, the lipophilic initiator may include any one or more of hydroperoxide, benzoyl peroxide, dialkyl peroxide, diacyl peroxide, tertiary amine, naphthenate, thiol, organoboronide, organometallic compound, and azo oil-soluble initiator.

[0023] In exemplary embodiments of this disclosure, the organoboride may include triethylboron.

[0024] In exemplary embodiments of this disclosure, the organometallic compound may include any one or more of aluminum-containing organometallic compounds and copper-containing organometallic compounds.

[0025] In exemplary embodiments of this disclosure, the aluminum-containing organometallic compound may include triethylaluminum.

[0026] In exemplary embodiments of this disclosure, the copper-containing organometallic compound may include cuprous naphthate.

[0027] In exemplary embodiments of this disclosure, the azo oil-soluble initiator may include any one or more of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, and dimethyl azobisisobutyrate.

[0028] In an exemplary embodiment of this disclosure, the initiator may be an oleophilic photoinitiator.

[0029] In an exemplary embodiment of this disclosure, the surfactant may account for 0.5% to 2% of the weight of the oil phase.

[0030] In an exemplary embodiment of this disclosure, the weight of the initiator may be 0.1% to 1% of the weight of the monomer used to prepare the surfactant.

[0031] In exemplary embodiments of this disclosure, the method used to form aqueous droplets containing biomolecules can be an active method or a passive method.

[0032] In exemplary embodiments of this disclosure, the active method may include electrowetting, dielectric electrophoresis, and thermocapillary methods.

[0033] In exemplary embodiments of this disclosure, the passive method may include the T-channel method, the flow focusing method, and the coaxial flow focusing method.

[0034] In an exemplary embodiment of this disclosure, the method for preparing the microdroplets can be performed on a microfluidic chip.

[0035] This disclosure also provides the applications of the microdroplets described above in molecular diagnostics, immunobiochemistry, cell culture, polymer synthesis, single-cell analysis, single-cell sorting, single-cell sequencing library construction, and drug delivery.

[0036] In an exemplary embodiment of this disclosure, the application may be an application in microdroplet digital PCR.

[0037] The microdroplets of this disclosure have the advantages of good stability and uniformity. On the one hand, by encapsulating the aqueous droplet as a core inside the shell, the breakage and fusion of the aqueous droplet can be avoided. On the other hand, the cross-linked polymer formed by cross-linking or polymerization with surfactants adjusts the interfacial tension between the oil phase and the aqueous phase. After the surfactant is cross-linked, its flow / exchange capacity decreases or even disappears, so it will hardly connect with the cross-linked polymer on the surface of other aqueous droplets. This is equivalent to fixing the cross-linked polymer to the aqueous droplet it is connected to, which can further improve the stability and uniformity of the microdroplets.

[0038] Other features and advantages of this disclosure will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the disclosure. Other advantages of this disclosure may be realized and obtained by means of the methods described in the description and the accompanying drawings. Attached Figure Description

[0039] The accompanying drawings are used to provide an understanding of the technical solutions of this disclosure and form part of the specification. They are used together with the embodiments of this disclosure to explain the technical solutions of this disclosure and do not constitute a limitation on the technical solutions of this disclosure.

[0040] Figure 1 A schematic diagram of the structure of a microdroplet, which is an exemplary embodiment of this disclosure;

[0041] Figure 2 This is a schematic diagram illustrating the formation process of microdroplets, which is an exemplary embodiment of the present disclosure.

[0042] Figure 3 A schematic diagram illustrating the stabilization process of microdroplets as an exemplary embodiment of this disclosure;

[0043] Figure 4 This is a schematic diagram illustrating the formation process of microdroplets, which is another exemplary embodiment of this disclosure.

[0044] Figure 5 This is a schematic diagram of the stabilization process of microdroplets, which is another exemplary embodiment of this disclosure.

[0045] The symbols in the attached diagram have the following meanings:

[0046] 1-Biomolecule; 2-Aqueous phase; 3-Surfactant; 4-Oil phase; 5-Aqueous droplet; 6-Microdroplet; 7-Hydrophilic monomer; 8-Oleophilic initiator; 10-Core; 20-Shell; 21-First shell; 22-Second shell. Detailed Implementation

[0047] The embodiments described herein can be implemented in many different forms. Those skilled in the art will readily understand that the implementation methods and content can be varied in many ways without departing from the spirit and scope of this disclosure. Therefore, this disclosure should not be construed as limited to the contents described in the following embodiments. Unless otherwise specified, the embodiments and features described in this disclosure can be arbitrarily combined with each other.

[0048] In the accompanying drawings, the size of constituent elements, the thickness of layers, or areas may sometimes be exaggerated for clarity. Therefore, any implementation of this disclosure is not necessarily limited to the dimensions shown in the drawings, and the shapes and sizes of components in the drawings do not reflect true proportions. Furthermore, the drawings schematically illustrate ideal examples, and any implementation of this disclosure is not limited to the shapes or values ​​shown in the drawings.

[0049] Microfluidics refers to the technology of manipulating fluids at the micrometer scale. This technology can miniaturize the basic functions of chemical and biological laboratories onto a chip of just a few square centimeters, hence the name "lab-on-a-chip." Droplet microfluidics technology was developed based on traditional continuous flow microfluidic systems. Droplet microfluidics has wide applications in biomedicine. For example, by precisely manipulating microdroplets in a reaction, it is possible to reduce the consumption of reaction reagents and improve reagent utilization.

[0050] In recent years, with the rapid development of droplet microfluidics technology, its application in single-cell analysis has attracted increasing attention. Droplets, as single-cell microreactors, can effectively control diffusion, accelerate mixing, and improve detection sensitivity, and have been successfully applied in various single-cell analyses. The single-cell analysis process based on microfluidic droplet technology involves forming microdroplets in the oil phase, ensuring each droplet contains one cell through microfluidic channels, and then using techniques such as fluorescence signals and microscopic imaging to sort the cell-containing microdroplets. Further analysis of single cells is achieved through intradroplet digital polymerase chain reaction (PCR), sequencing, and cell expression antibody analysis. This method can be applied not only to single-cell analysis, sorting, and library construction, but also extended to digital PCR, liposome drug preparation, and other fields. This method requires surfactants to have the ability to rapidly diffuse, assemble, and stabilize at the oil-water interface to form a structurally stable and uniformly sized water-in-oil structure. Otherwise, the microdroplets are prone to breakage and fusion after formation, affecting the microdroplet size and reducing the accuracy of the final bioanalytical data. Furthermore, cell sorting is often achieved through high-pressure liquid jets or electric fields, which poses a significant challenge to the stability of microdroplets. Reducing the lateral forces (jet or electric field) during sorting to minimize damage to microdroplets would decrease sorting efficiency and analytical throughput. Therefore, developing rapid microdroplet stabilization techniques is crucial for expanding the applications of droplet microfluidic platforms.

[0051] This disclosure provides a microdroplet that may have a core-shell structure, wherein the core is an aqueous droplet containing biomolecules, and the shell is a cross-linked polymer of a surfactant, the shell covering the core, and the microdroplet is able to move freely in the oil phase in which the microdroplet is prepared.

[0052] The microdroplets of this disclosure have the advantages of good stability and uniformity. On the one hand, by encapsulating the aqueous droplet as a core inside the shell, the breakage and fusion of the aqueous droplet can be avoided. Moreover, the overall elasticity and deformability of the microdroplet are reduced compared to the aqueous droplet, which is beneficial for data analysis. On the other hand, surfactants flow or exchange at the oil-water interface, causing the surfactant on the surface of one aqueous droplet to easily connect with the surfactant on the surface of another aqueous droplet, thereby causing the two aqueous droplets to fuse together and affecting the stability and uniformity of the aqueous droplets. However, the microdroplets of this disclosure use cross-linked polymers formed by cross-linking or polymerization of surfactants to regulate the interfacial tension between the oil and water phases. After the surfactant is cross-linked, its flow / exchange capacity decreases or even disappears. Therefore, it will hardly connect with the cross-linked polymers on the surface of other aqueous droplets, which is equivalent to fixing the cross-linked polymer to the aqueous droplet it is connected to, which can further improve the stability and uniformity of the microdroplet.

[0053] When using microdroplets from the embodiments of this disclosure for cell analysis and sorting, increasing the analysis speed and throughput, or increasing the lateral force of sorting, can effectively reduce the impact on the stability of the microdroplets, which is of great practical significance.

[0054] The microdroplets in this embodiment can move freely within their preparation system. Therefore, when processing the microdroplets using fluorescence signals, either flow cytometry or imaging can be employed. Theoretically, flow cytometry has a lower detection limit and a significantly higher throughput than imaging. If further processing is required, such as droplet demulsification, droplet re-sorting, and recollection of some target droplets, flow cytometry is more advantageous.

[0055] In an exemplary embodiment of this disclosure, the shell may include a hydrophilic first shell and an oleophilic second shell, the first shell being formed by the hydrophilic end of the crosslinked polymer of the surfactant, the second shell being formed by the oleophilic end of the crosslinked polymer of the surfactant, the first shell extending into the core, the second shell covering the core and connected together with the first shell, the second shell having a crosslinked structure.

[0056] Figure 1 This is a schematic diagram of the structure of a microdroplet, which is an exemplary embodiment of this disclosure. Figure 1 As shown, the microdroplet has a core-shell structure. The aqueous droplet containing biomolecules is the core 10, and the cross-linked polymer of the surfactant is the shell 20. The shell 20 includes a hydrophilic first shell 21 and an oleophilic second shell 22. The first shell 21 is formed by the hydrophilic end of the cross-linked polymer of the surfactant, and the second shell 22 is formed by the oleophilic end of the cross-linked polymer of the surfactant. The first shell 21 extends into the core, and the second shell 22 covers the core and is connected to the first shell 21. The second shell 22 has a cross-linked structure.

[0057] In exemplary embodiments of this disclosure, the biomolecules may include any one or more of cells, nucleic acids, proteins, carbohydrates, and fats.

[0058] In the exemplary embodiments of this disclosure, the aqueous droplet containing biomolecules refers to a droplet formed from an aqueous phase containing biomolecules. For example, the aqueous droplet may contain biomolecules and water, and may also contain other aqueous additives, such as biomolecule stabilizers; or, the aqueous droplet may contain only aqueous biomolecules.

[0059] In exemplary embodiments of this disclosure, the shell may be comb-shaped; for example, the first shell layer may be comb-shaped, and the second shell layer may be comb-shaped.

[0060] This disclosure also provides a method for preparing microdroplets, comprising:

[0061] The oil phase, surfactant, and aqueous phase containing biomolecules are mixed to form aqueous droplets containing biomolecules.

[0062] The surfactant is initiated by an initiator to undergo a crosslinking or polymerization reaction, forming a crosslinked polymer of the surfactant coating the aqueous droplet containing biomolecules on the surface of the droplet, thus obtaining the microdroplet.

[0063] The microdroplet preparation method of this disclosure first forms an aqueous droplet, and then performs a cross-linking or polymerization reaction on a surfactant. The resulting cross-linked polymer of the surfactant encapsulates the aqueous droplet internally, resulting in a core-shell structure microdroplet with the aqueous droplet as the core and the cross-linked polymer of the surfactant as the shell. On the one hand, the aqueous droplet being encapsulated internally prevents it from breaking down and fusing. On the other hand, since the surfactant has already undergone a cross-linking or polymerization reaction, its flow / exchange capacity decreases or even disappears. Therefore, the resulting cross-linked polymer is unlikely to connect with cross-linked polymers on the surface of other aqueous droplets, effectively fixing the cross-linked polymer to the aqueous droplet it is connected to, which can further improve the stability and uniformity of the microdroplet.

[0064] Because the surfactants in the raw materials for microdroplet preparation can rapidly polymerize or crosslink, they can quickly form microdroplet structures even if they do not complete the diffusion, equilibrium, and assembly process at the interface between the oil and water phases.

[0065] In an exemplary embodiment of this disclosure, the preparation method may include:

[0066] The oil phase, the monomer used to prepare the surfactant, the initiator, and the aqueous phase containing biomolecules are mixed.

[0067] The initiator initiates the polymerization reaction of the monomers used to prepare the surfactant to form the surfactant;

[0068] In the presence of the surfactant, aqueous droplets containing biomolecules are formed;

[0069] The initiator initiates the surfactant to undergo a crosslinking or polymerization reaction, forming a crosslinked polymer of the surfactant coating the aqueous droplet containing the biomolecule on the surface of the droplet, thus obtaining the microdroplet.

[0070] In exemplary embodiments of this disclosure, the crosslinking or polymerization reaction of the surfactant can be carried out on the oil phase side of the interface between the aqueous phase and the oil phase. Carrying the crosslinking or polymerization reaction of the surfactant on the oil phase side of the interface can maintain the activity of biomolecules in the aqueous droplets and reduce the impact of the crosslinking or polymerization reaction or the initiator on the biomolecules. Furthermore, the polymerization reaction carried out on the oil phase side of the interface is a random polymerization, and the resulting polymer is essentially a crosslinked polymer, capable of encapsulating the aqueous droplets containing biomolecules.

[0071] In an exemplary embodiment of this disclosure, the surfactant may be of type AB or type ABA, where A is the lipophilic end of the surfactant containing unsaturated bonds and B is the hydrophilic end of the surfactant.

[0072] In the description of this disclosure, the two A's in "ABA type" indicate that both ends of the surfactant are lipophilic ends containing unsaturated bonds, but it is not limited to the two ends being the same lipophilic ends. The two ends can be different, for example, the groups or unsaturated bonds of the two ends can be different.

[0073] When microdroplets of the present invention are prepared using surfactants with unsaturated bonds at their lipophilic ends, it is advantageous for the crosslinking or polymerization reactions of the surfactant to occur on the oil phase side of the interface between the oil and aqueous phases. This helps maintain the activity of the biomolecules in the core and reduces the impact of polymerization reactions or initiators on the biomolecules. Moreover, surfactants containing unsaturated bonds can undergo crosslinking reactions in the presence of an initiator without the need for additional crosslinking agents.

[0074] In exemplary embodiments of this disclosure, the unsaturated bond may include any one or more of carbon-carbon double bonds and carbon-carbon triple bonds.

[0075] In an exemplary embodiment of this disclosure, the monomer used to prepare the surfactant may be a hydrophilic monomer.

[0076] In exemplary embodiments of this disclosure, the hydrophilic monomer may include any one or more hydrophilic vinyl monomers.

[0077] In exemplary embodiments of this disclosure, the hydrophilic vinyl monomer may include any one or more of acrylamide, acrylic acid, methacrylic acid, vinylpyridine, styrene, N-vinylpyrrolidone, a tertiary ammonium salt of dimethylaminoethyl methacrylate, a quaternary ammonium salt of dimethylaminoethyl methacrylate, and hydroxyethyl acrylate.

[0078] In exemplary embodiments of this disclosure, the initiator may be a lipophilic initiator. When a lipophilic initiator is used to prepare microdroplets according to embodiments of this disclosure, it facilitates the occurrence of crosslinking or polymerization reactions of the surfactant on the oil phase side of the interface between the oil and water phases, thereby maintaining the activity of biomolecules in the core and reducing the impact of polymerization reactions or initiators on biomolecules.

[0079] In exemplary embodiments of this disclosure, the lipophilic initiator may include any one or more of hydroperoxide, benzoyl peroxide, dialkyl peroxide, diacyl peroxide, tertiary amine, naphthenate, thiol, organoboronide, organometallic compound, and azo oil-soluble initiator.

[0080] In exemplary embodiments of this disclosure, the organoboride may include triethylboron.

[0081] In exemplary embodiments of this disclosure, the organometallic compound may include any one or more of aluminum-containing organometallic compounds and copper-containing organometallic compounds.

[0082] In exemplary embodiments of this disclosure, the aluminum-containing organometallic compound may include triethylaluminum.

[0083] In exemplary embodiments of this disclosure, the copper-containing organometallic compound may include cuprous naphthate.

[0084] In exemplary embodiments of this disclosure, the azo oil-soluble initiator may include any one or more of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, and dimethyl azobisisobutyrate.

[0085] In an exemplary embodiment of this disclosure, the initiator may be an oleophilic photoinitiator.

[0086] In exemplary embodiments of this disclosure, the surfactant may account for approximately 0.5% to 2% of the weight of the oil phase. For example, the surfactant may account for approximately 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, or 2% of the weight of the oil phase.

[0087] In exemplary embodiments of this disclosure, the weight of the initiator may account for approximately 0.1% to 1% of the weight of the monomer used to prepare the surfactant. For example, the weight of the initiator may account for approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the weight of the monomer used to prepare the surfactant.

[0088] In exemplary embodiments of this disclosure, the method used to form aqueous droplets containing biomolecules can be an active method or a passive method.

[0089] In exemplary embodiments of this disclosure, the active method may include electrowetting, dielectric electrophoresis, and thermocapillary methods.

[0090] In exemplary embodiments of this disclosure, the passive method may include the T-channel method, the flow focusing method, and the coaxial flow focusing method.

[0091] In exemplary embodiments of this disclosure, the microdroplet preparation method can be performed in a microfluidic chip. For example, the microdroplets can be formed in a microfluidic chip with a cross-shaped microfluidic channel using a flow focusing method. When microdroplets of this embodiment are formed using a microfluidic chip, the basic characteristics such as the size of the microdroplets are mainly determined by the structure of the microfluidic chip, which can reduce the impact of breakage or fusion on the size of the microdroplets and improve the stability and uniformity of the microdroplets.

[0092] The microdroplets provided in this embodiment can be obtained by the microdroplet preparation method provided in the above embodiment.

[0093] Figure 2 This is a schematic diagram illustrating the formation process of microdroplets, as an exemplary embodiment of this disclosure. Figure 2 As shown, an aqueous phase 2 containing biomolecules 1 (e.g., cells) comes into contact with an oil phase 4 containing an AB-type surfactant 3 (A is the lipophilic end and B is the hydrophilic end). The lipophilic end A of the surfactant 3 extends into the oil phase 4, and the hydrophilic end B of the surfactant 3 extends into the aqueous phase 2. At the interface between the oil phase 4 and the aqueous phase 2, an aqueous droplet 5 containing biomolecules 1 is rapidly formed. Under the conditions of light and the presence of a lipophilic photoinitiator (not shown in the figure), the surfactant 3 undergoes a cross-linking reaction or polymerization reaction on the oil phase side of the interface between the oil phase 4 and the aqueous phase 2 to form a cross-linked polymer. This cross-linked polymer encapsulates the aqueous droplet containing biomolecules inside, resulting in a core-shell structured microdroplet 6 with the aqueous droplet containing biomolecules as the core and the cross-linked polymer of the surfactant as the shell.

[0094] Figure 3 This is a schematic diagram illustrating the formation and stabilization process of microdroplets, as an exemplary embodiment of this disclosure. Figure 3As shown, the microdroplet preparation method of this embodiment first forms aqueous droplets 5 in an aqueous phase 2 containing biomolecules 1 under the action of AB-type surfactant 3. Then, the AB-type surfactant 3 undergoes a crosslinking or polymerization reaction on the oil phase side of the interface between the oil phase 4 and the aqueous phase 2. The resulting crosslinked polymer of the surfactant encapsulates the aqueous droplets internally, resulting in a core-shell structure microdroplet 6 with the aqueous droplet as the core and the crosslinked polymer of the surfactant as the shell. On the one hand, the aqueous droplet being encapsulated internally prevents it from breaking down and fusing. On the other hand, since the surfactant has already undergone a crosslinking or polymerization reaction, its flow / exchange capacity decreases or even disappears. Therefore, the resulting crosslinked polymer is unlikely to connect with crosslinked polymers on the surface of other aqueous droplets, effectively fixing the crosslinked polymer to the connected aqueous droplets, further improving the stability and uniformity of the microdroplets. Figure 2 and Figure 3 The dashed circles on the surface of the aqueous phase droplet 5 and the solid circles on the surface of the microdroplet 6 represent the states of the surfactant before and after crosslinking.

[0095] Figure 4 This is a schematic diagram illustrating the formation process of microdroplets, as shown in another exemplary embodiment of this disclosure. Figure 4 As shown, in the microdroplet formation process of this exemplary embodiment, instead of directly adding a surfactant, a hydrophilic monomer is polymerized to form a surfactant. This includes: an aqueous phase 2 containing biomolecules 1 (e.g., cells) and hydrophilic monomers 7 is contacted with an oil phase 4 containing a lipophilic initiator 8. The lipophilic initiator 8 initiates the polymerization of the hydrophilic monomers 7 at the interface between the oil phase 4 and the aqueous phase 2 to form a surfactant 3. Subsequently, an aqueous droplet 5 containing biomolecules 1 is rapidly formed at the interface between the oil phase 4 and the aqueous phase 2. The surfactant 3 continues to undergo a crosslinking reaction or polymerization reaction to form a crosslinked polymer. This crosslinked polymer encapsulates the aqueous droplet containing biomolecules inside, resulting in a core-shell structure microdroplet 6 with the aqueous droplet containing biomolecules as the core and the crosslinked polymer of the surfactant as the shell.

[0096] Figure 5 This is a schematic diagram illustrating the formation and stabilization process of microdroplets, as shown in another exemplary embodiment of this disclosure. Figure 5As shown, the microdroplet preparation method of this embodiment first brings an aqueous phase 2 containing biomolecules 1 and hydrophilic monomers 7 into contact with an oil phase 4 containing a lipophilic initiator 8. The hydrophilic monomers 7 aggregate at the interface between the oil phase 4 and the aqueous phase 2. The hydrophilic monomers 7 near the oil phase 4 are initiated by the lipophilic initiator 8 to undergo a polymerization reaction at the interface between the oil phase 4 and the aqueous phase 2, forming a surfactant 3. Subsequently, an aqueous droplet 5 containing biomolecules 1 is rapidly formed at the interface between the oil phase 4 and the aqueous phase 2. The surfactant 3 continues to undergo a cross-linking reaction or polymerization reaction to form a cross-linked polymer. This cross-linked polymer encapsulates the aqueous droplet containing biomolecules inside, resulting in a core-shell structure microdroplet 6 with the aqueous droplet containing biomolecules as the core and the cross-linked polymer of the surfactant as the shell. On the one hand, the aqueous droplet being encapsulated inside can prevent the aqueous droplet from breaking and fusing. On the other hand, since the surfactant has already undergone cross-linking or polymerization reactions, its flow / exchange capacity decreases or even disappears. Therefore, the resulting cross-linked polymer will hardly be connected to the cross-linked polymers on the surface of other aqueous droplets. This is equivalent to fixing the cross-linked polymer to the aqueous droplet it is connected to, which can further improve the stability and uniformity of the microdroplets. Figure 3 and Figure 4 The dashed circles on the surface of the aqueous phase droplet 5 and the solid circles on the surface of the microdroplet 6 represent the states of the surfactant before and after crosslinking.

[0097] This disclosure also provides the applications of the microdroplets described above in molecular diagnostics, immunobiochemistry, cell culture, polymer synthesis, single-cell analysis, single-cell sorting, single-cell sequencing library construction, and drug delivery.

[0098] In an exemplary embodiment of this disclosure, the application may be an application in microdroplet digital PCR.

[0099] While the embodiments disclosed herein are as described above, the content is merely for the purpose of facilitating understanding of this disclosure and is not intended to limit this disclosure. Any person skilled in the art may make any modifications and changes to the form and details of the implementation without departing from the spirit and scope of this disclosure; however, the scope of patent protection of this application shall still be determined by the scope defined in the appended claims.

Claims

1. A microdroplet, characterized in that, It has a core-shell structure, with the core being an aqueous droplet containing biomolecules and the shell being a cross-linked polymer of a surfactant. The shell encapsulates the core, and the microdroplet can move freely in the oil phase in which the microdroplet is prepared.

2. The microdroplet according to claim 1, wherein, The shell comprises a hydrophilic first shell and an oleophilic second shell. The first shell is formed by the hydrophilic end of the crosslinked polymer of the surfactant, and the second shell is formed by the oleophilic end of the crosslinked polymer of the surfactant. The first shell extends into the core, and the second shell covers the core and is connected to the first shell. The second shell has a crosslinked structure.

3. The microdroplet according to claim 1 or 2, wherein, The biomolecules include any one or more of cells, nucleic acids, proteins, carbohydrates, and fats.

4. The method for preparing microdroplets according to any one of claims 1 to 3, characterized in that, include: The oil phase, surfactant, and aqueous phase containing biomolecules are mixed to form aqueous droplets containing biomolecules. The surfactant is initiated by an initiator to undergo a crosslinking or polymerization reaction, forming a crosslinked polymer of the surfactant coating the aqueous droplet containing biomolecules on the surface of the droplet, thus obtaining the microdroplet.

5. The preparation method according to claim 4, comprising: The oil phase, the monomer used to prepare the surfactant, the initiator, and the aqueous phase containing biomolecules are mixed. The initiator initiates the polymerization reaction of the monomers used to prepare the surfactant to form the surfactant; In the presence of the surfactant, aqueous droplets containing biomolecules are formed; The initiator initiates the surfactant to undergo a crosslinking or polymerization reaction, forming a crosslinked polymer of the surfactant coating the aqueous droplet containing the biomolecule on the surface of the droplet, thus obtaining the microdroplet.

6. The preparation method according to claim 4 or 5, wherein, The crosslinking or polymerization reaction of the surfactant occurs on the oil phase side of the interface between the aqueous phase and the oil phase.

7. The preparation method according to claim 6, wherein, The surfactant is of type AB or type ABA, where A is the lipophilic end of the surfactant containing unsaturated bonds and B is the hydrophilic end of the surfactant.

8. The preparation method according to claim 7, wherein, The unsaturated bonds include any one or more of carbon-carbon double bonds and carbon-carbon triple bonds.

9. The preparation method according to claim 5, wherein, The monomer used to prepare the surfactant is a hydrophilic monomer.

10. The preparation method according to claim 9, wherein, The hydrophilic monomer includes any one or more hydrophilic vinyl monomers.

11. The preparation method according to claim 10, wherein, The hydrophilic vinyl monomers include any one or more of acrylamide, acrylic acid, methacrylic acid, vinylpyridine, styrene, N-vinylpyrrolidone, tertiary ammonium salts of dimethylaminoethyl methacrylate, quaternary ammonium salts of dimethylaminoethyl methacrylate, and hydroxyethyl acrylate.

12. The preparation method according to claim 6, wherein, The initiator is a lipophilic initiator.

13. The preparation method according to claim 12, wherein, The lipophilic initiator includes any one or more of hydroperoxide, benzoyl peroxide, dialkyl peroxide, diacyl peroxide, tertiary amine, naphthenate, thiol, organoboronide, organometallic compound, and azo oil-soluble initiator.

14. The preparation method according to claim 13, wherein, The organoborides include triethylboron; The organometallic compound includes any one or more of aluminum-containing organometallic compounds and copper-containing organometallic compounds, wherein the aluminum-containing organometallic compound includes triethylaluminum and the copper-containing organometallic compound includes cuprous naphthate; The azo oil-soluble initiator includes any one or more of azobisisobutyronitrile, azobisisovalerate, azobisisoheptanenitrile, and dimethyl azobisisobutyrate.

15. The preparation method according to claim 6, wherein, The initiator is a lipophilic photoinitiator.

16. The preparation method according to claim 4 or 5, wherein, The surfactant accounts for 0.5% to 2% of the weight of the oil phase.

17. The preparation method according to claim 5, wherein, The initiator accounts for 0.1%-1% of the weight of the monomer used to prepare the surfactant.

18. The preparation method according to any one of claims 4, 5, 7 to 15, 17, wherein, The method used to form aqueous droplets containing biomolecules is either an active method or a passive method; the active method includes electrowetting, dielectric electrophoresis, and thermocapillary method; the passive method includes T-channel method, flow focusing method, and coaxial flow focusing method.

19. The preparation method according to claim 6, wherein, The method used to form aqueous droplets containing biomolecules is either an active method or a passive method; the active method includes electrowetting, dielectric electrophoresis, and thermocapillary method; the passive method includes T-channel method, flow focusing method, and coaxial flow focusing method.

20. The preparation method according to claim 16, wherein, The method used to form aqueous droplets containing biomolecules is either an active method or a passive method; the active method includes electrowetting, dielectric electrophoresis, and thermocapillary method; the passive method includes T-channel method, flow focusing method, and coaxial flow focusing method.

21. The preparation method according to any one of claims 4, 5, 7 to 15, 17, is carried out in a microfluidic chip.

22. The preparation method according to claim 6 is carried out in a microfluidic chip.

23. The preparation method according to claim 16 is carried out in a microfluidic chip.

24. The application of microdroplets according to any one of claims 1 to 3 in molecular diagnostics, immunobiochemistry, cell culture, polymer synthesis, single-cell analysis, single-cell sorting, single-cell sequencing library construction, and drug delivery.

25. The application according to claim 24, for use in microdroplet digital PCR.

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