Droplet array generation chip, method of making and use thereof

Abstract

A droplet array generation chip, its fabrication method, and its application are disclosed. The droplet array generation chip includes: a substrate having a first surface; and a plurality of silica spherical shells arranged in a single layer on the first surface of the substrate at intervals. Each silica spherical shell is a hollow shell structure with mesopores. Each silica spherical shell has an inner surface and an outer surface, and the mesopores connect the inner and outer surfaces. The outer surface of the silica spherical shell is hydrophobic, while the inner surface of the silica spherical shell and the pore surface of the mesopores are hydrophilic. The droplet array generation chip of this disclosure has a simple structure, is easy to operate, can quickly generate droplet arrays, and produces stable droplet sizes with minimal cross-contamination between droplets.

Description

TECHNICAL FIELD

[0001] The embodiment of the present disclosure relates to, but is not limited to, the technical field of biochips, and particularly relates to a droplet array generation chip and a preparation method and application thereof. BACKGROUND

[0002] Polymerase Chain Reaction (PCR) technology is a practical biological technology for artificially amplifying Deoxyribonucleic Acid (DNA) that was born in the 1990s. Generally, template DNA, primers, DNA polymerase and deoxy-ribonucleotide triphosphate (dNTP) are mixed in a buffer solution, and after multiple thermal cycle (denaturation, annealing and extension) reactions, the template DNA product is exponentially increased.

[0003] After more than 30 years of development, PCR technology has been widely used in biomedical diagnostic testing, cell and molecular biology, genetic engineering, pathology and drug science research fields. Digital Polymerase Chain Reaction (dPCR) technology is a new generation of DNA amplification technology developed from PCR. It divides the PCR reaction solution into thousands of individual reaction zones for amplification reaction, quantifies the number of positive and negative reaction zones, and calculates the initial concentration of target DNA molecules by using Poisson distribution statistics. For the partitioning method of the reaction solution in the current dPCR technology, the most commonly used and low-cost method is to generate small droplet reaction zones in the oil phase through T-junction or fluid focusing injection, i.e., droplet digital Polymerase Chain Reaction (ddPCR) technology. This often requires complex punching technology and equipment, and often also faces problems such as unstable droplet generation frequency and droplet size, easy cross-contamination between droplets, and overly complex signal detection system. SUMMARY

[0004] The following is a summary of the subject matter of the detailed description herein. This summary is not intended to limit the scope of protection of the present disclosure.

[0005] The embodiment of the present disclosure provides a droplet array generation chip, which comprises:

[0006] a substrate, the substrate having a first surface;

[0007] a plurality of silica shells, a plurality of the silica shells are arranged in a single layer on the first surface of the substrate at intervals, the silica shells are hollow shell structures, and the shell structures have mesopores, the silica shells have inner surfaces and outer surfaces, the mesopores communicate the inner surfaces and the outer surfaces of the silica shells, the outer surfaces of the silica shells are hydrophobic, and the inner surfaces of the silica shells and the pore surfaces of the mesopores are hydrophilic.

[0008] In an exemplary embodiment of the present disclosure, the outer diameter of the silica shell can be 20 μm to 30 μm, and the shell layer thickness of the silica shell can be 1.5 μm to 3 μm.

[0009] In an exemplary embodiment of the present disclosure, the outer surface of the silica shell can have a first hydrophobic functional group.

[0010] In an exemplary embodiment of the present disclosure, the first hydrophobic functional group can be selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group.

[0011] In an exemplary embodiment of the present disclosure, the first hydrophobic functional group can be connected to the outer surface of the silica shell through a Si-O-Si chemical bond.

[0012] In an exemplary embodiment of the present disclosure, the inner surface of the silica shell and the pore surface of the mesopore can have a hydrophilic functional group.

[0013] In an exemplary embodiment of the present disclosure, the pore size of the mesopore can be 0.8 μm to 1.5 μm.

[0014] In an exemplary embodiment of the present disclosure, the distribution density of the mesopore on the outer surface of the silica shell can be 1 mesopore per 1.13 μm 2 to 11.04 μm 2 of the outer surface.

[0015] In an exemplary embodiment of the present disclosure, the distance between two adjacent silica shells is D, the outer diameter of the silica shell is R, and D and R can satisfy: 0 < D ≤ R.

[0016] In an exemplary embodiment of the present disclosure, the first surface of the substrate can be hydrophobic.

[0017] In an exemplary embodiment of the present disclosure, the first surface of the substrate can have a second hydrophobic functional group.

[0018] In an exemplary embodiment of the present disclosure, the plurality of silica shells and the substrate can be combined together by a Si-O chemical bond.

[0019] In the example embodiment of the present disclosure, the substrate further has a second surface, and the first surface or the second surface of the substrate can be provided with a fluorescent signal blocking layer.

[0020] The present disclosure further provides a preparation method of a droplet array generation chip, the preparation method comprising:

[0021] providing a substrate, the substrate having a first surface;

[0022] forming a plurality of silica shell spheres on the first surface of the substrate, the silica shell spheres being hollow shell structures and having mesopores on the shell structures, the silica shell spheres having inner surfaces and outer surfaces, the mesopores being in communication with the inner surfaces and the outer surfaces of the silica shell spheres, the outer surfaces of the silica shell spheres being hydrophobic, and the inner surfaces of the silica shell spheres and the pore surfaces of the mesopores being hydrophilic.

[0023] In the example embodiment of the present disclosure, the forming of the plurality of silica shell spheres on the first surface of the substrate can comprise:

[0024] performing a first modification on the first surface of the substrate so as to introduce at least one of a hydroxyl group and a functional group capable of being hydrolyzed into a hydroxyl group on the first surface of the substrate;

[0025] preparing a plurality of core-shell microspheres having a core-shell structure, the shell of the core-shell microspheres being an initial silica shell sphere without mesopores, and the core of the core-shell microspheres being configured to be capable of providing support for the initial silica shell sphere and capable of being removed;

[0026] arranging the plurality of core-shell microspheres on the first surface of the substrate subjected to the first modification in a single layer and at intervals from each other;

[0027] performing a hydrophobic modification on the outer surfaces of the core-shell microspheres so as to make the outer surfaces of the core-shell microspheres hydrophobic;

[0028] forming mesopores on the initial silica shell spheres, and removing the core of the core-shell microspheres, to obtain silica shell spheres with mesopores, and the mesopores being in communication with the inner surfaces and the outer surfaces of the silica shell spheres;

[0029] the hydroxyl groups possessed by the silica shell spheres themselves make the inner surfaces of the silica shell spheres and the pore surfaces of the mesopores exhibit hydrophilicity.

[0030] In the example embodiment of the present disclosure, the arranging of the plurality of core-shell microspheres on the first surface of the substrate subjected to the first modification in a single layer and at intervals from each other can comprise:

[0031] dispersing a plurality of the core-shell microspheres in a solvent to obtain a solution containing core-shell microspheres;

[0032] placing the once-modified substrate in the solution containing core-shell microspheres, and using a gravity deposition method to arrange a plurality of the core-shell microspheres in a single layer and spaced apart from each other on the first surface of the once-modified substrate;

[0033] under vacuum drying conditions, performing a dehydration condensation reaction between the hydroxyl groups inherently present on the outer surface of the initial silica shell of the core-shell microspheres and the hydroxyl groups on the first surface of the once-modified substrate or the hydroxyl groups obtained by hydrolysis of a functional group capable of being hydrolyzed into a hydroxyl group, so that the core-shell microspheres and the substrate are combined together by a chemical bond.

[0034] In exemplary embodiments of the present disclosure, the preparation method can further include:

[0035] performing secondary hydrophobic modification on the first surface of the once-modified substrate to make the first surface of the substrate hydrophobic;

[0036] forming a hydrophobic interaction between the outer surface of the hydrophobically modified core-shell microspheres (i.e., the outer surface of the silica shell) and the first surface of the substrate that has been subjected to secondary hydrophobic modification, so that the core-shell microspheres and the substrate are combined together by the chemical bond and the hydrophobic interaction.

[0037] In exemplary embodiments of the present disclosure, the first hydrophobic modifier used for hydrophobically modifying the outer surface of the silica shell can be a silane coupling agent containing a first hydrophobic functional group.

[0038] In exemplary embodiments of the present disclosure, the second hydrophobic modifier used for performing secondary hydrophobic modification on the first surface of the once-modified substrate can be a silane coupling agent containing a second hydrophobic functional group.

[0039] In exemplary embodiments of the present disclosure, the first hydrophobic functional group and the second hydrophobic functional group can be selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group.

[0040] In exemplary embodiments of the present disclosure, the first hydrophobic modifier and the second hydrophobic modifier can be selected from any one or more of methyltriethoxysilane, octadecyltrichlorosilane, octadecyltrimethoxysilane, trifluoropropyltriethoxysilane, perfluorooctyltriethoxysilane, chloromethyltrimethoxysilane, and (chloromethyl)methyldiethoxysilane.

[0041] In exemplary embodiments of the present disclosure, the substrate can further have a second surface;

[0042] The preparation method can further include disposing a fluorescent signal blocking layer on the first surface or the second surface of the substrate.

[0043] The present disclosure also provides a droplet array generation method, which includes:

[0044] A droplet array generation chip is provided, which is a droplet array generation chip as described above or obtained by a preparation method of a droplet array generation chip as described above.

[0045] The droplet array generation chip is immersed in an aqueous phase containing a reaction solution for generating a droplet array, the aqueous phase containing the reaction solution enters the interior of the silica shell through the mesopore on the plurality of silica shells of the droplet array generation chip, and an aqueous phase droplet is generated in the interior of the silica shell.

[0046] An oil phase is covered on the surface of the aqueous phase.

[0047] The droplet array generation chip is moved so that the silica shells of the droplet array generation chip enter the oil phase, and when the droplet array generation chip passes through the interface between the aqueous phase and the oil phase, a plurality of silica shells containing the aqueous phase droplets in the interior are enclosed and arranged at intervals on the substrate to form a droplet array.

[0048] The present disclosure also provides a fluorescence detection method, which includes:

[0049] A droplet array generation chip is provided, which is a droplet array generation chip as described above or obtained by a preparation method of a droplet array generation chip as described above.

[0050] The droplet array generation chip is immersed in an aqueous phase containing a reaction solution for generating a droplet array, the aqueous phase containing the reaction solution enters the interior of the silica shell through the mesopore on the plurality of silica shells of the droplet array generation chip, and an aqueous phase droplet is generated in the interior of the silica shell.

[0051] An oil phase is covered on the surface of the aqueous phase.

[0052] The droplet array generation chip is moved so that the silica shells of the droplet array generation chip enter the oil phase, and when the droplet array generation chip passes through the interface between the aqueous phase and the oil phase, a plurality of silica shells containing the aqueous phase droplets in the interior are enclosed and arranged at intervals on the substrate to form a droplet array.

[0053] The droplet array generation chip is heated and amplification reactions are performed in the droplets of the droplet array, and the droplet array generation chip after the amplification reactions is subjected to fluorescence detection.

[0054] Additional features and advantages of the present disclosure will be set forth in the description that follows, and in part will be apparent from the description, or can be learned by practice of the present disclosure. Other advantages of the present disclosure will be realized and attained by the solution described in the description, and the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0055] The accompanying drawings are included to provide an understanding of the present solution and constitute a part of the specification, together with the description, to explain the principles of the present solution, and do not limit the present solution.

[0056] Figure 1 A structure schematic diagram of a droplet array generation chip according to an exemplary embodiment of the present disclosure;

[0057] Figure 2 A structure schematic diagram of a droplet array generation chip according to an exemplary embodiment of the present disclosure; Figure 1 A structure schematic diagram of a droplet array generation chip according to an exemplary embodiment of the present disclosure;

[0058] Figure 3 A structure schematic diagram of a droplet array generation chip according to an exemplary embodiment of the present disclosure; Figure 1 A structure schematic diagram of a droplet array generation chip according to an exemplary embodiment of the present disclosure;

[0059] Figure 4 Corresponding drawings of formulas (1) to (4);

[0060] Figure 5A A droplet distribution schematic diagram of a planar microstructure model surface having similar size and hydrophilic / hydrophobic properties to the silica shell of the exemplary embodiment of the present disclosure;

[0061] Figure 5B A distribution state schematic diagram of an aqueous phase in a planar microstructure model according to an exemplary embodiment of the present disclosure; Figure 5A A distribution state schematic diagram of an aqueous phase in a planar microstructure model according to an exemplary embodiment of the present disclosure;

[0062] Figure 6 A structure schematic diagram of a droplet array generation chip according to another exemplary embodiment of the present disclosure;

[0063] Figure 7 A reaction mechanism schematic diagram of a dehydration condensation reaction when the core-shell microspheres are arranged and fixed on the surface of the substrate in the preparation method according to an exemplary embodiment of the present disclosure;

[0064] Figure 8 A reaction mechanism schematic diagram of hydrophobic modification of the outer surface of the core-shell microspheres and the first surface of the substrate in the preparation method according to an exemplary embodiment of the present disclosure;

[0065] Figure 9 A flowchart of a droplet array generation method according to an exemplary embodiment of the present disclosure;

[0066] Figure 10 A schematic diagram of silica shell loaded with an aqueous phase (reaction solution) arranged in an array on a substrate formed by a droplet array generation method according to an exemplary embodiment of the present disclosure;

[0067] Figure 11 A structure of silica shell loaded with an aqueous phase according to Figure 10

[0068] Figure 12A A top view of a droplet array generation device according to an exemplary embodiment of the present disclosure;

[0069] Figure 12B A front view of a droplet array generation device according to Figure 12A

[0070] Meanings of the reference symbols in the drawings are as follows:

[0071] 10 - substrate; 20 - silica shell; 21 - inner surface; 22 - outer surface; 30 - fluorescent signal blocking layer; 40 - mesopore; 50 - outer shell; 60 - push rod; 70 - aqueous phase; 80 - oil phase; 90 - aqueous phase droplet. DETAILED DESCRIPTION

[0072] In order to make the objects, technical solutions, and advantages of the present disclosure clearer, below the embodiments of the present disclosure will be described in detail with reference to the drawings. It should be explained that the embodiments in the present disclosure and the features in the embodiments can be combined with each other in any manner without conflict.

[0073] The implementation manner herein can be implemented in multiple different forms. One of ordinary skill in the art can easily understand the fact that the implementation manner and content can be transformed into various forms without departing from the purpose of the present disclosure and the scope thereof. Therefore, the present disclosure should not be interpreted as being limited in the content described in the following embodiments. The embodiments in the present disclosure and the features in the embodiments can be combined with each other in any manner without conflict.

[0074] The drawing proportions in the present disclosure can be used as a reference in the actual process, but are not limited thereto. For example, the width-length ratio of the channel, the thickness and spacing of each film layer, and the width and spacing of each signal line can be adjusted according to actual needs. The number of pixels in the display substrate and the number of sub-pixels in each pixel are also not limited to the numbers shown in the drawings. The drawings described in the present disclosure are only schematic diagrams, and one embodiment of the present disclosure is not limited to the shapes or values shown in the drawings.

[0075] ​​In the present specification, the words of "middle", "upper", "lower", "front", "back", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicating the positional or locational relationship are used to describe the positional relationship of the components with reference to the drawings for the purpose of convenience and simplification of the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present disclosure. The positional relationship of the components is appropriately changed according to the direction in which each component is described. Therefore, it is not limited to the words described in the specification, and can be appropriately changed according to the situation.

[0076] In the present specification, unless explicitly specified and limited otherwise, the terms "provided", "connected" should be understood broadly. For example, it can be fixedly connected, or detachably connected, or integrally connected; it can be mechanically connected, or electrically connected; it can be directly connected, or indirectly connected through an intermediate piece, or the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present disclosure can be understood according to the specific circumstances.

[0077] In the description of the present disclosure, the ordinal numbers "first", "second" and the like are set to avoid confusion of the components, rather than to limit in quantity.

[0078] The embodiment of the present disclosure provides a droplet array generation chip, which comprises:

[0079] a substrate, the substrate has a first surface;

[0080] a plurality of silica shell, a plurality of the silica shell is arranged on the first surface of the substrate in a single layer with mutual spacing, the silica shell is a hollow shell structure, and the shell structure has a mesopore, the silica shell has an inner surface and an outer surface, the mesopore communicates the inner surface and the outer surface of the silica shell, the outer surface of the silica shell is hydrophobic, and the inner surface of the silica shell and the pore surface of the mesopore are hydrophilic.

[0081] In the description of the present disclosure, "hydrophobic" refers to a water contact angle greater than 90°; "hydrophilic" refers to a water contact angle less than 90°.

[0082] In the embodiment of the present disclosure, the shape of "silica shell" is not required, and the shape of "silica shell" is not limited to a regular sphere, but can also be an ellipsoid, a regular tetrahedron, a regular hexahedron and the like, as long as the silica shell can physically separate the liquid droplets contained therein.

[0083] The mesoporous silica shell of the droplet array generation chip in the embodiments of the present disclosure can serve as a guiding template, and the rapid generation of the droplet array can be realized through simple spatial displacement between the interface of the water phase and the oil phase, which can avoid the use of complex punching technology and equipment. Moreover, the water phase droplets formed by the droplet array generation chip in the embodiments of the present disclosure are encapsulated and spaced by the silica shell and fixed in position with the substrate, which can avoid cross contamination between the droplets and facilitate efficient PCR amplification reaction and signal detection analysis. In addition, the size of the droplets can be regulated by adjusting the inner diameter of the silica shell, thereby solving the problem of unstable droplet size.

[0084] In addition, the droplet array generation chip in the embodiments of the present disclosure has a simple structure and is easy to operate, which can avoid the use of complex surface microstructure processing equipment and process, further reducing the production cost. Figure 1 FIG. 1 is a structural schematic diagram of a droplet array generation chip according to an exemplary embodiment of the present disclosure; Figure 2 FIG. 2 is a longitudinal sectional structural schematic diagram of the droplet array generation chip shown in FIG. 1; Figure 1 FIG. 3 is a structural schematic diagram of the silica shell of the droplet array generation chip shown in FIG. 1. As shown in FIG. 3, the droplet array generation chip comprises a substrate 10 and a plurality of silica shells 20; Figure 3 FIG. 4 is a structural schematic diagram of the silica shell of the droplet array generation chip shown in FIG. 1. As shown in FIG. 4, the droplet array generation chip comprises a substrate 10 and a plurality of silica shells 20; Figure 1 FIG. 3 is a structural schematic diagram of the silica shell of the droplet array generation chip shown in FIG. 1. As shown in FIG. 3, the droplet array generation chip comprises a substrate 10 and a plurality of silica shells 20; Figures 1 to 3 FIG. 3 is a structural schematic diagram of the silica shell of the droplet array generation chip shown in FIG. 1. As shown in FIG. 3, the droplet array generation chip comprises a substrate 10 and a plurality of silica shells 20;

[0085] The substrate 10 has a first surface and a second surface, and a fluorescent signal blocking layer 30 is arranged on the second surface of the substrate 10. The main function of the substrate 10 is to provide a fixed base for the plurality of silica shells 20, so that the plurality of silica shells 20 can form a randomly dispersed microsphere array, and the silica shells 20 can be lifted and displaced when generating the droplet array. The function of the fluorescent signal blocking layer 30 is to block the fluorescent signal from passing through the second surface of the substrate 10, otherwise the fluorescent signal scanning detection cannot be performed. In other embodiments, the fluorescent signal blocking layer 30 can also be arranged on the first surface of the substrate 10, for example, between the first surface of the substrate 10 and the plurality of silica shells 20;

[0086] A plurality of silica shells 20 are arranged in a single layer on the first surface of the substrate 10, the silica shells 20 are hollow shell structures with a plurality of mesopores 40 on the shell structure, the silica shells 20 have an inner surface 21 and an outer surface 22, the mesopores 40 communicate the inner surface 21 and the outer surface 22 of the silica shells 20, the outer surface 22 of the silica shells 20 is hydrophobic, and the inner surface 21 of the silica shells 20 and the pore surface of the mesopores 40 are hydrophilic; the silica shells 20 are mainly used to wrap the droplets and maintain the shape of the droplets when the droplet array is generated, and form rigid physical isolation on the surface of the droplets to avoid mutual contact between the droplets; the silica shell has a low intrinsic fluorescence intensity, which can effectively reduce the interference on the detection signal.

[0087] In the example embodiments of the present disclosure, the first surface and the second surface of the substrate can be located on opposite sides of the substrate, respectively.

[0088] According to the report in the reference (J. Phys. Chem. B 2021, 125, 3, 883-894), the key to the stable maintenance of the monostable Cassie state (non-wetting state) of the hydrophobic material surface is that the apparent contact angle θ e of the micro-porous inclined wall surface is much larger than the initial contact angle θ t0 of the liquid with the material surface, and the former is mainly determined by the hydrophilic / hydrophobic properties of the material surface itself, while the latter is further affected by the geometric shape and spacing of the surface microstructure. Figure 4 The following formulas (1) to (4) correspond to the drawings.

[0089]

[0090] θ e ∈(0, θt0) (Wenzel state) (2)

[0091]

[0092]

[0093] On the contrary, according to formula (2), when θ e is less than θ t0 , the liquid tends to form a stable Wenzel state on the porous material surface, i.e., a spontaneous wetting state. For the droplet array generation chip of the present disclosure, the inner surface of the silica shell and the pore of the mesopore are hydrophilic, i.e., the apparent contact angle θ e of the pore wall is much smaller than 90°, and according to formula (1), θ t0 is always greater than 90°, so θ e is always less than θ t0Based on the above literature references and corresponding analyses, it can be seen that the aqueous liquid can spontaneously wet the mesoporous channels and thus smoothly enter the interior of the silica spherical shell. Furthermore, the liquid's own gravity, internal hydraulic pressure, and capillary action (each mesoporous channel can be analogized to a capillary tube) all promote its entry along the channels into the interior of the silica spherical shell.

[0094] Figure 5A A schematic diagram of droplet distribution on the surface of a planar microstructure model having similar size and hydrophilic / hydrophobic properties to the silica spherical shell of an exemplary embodiment of the present disclosure; Figure 5B For water phase Figure 5A A schematic diagram of the distribution state in the planar microstructure model shown. From... Figure 5A and Figure 5B As can be seen, the aqueous phase can successfully enter the interior of the silica sphere shell of the droplet array generation chip in the exemplary embodiment of this disclosure. At the same time, the outer surface of the silica sphere shell (except for the surface pores) is hydrophobic, which ensures that when the silica sphere shell containing the PCR reaction solution enters the oil phase from the aqueous phase, it will be quickly wrapped and sealed by the oil phase, forming a droplet array arranged on the substrate surface.

[0095] In exemplary embodiments of this disclosure, the outer diameter of the silica spherical shell can be from 20 μm to 30 μm, for example, it can be 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm or 30 μm. When the outer diameter of the silica spherical shell is from 20 μm to 30 μm, it is beneficial for the preparation of the silica spherical shell and for arranging silica spherical shells on a suitable substrate area with a number of partitions comparable to those of currently commercially available dPCR instruments (e.g., Qiagen, Thermo Fisher Scientific, Fruda and Bio-Rad, etc.), a partition volume comparable to that of the partitions (approximately pL-nL level), and dispersed among them.

[0096] In exemplary embodiments of this disclosure, the shell thickness of the silica spherical shell can be from 1.5 μm to 3 μm, for example, it can be 1.5 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, or 3 μm. When the shell thickness of the silica spherical shell is from 1.5 μm to 3 μm, the internal hollow region in the silica spherical shell used for storing droplets will not be too small, and the collapse of the hollow structure is less likely to occur.

[0097] In an exemplary embodiment of this disclosure, the outer surface of the silica spherical shell may have a first hydrophobic functional group.

[0098] In exemplary embodiments of the present disclosure, the outer surface of the silica shell can be rendered hydrophobic by introducing a first hydrophobic functional group, for example, the outer surface of the silica shell can be provided with a first hydrophobic functional group.

[0099] In exemplary embodiments of the present disclosure, the first hydrophobic functional group can be selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group, for example, can be selected from any one or more of a C10 to C20 hydrocarbon group, a C10 to C20 fluorocarbon group, a C10 to C20 chlorocarbon group, a hydrocarbon group containing an aryl group, an ester group, an ether group, a nitro group, an amide group, and a hydrocarbon group containing a double bond.

[0100] In exemplary embodiments of the present disclosure, the first hydrophobic functional group can be attached to the outer surface of the silica shell via a Si-O-Si chemical bond.

[0101] Further, in exemplary embodiments of the present disclosure, the outer surface of the silica shell can also be rendered hydrophobic by physically adsorbing a hydrophobic material or chemically modifying a first hydrophobic functional group, a hydrophobic monolayer, and a hydrophobic polymer.

[0102] The hydrophobic material can be selected from any one or more of polytetrafluoroethylene, polystyrene, polymethyl methacrylate, and polyhexafluorobutyl acrylate.

[0103] The hydrophobic monolayer can be formed from a material containing a first hydrophobic functional group, and the hydrophobic monolayer can be formed on the outer surface of the silica shell by a self-assembly method.

[0104] The hydrophobic polymer can be formed on the outer surface of the silica shell by a chemical grafting method.

[0105] In exemplary embodiments of the present disclosure, the inner surface of the silica shell and the pore surface of the mesopore can be provided with a hydrophilic functional group.

[0106] In exemplary embodiments of the present disclosure, the inner surface of the silica shell and the pore surface of the mesopore can be rendered hydrophilic by introducing a hydrophilic functional group, for example, the inner surface of the silica shell and the pore surface of the mesopore can be provided with a hydrophilic functional group.

[0107] In exemplary embodiments of the present disclosure, the hydrophilic functional group is a hydroxyl group.

[0108] In the exemplary embodiments of the present disclosure, the pore size of the mesopores can be 0.8 μm to 1.5 μm. When the pore size of the mesopores is selected to be the minimum value in the range of 0.8 μm to 1.5 μm, the pore size is still greater than the wavelength of visible light (390 nm to 780 nm), and the influence of small hole diffraction on the fluorescence signal intensity can be reduced. In addition, when the pore size of the mesopores is 0.8 μm to 1.5 μm, the stability of the hollow structure of the silica shell can be maintained, and the liquid droplets formed inside the silica shell can be prevented from leaking out of the silica shell encapsulated by the oil phase.

[0109] In the exemplary embodiments of the present disclosure, the distribution density of the mesopores on the outer surface of the silica shell can be 1 mesopore per 1.13 μm 2 to 11.04 μm 2 of the outer surface.

[0110] In the exemplary embodiments of the present disclosure, the outer diameter of the silica shell can be 20 μm to 30 μm, and the number of the mesopores on each silica shell can be 110 to 2500.

[0111] In the exemplary embodiments of the present disclosure, the distance between the channels of two adjacent mesopores is d, and the pore size of the mesopores is r, and 0.5r≤d≤1.5r. Here, the "distance between the channels of two adjacent mesopores" refers to the distance between the edges of the opposite sides of the channels of two adjacent mesopores, rather than the distance between the centers of the channels of two adjacent mesopores.

[0112] When the distribution density of the mesopores on the outer surface of the silica shell is 1 mesopore per 1.13 μm 2 to 11.04 μm 2 of the outer surface, or d and r satisfy 0.5r≤d≤1.5r, the stability of the hollow structure of the silica shell can be maintained, and the liquid droplets formed inside the silica shell can be prevented from leaking out of the silica shell encapsulated by the oil phase.

[0113] In the exemplary embodiments of the present disclosure, the distance between two adjacent silica shells is D, and the outer diameter of the silica shell is R, and D and R can satisfy 0

[0114] In the exemplary embodiments of the present disclosure, the distribution density of the silica shells on the first surface of the substrate can be 250 / mm 2up to 2500 / mm 2 .

[0115] In an exemplary embodiment of the present disclosure, the substrate can have a size selected according to actual detection requirements, for example, can have a size of length x width x thickness = 5 mm x 4 mm x 0.2 mm.

[0116] In an exemplary embodiment of the present disclosure, the substrate can have a size of length x width x thickness = 5 mm x 4 mm x 0.2 mm, the silica shell can have an outer diameter of 30 μm, the number of silica shells on the first surface of the substrate can be 5000 to 50000, and the silica shells can be randomly dispersed on the first surface of the substrate.

[0117] In an exemplary embodiment of the present disclosure, the first surface of the substrate can be hydrophobic.

[0118] In an exemplary embodiment of the present disclosure, the first surface of the substrate can have a second hydrophobic functional group.

[0119] In an exemplary embodiment of the present disclosure, the first surface of the substrate can be hydrophobic by introducing a second hydrophobic functional group, for example, the first surface of the substrate can have a second hydrophobic functional group.

[0120] In an exemplary embodiment of the present disclosure, the second hydrophobic functional group can be selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group, for example, can be selected from any one or more of a C10 to C20 hydrocarbon group, a C10 to C20 fluorocarbon group, a C10 to C20 chlorocarbon group, a hydrocarbon group containing an aryl group, an ester group, an ether group, a nitro group, an amide group, and a hydrocarbon group containing a double bond.

[0121] In an exemplary embodiment of the present disclosure, the first hydrophobic functional group and the second hydrophobic functional group are the same.

[0122] In an exemplary embodiment of the present disclosure, the plurality of silica shells and the substrate can be combined together by a Si-O chemical bond.

[0123] In an exemplary embodiment of the present disclosure, the plurality of silica shells and the substrate can be combined together by a Si-O chemical bond and a hydrophobic interaction.

[0124] The first surface of the substrate can be modified by at least one of hydroxylation and alkoxysilaneation. The first surface of the substrate after such modification can contain hydroxyl or alkoxy groups, and the alkoxy groups can be hydrolyzed to produce hydroxyl groups. The hydroxyl groups on the first surface of the substrate, or the hydroxyl groups obtained from the hydrolysis of alkoxy groups, can undergo dehydration condensation with the hydroxyl groups on the outer surface of the silica spheres to form stable Si-O chemical bonds, allowing multiple silica spheres to be chemically bonded to the substrate. If both the outer surface of the silica spheres and the first surface of the substrate are hydrophobic, they tend to aggregate in an aqueous solution because they simultaneously "avoid" water; that is, the hydrophobic materials attract each other, forming a hydrophobic interaction. These two forces, chemical bonding and hydrophobic interaction, maintain the stable adhesion of the silica spheres to the substrate surface.

[0125] In exemplary embodiments of this disclosure, the substrate can be a glass substrate or a plastic substrate with good heat resistance and light transmittance, for example, a glass sheet or a plastic sheet. The substrate material can be polymethyl methacrylate (PMMA), polycarbonate (PC), or cyclic olefin copolymer (COC), etc. A substrate with good heat resistance and light transmittance is beneficial to the stability of the overall structure of the droplet array generation chip during subsequent PCR reactions and minimizes adverse effects on fluorescence signal detection.

[0126] In an exemplary embodiment of this disclosure, the fluorescence signal blocking layer may be a black film layer, for example, a black matrix film layer.

[0127] Figure 6 This is a schematic diagram of a longitudinal cross-sectional structure of another droplet array generation chip according to an exemplary embodiment of this disclosure. Figure 6 As shown, the droplet array generation chip of the exemplary embodiment of this disclosure may further include a housing 50. The housing 50 may accommodate the substrate, the plurality of silica spheres, and the oil and water phases for detection. The housing 50 may be a rectangular transparent glass housing with a wall thickness of 0.2 mm, inner wall dimensions of length × width × height = 5.1 mm × 4.1 mm × 2 mm, and outer wall dimensions of length × width × height = 5.5 mm × 4.5 mm × 2.5 mm. The housing 50 may include a lower plate and peripheral walls, the lower plate being removable to accommodate the substrate 10 loaded with silica spheres 20.

[0128] The droplet array generation chip may further include a push rod 60, which is configured to move the substrate vertically. The push rod 60 can pass through a hole in the lower plate of the housing 50 and connect to the substrate 10. Both the perimeter of the chip and the perimeter of the removable lower plate are well sealed with rubber.

[0129] The present disclosure also provides a method for preparing a droplet array generation chip. The droplet array generation chip provided by the present disclosure can be prepared by the method. The method comprises:

[0130] providing a substrate, the substrate having a first surface;

[0131] forming a plurality of silica shell spheres on the first surface of the substrate, the silica shell spheres being hollow shell structures and having mesopores on the shell structures, the silica shell spheres having inner surfaces and outer surfaces, the mesopores being in communication with the inner surfaces and the outer surfaces of the silica shell spheres, the outer surfaces of the silica shell spheres being hydrophobic, and the inner surfaces of the silica shell spheres and the pore surfaces of the mesopores being hydrophilic.

[0132] In an exemplary embodiment of the present disclosure, the forming a plurality of silica shell spheres on the first surface of the substrate can comprise:

[0133] performing a first modification on the first surface of the substrate so as to introduce at least one of a hydroxyl group and a functional group capable of being hydrolyzed into a hydroxyl group on the first surface of the substrate;

[0134] preparing a plurality of core-shell microspheres having a core-shell structure, the shell of the core-shell microspheres being an initial silica shell sphere without mesopores, and the core of the core-shell microspheres being configured to be capable of providing support for the initial silica shell sphere and capable of being removed;

[0135] arranging the plurality of core-shell microspheres on the first surface of the substrate subjected to the first modification in a single layer and at intervals from each other;

[0136] performing a hydrophobic modification on the outer surface of the core-shell microspheres (i.e. the outer surface of the initial silica shell sphere) so as to make the outer surface of the core-shell microspheres hydrophobic;

[0137] forming mesopores on the initial silica shell sphere and removing the core of the core-shell microspheres to obtain a silica shell sphere with mesopores, and the mesopores being in communication with the inner surfaces and the outer surfaces of the silica shell sphere;

[0138] the hydroxyl groups possessed by the silica shell sphere itself make the inner surfaces of the silica shell sphere and the pore surfaces of the mesopores exhibit hydrophilicity.

[0139] In an exemplary embodiment of the present disclosure, the performing a first modification on the first surface of the substrate so as to introduce at least one of a hydroxyl group and a functional group capable of being hydrolyzed into a hydroxyl group on the first surface of the substrate can comprise:

[0140] at least one of a hydroxylation modification and an alkoxysilane modification is performed on the first surface of the substrate, such that at least one of a hydroxyl group and an alkoxy group is introduced on the first surface of the substrate.

[0141] In an exemplary embodiment of the present disclosure, the alkoxysilane modification performed on the first surface of the substrate can include performing a graft polymerization reaction on the first surface of the substrate using a double-bonded silane coupling agent, such that an alkoxyl functional group having hydrolysis activity is introduced on the first surface of the substrate; and wherein the double-bonded silane coupling agent can include any one or more of γ-methacryloxypropyltrimethoxysilane, γ-methacryloxypropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, or the like.

[0142] In an exemplary embodiment of the present disclosure, the hydroxylation modification performed on the first surface of the substrate can include introducing a hydroxyl group on the first surface of the substrate using a plasma treatment, an otter solution (a mixture of concentrated sulfuric acid and hydrogen peroxide) washing, or a confined photo-catalytic oxidation (CPO) method.

[0143] In an exemplary embodiment of the present disclosure, the preparing of the plurality of core-shell microspheres having a core-shell structure can include:

[0144] mixing a raw material for preparing the initial silica shell, a microsphere template, a porogen, and a catalyst, depositing silica and the porogen on a surface of the microsphere template using a template method, forming an initial silica shell formed of silica and the porogen on the surface of the microsphere template, and obtaining the plurality of core-shell microspheres having a core-shell structure, the microsphere template serving as a core of the core-shell microspheres and the initial silica shell serving as a shell of the core-shell microspheres;

[0145] The forming of mesopores on the initial silica shell and the removing of the core of the core-shell microspheres can include:

[0146] The removing of the porogen on the initial silica shell forms mesopores on the initial silica shell, and the mesopores are used to remove the core of the core-shell microspheres.

[0147] In an exemplary embodiment of the present disclosure, the raw material for preparing the initial silica shell can be tetraethyl orthosilicate (TEOS), and the catalyst can be aqueous ammonia (NH3·H2O).

[0148] In exemplary embodiments of the present disclosure, the microsphere template can be selected from any one of a soluble (e.g., dissolvable in water or other solvent) polymer microsphere, inorganic nanoparticle, inorganic microparticle, polyelectrolyte, and oil-in-water microemulsion. These targets can be removed by using a mild dissolution and washing method in the subsequent process, without the need for long-time high-temperature calcination removal, which can avoid affecting the hydrophilicity of the outer surface of the silica shell and the pore surface of the mesopore.

[0149] In exemplary embodiments of the present disclosure, the porogen can be selected from any one or more of a cationic surfactant porogen, an alcohol porogen, and a lipid porogen.

[0150] In exemplary embodiments of the present disclosure, the preparation of the plurality of core-shell microspheres having a core-shell structure can include: stepwise hydrolysis and condensation of tetraethyl orthosilicate (TEOS) on the surface of the microsphere template by catalysis of ammonia (NH3·H2O) and deposition of silica, in which a porogen is additionally added and uniformly dispersed in the reaction system, and the porogen is also randomly dispersed and deposited on the surface of the microsphere template together with the silica, to obtain an initial silica shell with a certain shell thickness, the microsphere template being a core, and the silica shell on the surface of the microsphere template being a shell, to form a core-shell microsphere.

[0151] In exemplary embodiments of the present disclosure, the spacing and single-layer arrangement of the plurality of core-shell microspheres on the first surface of the substrate subjected to one modification can include:

[0152] dispersing the plurality of core-shell microspheres in a solvent to obtain a solution containing core-shell microspheres;

[0153] placing the substrate subjected to one modification in the solution containing core-shell microspheres, and using a gravity deposition method to space and arrange the plurality of core-shell microspheres in a single layer on the first surface of the substrate subjected to one modification;

[0154] under vacuum drying conditions, the hydroxyl groups on the outer surface of the initial silica shell of the core-shell microspheres and the hydroxyl groups on the first surface of the substrate subjected to one modification or the hydroxyl groups obtained by hydrolysis of a functional group capable of being hydrolyzed to a hydroxyl group are subjected to dehydration condensation reaction, so that the core-shell microspheres and the substrate are chemically bonded together.

[0155] In exemplary embodiments of the present disclosure, the mass fraction of the core-shell microspheres in the solvent can be 0.25% to 1%, for example, 0.25%, 0.5%, 0.75%, or 1%.

[0156] In an exemplary embodiment of the present disclosure, the solvent for dispersing the core-shell microspheres can be a mixed solution of any one or more of methanol and ethanol and water, and the volume ratio of any one of methanol and ethanol to water can be 1:1; the pH of the solution containing the core-shell microspheres can be 4.

[0157] In an exemplary embodiment of the present disclosure, after the core-shell microspheres completely deposit on the surface of the substrate by gravity, the substrate can be continuously soaked for a period of time, for example, 24 h, so that the functional groups on the first surface of the substrate capable of being hydrolyzed to hydroxyl groups can be hydrolyzed to hydroxyl groups. If the first surface of the substrate does not have functional groups capable of being hydrolyzed to hydroxyl groups, but directly has hydroxyl groups, after the core-shell microspheres completely deposit on the surface of the substrate, the substrate does not need to be continuously soaked in the solution containing the core-shell microspheres, and can be directly subjected to vacuum drying.

[0158] In an exemplary embodiment of the present disclosure, the vacuum drying conditions can include that the drying temperature is 37°C and the drying time is 4 h.

[0159] Figure 7 A schematic diagram of the reaction mechanism of the dehydration condensation reaction of the core-shell microspheres when arranged and fixed on the surface of the substrate in the preparation method of the exemplary embodiment of the present disclosure is shown. As shown in the figure, the dehydration condensation reaction between the hydroxyl groups on the outer surface of the core-shell microspheres (i.e., the outer surface of the initial silica shell) and the hydroxyl groups on the first surface of the substrate can be completed by vacuum drying, and the arrangement and fixation of the core-shell microspheres are completed. Figure 7

[0160] In an exemplary embodiment of the present disclosure, the preparation method can further include:

[0161] The first surface of the substrate subjected to the first modification is subjected to secondary hydrophobic modification, so that the first surface of the substrate is hydrophobic;

[0162] The outer surface of the core-shell microspheres subjected to the hydrophobic modification (i.e., the outer surface of the initial silica shell) and the first surface of the substrate subjected to the secondary hydrophobic modification form a hydrophobic interaction, and the core-shell microspheres and the substrate are combined together by the chemical bond and the hydrophobic interaction.

[0163] In an exemplary embodiment of the present disclosure, the first hydrophobic modifier used for the hydrophobic modification of the outer surface of the core-shell microspheres can be a silane coupling agent containing a first hydrophobic functional group.

[0164] In an exemplary embodiment of the present disclosure, the second hydrophobic modifier used for the secondary hydrophobic modification of the first surface of the substrate subjected to the first modification can be a silane coupling agent containing a second hydrophobic functional group.

[0165] ​In exemplary embodiments of the present disclosure, the first and second hydrophobic functional groups can be selected from any one or more of saturated or unsaturated hydrocarbon groups, fluorocarbon groups and chlorocarbon groups.

[0166] In exemplary embodiments of the present disclosure, the first and second hydrophobic functional groups are the same.

[0167] In exemplary embodiments of the present disclosure, the first and second hydrophobic modifiers can be selected from any one or more of methyltriethoxysilane, octadecyltrichlorosilane, octadecyltrimethoxysilane, trifluoropropyltriethoxysilane, perfluorooctyltriethoxysilane, chloromethyltrimethoxysilane and (chloromethyl)methyldiethoxysilane.

[0168] In exemplary embodiments of the present disclosure, the first and second hydrophobic modifiers can be the same, in which case the same silane coupling agent can be used to modify both the outer surface of the core-shell microspheres and the first surface of the substrate.

[0169] Figure 8 A schematic diagram of the reaction mechanism for hydrophobic modification of the outer surface of the core-shell microspheres and the first surface of the substrate in exemplary embodiments of the present disclosure is shown. When the first and second hydrophobic modifiers are silane coupling agents containing hydrocarbon groups, fluorocarbon groups and chlorocarbon groups, the molecular fragments other than the hydrocarbon groups, fluorocarbon groups and chlorocarbon groups in the silane coupling agents can hydrolyze to form hydroxyl groups, which can then undergo dehydration condensation reactions with the hydroxyl groups on the outer surface of the core-shell microspheres and the first surface of the substrate, thereby introducing the first and second hydrophobic functional groups such as hydrocarbon groups, fluorocarbon groups and chlorocarbon groups, and completing the hydrophobic modification of both.

[0170] In exemplary embodiments of the present disclosure, the microsphere template inside the silica shell can be removed by solvent dissolution and etching. After the outer surface of the silica shell and the first surface of the substrate are hydrophobically modified, the microsphere template is removed, which is advantageous for maintaining the hydrophobic state of the outer surface of the silica shell (except for the mesoporous channels on the surface) and the first surface of the substrate, while the inner surface of the silica shell and the mesoporous channels can exhibit good hydrophilicity due to the presence of a large number of hydroxyl groups on the silica shell, thereby obtaining the droplet array generation chip of the embodiments of the present disclosure.

[0171] In exemplary embodiments of the present disclosure, the substrate can further have a second surface;

[0172] The preparation method can further include disposing a fluorescent signal blocking layer on the first surface or the second surface of the substrate.

[0173] The embodiments of the present disclosure also provide a droplet array generation method. Figure 9A flowchart of a process of forming a droplet array by a droplet array generation method of an exemplary embodiment of the present disclosure; Figure 10 A schematic diagram of an array of silica shell loaded with an aqueous phase formed by a droplet array generation method of an exemplary embodiment of the present disclosure; Figure 11 A schematic diagram of an array of silica shell loaded with an aqueous phase (reaction solution) formed by a droplet array generation method of an exemplary embodiment of the present disclosure. Figure 10 A schematic diagram of an array of silica shell loaded with an aqueous phase (reaction solution) formed by a droplet array generation method of an exemplary embodiment of the present disclosure.

[0174] As shown in Figures 9 to 11 The droplet array generation method comprises:

[0175] A droplet array generation chip is provided, which is a droplet array generation chip as described above or obtained by a preparation method of a droplet array generation chip as described above;

[0176] The droplet array generation chip is immersed in an aqueous phase 70 containing a reaction solution for generating a droplet array, the aqueous phase 70 containing the reaction solution enters the interior of the silica shell 20 through the mesopore 40 on the plurality of silica shells 20 of the droplet array generation chip, and an aqueous phase droplet 90 is generated in the interior of the silica shell 20;

[0177] An oil phase 80 is covered on the surface of the aqueous phase 70;

[0178] The droplet array generation chip is moved so that the silica shells 20 of the droplet array generation chip enter the oil phase 80, and when the droplet array generation chip passes through the interface between the aqueous phase 70 and the oil phase 80, a plurality of silica shells 20 containing the aqueous phase droplet 90 are rapidly enclosed and arranged in an array on the substrate 10 by the oil phase 80 to form a droplet array.

[0179] The droplet array generation method of the present disclosure only needs to move the substrate of the droplet array generation chip from the aqueous phase to the oil phase to quickly generate a droplet array, without additional process technology requirements, and is simple, fast and easy to implement; the droplet array enclosed in the oil phase does not need to be transferred and can be directly subjected to subsequent amplification reaction and signal detection.

[0180] From the detection and analysis of ddPCR results, the single-layer droplet array physically separated from each other can be directly subjected to camera-based fluorescence signal scanning detection in the oil phase, without the need for collecting the reaction droplets in advance or using expensive and time-consuming photomultiplier tubes (PMT) for detection; the rigid silica shell can avoid cross contamination caused by droplet fusion and ensure the accuracy of the experiment; in addition, the particle size and quantity of the silica shell used can be adjusted according to actual detection needs to cover a wider detection accuracy range.

[0181] The droplet array generation method provided in this disclosure can be applied to detection applications that require droplet array generation, such as droplet polymerase chain reaction detection, surface plasmon resonance (SPR) enhanced fluorescence signal detection, and surface enhanced Raman spectroscopy (SERS) detection.

[0182] This disclosure also provides a fluorescence detection method, the fluorescence detection method comprising:

[0183] A droplet array generation chip is provided, wherein the droplet array generation chip is as described above or is obtained by the droplet array generation chip fabrication method described above;

[0184] The droplet array generating chip is immersed in an aqueous phase containing a reaction solution for generating droplet arrays. The aqueous phase containing the reaction solution enters the interior of the silica spheres through mesopores on the multiple silica spheres of the droplet array generating chip, and aqueous droplets are generated inside the silica spheres.

[0185] An oil phase is coated on the surface of the aqueous phase;

[0186] The droplet array generating chip is moved so that the silica spherical shell of the droplet array generating chip enters the oil phase. When the droplet array generating chip passes through the interface between the aqueous phase and the oil phase, multiple silica spherical shells containing aqueous phase droplets are sealed and wrapped by the oil phase and arranged at intervals on the substrate to form a droplet array.

[0187] The droplet array generation chip is heated and an amplification reaction is carried out in the droplets of the droplet array, and the droplet array generation chip after the amplification reaction is subjected to fluorescence detection.

[0188] In exemplary embodiments of this disclosure, the fluorescence detection method includes, but is not limited to, detection methods such as polymerase chain reaction (PCR) detection.

[0189] This disclosure also provides a droplet array generation device, which includes a plurality of droplet array generation chips as described above. Figure 12A This is a top view of a droplet array generation apparatus according to an exemplary embodiment of the present disclosure; Figure 12B for Figure 12A The front view of the droplet array generation device shown. Figure 12A and Figure 12B As shown, the droplet array generation device integrates multiple, for example, 16 identical droplet array generation chips onto a glass plate with a size of 24mm×20mm×0.2mm.

[0190] Each of the droplet array generation chips can be independently injected, and rapid generation of the droplet array and nucleic acid amplification reaction can be achieved. Therefore, the droplet array generation device adopting the embodiments of the present disclosure can perform synchronous operation on 16 samples in a smaller area, and high-throughput detection of the nucleic acid molecules to be detected can be achieved.

[0191] Although the embodiments disclosed in the present disclosure are as above, the content described is only the embodiments adopted for the purpose of facilitating understanding of the present disclosure, and is not intended to limit the present disclosure. Any person skilled in the art can make any modification and change in the form and details without departing from the spirit and scope of the present disclosure, but the protection scope of the present disclosure shall be subject to the scope defined by the appended claims.

Claims

1. A droplet array generation chip, characterized by, Comprising: a substrate having a first surface; a plurality of silica shell balls arranged in a single layer on the first surface of the substrate at intervals, the silica shell balls being hollow shell structures, and having mesopores on the shell structures, the silica shell balls having inner surfaces and outer surfaces, the mesopores communicating the inner surfaces and the outer surfaces of the silica shell balls, the outer surfaces of the silica shell balls being hydrophobic, and the inner surfaces of the silica shell balls and the pore surfaces of the mesopores being hydrophilic.

2. The droplet array generation chip of claim 1, wherein, The outer diameter of the silica shell balls is 20 μm to 30 μm, and the shell layer thickness of the silica shell balls is 1.5 μm to 3 μm.

3. The droplet array generation chip of claim 1, wherein, The outer surface of the silica shell balls has a first hydrophobic functional group.

4. The droplet array generation chip of claim 2, wherein, The outer surface of the silica shell balls has a first hydrophobic functional group.

5. The droplet array generation chip of claim 3, wherein, The first hydrophobic functional group is selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group.

6. The droplet array generation chip of claim 4, wherein, The first hydrophobic functional group is selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group.

7. The droplet array generation chip of claim 5, wherein, The first hydrophobic functional group is attached to the outer surface of the silica shell balls via a Si-O-Si chemical bond.

8. The droplet array generation chip of claim 6, wherein, The first hydrophobic functional group is attached to the outer surface of the silica shell balls via a Si-O-Si chemical bond.

9. The droplet array generation chip of any one of claims 1 to 8, wherein, The inner surfaces of the silica shell balls and the pore surfaces of the mesopores have a hydrophilic functional group.

10. The droplet array generation chip of any one of claims 1 to 8, wherein, The pore diameter of the mesopores is 0.8 μm to 1.5 μm.

11. The droplet array generation chip of claim 9, wherein, The pore diameter of the mesopores is 0.8 μm to 1.5 μm.

12. The droplet array generation chip of claim 10, wherein, The distribution density of the mesopores on the outer surface of the silica shell is 1 mesopore per 1.13 pm 2 to 11.04 pm 2 1 mesopore is present on the outer surface.

13. The droplet array generation chip of claim 11, wherein, The distribution density of the mesopores on the outer surface of the silica shell is 1 mesopore per 1.13 pm 2 to 11.04 pm 2 1 mesopore is present on the outer surface.

14. The droplet array generation chip of any one of claims 1 to 8, 11 to 13, wherein, The distance between adjacent two of the silica shell balls is D, and the outer diameter of the silica shell balls is R, 0 < D ≤ R.

15. The droplet array generation chip of claim 9, wherein, The distance between adjacent two of the silica shell balls is D, and the outer diameter of the silica shell balls is R, 0 < D ≤ R.

16. The droplet array generation chip of claim 10, wherein, The distance between adjacent two of the silica shell balls is D, and the outer diameter of the silica shell balls is R, 0 < D ≤ R.

17. The droplet array generation chip of any one of claims 1 to 8, 11 to 13, 15 to 16, wherein, The first surface of the substrate is hydrophobic.

18. The droplet array generation chip of claim 9, wherein, The first surface of the substrate is hydrophobic.

19. The droplet array generation chip of claim 10, wherein, The first surface of the substrate is hydrophobic.

20. The droplet array generation chip of claim 14, wherein, The first surface of the substrate is hydrophobic.

21. The droplet array generation chip of claim 17, wherein, The first surface of the substrate has a second hydrophobic functional group.

22. The droplet array generation chip of any one of claims 18 to 20, wherein, The first surface of the substrate has a second hydrophobic functional group.

23. The droplet array generation chip of any one of claims 1-8, 11-13, 15-16, 18-21, wherein, The plurality of silica shell balls and the substrate are bonded together using a Si-O chemical bond.

24. The droplet array generation chip of claim 9, wherein, The plurality of silica shell balls and the substrate are bonded together using a Si-O chemical bond.

25. The droplet array generation chip of claim 10, wherein, The plurality of silica shell balls and the substrate are bonded together using a Si-O chemical bond.

26. The droplet array generation chip of claim 14, wherein, The plurality of silica shell balls and the substrate are bonded together using a Si-O chemical bond.

27. The droplet array generation chip of claim 17, wherein, The plurality of silica shell balls and the substrate are bonded together using a Si-O chemical bond.

28. The droplet array generation chip of claim 22, wherein, The plurality of silica shell balls and the substrate are bonded together using a Si-O chemical bond.

29. The droplet array generation chip of any one of claims 1-8, 11-13, 15-16, 18-21, 24-28, wherein, The substrate further has a second surface, and a fluorescent signal blocking layer is provided on the first surface or the second surface of the substrate.

30. The droplet array generation chip of claim 9, wherein, The substrate further has a second surface, and a fluorescent signal blocking layer is provided on the first surface or the second surface of the substrate.

31. The droplet array generation chip of claim 10, wherein, The substrate further has a second surface, and a fluorescent signal blocking layer is arranged on the first surface or the second surface of the substrate.

32. The droplet array generation chip of claim 14, wherein, The substrate further has a second surface, and a fluorescent signal blocking layer is arranged on the first surface or the second surface of the substrate.

33. The droplet array generation chip of claim 17, wherein, The substrate further has a second surface, and a fluorescent signal blocking layer is arranged on the first surface or the second surface of the substrate.

34. The droplet array generation chip of claim 22, wherein, The substrate further has a second surface, and a fluorescent signal blocking layer is arranged on the first surface or the second surface of the substrate.

35. The droplet array generation chip of claim 23, wherein, The substrate further has a second surface, and a fluorescent signal blocking layer is arranged on the first surface or the second surface of the substrate.

36. A method of fabricating a droplet array generation chip, comprising: Comprising: a substrate is provided, the substrate having a first surface; a plurality of silica shell spheres are formed on the first surface of the substrate, the silica shell spheres being hollow shell structures and having mesopores on the shell structures, the silica shell spheres having inner surfaces and outer surfaces, the mesopores communicating the inner surfaces and the outer surfaces of the silica shell spheres, the outer surfaces of the silica shell spheres being hydrophobic, and the inner surfaces of the silica shell spheres and the pore surfaces of the mesopores being hydrophilic.

37. The preparation method according to claim 36, wherein the forming a plurality of silica shell spheres on the first surface of the substrate comprises: a first modification is performed on the first surface of the substrate, so that at least one of a hydroxyl group and a functional group capable of being hydrolyzed into a hydroxyl group is introduced on the first surface of the substrate; a plurality of core-shell microspheres having a core-shell structure are prepared, the shell of the core-shell microspheres being an initial silica shell sphere without mesopores, and the core of the core-shell microspheres being configured to be capable of providing support for the initial silica shell sphere and capable of being removed; the plurality of core-shell microspheres are arranged on the first surface of the substrate subjected to the first modification in a manner of being spaced apart from each other and in a single layer; a hydrophobic modification is performed on the outer surface of the core-shell microspheres, so that the outer surface of the core-shell microspheres is hydrophobic; mesopores are formed on the initial silica shell sphere, and the core of the core-shell microspheres is removed, so that a silica shell sphere with mesopores is obtained, and the mesopores communicate the inner surfaces and the outer surfaces of the silica shell sphere; the hydroxyl group possessed by the silica shell sphere itself makes the inner surfaces of the silica shell sphere and the pore surfaces of the mesopores exhibit hydrophilicity.

38. The method of manufacturing according to claim 37, wherein, The arranging the plurality of core-shell microspheres on the first surface of the substrate subjected to the first modification in a manner of being spaced apart from each other and in a single layer comprises: the plurality of core-shell microspheres are dispersed in a solvent to obtain a solution containing core-shell microspheres; the substrate subjected to the first modification is placed in the solution containing core-shell microspheres, and the plurality of core-shell microspheres are arranged on the first surface of the substrate subjected to the first modification in a manner of being spaced apart from each other and in a single layer by using a gravity deposition method; under vacuum drying conditions, the hydroxyl group possessed by the outer surface of the initial silica shell sphere of the core-shell microspheres itself and the hydroxyl group on the first surface of the substrate subjected to the first modification or the hydroxyl group obtained by hydrolysis of the functional group capable of being hydrolyzed into a hydroxyl group perform a dehydration condensation reaction, so that the core-shell microspheres and the substrate are combined together by using a chemical bond.

39. The method of claim 38, further comprising: subjecting the first surface of the once-modified substrate to a second hydrophobic modification to render the first surface of the substrate hydrophobic; forming a hydrophobic interaction between the outer surface of the hydrophobically-modified core-shell microspheres and the first surface of the substrate subjected to the second hydrophobic modification, the core-shell microspheres and the substrate being bound together using the chemical bond and the hydrophobic interaction.

40. The method of manufacturing according to claim 39, wherein, the first hydrophobic modification agent used to hydrophobically modify the outer surface of the core-shell microspheres is a silane coupling agent containing a first hydrophobic functional group; the second hydrophobic modification agent used to hydrophobically modify the first surface of the once-modified substrate is a silane coupling agent containing a second hydrophobic functional group; the first and second hydrophobic functional groups are selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group; the first and second hydrophobic modification agents are selected from any one or more of methyltriethoxysilane, octadecyltrichlorosilane, octadecyltrimethoxysilane, trifluoropropyltriethoxysilane, perfluorooctyltriethoxysilane, chloromethyltrimethoxysilane, and (chloromethyl)methyldiethoxysilane.

41. The method of manufacturing according to any one of claims 36 to 40, wherein, the substrate further has a second surface; the method further comprises disposing a fluorescent signal blocking layer on the first or second surface of the substrate.

42. A method of droplet array generation, comprising: comprising: providing a droplet array generation chip, the droplet array generation chip being according to any one of claims 1 to 35 or being obtained by the method of preparation of a droplet array generation chip according to any one of claims 36 to 41 ; immersing the droplet array generation chip in an aqueous phase containing a reaction solution for generating a droplet array, the aqueous phase containing the reaction solution entering the interior of the plurality of silica shell through the mesopores on the silica shell of the droplet array generation chip, generating aqueous phase droplets in the interior of the silica shell; covering the aqueous phase with an oil phase; moving the droplet array generation chip so that the silica shell of the droplet array generation chip enters the oil phase, a plurality of the silica shell containing the aqueous phase droplets in the interior being enclosed by the oil phase and arranged at intervals on the substrate to form a droplet array as the droplet array generation chip passes through the interface of the aqueous phase and the oil phase.

43. A method of fluorescent detection, comprising: comprising: providing a droplet array generation chip, the droplet array generation chip being according to any one of claims 1 to 35 or being obtained by the method of preparation of a droplet array generation chip according to any one of claims 36 to 41 ; immersing the droplet array generation chip in an aqueous phase containing a reaction solution for generating a droplet array, the aqueous phase containing the reaction solution entering the interior of the plurality of silica shell through the mesopores on the silica shell of the droplet array generation chip, generating aqueous phase droplets in the interior of the silica shell; covering the aqueous phase with an oil phase; moving the droplet array generation chip so that the silica shell of the droplet array generation chip enters the oil phase, when the droplet array generation chip passes through the interface between the water phase and the oil phase, a plurality of the silica shells containing the water phase droplets inside are enclosed and arranged at intervals by the oil phase on the substrate to form a droplet array; heating the droplet array generation chip and performing amplification reaction in the droplets of the droplet array, and performing fluorescence detection on the droplet array generation chip after the amplification reaction.

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