CN115895860A

CN115895860A - Droplet array generation chip and preparation method and application thereof

Info

Publication number: CN115895860A
Application number: CN202211366142.0A
Authority: CN
Inventors: 鄢健
Original assignee: BOE Technology Group Co Ltd
Current assignee: BOE Technology Group Co Ltd
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2023-04-04

Abstract

A liquid drop array generating chip and a preparation method and application thereof are provided, the liquid drop array generating chip comprises: a substrate having a first surface; the silicon dioxide spherical shells are arranged on the first surface of the substrate at intervals in a single layer mode, the silicon dioxide spherical shells are of a shell-shaped structure with a hollow interior and are provided with mesopores, the silicon dioxide spherical shells are provided with inner surfaces and outer surfaces, the mesopores are communicated with the inner surfaces and the outer surfaces of the silicon dioxide spherical shells, the outer surfaces of the silicon dioxide spherical shells are hydrophobic, and the inner surfaces of the silicon dioxide spherical shells and the pore channel surfaces of the mesopores are hydrophilic. The liquid drop array generating chip disclosed by the embodiment of the disclosure has the advantages of simple structure, simplicity and convenience in operation, capability of quickly generating the liquid drop array, stable liquid drop size and difficulty in cross contamination among liquid drops.

Description

Droplet array generation chip and preparation method and application thereof

Technical Field

The disclosed embodiments relate to, but are not limited to, the technical field of biochips, and in particular, to a droplet array generation chip, and a preparation method and application thereof.

Background

The Polymerase Chain Reaction (PCR) technique is a practical biological technique for artificially amplifying Deoxyribonucleotide (DNA) produced in the 90's of the last century, and generally, template DNA, primers, DNA Polymerase and deoxyribonucleotide triphosphate (dNTP) are mixed in a buffer solution, and an exponentially increased amount of template DNA products are generated after a plurality of thermal cycling (denaturation, annealing and extension) reactions.

After more than thirty years of development, the PCR technology has been widely applied in the research fields of biomedical diagnosis and detection, cell molecular biology, genetic engineering, pathology, pharmaceutical science and the like. The digital Polymerase Chain Reaction (dPCR) technology is a new generation of DNA amplification technology developed from PCR, which divides a PCR Reaction solution into thousands of individual Reaction regions to perform amplification reactions, respectively, and calculates the initial concentration of a target DNA molecule by quantifying the number of positive and negative Reaction regions and using poisson distribution statistics. For the partitioning method of the Reaction solution in the current dPCR technology, a small droplet Reaction zone is generated in an oil phase through a T-junction or fluid focused injection, which is a droplet digital PCR (ddPCR) technology, is adopted in a large amount and at a low cost, and often requires complicated punching technology and equipment, and often faces problems of unstable droplet generation frequency and droplet size, easy cross contamination among droplets, an excessively complicated signal detection system, and the like.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the present disclosure.

The disclosed embodiments provide a droplet array generation chip, which includes:

a substrate having a first surface;

the silicon dioxide spherical shells are arranged on the first surface of the substrate at intervals in a single layer mode, each silicon dioxide spherical shell is of a shell-shaped structure with a hollow interior and is provided with a mesoporous hole, each silicon dioxide spherical shell is provided with an inner surface and an outer surface, each mesoporous hole is communicated with the inner surface and the outer surface of each silicon dioxide spherical shell, the outer surfaces of the silicon dioxide spherical shells are hydrophobic, and the inner surfaces of the silicon dioxide spherical shells and the pore channel surfaces of the mesoporous holes are hydrophilic.

In an exemplary embodiment of the present disclosure, the outer diameter of the silica spherical shell may be 20 to 30 μm, and the shell thickness of the silica spherical shell may be 1.5 to 3 μm.

In exemplary embodiments of the present disclosure, the outer surface of the silica spherical shell may have a first hydrophobic functional group thereon.

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

In exemplary embodiments of the present disclosure, the first hydrophobic functional group may be attached on the outer surface of the silica spherical shell through a Si-O-Si chemical bond.

In exemplary embodiments of the present disclosure, the inner surface of the silica spherical shell and the pore channel surface of the mesopores may be provided with a hydrophilic functional group.

In exemplary embodiments of the present disclosure, the pore size of the mesopores may be 0.8 μm to 1.5 μm.

In exemplary embodiments of the present disclosure, the distribution density of the mesopores on the outer surface of the silica spherical shell may be 1.13 μm per ² To 11.04 μm ² There are 1 mesopore on the outer surface of (a).

In an exemplary embodiment of the present disclosure, a distance between two adjacent silica spherical shells is D, an outer diameter of the silica spherical shell is R, and D and R may satisfy: d is more than 0 and less than or equal to R.

In exemplary embodiments of the present disclosure, the first surface of the substrate may be hydrophobic.

In exemplary embodiments of the present disclosure, the first surface of the substrate may have a second hydrophobic functional group thereon.

In an exemplary embodiment of the present disclosure, the silica spherical shells and the substrate may be bonded together using Si — O chemical bonds.

In an exemplary embodiment of the present disclosure, the substrate further has a second surface, and a fluorescent signal blocking layer may be disposed on the first surface or the second surface of the substrate.

The embodiment of the present disclosure further provides a method for manufacturing a droplet array generation chip, where the method includes:

providing a substrate having a first surface;

the method comprises the steps of forming a plurality of silica spherical shells which are arranged at intervals in a single layer on a first surface of a substrate, wherein the silica spherical shells are of a shell-shaped structure with a hollow interior and are provided with mesopores, each silica spherical shell is provided with an inner surface and an outer surface, the mesopores are communicated with the inner surface and the outer surface of each silica spherical shell, the outer surfaces of the silica spherical shells are hydrophobic, and the inner surfaces of the silica spherical shells and the pore channel surfaces of the mesopores are hydrophilic.

In an exemplary embodiment of the present disclosure, the forming of the plurality of silica spherical shells spaced apart from each other and arranged in a single layer on the first surface of the substrate may include:

performing primary modification 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;

preparing a plurality of core-shell microspheres having a core-shell structure, the shells of the core-shell microspheres being initial silica spherical shells without mesopores, the cores of the core-shell microspheres being configured to be capable of providing support to the initial silica spherical shells and to be removable;

arranging a plurality of core-shell microspheres on the first surface of the substrate subjected to primary modification at intervals in a single layer;

performing hydrophobic modification on the outer surface of the core-shell microsphere to make the outer surface of the core-shell microsphere hydrophobic;

forming mesopores on the initial silica spherical shell, removing the core of the core-shell microsphere, and obtaining a silica spherical shell with mesopores, wherein the mesopores are communicated with the inner surface and the outer surface of the silica spherical shell;

the inner surface of the silicon dioxide spherical shell and the pore channel surface of the mesopore are rendered hydrophilic by the hydroxyl group carried by the silicon dioxide spherical shell.

In an exemplary embodiment of the present disclosure, the arranging the plurality of core-shell microspheres spaced apart from each other and in a monolayer on the first surface of the once-modified substrate may include:

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

placing the once-modified substrate in the solution containing the core-shell microspheres, and arranging a plurality of the core-shell microspheres on the first surface of the once-modified substrate at intervals in a single layer by adopting a gravity deposition method;

under the vacuum drying condition, hydroxyl groups carried by the outer surface of the initial silicon dioxide spherical shell of the core-shell microsphere and hydroxyl groups on the first surface of the substrate subjected to primary modification or hydroxyl groups obtained by hydrolysis of functional groups capable of being hydrolyzed into hydroxyl groups are subjected to dehydration condensation reaction, so that the core-shell microsphere and the substrate are bonded together by chemical bonds.

In an exemplary embodiment of the present disclosure, the preparation method may further include:

performing secondary hydrophobic modification on the first surface of the substrate subjected to the primary modification to make the first surface of the substrate hydrophobic;

hydrophobic interaction is formed between the outer surface of the core-shell microsphere subjected to hydrophobic modification (namely the outer surface of the silica spherical shell) and the first surface of the substrate subjected to secondary hydrophobic modification, and the core-shell microsphere and the substrate are combined together by utilizing the chemical bond and the hydrophobic interaction.

In exemplary embodiments of the present disclosure, the first hydrophobic modifier used to hydrophobically modify the outer surface of the silica spherical shell may be a silane coupling agent containing a first hydrophobic functional group.

In an exemplary embodiment of the present disclosure, the second hydrophobic modifier used to hydrophobically modify the first surface of the substrate, which has been once modified, twice may be a silane coupling agent containing a second hydrophobic functional group.

In exemplary embodiments of the present disclosure, the first and second hydrophobic functional groups may be selected from any one or more of saturated or unsaturated alkyl groups, fluoroalkyl groups, and chloroalkyl groups.

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

In an exemplary embodiment of the present disclosure, the substrate may further have a second surface;

the preparation method can also comprise the following steps: a fluorescent signal blocking layer is disposed on the first surface or the second surface of the substrate.

The embodiment of the present disclosure also provides a droplet array generation method, which includes:

providing a droplet array generation chip, wherein the droplet array generation chip is the droplet array generation chip or is obtained by the preparation method of the droplet array generation chip;

immersing the droplet array generation chip in an aqueous phase containing a reaction solution for generating a droplet array, wherein the aqueous phase containing the reaction solution enters the interior of the silica spherical shell through the mesopores on the plurality of silica spherical shells of the droplet array generation chip, and aqueous phase droplets are generated in the interior of the silica spherical shell;

covering the surface of the water phase with an oil phase;

and moving the liquid drop array generating chip to enable the silica spherical shell of the liquid drop array generating chip to enter the oil phase, wherein when the liquid drop array generating chip passes through the interface between the water phase and the oil phase, a plurality of silica spherical shells containing the liquid drops of the water phase are enclosed and wrapped by the oil phase and are arranged on the substrate at intervals to form a liquid drop array.

The embodiment of the present disclosure also provides a fluorescence detection method, including:

covering the surface of the water phase with an oil phase;

moving the droplet array generation chip to enable the silica spherical shell of the droplet array generation chip to enter the oil phase, wherein when the droplet array generation chip passes through the interface between the water phase and the oil phase, a plurality of silica spherical shells containing the water phase droplets are enclosed and wrapped by the oil phase and are arranged on the substrate at intervals to form a droplet array;

heating the droplet array generation chip, carrying out amplification reaction in the droplets of the droplet array, and carrying out fluorescence detection on the droplet array generation chip after the amplification reaction.

Additional features and advantages of the disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the disclosure. Other advantages of the disclosure may be realized and attained by the instrumentalities and combinations particularly pointed out in the specification and the drawings.

Drawings

The accompanying drawings are included to provide an understanding of the disclosed embodiments and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the example serve to explain the principles of the disclosure and not to limit the disclosure.

FIG. 1 is a schematic diagram of a droplet array generation chip according to an exemplary embodiment of the present disclosure;

FIG. 2 is a schematic diagram of a longitudinal cross-sectional structure of the droplet array generating chip shown in FIG. 1;

FIG. 3 is a schematic structural diagram of a silica spherical shell of the droplet array generating chip shown in FIG. 1;

FIG. 4 is a diagram corresponding to equations (1) to (4);

FIG. 5A is a schematic of a droplet distribution with a planar microstructure model surface of similar size and hydrophilic/hydrophobic nature to the silica spherical shell of an exemplary embodiment of the present disclosure;

FIG. 5B is a schematic diagram showing the distribution of the aqueous phase in the planar microstructure model shown in FIG. 5A;

FIG. 6 is a schematic diagram of a longitudinal cross-sectional structure of another droplet array generation chip according to an exemplary embodiment of the disclosure;

fig. 7 is a reaction mechanism diagram illustrating a dehydration condensation reaction when core-shell microspheres are aligned and fixed on a substrate surface in a manufacturing method according to an exemplary embodiment of the present disclosure;

FIG. 8 is a schematic diagram illustrating a reaction mechanism of hydrophobic modification of the outer surface of the core-shell microsphere and the first surface of the substrate in a method of making an exemplary embodiment of the present disclosure;

FIG. 9 is a schematic flow chart of a droplet array formation method of an exemplary embodiment of the present disclosure;

FIG. 10 is a schematic diagram of silica spherical shells loaded with an aqueous phase (reaction solution) formed by a droplet array generating method according to an exemplary embodiment of the present disclosure arranged in an array on a substrate;

FIG. 11 is a structural schematic of the silica sphere shell containing the aqueous phase of FIG. 10;

FIG. 12A is a top view of a droplet array generation apparatus according to an exemplary embodiment of the present disclosure;

fig. 12B is a front view of the droplet array generating device shown in fig. 12A.

The reference symbols in the drawings have the following meanings:

10-a substrate; 20-silica spherical shell; 21-an inner surface; 22-an outer surface; 30-a fluorescent signal blocking layer; 40-mesopores; 50-a housing; 60-a push rod; 70-aqueous phase; 80-oil phase; 90-aqueous phase droplets.

Detailed Description

To make the objects, technical solutions and advantages of the present disclosure more apparent, embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. It should be noted that, in the present disclosure, the embodiments and features of the embodiments may be arbitrarily combined with each other without conflict.

The embodiments herein may be embodied in a number of different forms. Those skilled in the art can readily appreciate the fact that the present implementations and teachings can be modified into a variety of forms without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure should not be construed as being limited to the contents described in the following embodiments. The embodiments and features of the embodiments in the present disclosure may be arbitrarily combined with each other without conflict.

The scale of the drawings in this disclosure may be referenced in actual processing, but is not limited to such. For example: the width-length ratio of the channel, the thickness and the interval of each film layer and the width and the interval 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 not limited to the numbers shown in the drawings, and the drawings described in the present disclosure are only schematic structural views, and one embodiment of the present disclosure is not limited to the shapes, numerical values, or the like shown in the drawings.

In this specification, for convenience, terms indicating orientation or positional relationship such as "middle", "upper", "lower", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like are used to explain positional relationship of constituent elements with reference to the drawings, only for convenience of description and simplification of description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present disclosure. The positional relationship of the components is changed as appropriate in accordance with the direction in which each component is described. Therefore, the words and phrases described in the specification are not limited thereto, and may be replaced as appropriate depending on the case.

In this specification, the terms "disposed" and "connected" are to be construed broadly unless otherwise explicitly stated or limited. For example, it may be a fixed connection, or a removable connection, or an integral connection; can be a mechanical connection, or an electrical connection; either directly or indirectly through intervening components, or both may be interconnected. The specific meaning of the above terms in the present disclosure can be understood in specific instances by those of ordinary skill in the art.

In the description of the present disclosure, ordinal numbers such as "first", "second", and the like are provided to avoid confusion of constituent elements, and are not limited in number.

a substrate having a first surface;

In the description of the present disclosure, "hydrophobic" means that the water contact angle is greater than 90 °; by "hydrophilic" is meant that the water contact angle is less than 90 °.

In the embodiment of the present disclosure, there is no requirement for the shape of the "silica spherical shell," which is not limited to a regular sphere, but may be an ellipsoid, a regular tetrahedron, a regular hexahedron, or the like, as long as the silica spherical shell can physically space the droplets contained therein.

The silicon dioxide spherical shell with the mesopores of the liquid drop array generating chip disclosed by the embodiment of the disclosure can be used as a guide template, the liquid drop array can be quickly generated through simple space displacement between the interfaces of the water phase and the oil phase, and the complex punching technology and equipment can be avoided; moreover, the water phase liquid drops formed by the liquid drop array generating chip of the embodiment of the disclosure are wrapped and spaced by the silicon dioxide spherical shell and fixed with the substrate, so that cross contamination among the liquid drops can be avoided, and efficient PCR amplification reaction and signal detection and analysis can be conveniently realized; in addition, the size of the liquid drop can be regulated and controlled by adjusting the inner diameter of the silicon dioxide spherical shell, so that the problem of unstable size of the liquid drop is solved.

In addition, the liquid drop array generating chip disclosed by the embodiment of the disclosure has the advantages of simple structure and simplicity and convenience in operation, and can avoid the use of complex surface microstructure processing equipment and process, so that the production and manufacturing cost is further reduced. FIG. 1 is a schematic diagram of a droplet array generation chip according to an exemplary embodiment of the disclosure; FIG. 2 is a schematic diagram of a longitudinal cross-sectional structure of the droplet array generating chip shown in FIG. 1; fig. 3 is a schematic structural view of a silica spherical shell of the droplet array generating chip shown in fig. 1. As shown in fig. 1 to 3, the droplet array generating chip includes a substrate 10 and a plurality of silica spherical shells 20;

the substrate 10 has a first surface and a second surface, and the fluorescent signal blocking layer 30 is disposed on the second surface of the substrate 10; the substrate 10 mainly functions to provide a substrate for arranging and fixing the plurality of silica spherical shells 20, so that the plurality of silica spherical shells 20 can form a randomly dispersed microsphere array, and is responsible for driving the silica spherical shells 20 to lift and displace when a droplet array is generated; the fluorescent signal blocking layer 30 is used for blocking fluorescent signals from passing through the second surface of the substrate 10, otherwise, the scanning detection of the fluorescent signals cannot be carried out; in other embodiments, the fluorescence signal blocking layer 30 may also be disposed on the first surface of the substrate 10, for example, may be located between the first surface of the substrate 10 and the plurality of silica spherical shells 20;

a plurality of silica spherical shells 20 are arranged on the first surface of the substrate 10 at intervals in a single layer, the silica spherical shells 20 are of a shell-shaped structure with a hollow interior, and the shell-shaped structure has a plurality of mesopores 40, each silica spherical shell 20 has an inner surface 21 and an outer surface 22, the mesopores 40 communicate with the inner surface 21 and the outer surface 22 of the silica spherical shell 20, the outer surface 22 of the silica spherical shell 20 is hydrophobic, and the pore surfaces of the inner surface 21 of the silica spherical shell 20 and the mesopores 40 are hydrophilic; when the liquid drop array is generated, the silicon dioxide spherical shell 20 is mainly used for wrapping the liquid drops and maintaining the shape of the liquid drops, and rigid physical isolation is formed on the surfaces of the liquid drops so as to avoid the liquid drops from contacting with each other; the spherical shell formed by the silicon dioxide material has lower inherent fluorescence intensity, and can effectively reduce the interference on detection signals.

In an exemplary embodiment of the present disclosure, the first surface and the second surface of the substrate may be respectively located at opposite sides of the substrate.

From the report of the reference (J.Phys.chem.B. 2021,125,3,883-894), it is known that the stable maintenance of the monostable Cassie state (non-wetting state) of the surface of the hydrophobic material is critical to the apparent contact angle theta of the inclined wall surface of the micropore _e Much larger than the initial contact angle theta of the liquid with the surface of the material _t0 The former is mainly determined by the hydrophilic/hydrophobic properties of the material surface, and the latter is further influenced by the geometrical shape and spacing of the surface microstructure. Fig. 4 is a diagram corresponding to the following equations (1) to (4).

θ _e ∈(0，θt ₀ )(Wenzel state) (2)

On the contrary, when θ is expressed by the formula (2) _e Less than theta _t0 In time, the liquid tends to spontaneously form a stable warm zel state, i.e., a spontaneous wetting state, on the surface of the porous material. For the droplet array generation chip of the embodiment of the present disclosure, the inner surface of the silica spherical shell and the pore of the mesopores are hydrophilic, that is, the apparent contact angle θ of the pore wall is _e Is much less than 90 DEG, and theta can be known according to the formula (1) _t0 Is always greater than 90 degrees, theta can be obtained _e Is always less than theta _t0 . According to the above literature references and corresponding analysis, the aqueous phase liquid can spontaneously wet the pore channels of the mesopores and thus smoothly enter the interior of the silica spherical shell. In addition, the gravity, internal hydraulic pressure and capillary phenomenon (each mesoporous channel can be similar to a capillary) of the aqueous phase liquid can promote the aqueous phase liquid to enter the inside of the silica spherical shell along the channel.

FIG. 5A is a schematic of a droplet distribution with a planar microstructure model surface of similar size and hydrophilic/hydrophobic nature to the silica spherical shell of an exemplary embodiment of the present disclosure; FIG. 5B is a schematic diagram of the distribution of the water phase in the planar microstructure model shown in FIG. 5A. As can be seen from fig. 5A and 5B, the aqueous phase can successfully enter the interior of the silica spherical shell of the droplet array generating chip of the exemplary embodiment of the present disclosure. Meanwhile, the outer surface (except surface pore channels) of the silicon dioxide spherical shell is in a hydrophobic state, so that the silicon dioxide spherical shell containing the PCR reaction solution is quickly wrapped and sealed by the oil phase when entering the oil phase from the water phase, and a liquid drop array arranged on the surface of the substrate is formed.

In exemplary embodiments of the present disclosure, the outer diameter of the silica spherical shell may be 20 μm to 30 μm, for example, may 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 20 μm to 30 μm, it is advantageous to both prepare the silica spherical shell and arrange the silica spherical shells on a suitable substrate area in an amount equivalent to the number of partitions, a volume of partitions (about pL-nL level) and dispersed with each other of a currently commercially available dPCR apparatus (e.g., ketjen, seemer fly, fuluda, berle, etc.).

In exemplary embodiments of the present disclosure, the shell thickness of the silica spherical shell may be 1.5 μm to 3 μm, for example, may 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 1.5 to 3 μm, the internal hollow region for storing liquid droplets in the silica spherical shell is not small and collapse of the hollow structure is not easily caused.

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

In exemplary embodiments of the present disclosure, the first hydrophobic functional group may be selected from any one or more of a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group, for example, may 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.

In exemplary embodiments of the present disclosure, the first hydrophobic functional group may be attached on the outer surface of the silica sphere shell through a Si-O-Si chemical bond.

Furthermore, in exemplary embodiments of the present disclosure, the outer surface of the silica spherical shell may also be rendered hydrophobic by physically adsorbing a hydrophobic material or chemically modifying the first hydrophobic functional group, the hydrophobic monolayer, and the hydrophobic polymer.

The hydrophobic material may be selected from any one or more of polytetrafluoroethylene, polystyrene, polymethyl methacrylate and hexafluorobutyl acrylate.

The hydrophobic monolayer may be formed of a material including a first hydrophobic functional group, and the hydrophobic monolayer may be formed on the outer surface of the silica spherical shell by a self-assembly method.

The hydrophobic polymer may be formed on the outer surface of the silica spherical shell by a chemical grafting method.

In exemplary embodiments of the present disclosure, the inner surface of the silica spherical shell and the pore channel surface of the mesopores may carry hydrophilic functional groups.

In exemplary embodiments of the present disclosure, the inner surface of the silica spherical shell and the pore surface of the mesopores may be rendered hydrophilic by introducing hydrophilic functional groups, for example, the inner surface of the silica spherical shell and the pore surface of the mesopores may be provided with hydrophilic functional groups.

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

In exemplary embodiments of the present disclosure, the pore size of the mesopores may be 0.8 μm to 1.5 μm. When the pore diameter of the mesopores is selected to be the minimum value in the range of 0.8-1.5 μm, the pore diameter is still larger than the wavelength of visible light (390-780 nm), and the influence of the diffraction of the micropores on the intensity of a fluorescence signal can be reduced; in addition, when the pore diameter of the mesopores is 0.8 μm to 1.5 μm, it is advantageous to maintain the stability of the hollow structure of the silica spherical shell, and it is possible to prevent the liquid droplets formed inside the silica spherical shell from leaking out from the inside of the silica spherical shell coated with the oil phase.

In an exemplary embodiment of the present disclosure, the outer diameter of the silica spherical shell may be 20 μm to-30 μm, and the number of the mesopores on each of the silica spherical shells may be 110 to 2500.

In the exemplary embodiment of the disclosure, the distance between the channels of two adjacent mesopores is d, the pore diameter of the mesopores is r, and d is greater than or equal to 0.5r and less than or equal to 1.5r. Here, the "interval between the channels of the adjacent two mesopores" refers to a distance between edges of opposite sides of the channels of the adjacent two mesopores, not a distance between the centers of the channels of the adjacent two mesopores.

When the distribution density of the mesopores on the outer surface of the silica spherical shell is 1.13 mu m ² To 11.04 μm ² Or d and r satisfy: when d is more than or equal to 0.5r and less than or equal to 1.5r, the stability of the hollow structure of the silica spherical shell is favorably maintained, and liquid drops formed inside the silica spherical shell can be prevented from leaking out of the interior of the silica spherical shell wrapped by the oil phase.

In an exemplary embodiment of the present disclosure, a distance between two adjacent silica spherical shells is D, an outer diameter of the silica spherical shell is R, and D and R may satisfy: d is more than 0 and less than or equal to R. D and R satisfy: when D is more than 0 and less than or equal to R, two adjacent silicon dioxide spherical shells can be well spaced, and cross contamination among liquid drops formed in the silicon dioxide spherical shells during fluorescence detection can be avoided.

In an exemplary embodiment of the present disclosure, the distribution density of the silica spherical shells on the first surface of the substrate may be 250/mm ² To 2500 pieces/mm ² 。

In an exemplary embodiment of the present disclosure, the size of the substrate may be selected according to actual detection requirements, for example, may be length × width × thickness =5mm × 4mm × 0.2mm.

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

In an exemplary embodiment of the present disclosure, the first surface of the substrate may be hydrophobic.

In exemplary embodiments of the present disclosure, the first surface of the substrate may be rendered hydrophobic by introducing a second hydrophobic functional group, for example, the first surface of the substrate may be provided with the second hydrophobic functional group thereon.

In exemplary embodiments of the present disclosure, the second hydrophobic functional group may be selected from any one or more of a hydrocarbon group, a saturated or unsaturated hydrocarbon group, a fluorocarbon group, and a chlorocarbon group, for example, may 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.

In exemplary embodiments of the present disclosure, the first hydrophobic functional group and the second hydrophobic functional group are the same.

In exemplary embodiments of the present disclosure, the plurality of silica spherical shells and the substrate may be bonded together using Si — O chemical bonds.

In exemplary embodiments of the present disclosure, a plurality of the silica spherical shells and the substrate may be bonded together using Si — O chemical bonds and hydrophobic interactions.

The first surface of the substrate can be subjected to at least one of hydroxylation modification and alkoxy silanization modification, the first surface of the substrate subjected to the modification can be provided with hydroxyl or alkoxy, and the alkoxy can be hydrolyzed to generate hydroxyl; hydroxyl groups on the first surface of the substrate or hydroxyl groups obtained by alkoxy hydrolysis and hydroxyl groups on the outer surface of the silicon dioxide spherical shell can be subjected to dehydration condensation to form stable Si-O chemical bonds, so that a plurality of silicon dioxide spherical shells and the substrate can be bonded together by using the chemical bonds; if the outer surface of the silica spherical shell and the first surface of the substrate are hydrophobic, the two surfaces tend to aggregate in an aqueous solution by simultaneously avoiding water, i.e., the two surfaces with hydrophobicity are mutually attracted to form hydrophobic interaction. The stable adhesion of the silica spherical shell on the surface of the substrate can be maintained by two acting forces of chemical bonds and hydrophobic interaction.

In an exemplary embodiment of the present disclosure, the substrate may be a glass substrate or a plastic substrate, for example, a glass sheet or a plastic sheet, which has good heat resistance and light transmittance. The substrate may be made of polymethyl methacrylate (PMMA), polycarbonate (PC), cyclic Olefin Copolymer (COC), or the like. The substrate with good heat resistance and light transmittance is beneficial to the stability of the whole structure of the droplet array generation chip in the subsequent PCR reaction process, and the adverse effect on the fluorescent signal detection is reduced as much as possible.

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

Fig. 6 is a schematic longitudinal sectional structure diagram of another droplet array generation chip according to an exemplary embodiment of the disclosure. As shown in fig. 6, the droplet array generating chip of the exemplary embodiment of the present disclosure may further include a housing 50. The housing 50 may contain the substrate, a plurality of the silica spheres, and an oil phase and an aqueous phase for detection. The housing 50 may be a cuboid clear glass housing, the wall thickness of which may be 0.2mm, and the inner wall dimensions may be: length × width × height =5.1mm × 4.1mm × 2mm, and the outer wall dimensions may be length × width × height =5.5mm × 4.5mm × 2.5mm. The housing 50 may include a lower plate detachable to place the substrate 10 loaded with the silica spherical shell 20 and a peripheral wall.

The droplet array generating chip may further include a push rod 60, and the push rod 60 may be configured to drive the substrate to move up and down. The push rod 60 may be coupled to the base plate 10 through a hole provided on a lower plate of the housing 50. The periphery and the periphery of the detachable lower plate are well sealed by rubber.

The embodiment of the disclosure also provides a preparation method of the droplet array generation chip, and the droplet array generation chip provided by the embodiment of the disclosure can be prepared by the method. The preparation method comprises the following steps:

providing a substrate having a first surface;

preparing a plurality of core-shell microspheres having a core-shell structure, the shell of the core-shell microspheres being an initial silica spherical shell without mesopores, the core of the core-shell microspheres being configured to provide support for the initial silica spherical shell and to be removable;

performing hydrophobic modification on the outer surface of the core-shell microsphere (namely the outer surface of the initial silica spherical shell) to make the outer surface of the core-shell microsphere hydrophobic;

forming mesopores on the initial silica spherical shell, removing the core of the core-shell microsphere to obtain a silica spherical shell with mesopores, wherein the mesopores are communicated with the inner surface and the outer surface of the silica spherical shell;

In an exemplary embodiment of the present disclosure, the primarily modifying the first surface of the substrate such 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 may include:

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.

In exemplary embodiments of the present disclosure, the alkoxysilane modification of the first surface of the substrate may include: performing graft polymerization reaction on the first surface of the substrate by using a silane coupling agent with double bonds, so that an alkoxy functional group with hydrolytic activity is introduced into the first surface of the substrate; wherein, the silane coupling agent with double bonds can comprise any one or more of gamma-methacryloxypropyltrimethoxysilane, gamma-methacryloxypropyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane and the like.

In exemplary embodiments of the present disclosure, the hydroxylating modification of the first surface of the substrate may include: introducing hydroxyl on the first surface of the substrate by adopting a plasma treatment, washing with piranha solution (a mixture of concentrated sulfuric acid and hydrogen peroxide) or a limited photocatalytic oxidation (CPO) method.

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

mixing a raw material for preparing the initial silicon dioxide spherical shell, a microsphere template, a pore-forming agent and a catalyst, depositing silicon dioxide and the pore-forming agent on the surface of the microsphere template by adopting a template method, and forming the initial silicon dioxide spherical shell formed by the silicon dioxide and the pore-forming agent on the surface of the microsphere template to obtain a plurality of core-shell microspheres with core-shell structures, wherein the microsphere template is used as the core of the core-shell microspheres, and the initial silicon dioxide spherical shell is used as the shell of the core-shell microspheres;

the forming mesopores on the initial silica spherical shell and removing the core of the core-shell microsphere may include:

and removing the pore-forming agent on the initial silicon dioxide spherical shell, forming mesopores on the initial silicon dioxide spherical shell, and removing the core of the core-shell microsphere by using the mesopores.

In exemplary embodiments of the present disclosure, the raw material for preparing the initial silica spherical shell may be tetraethyl orthosilicate (TEOS); the catalyst may be ammonia (NH) ₃ ·H ₂ O)。

In exemplary embodiments of the present disclosure, the microsphere template may be selected from any one of soluble (e.g., soluble in water or other solvents) polymeric microspheres, inorganic nanoparticles, inorganic microparticles, polyelectrolytes, and oil-in-water microemulsions. The targets can be removed by adopting a dissolving and washing method with mild conditions, and the targets do not need to be removed by long-time high-temperature calcination, so that the influence on the hydrophilicity of the outer surface of the silicon dioxide spherical shell and the surface of the mesoporous pore channel can be avoided.

In exemplary embodiments of the present disclosure, the porogen may be selected from any one or more of cationic surfactant-based porogens, alcohol porogens and lipid porogens.

In an exemplary embodiment of the present disclosure, the preparing a plurality of core-shell microspheres having a core-shell structure may include: tetraethyl orthosilicate (TEOS) is put through ammonia water (NH) ₃ ·H ₂ And O) gradually hydrolyzing and condensing the surface of the microsphere template under the catalytic action of the catalyst, and depositing the hydrolyzed and condensed microsphere template into silicon dioxide, wherein a pore-forming agent is additionally added in the process and uniformly dispersed in a reaction system, the pore-forming agent is randomly dispersed and deposited on the surface of the microsphere template along with the silicon dioxide to obtain an initial silicon dioxide spherical shell with a certain shell thickness, the microsphere template is a core, and the silicon dioxide spherical shell on the surface of the microsphere template is a shell to form the core-shell microsphere.

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

In an exemplary embodiment of the present disclosure, the solvent for dispersing the core-shell microspheres may 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 may be 1:1; the pH of the solution containing core-shell microspheres may be 4.

In an exemplary embodiment of the present disclosure, after the core-shell microspheres completely deposit on the substrate surface by gravity, the substrate may be continuously soaked for a period of time, for example, 24 hours, so that the functional group capable of being hydrolyzed into a hydroxyl group on the first surface of the substrate may be hydrolyzed into a hydroxyl group. If the first surface of the substrate does not have a functional group which can be hydrolyzed into hydroxyl but directly has hydroxyl, the substrate can be directly subjected to vacuum drying without continuously soaking the substrate in a solution containing the core-shell microspheres after the core-shell microspheres are completely deposited on the surface of the substrate.

In exemplary embodiments of the present disclosure, the vacuum drying condition may include: the drying temperature is 37 ℃ and the drying time is 4h.

Fig. 7 is a reaction mechanism diagram illustrating a dehydration condensation reaction when core-shell microspheres are aligned and fixed on a substrate surface in a manufacturing method according to an exemplary embodiment of the present disclosure. As shown in fig. 7, the hydroxyl groups on the outer surface of the core-shell microspheres (i.e., the outer surface of the initial silica spherical shell) can undergo a dehydration condensation reaction with the hydroxyl groups on the first surface of the substrate by vacuum drying, thereby completing the alignment and fixation of the core-shell microspheres.

hydrophobic interaction is formed between the outer surface of the core-shell microsphere (namely the initial silicon dioxide spherical shell) subjected to hydrophobic modification and the first surface of the substrate subjected to secondary hydrophobic modification, and the core-shell microsphere and the substrate are combined together by utilizing the chemical bond and the hydrophobic interaction.

In an exemplary embodiment of the present disclosure, the first hydrophobic modifier used for hydrophobically modifying the outer surface of the core-shell microsphere may be a silane coupling agent containing a first hydrophobic functional group.

In an exemplary embodiment of the present disclosure, the first hydrophobic modifier and the second hydrophobic modifier may be the same, and in this case, the same silane coupling agent may be used to modify the outer surface of the core-shell microsphere and the first surface of the substrate that is once modified.

FIG. 8 is a schematic illustration of a reaction mechanism for hydrophobically modifying the outer surface of the core-shell microspheres and the first surface of the substrate in accordance with an exemplary embodiment of the disclosure. When the first hydrophobic modifier and the second hydrophobic modifier are silane coupling agents containing alkyl, fluorocarbon and chlorocarbon, molecular fragments except the alkyl, fluorocarbon and chlorocarbon in the silane coupling agents can be hydrolyzed to generate hydroxyl groups, and then the hydroxyl groups can be subjected to dehydration condensation reaction with the outer surface of the core-shell microsphere and the hydroxyl groups on the first surface of the substrate, so that the hydrophobic modification of the alkyl, fluorocarbon and chlorocarbon can be completed by introducing the first hydrophobic functional group and the second hydrophobic functional group.

In an exemplary embodiment of the present disclosure, the microsphere template inside the silica spherical shell may be dissolved and removed by etching using a solvent. The outer surface of the silica spherical shell and the first surface of the substrate are subjected to hydrophobic modification, and then the microsphere template is removed, so that the outer surface of the silica spherical shell (except mesoporous channels on the surface) and the first surface of the substrate are kept in a hydrophobic state, and the inner surface of the silica spherical shell and the mesoporous channels can present better hydrophilicity due to the existence of a large amount of hydroxyl groups of the silica spherical shell, so that the droplet array generating chip disclosed by the embodiment of the disclosure can be obtained.

The embodiment of the disclosure also provides a liquid drop array generation method. FIG. 9 is a schematic flow chart of a droplet array formation method of an exemplary embodiment of the present disclosure; FIG. 10 is a schematic illustration of silica spheres shells loaded with an aqueous phase formed by a droplet array generating method according to an exemplary embodiment of the disclosure arranged in an array on a substrate; fig. 11 is a schematic view of the structure of the silica spherical shell filled with the aqueous phase (reaction solution) of fig. 10.

As shown in fig. 9 to 11, the droplet array generating method includes:

providing a liquid drop array generating chip, wherein the liquid drop array generating chip is the liquid drop array generating chip or is obtained by the preparation method of the liquid drop array generating chip;

immersing the droplet array generation chip in an aqueous phase 70 containing a reaction solution for generating a droplet array, wherein the aqueous phase 70 containing the reaction solution enters the interior of the silica spherical shell 20 through the mesopores 40 on the plurality of silica spherical shells 20 of the droplet array generation chip, and generates aqueous phase droplets 90 in the interior of the silica spherical shell 20;

covering the surface of the water phase 70 with an oil phase 80;

and moving the droplet array generation chip to enable the silica spherical shell 20 of the droplet array generation chip to enter the oil phase 80, wherein when the droplet array generation chip passes through the interface between the water phase 70 and the oil phase 80, a plurality of silica spherical shells 20 containing the water phase droplets 90 are rapidly enclosed and wrapped by the oil phase 80 and are arranged on the substrate 10 at intervals to form a droplet array.

The method for generating the liquid drop array can quickly generate the liquid drop array only by moving the substrate of the liquid drop array generating chip from the water phase to the oil phase, has no additional technical requirements, and is simple, quick and convenient to implement; the closed liquid drop array in the oil phase does not need to be transferred, and subsequent amplification reaction and signal detection can be directly carried out.

For ddPCR result detection and analysis, a single-layer droplet array physically divided from each other can be directly used for camera-based fluorescence signal scanning detection in an oil phase, and reaction droplets do not need to be collected in advance or detected by using an expensive and time-consuming photomultiplier tube (PMT); the rigid silicon dioxide spherical shell can avoid cross contamination caused by liquid drop fusion, and the accuracy of an experiment is ensured; in addition, the particle size and the number of the used silicon dioxide spherical shells can be adjusted according to the actual detection requirement so as to cover a wider detection precision range.

The method for generating the liquid drop array provided by the embodiment of the disclosure can be applied to detection needing to generate the liquid drop array, for example, can be applied to liquid drop polymerase chain reaction detection, surface Plasmon Resonance (SPR) enhanced fluorescence signal detection, and Surface Enhanced Raman Spectroscopy (SERS) detection.

The embodiment of the present disclosure further provides a fluorescence detection method, including:

covering the surface of the water phase with an oil phase;

In exemplary embodiments of the present disclosure, the fluorescence detection method includes, but is not limited to, polymerase chain reaction detection and the like.

The embodiment of the disclosure also provides a liquid drop array generating device, which comprises a plurality of liquid drop array generating chips as described above. FIG. 12A is a top view of a droplet array generation apparatus according to an exemplary embodiment of the present disclosure; fig. 12B is a front view of the droplet array generating device shown in fig. 12A. As shown in fig. 12A and 12B, the droplet array generating apparatus integrates a plurality of, for example, 16 identical droplet array generating chips on a glass sheet having a size of 24mm × 20mm × 0.2mm.

Each droplet array generation chip can independently sample, and realize the rapid generation of the droplet array and the nucleic acid amplification reaction. Therefore, the droplet array generating device of the embodiment of the disclosure can synchronously operate 16 samples in a smaller area, and realize high-throughput detection of nucleic acid molecules to be detected.

Although the embodiments disclosed in the present disclosure are described above, the descriptions are only for the convenience of understanding the present disclosure, and are not intended to limit the present disclosure. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure, and that the scope of the disclosure is to be limited only by the terms of the appended claims.

Claims

1. A droplet array generation chip, comprising:

a substrate having a first surface;

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

3. The droplet array generation chip of claim 1 or 2, wherein the silica spherical shell has a first hydrophobic functional group on an outer surface thereof.

4. 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.

5. The droplet array generation chip of claim 4, wherein the first hydrophobic functional group is attached to the outer surface of the silica spherical shell by a Si-O-Si chemical bond.

6. The droplet array generation chip according to any one of claims 1 to 5, wherein the inner surface of the silica spherical shell and the pore surface of the mesopores carry hydrophilic functional groups.

7. The droplet array generation chip according to any one of claims 1 to 6, wherein the pore size of the mesopores is 0.8 μm to 1.5 μm.

8. The droplet array generation chip of claim 7, wherein the distribution density of the mesopores on the outer surface of the silica spherical shell is 1.13 μm per ² To 11.04 μm ² Has 1 mesopore on the outer surface of (a).

9. The droplet array generation chip according to any one of claims 1 to 8, wherein a distance between two adjacent silica spherical shells is D, an outer diameter of the silica spherical shells is R, and 0 < D ≦ R.

10. The droplet array generation chip of any one of claims 1 to 9, wherein the first surface of the substrate is hydrophobic.

11. The droplet array generation chip of claim 10, wherein the first surface of the substrate bears a second hydrophobic functional group thereon.

12. The droplet array generation chip of any one of claims 1 to 11, wherein a plurality of the silica spherical shells and the substrate are bonded together with Si-O chemical bonds.

13. The droplet array generation chip of any one of claims 1 to 12, wherein the substrate further has a second surface, and a fluorescence signal blocking layer is disposed on the first or second surface of the substrate.

14. A method of fabricating a droplet array generating chip, comprising:

providing a substrate having a first surface;

15. The manufacturing method according to claim 14, wherein the forming of the plurality of silica spherical shells spaced apart from each other and arranged in a single layer on the first surface of the substrate comprises:

16. The preparation method according to claim 15, wherein the arranging the plurality of core-shell microspheres in a single layer spaced apart from each other on the first surface of the once-modified substrate comprises:

placing the substrate subjected to primary modification in the solution containing the core-shell microspheres, and arranging a plurality of the core-shell microspheres at intervals on the first surface of the substrate subjected to primary modification in a single layer manner by adopting a gravity deposition method;

17. The method of manufacturing of claim 16, further comprising:

hydrophobic interaction is formed between the outer surface of the core-shell microsphere subjected to hydrophobic modification and the first surface of the substrate subjected to secondary hydrophobic modification, and the core-shell microsphere and the substrate are combined together by utilizing the chemical bond and the hydrophobic interaction.

18. The preparation method of claim 17, wherein the first hydrophobic modifier for hydrophobically modifying the outer surface of the core-shell microsphere is a silane coupling agent containing a first hydrophobic functional group;

the second hydrophobic modifier adopted for carrying out the second hydrophobic modification on the first surface of the substrate subjected to the first modification is a silane coupling agent containing a second hydrophobic functional group;

the first hydrophobic functional group and the second hydrophobic functional group are selected from any one or more of saturated or unsaturated alkyl, fluorine alkyl and chlorine alkyl;

the first hydrophobic modifier and the second hydrophobic modifier are selected from any one or more of methyl triethoxysilane, octadecyl trichlorosilane, octadecyl trimethoxysilane, trifluoropropyl triethoxysilane, perfluorooctyl triethoxysilane, chloromethyl trimethoxysilane and (chloromethyl) methyldiethoxysilane.

19. The production method according to any one of claims 14 to 18, wherein the substrate further has a second surface;

the preparation method further comprises the following steps: a fluorescent signal blocking layer is disposed on the first surface or the second surface of the substrate.

20. A method of droplet array generation comprising:

providing a droplet array generating chip according to any one of claims 1 to 13 or obtained by a method of making a droplet array generating chip according to any one of claims 14 to 19;

covering the surface of the water phase with an oil phase;

21. A method of fluorescence detection, comprising:

covering the surface of the water phase with an oil phase;