CN113814014A

CN113814014A - Digital polymerase chain reaction microfluidic device and preparation method thereof

Info

Publication number: CN113814014A
Application number: CN202111117340.9A
Authority: CN
Inventors: 邓睿君; 邓林; 刘祝凯; 丁丁
Original assignee: BOE Technology Group Co Ltd; Beijing BOE Technology Development Co Ltd
Current assignee: BOE Technology Group Co Ltd; Beijing BOE Technology Development Co Ltd
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2021-12-21
Anticipated expiration: 2041-09-23
Also published as: CN113814014B

Abstract

The disclosed exemplary embodiments provide a digital polymerase chain reaction microfluidic device and a method for preparing the same. The digital polymerase chain reaction microfluidic device comprises a first substrate and a second substrate which are oppositely arranged; the first substrate comprises a first substrate, a microcavity limiting layer arranged on one side of the first substrate, which faces the second substrate, and a first surface modification layer arranged on one side of the microcavity limiting layer, which is far away from the first substrate; the microcavity defining layer includes a plurality of grooves as micro-reaction chambers and defining dams between adjacent grooves, and the first surface modification layer in at least one of the grooves is provided with a first modification structure configured to increase hydrophilic properties inside the micro-reaction chambers. This is disclosed through carry out hydrophilic structure and hydrophobic structure design respectively in the inside and the outside of little reaction chamber, has improved from inhaling liquid and has advanced kind and oil blanket performance.

Description

Digital polymerase chain reaction microfluidic device and preparation method thereof

Technical Field

The disclosure relates to, but is not limited to, the technical field of digital fluorescence detection, and in particular relates to a digital polymerase chain reaction microfluidic device and a preparation method thereof.

Background

Digital Polymerase Chain Reaction (dPCR) is a quantitative analysis method for providing quantitative information of Digital DNA, and a detection strategy of divide and conquer (divide and conquer) is adopted, and a mixture of a sample and a PCR reagent is dispersed in a chip in micro-Reaction chambers by using a microfluidic technology, so as to perform independent PCR amplification on target molecules in each chamber.

The research of the inventor of the application finds that the existing digital polymerase chain reaction microfluidic device has the problems that the reaction liquid is not completely filled or the reaction liquid is disturbed after being filled in the process of sample introduction and amplification, and the like.

BRIEF SUMMARY OF THE PRESENT DISCLOSURE

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

The technical problem to be solved by the exemplary embodiments of the present disclosure is to provide a digital polymerase chain reaction microfluidic device and a method for manufacturing the same, so as to solve the problems of incomplete filling of a reaction solution or occurrence of crosstalk after filling in the existing structure.

In order to solve the above technical problems, exemplary embodiments of the present disclosure provide a digital polymerase chain reaction microfluidic device including a first substrate and a second substrate that are oppositely disposed; the first substrate comprises a first substrate, a microcavity limiting layer arranged on one side of the first substrate, which faces the second substrate, and a first surface modification layer arranged on one side of the microcavity limiting layer, which is far away from the first substrate; the microcavity defining layer includes a plurality of grooves as micro-reaction chambers and defining dams between adjacent grooves, and the first surface modification layer in at least one of the grooves is provided with a first modification structure configured to increase hydrophilic properties inside the micro-reaction chambers.

In an exemplary embodiment, the first surface modification layer includes a bottom wall modification layer covering the bottom wall of the groove in the groove and a side wall modification layer covering the side wall of the groove in the groove, and the first modification structure is disposed on a surface of the bottom wall modification layer on a side away from the first substrate.

In an exemplary embodiment, the first modification structure includes at least one first protrusion provided on a side surface of the groove bottom wall modification layer away from the first substrate.

In an exemplary embodiment, in a plane parallel to the first substrate, the first protrusions have a first feature length of 0.5 μm to 2 μm, and a first pitch between adjacent first protrusions is 0.5 times to 2.0 times the first feature length, which is a maximum dimension of the first protrusions; the first protrusions have a first height of 0.1 to 20 μm in a plane perpendicular to the first substrate.

In an exemplary embodiment, a side of the limiting dam facing away from the first substrate is provided with a second modification structure configured to increase a hydrophobic property of an outside of the micro reaction chamber.

In an exemplary embodiment, the first surface modification layer includes a groove bottom wall modification layer covering the groove-in-groove bottom wall, a groove side wall modification layer covering the groove-in-groove side wall, and a dam top wall modification layer covering the limiting dam-in-dam top wall; and a second surface modification layer is arranged on one side, far away from the first substrate, of the at least one dam top wall modification layer, and the second modification structure is arranged on the surface of one side, far away from the first substrate, of the second surface modification layer.

In an exemplary embodiment, the first surface modification layer includes a groove bottom wall modification layer covering the groove bottom wall in the groove and a groove side wall modification layer covering the groove side wall in the groove; the second modifying structure is disposed on a surface of a side of the dam apex wall away from the first base in the defining dam.

In an exemplary embodiment, the second modifying structure includes at least one second protrusion provided on a surface of a side of the dam apex wall facing away from the first substrate in the second surface modifying layer or defining dam.

In an exemplary embodiment, in a plane parallel to the first substrate, the second protrusions have a second feature length of 0.5 μm to 2 μm, and a second pitch between adjacent second protrusions is 0.5 times to 2.0 times the first feature length, the second feature length being a maximum dimension of the second protrusions; the second protrusions have a second height of 0.1 to 20 μm in a plane perpendicular to the first substrate.

In an exemplary embodiment, the first surface modification layer includes a groove bottom wall modification layer covering the groove-in-groove bottom wall, a groove side wall modification layer covering the groove-in-groove side wall, and a dam top wall modification layer covering the limiting dam-in-dam top wall; and a flow guide structure is arranged on the surface of one side, far away from the first substrate, of at least one dam top wall modification layer, the flow guide structure is positioned on one side, close to the groove, of the dam top wall modification layer, and the flow guide structure is configured to improve the efficiency of reaction liquid entering the micro-reaction cavity.

In an exemplary embodiment, the flow guiding structure includes at least one flow guiding pillar disposed on a side surface of the dam crest wall modification layer away from the first substrate.

In an exemplary embodiment, in a plane parallel to the first substrate, the flow guide columns have a flow guide characteristic length of 0.5 to 2 μm, and a flow guide spacing between adjacent flow guide columns is 0.5 to 2.0 times the flow guide characteristic length, which is the largest dimension of the flow guide columns; the flow guide height of the flow guide column in a plane perpendicular to the first substrate is 0.1-20 μm.

In an exemplary embodiment, the flow guiding height of the flow guiding columns in the flow guiding structure is greater than the first height of the first protrusions in the first modifying structure, and/or the flow guiding interval of the flow guiding columns in the flow guiding structure is greater than the first interval of the first protrusions in the first modifying structure.

In an exemplary embodiment, the second substrate includes a second substrate and a third surface modification layer disposed on a side of the second substrate facing the first substrate, and a third modification structure is disposed on a surface of the third surface modification layer facing the first substrate, and the third modification structure is configured to increase a hydrophobic property of an exterior of the micro reaction chamber.

In an exemplary embodiment, the third modifying structure includes at least one third protrusion provided on a surface of the third surface modifying layer facing the first substrate side.

In an exemplary embodiment, a third feature length of the third protrusions is 0.5 μm to 2 μm, a second pitch between adjacent third protrusions is 0.5 times to 2.0 times the third feature length, the third feature length being a maximum dimension of the third protrusions, in a plane parallel to the first substrate; a third height of the third protrusion in a plane perpendicular to the first substrate is 0.1 to 20 μm.

In exemplary embodiments, the third height of the third protrusions in the third trim structure is greater than the second height of the second protrusions in the second trim structure, and/or the third pitch of the third protrusions in the third trim structure is greater than the second pitch of the second protrusions in the second trim structure.

The disclosed exemplary embodiments also provide a method for preparing a digital polymerase chain reaction microfluidic device, including:

respectively preparing a first substrate and a second substrate; the first substrate comprises a first substrate, a microcavity limiting layer arranged on one side of the first substrate, which faces the second substrate, and a first surface modification layer arranged on one side of the microcavity limiting layer, which is far away from the first substrate; the micro-cavity limiting layer comprises a plurality of grooves serving as micro-reaction cavities and limiting dams positioned between the adjacent grooves, the first surface modification layer in at least one groove is provided with a first modification structure, and the first modification structure is configured to increase the hydrophilic property inside the micro-reaction cavities;

and packaging the first substrate and the second substrate in a box through a packaging process.

In an exemplary embodiment, preparing the first substrate includes:

forming a microcavity-defining layer on the first substrate, the microcavity-defining layer including a plurality of recesses as microreaction cavities and defining dams between adjacent recesses;

forming a first surface modification layer, wherein the first surface modification layer comprises a groove bottom wall modification layer covering the groove bottom wall in the groove, a groove side wall modification layer covering the groove side wall in the groove and a dam top wall modification layer covering one side, far away from the first substrate, of the limiting dam, and a first modification structure is formed on the surface of one side, far away from the first substrate, of at least one groove bottom wall modification layer and comprises at least one first protrusion;

and forming a second surface modification layer, wherein the second surface modification layer is formed on one side of the at least one dam crest wall modification layer far away from the first substrate, a second modification structure is formed on the surface of one side of the second surface modification layer far away from the first substrate, and the second modification structure comprises at least one second protrusion.

In an exemplary embodiment, preparing the second substrate includes:

and forming a third surface modification layer on one side of the second substrate facing the first substrate, wherein a third modification structure is formed on the surface of the third surface modification layer facing one side of the first substrate, and the third modification structure comprises at least one third protrusion.

The disclosed exemplary embodiment provides a digital polymerase chain reaction microfluidic device and a preparation method thereof, by respectively designing a hydrophilic structure and a hydrophobic structure inside and outside a micro-reaction cavity, the defects of incomplete filling of reaction liquid or crosstalk after filling in the processes of sample injection and amplification are effectively avoided, and the sample injection and oil sealing performance of self-priming liquid is improved.

Of course, not all advantages described above need to be achieved at the same time to practice any one product or method of the present disclosure. 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. The objectives and other advantages of the disclosed exemplary embodiments may be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

Other aspects will be apparent upon reading and understanding the attached drawings and detailed description.

Drawings

The accompanying drawings are included to provide a further 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. The shapes and sizes of the various elements in the drawings are not to be considered as true proportions, but are merely intended to illustrate the present disclosure.

Fig. 1 is a schematic structural diagram of a dPCR microfluidic device according to an exemplary embodiment of the present disclosure;

FIG. 2 is a schematic structural diagram illustrating a first conductive layer pattern formed according to an embodiment of the disclosure;

FIG. 3 is a schematic structural diagram illustrating a first insulating layer pattern formed according to an embodiment of the present disclosure;

FIG. 4 is a schematic structural diagram illustrating a second conductive layer pattern formed according to an embodiment of the disclosure;

FIG. 5 is a schematic structural diagram illustrating a second insulating layer pattern formed according to an embodiment of the present disclosure;

FIGS. 6a and 6b are schematic structural views after patterning a microcavity defining layer according to an embodiment of the disclosure;

fig. 7a and 7b are schematic structural diagrams after the first surface modification layer pattern is formed according to the present disclosure;

fig. 8a and 8b are schematic structural diagrams illustrating the second surface modification layer pattern formed according to the present disclosure;

FIG. 9 is a schematic diagram illustrating a third surface modification layer pattern formed according to an embodiment of the disclosure;

FIGS. 10a and 10b are schematic structural views of another dPCR microfluidic device according to an embodiment of the disclosure;

FIGS. 11a and 11b are schematic structural views of another dPCR microfluidic device according to an embodiment of the disclosure;

FIG. 12 is a schematic plan view of a reaction zone according to an exemplary embodiment of the present disclosure.

Description of reference numerals:

10 — a first substrate; 11-a control electrode; 12 — a first insulating layer;

13-heating electrodes; 14 — a second insulating layer; 20 — a second substrate;

30-a microcavity-defining layer; 31-a confining dam; 32-a dam top wall;

33-dam side wall; 40-a first surface modification layer; 41-first modified structure;

50-a second surface modification layer; 51-a second modified structure; 60, forming a groove;

61-tank bottom wall; 62-a trough side wall; 70, a flow guide structure;

80-a third surface modifying layer; 81-a third modified structure; 82-liquid inlet;

90-frame sealing glue; 100 — a first substrate; 110-a reaction zone;

120-a peripheral region; 200 — a second substrate.

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. Note that the embodiments may be implemented in a plurality of different forms. Those skilled in the art can readily appreciate the fact that the forms and details may be varied 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 drawing scale in this disclosure may be referenced in the actual process, but is not limited thereto. The drawings described in the present disclosure are only schematic structural views, and one mode of the present disclosure is not limited to the shapes, numerical values, or the like shown in the drawings.

The ordinal numbers such as "first", "second", "third", and the like in the present specification are provided for avoiding confusion among the constituent elements, and are not limited in number.

In this specification, for convenience, words such as "middle", "upper", "lower", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicating orientations or positional relationships are used to explain positional relationships 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 referred device or element 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 described in the specification are not limited to the words described in the specification, and may be replaced as appropriate.

In this specification, the terms "mounted," "connected," and "connected" are to be construed broadly unless otherwise specifically indicated and 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 present specification, "parallel" means a state in which an angle formed by two straight lines is-10 ° or more and 10 ° or less, and therefore, includes a state in which the angle is-5 ° or more and 5 ° or less. The term "perpendicular" refers to a state in which the angle formed by two straight lines is 80 ° or more and 100 ° or less, and therefore includes a state in which the angle is 85 ° or more and 95 ° or less.

In this specification, a triangle, a rectangle, a trapezoid, a pentagon, a hexagon, or the like is not strictly defined, and may be an approximate triangle, a rectangle, a trapezoid, a pentagon, a hexagon, or the like, and some small deformations due to tolerances may exist, and a lead angle, a curved edge, deformation, or the like may exist.

"about" in this disclosure means that the limits are not strictly defined, and that the numerical values are within the tolerances allowed for the process and measurement.

The digital polymerase chain reaction (dPCR) is a quantitative analysis method for providing digital DNA quantitative information, is an important technology for molecular biology detection and analysis, and the accurate quantification of the DNA copy number is one of important applications in modern molecular biology and medicine. In recent years, with the rapid development of microfluidic (MicroFluidics) technology, the sensitivity and accuracy of a dPCR microfluidic device combined with the microfluidic technology are greatly improved. The dPCR microfluidic device adopts a divide-and-conquer detection strategy, utilizes a microfluidic technology to disperse a mixture of a sample and a PCR reagent in each micro-reaction cavity in a chip, and carries out independent PCR amplification on target molecules in each micro-reaction cavity. Generally, the dPCR adopts secondary sample injection to carry out oil sealing, namely a sample and a dPCR reagent are positioned in a micro-reaction cavity, and the sample and the dPCR reagent are filled in a flow channel outside the micro-reaction cavity after the oil phase is injected by utilizing the hydrophilic and hydrophobic properties and the capillary action of a micro-flow channel, so that the sample liquid in the micro-reaction cavity is segmented. The research of the inventor of the application discovers that the oil phase liquid sealing mode needs hydrophilic and hydrophobic design on the inside and the outside of the micro reaction cavity to ensure better secondary sample introduction effect and ensure that the oil phase completely fills a flow channel outside the micro reaction cavity, and the conventional device usually only carries out hydrophilic design on the inside of the micro reaction cavity, so that the problems of incomplete filling or crosstalk after filling of reaction liquid in the sample introduction and amplification processes of the conventional structure are caused.

In order to solve the problems of incomplete filling of reaction liquid in the sample introduction and amplification processes or crosstalk after filling and the like in the existing structure, the exemplary embodiment of the disclosure provides a digital polymerase chain reaction microfluidic device. The digital polymerase chain reaction microfluidic device may include a first substrate and a second substrate which are oppositely disposed, the first substrate may include a first substrate, a microcavity defining layer disposed on a side of the first substrate facing the second substrate, and a first surface modification layer disposed on a side of the microcavity defining layer away from the first substrate, the microcavity defining layer may include a plurality of grooves as micro-reaction chambers and defining dams between adjacent grooves, and the first surface modification layer in at least one groove may be provided with a first modification structure configured to increase hydrophilic properties inside the micro-reaction chamber.

In an exemplary embodiment, a side of the limiting dam facing away from the first substrate may be provided with a second modification structure configured to increase a hydrophobic property of an outside of the micro reaction chamber.

In an exemplary embodiment, the second substrate may include a second substrate and a third surface modification layer disposed on a side of the second substrate facing the first substrate, and a third modification structure disposed on a surface of the third surface modification layer facing the first substrate, the third modification structure being configured to increase a hydrophobic property of an outside of the micro reaction chamber.

Fig. 1 is a schematic structural diagram of a dPCR microfluidic device according to an exemplary embodiment of the present disclosure. In exemplary embodiments, the digital polymerase chain reaction microfluidic device of the present disclosure may be used to perform digital polymerase chain reaction, and may be used for detection processes after the reaction. As shown in fig. 1, the main structure of the digital pcr microfluidic device may include a first substrate 100 and a second substrate 200 disposed opposite to each other, and the first substrate 100 may include a first substrate 10, a microcavity defining layer 30 disposed on a side of the first substrate 10 close to the second substrate 200, and a first surface modification layer 40 disposed on a side of the microcavity defining layer 30 close to the second substrate 200. The second substrate 200 may include a second substrate 20 and a third surface modification layer 80 disposed on a side of the second substrate 20 adjacent to the first substrate 10. In an exemplary embodiment, a sealant 90 is disposed between the first substrate 100 and the second substrate 200, and the first substrate 100 and the second substrate 200 are connected together by the sealant 90.

In an exemplary embodiment, the microcavity defining layer 30 may include a plurality of grooves 60 and defining dams 31 between adjacent grooves 60, the defining dams 31 defining the plurality of grooves 60, and the plurality of grooves 60 may serve as micro reaction chambers for performing a digital polymerase chain reaction.

In an exemplary embodiment, the first surface modification layer 40 has a hydrophilic characteristic, and the first surface modification layer 40 in the at least one groove 60 may be provided with a first modification structure 41, the first modification structure 41 being configured to increase the hydrophilic characteristic inside the micro reaction chamber.

In an exemplary embodiment, the groove 60 may include a groove bottom wall adjacent to one side of the first substrate and a groove side wall at the periphery of the groove bottom wall. In at least one of the grooves 60, the first surface modification layer 40 may include a groove bottom wall modification layer covering a groove bottom wall in the groove 60 and a groove side wall modification layer covering a groove side wall in the groove 60, and the first modification structure 41 may be disposed on a surface of the groove bottom wall modification layer on a side away from the first substrate.

In an exemplary embodiment, the first modification structure 41 may include at least one first protrusion disposed on a surface of the bottom wall modification layer on a side away from the first substrate, the at least one first protrusion being configured to increase roughness of the surface of the bottom wall modification layer on the side away from the first substrate.

In an exemplary embodiment, the defining dam 31 may include a dam top wall at a side away from the first substrate and a dam side wall at a periphery. The first surface modification layer 40 may further include a dam crest wall modification layer covering at least one dam crest wall of the limiting dam 31, a second surface modification layer 50 is disposed on a side of the dam crest wall modification layer away from the first substrate, the second surface modification layer 50 may be provided with a second modification structure 51, and the second modification structure 51 is configured to increase a hydrophobic property outside the micro reaction chamber.

In an exemplary embodiment, the second surface modification layer 50 has a hydrophobic property, and the second modification structure 51 may be disposed on a surface of the second surface modification layer 50 on a side away from the first substrate.

In an exemplary embodiment, the second modifying structure 51 may include at least one second protrusion disposed on a surface of the second surface modifying layer 50 on a side away from the first substrate, the at least one second protrusion being configured to increase roughness of a surface of the second surface modifying layer 50 on a side away from the first substrate.

In an exemplary embodiment, the third surface modification layer 80 may be provided with a third modification structure 81, and the third modification structure 81 is configured to increase a hydrophobic property outside the micro reaction chamber.

In an exemplary embodiment, the third surface modification layer 80 has a hydrophobic property, and the third modification structure 81 may be disposed on a surface of the third surface modification layer 80 on a side away from the second substrate (a side toward the first substrate).

In an exemplary embodiment, the third modification structure 81 may include at least one third protrusion disposed on a side surface of the third surface modification layer 80 away from the second substrate, the at least one third protrusion being configured to increase roughness of the side surface of the third surface modification layer 80 away from the second substrate.

In an exemplary embodiment, the first substrate 10 may include a reaction region 110 and a peripheral region 120, and the peripheral region 120 may surround the reaction region 110, i.e., the peripheral region 120 is located outside the reaction region 110. The second substrate 20 may be disposed opposite to the reaction region 110 on the first substrate 10, and a sealed reaction chamber is formed by the frame sealing glue 90 and the reaction region 110 on the first substrate 10, and a plurality of grooves serving as micro-reaction chambers are located in the sealed reaction chamber.

In an exemplary embodiment, the first substrate 100 may further include a heating structure layer, and the heating structure layer may be disposed between the first substrate 10 and the microcavity defining layer 30.

In an exemplary embodiment, the heating structure layer may include a control electrode 11 disposed on the first substrate 10, a first insulating layer 12 covering the control electrode 11, a heating electrode 13 disposed on the first insulating layer 12, and a second insulating layer 14 disposed on the heating electrode 13, the heating electrode 13 may be connected with the control electrode 11 through a connection via, and the microcavity defining layer 30 may be disposed on the second insulating layer 14.

In an exemplary embodiment, the second substrate 200 may further be provided with a liquid inlet 82, and the liquid inlet 82 is a through hole penetrating through the second substrate 20 and the third modification structure 81.

In an exemplary embodiment, the first and second substrates may employ glass, the first and second substrates may be rectangular in shape, and the second substrate may have a size smaller than that of the first substrate. For example, the first substrate may have dimensions of about 3.2cm by 4.5cm, and the second substrate may have dimensions of about 3.2cm by 3.0 cm.

In an exemplary embodiment, the shape of the plurality of grooves 60 in a plane parallel to the first substrate may include any one or more of: triangular, square, rectangular, pentagonal, hexagonal, polygonal, circular, and elliptical. The cross-sectional shape of the plurality of grooves in a plane perpendicular to the first substrate may include any one or more of: rectangular, trapezoidal and polygonal, the groove side walls of the groove may be straight, broken or curved.

In an exemplary embodiment, the characteristic length of the groove 60 may be about 1 μm to 100 μm in a plane parallel to the first substrate, and the characteristic length of the groove may be the maximum dimension of the groove.

In an exemplary embodiment, the depth of the groove 60 may be about 5 μm to 100 μm in a plane perpendicular to the first substrate. For example, the depth of the groove 60 may be about 9.8 μm.

In an exemplary embodiment, the number of the plurality of grooves 60 as the micro reaction chamber may be about 2000 to 1000000. For example, the number of grooves 60 may be about 40000 to 100000.

In an exemplary embodiment, the shape of the plurality of first protrusions, second protrusions, and third protrusions in a plane parallel to the first substrate may include any one or more of: triangular, square, rectangular, pentagonal, hexagonal, polygonal, circular, and elliptical. The cross-sectional shape of the plurality of first protrusions, second protrusions, and third protrusions in a plane perpendicular to the first substrate may include any one or more of: rectangular, trapezoidal and polygonal, the side walls of the protrusions may be straight, broken or curved.

In an exemplary embodiment, the characteristic length of the plurality of first, second, and third protrusions may be about 0.5 μm to 2 μm in a plane parallel to the first substrate, and the characteristic length of the protrusion may be a maximum size of the protrusion.

In an exemplary embodiment, the plurality of first, second, and third protrusions may have a height of about 0.1 μm to 20 μm in a plane perpendicular to the first substrate.

In an exemplary embodiment, the digital pcr microfluidic device may further include a temperature sensing device disposed on a side of the first substrate away from the second substrate, and a program control device connected to the temperature sensing device, the control electrode, and the heating electrode, respectively, and the disclosure is not limited herein.

The following is an exemplary description of the process for making the digital polymerase chain reaction microfluidic device. The "patterning process" referred to in the present disclosure includes processes of coating a photoresist, mask exposure, development, etching, stripping a photoresist, and the like, for a metal material, an inorganic material, or a transparent conductive material, and processes of coating an organic material, mask exposure, development, and the like, for an organic material. The deposition can be any one or more of sputtering, evaporation and chemical vapor deposition, the coating can be any one or more of spraying, spin coating and ink-jet printing, and the etching can be any one or more of dry etching and wet etching, and the disclosure is not limited. "thin film" refers to a layer of a material deposited, coated, or otherwise formed on a substrate. The "thin film" may also be referred to as a "layer" if it does not require a patterning process throughout the fabrication process. If the "thin film" requires a patterning process during the entire fabrication process, it is referred to as "thin film" before the patterning process and "layer" after the patterning process. The "layer" after the patterning process includes at least one "pattern". In the present disclosure, the term "a and B are disposed in the same layer" means that a and B are formed simultaneously by the same patterning process, and the "thickness" of the film layer is the dimension of the film layer in the direction perpendicular to the display substrate. In the exemplary embodiment of the present disclosure, "the forward projection of B is located within the range of the forward projection of a" or "the forward projection of a includes the forward projection of B" means that the boundary of the forward projection of B falls within the boundary range of the forward projection of a, or the boundary of the forward projection of a overlaps with the boundary of the forward projection of B.

In an exemplary embodiment, the preparation of the digital polymerase chain reaction microfluidic device may include three parts, a first substrate preparation, a second substrate preparation and an encapsulation process, respectively. The preparation of the first substrate and the preparation of the second substrate have no precedence requirement and can be carried out simultaneously, and the packaging treatment needs to be carried out after the preparation of the first substrate and the preparation of the second substrate are finished. The three parts of the preparation process are described below.

First part, first substrate preparation

In an exemplary embodiment, the first substrate preparation may include the following operations.

(11) A first conductive layer pattern is formed. In an exemplary embodiment, the forming of the first conductive layer pattern may include: a first conductive film is deposited on a first substrate, and the first conductive film is patterned through a patterning process to form a first conductive layer pattern disposed on the first substrate 10, and the first conductive layer pattern may include at least one control electrode 11, as shown in fig. 2.

In an exemplary embodiment, the at least one control electrode 11 may be located at the peripheral region 120. The control electrode 11 is configured to apply an electrical signal (e.g., a voltage signal) to a subsequently formed heating electrode, so that the heating electrode generates heat under the action of the electrical signal, thereby heating the micro-reaction chamber.

In an exemplary embodiment, the first conductive layer may employ a metal material, such as any one or more of silver (Ag), copper (Cu), aluminum (Al), titanium (Ti), and molybdenum (Mo), or an alloy material of the above metals, and may have a single-layer structure or a multi-layer composite structure. For example, the first conductive layer may be a multilayer composite structure, and include a first sublayer, a second sublayer and a third sublayer stacked on each other, the first sublayer and the third sublayer may be made of molybdenum metal, and the second sublayer may be made of aluminum metal, so as to form a Mo/Al/Mo multilayer composite structure. In an exemplary embodiment, the first sub-layer may have a thickness of about 10nm to about 30nm, the second sub-layer may have a thickness of about 200nm to about 400nm, and the third sub-layer may have a thickness of about 70nm to about 90 nm. For example, the first sub-layer may have a thickness of about 20nm, the second sub-layer may have a thickness of about 300nm, and the third sub-layer may have a thickness of about 80 nm.

In an exemplary embodiment, the temperature for depositing the first conductive film may be about 100 to 150 ℃. For example, the temperature for depositing the first conductive film may be about 125 ℃.

(12) A first insulating layer pattern is formed. In an exemplary embodiment, the forming of the first insulation layer pattern may include: depositing a first insulating film on the first substrate on which the patterns are formed, patterning the first insulating film through a patterning process to form a first insulating layer 12 covering the first conductive layer patterns, forming at least one connection via hole on the first insulating layer 12, wherein an orthographic projection of the connection via hole on the first substrate is within an orthographic projection range of the control electrode 11 on the first substrate, and removing the first insulating film in the connection via hole to expose a surface of the control electrode 11, as shown in fig. 3.

In an exemplary embodiment, the first insulating layer may employ an inorganic material, which may be any one or more of silicon oxide (SiOx), silicon nitride (SiNx), and silicon oxynitride (SiON), may be a single layer, a multi-layer, or a composite layer, or an organic material, which may be a resin, or the like.

In an exemplary embodiment, the first insulating layer may employ silicon dioxide (SiO)₂) The thickness of the first insulating layer may be about 250nm to 350 nm. For example, the thickness of the first insulating layer may be about 300 nm.

In an exemplary embodiment, the temperature for depositing the first insulating film may be about 150 to 250 ℃. For example, the temperature for depositing the first insulating film may be about 200 ℃.

(13) Forming a second conductive layer pattern. In an exemplary embodiment, the forming of the second conductive layer pattern may include: depositing a second conductive film on the first substrate on which the aforementioned pattern is formed, and patterning the second conductive film through a patterning process to form a second conductive layer pattern disposed on the first insulating layer 12, the second conductive layer pattern may include at least one heating electrode 13, and the heating electrode 13 is connected to the control electrode 11 through a connection via, as shown in fig. 4.

In an exemplary embodiment, the heating electrode 13 may be located at the reaction region 110 and the peripheral region 120, and the heating electrode 13 is configured to heat a plurality of micro reaction chambers to be formed subsequently. Since the heating electrode 13 is connected to the control electrode 11 through the via hole, the heating electrode 13 can receive an electrical signal applied by the control electrode 11, and the heating electrode 13 generates heat due to the flow of current, and the heat is conducted to a plurality of micro reaction chambers formed later.

In an exemplary embodiment, the heating electrode is integrated on the first substrate, so that the micro-reaction cavity can be effectively heated, the temperature of the micro-reaction cavity can be controlled, external heating equipment is not needed, and the micro-reaction cavity has the characteristic of high integration level. In addition, compared with some digital polymerase chain reaction microfluidic devices which need to drive liquid drops to move and sequentially pass through a plurality of temperature areas, the digital polymerase chain reaction microfluidic device disclosed by the invention can realize temperature circulation without driving the liquid drops, and is simple to operate and low in production cost.

In an exemplary embodiment, the second conductive layer may be made of a conductive material with a relatively high resistivity, so that the heating electrode generates relatively high heat when a relatively small electrical signal is provided, thereby improving the energy conversion rate.

In an exemplary embodiment, the second conductive layer may employ a transparent conductive material, such as Indium Tin Oxide (ITO) or Indium Zinc Oxide (IZO). The second conducting layer is made of transparent conducting materials, so that laser can be incident into the micro-reaction cavity from the side, far away from the second conducting layer, of the first substrate.

In an exemplary embodiment, the second conductive layer may employ a metal material, such as any one or more of silver (Ag), copper (Cu), aluminum (Al), titanium (Ti), and molybdenum (Mo). At this time, laser light may be incident into the micro reaction chamber from a side of the first substrate where the second conductive layer is disposed.

In an exemplary embodiment, the heating electrode 13 may be a planar electrode to uniformly heat the plurality of micro reaction chambers in the reaction region 110.

(14) A second insulating layer pattern is formed. In an exemplary embodiment, the forming of the second insulation layer pattern may include: a second insulating film is deposited on the first substrate on which the aforementioned pattern is formed, and the second insulating film is patterned through a patterning process to form a second insulating layer 14 on the second conductive layer pattern, as shown in fig. 5.

In an exemplary embodiment, the second insulating layer 14 may be located within the reaction region 110, the second insulating layer 14 is configured to protect the heating electrode 13 and has an insulating function, may prevent liquid from corroding the heating electrode 13, may slow down the aging of the heating electrode 13, and may perform a planarization function.

In an exemplary embodiment, the second insulating layer may employ an inorganic material, which may be any one or more of silicon oxide (SiOx), silicon nitride (SiNx), and silicon oxynitride (SiON), may be a single layer, a multi-layer, or a composite layer, or an organic material, which may be a resin, or the like.

In an exemplary embodiment, the second insulating layer may have a multi-layer structure, the second insulating layer may include a first insulating sub-layer and a second insulating sub-layer stacked on each other, and the first insulating sub-layer may be silicon dioxide (SiO)₂) The second insulating sub-layer may be silicon nitride (SiNx).

In an exemplary embodiment, the first insulating sublayer may have a thickness of about 50nm to about 150nm, and the second insulating sublayer may have a thickness of about 150nm to about 250 nm. For example, the first insulating sublayer may have a thickness of about 100nm and the second insulating sublayer may have a thickness of about 200 nm.

(15) A microcavity defining layer pattern is formed. In an exemplary embodiment, forming the microcavity-defining layer pattern may include: coating a microcavity-defining thin film on the first substrate on which the aforementioned pattern is formed, patterning the microcavity-defining thin film through a patterning process, and forming a pattern of the microcavity-defining layer 30 on the second insulating layer 14, the microcavity-defining layer 30 may include a plurality of grooves 60 and defining dams 31 between adjacent grooves 60, the plurality of grooves 60 may serve as micro-reaction chambers for performing a polymerase chain reaction, as shown in fig. 6a and 6b, and fig. 6b is an enlarged view of one of the grooves in fig. 6 a.

In an exemplary embodiment, the microcavity-defining layer 30 pattern may be located in the reaction region 110, and the number of recesses 60 formed on the microcavity-defining layer 30 may be about 2000 to 1000000. For example, the number of grooves 60 may be about 40000 to 100000.

In an exemplary embodiment, the orthographic projection of the plurality of grooves 60 on the first substrate may be within the range of the orthographic projection of the heating electrode 13 on the first substrate to ensure that the heating electrode 13 can heat all the micro-reaction chambers.

In an exemplary embodiment, a plurality of grooves 60 as micro reaction chambers may be sequentially arranged in a first direction and a second direction, respectively, the first direction and the second direction crossing each other. For example, the first direction and the second direction may be perpendicular to each other, and the plurality of grooves 60 are arranged in an array, which may make the images obtained in the subsequent detection stage regular and orderly, so as to obtain the detection result quickly and accurately.

In an exemplary embodiment, the groove 60 may include a groove bottom wall 61 adjacent to the first base side and a groove side wall 62 located at the periphery of the groove bottom wall 61. In one possible embodiment, the recess 60 may be a through-hole through the microcavity-defining layer 30, the entire thickness of the microcavity-defining film within the recess 60 being removed, the recess 60 exposing the surface of the second insulating layer 14, i.e., the recess bottom wall 61 is the surface of the second insulating layer 14 and the recess side walls 62 are the surfaces of the microcavity-defining layer 30. In another possible embodiment, the recess 60 may be a blind hole opened in the microcavity-defining layer 30, a portion of the thickness of the microcavity-defining film in the recess 60 is removed, and the recess 60 exposes the surface of the microcavity-defining layer 30, i.e., both the bottom wall 61 and the side wall 62 of the recess are the surface of the microcavity-defining layer 30.

In an exemplary embodiment, the shape of the groove 60 in a plane parallel to the first substrate may include any one or more of: triangular, square, rectangular, pentagonal, hexagonal, polygonal, circular, and elliptical.

In an exemplary embodiment, the characteristic length L of the groove 60 may be about 1 μm to 100 μm in a plane parallel to the first substrate. In the present disclosure, the characteristic length L may be the largest dimension of the groove shape. For example, when the shape of the groove 60 is a circle, the diameter of the circle may be about 1 μm to 100 μm. As another example, where the recess 60 is rectangular in shape, the long side of the rectangle may be about 1 μm to about 100 μm. For another example, when the shape of the groove 60 is an ellipse, the major axis of the ellipse may be about 1 μm to 100 μm.

In an exemplary embodiment, the shape of the groove 60 may be circular, and the diameter of the circle may be about 8 μm or so.

In an exemplary embodiment, the cross-sectional shape of the groove 60 in a plane perpendicular to the first substrate may include any one or more of: rectangular, trapezoidal, and polygonal, the groove side walls 62 of the groove 60 may be straight, broken, or arc-shaped.

In an exemplary embodiment, the depth H of the groove 60 may be about 5 μm to 100 μm in a plane perpendicular to the first substrate. For example, the depth of the groove 60 may be about 9.8 μm.

In an exemplary embodiment, it may be understood that the microcavity defining layer 30 includes a plurality of defining dams 31, the plurality of defining dams 31 may extend along a first direction and along a second direction, respectively, the plurality of defining dams 31 extending along the first direction and the plurality of defining dams 31 extending along the second direction intersect with each other to define a plurality of grooves 60. The dam 31 may include a dam top wall 32 on a side away from the first base and a dam side wall 33 on a periphery of the dam top wall 32, the dam side wall 33 on a side toward the groove 60 serving as a groove side wall 62 of the groove. It should be noted that the limiting dam may include not only the region between adjacent grooves, but also the region on the side of the edge groove away from the inner groove, and the limiting dam may surround not only the inner groove but also the edge groove.

In an exemplary embodiment, the microcavity-defining layer can be an inorganic material, such as a photoresist. The photoresist can be formed by spin coating, and has a large thickness. For example, the microcavity-defining layer can be about 5 μm to 100 μm thick, e.g., about 9.8 μm thick.

(16) Forming a first surface modification layer pattern. In an exemplary embodiment, forming the first surface modification layer may include: depositing a first surface modification film on the first substrate on which the pattern is formed, patterning the first surface modification film through a patterning process to form a first surface modification layer 40 covering the limiting dam 31 and the groove 60 in the microcavity limiting layer, wherein a first modification structure 41 is formed on the first surface modification layer 40, as shown in fig. 7a and 7b, and fig. 7b is an enlarged view of one groove in fig. 7 a.

In the exemplary embodiment, the first surface-modifying layer 40 covering the microcavity defining layer means that the first surface-modifying layer 40 covers, on the one hand, the groove bottom wall and the groove side wall of the groove 60 and, on the other hand, the dam top wall of the defining dam 31.

In an exemplary embodiment, the material of the first surface modification layer 40 may be an inorganic material or an organic material having hydrophilic characteristics, such as silicon oxide, etc., so as to ensure that the surface of the first surface modification layer 40 on the side away from the first substrate has hydrophilic characteristics, which may include hydrophilic and oleophobic characteristics. In an exemplary embodiment, the material of the first surface modification layer 40 may be silicon dioxide (SiO)₂)。

In an exemplary embodiment, the thickness of the first surface modification layer 40 may be about 250nm to 350 nm. For example, the thickness of the first surface modification layer 40 may be about 300 nm.

In an exemplary embodiment, the temperature for depositing the first surface modification film may be about 150 to 250 ℃. For example, the temperature for depositing the first surface finish film may be about 200 ℃.

In an exemplary embodiment, the first surface modification layer 40 may include: the groove bottom wall modification layer 40-1 covering the groove bottom wall of the groove 60, the groove side wall modification layer 40-2 covering the groove side wall of the groove 60, and the dam crest wall modification layer 40-3 covering the dam crest wall of the limiting dam 31, wherein the groove bottom wall modification layer 40-1, the groove side wall modification layer 40-2, and the dam crest wall modification layer 40-3 are connected into an integral structure.

In an exemplary embodiment, the first modification structure 41 may be located on a surface of the bottom wall modification layer 40-1 on a side away from the first substrate, and the first modification structure 41 is configured to increase the hydrophilic property inside the micro reaction chamber. In some possible exemplary embodiments, the first modification structure 41 may be located on the groove sidewall modification layer 40-2. In other possible exemplary embodiments, the first modification structure 41 may be located on both the groove bottom wall modification layer 40-1 and the groove side wall modification layer 40-2.

In an exemplary embodiment, the first modifying structure 41 may include a plurality of first protrusions formed on a surface of the bottom-wall modifying layer 40-1 on a side away from the first substrate, and the plurality of first protrusions may be sequentially arranged along the first direction and the second direction, respectively. For example, the first direction and the second direction may be perpendicular to each other, and the plurality of first protrusions are arranged in an array.

In an exemplary embodiment, the plurality of first protrusions form micro-nano structures (micro-nano structures) in the shape of a cylinder, a truncated cone, a prism, etc. on the surface of the bottom wall modification layer 40-1 on the side away from the first substrate, so that the roughness of the bottom wall modification layer 40-1 is increased, thereby increasing the hydrophilic property inside the micro-reaction chamber.

In an exemplary embodiment, the shape of the first protrusion in a plane parallel to the first substrate may include any one or more of: triangular, square, rectangular, pentagonal, hexagonal, polygonal, circular, and elliptical.

In an exemplary embodiment, the first characteristic length L1 of the first protrusion may be about 0.5 μm to 2 μm in a plane parallel to the first substrate. In the present disclosure, the first characteristic length L1 may be the largest dimension of the first convex shape. For example, when the shape of the first protrusions is a circle, the diameter of the circle may be about 0.5 μm to 2 μm. For another example, when the first protrusion has a rectangular shape, the long side of the rectangular shape may be about 0.5 μm to 2 μm. For another example, when the first protrusions have an elliptical shape, the major axis of the elliptical shape may be about 0.5 μm to about 2 μm.

In an exemplary embodiment, the first protrusion may have a circular shape, and the diameter of the circular shape may be about 1 μm.

In an exemplary embodiment, the cross-sectional shape of the first protrusion in a plane perpendicular to the first substrate may include any one or more of: rectangular, trapezoidal and polygonal, the side walls of the first protrusion may be straight, broken or curved.

In an exemplary embodiment, the first height H1 of the first protrusion may be about 0.1 μm to 20 μm in a plane perpendicular to the first substrate. For example, the first height H1 of the first bump may be about 1 μm.

In an exemplary embodiment, the first spacing M1 between adjacent first protrusions may be about 0.5 to 2.0 times the first characteristic length L1. For example, the first protrusions may have a circular shape, the diameter of the circular shape may be about 1 μm, and the pitch between adjacent first protrusions may be about 1 μm.

Contact angle is a direct criterion used to describe the wetting behavior of a solid surface, and for a flat surface, the magnitude of the contact angle is determined by the surface tension of a liquid drop at the equilibrium point of the solid-liquid-gas three phases on the solid surface, and the magnitude of the contact angle θ is usually expressed by the Young (Young) equation:

wherein, γ_sol-gas、γ_sol-liqAnd gamma_gas-liqThe surface tension coefficients between solid-gas, solid-liquid and gas-liquid, respectively. Based on young's equation, lyophilic means that the contact angle of a liquid drop on a solid surface is less than 90 °, and lyophobic means that the contact angle of a liquid drop on a solid surface is greater than 90 °. If a liquid is uniformly dispersed on a surface without forming droplets, it is believed that such a surface tends to be hydrophilic in nature, allowing for water dispersion. Conversely, water forms droplets on a liquid-repellent surface, and such a surface is considered to be inherently hydrophobic. For a rough surface, the actual contact area of solid and liquid is larger than that of a flat surface, and the magnitude of the contact angle is represented by a Wenzel model: cos θ_W＝rcosθ_Y，θ_WApparent contact angle of the asperity, θ_YIs the intrinsic contact angle of a smooth surface, r is the roughness factor, which indicates the ratio of the actual solid/liquid contact area to the apparent solid/liquid contact area of a rough surface, i.e. the ratio of the actual surface area to the projected area of the surface, r is always greater than 1.

From the Wenzel model, it is known that as the roughness of the hydrophilic surface increases, the contact angle decreases, i.e., hydrophilicity increases. This is disclosed through form the first modification structure including a plurality of first archs at the recess bottom, has increased the roughness on little reaction chamber bottom surface, and then has increased the hydrophilic characteristic on little reaction chamber internal surface, under the circumstances of drive power is not applyed to the external world, reaction liquid can be based on capillary phenomenon and the automatic micro-reaction intracavity that gets into, not only can realize autoinjection, and reaction liquid gets into little reaction chamber more easily moreover, and the sampling speed has been improved, has improved digital micro-fluidic chip from inhaling liquid sampling performance.

In an exemplary embodiment, the roughness of the inner surface of the micro-reaction cavity is increased, so that the specific surface area of the inner film layer of the micro-reaction cavity is increased, the heat dissipation performance of the film layer is improved, the stress of the film layer is released, the mass production process quality is improved, and the product quality and the service life are improved.

(17) And forming a second surface modification layer pattern. In an exemplary embodiment, forming the second surface modification layer may include: depositing a second surface modification film on the first substrate on which the pattern is formed, patterning the second surface modification film through a patterning process, forming a second surface modification layer 50 on a side of the dam crest wall modification layer 40-3 of the first surface modification layer 40 away from the first substrate, and forming a second modification structure 51 on the second surface modification layer 50, as shown in fig. 8a and 8b, where fig. 8b is an enlarged view of one groove in fig. 8 a.

In the exemplary embodiment, the second surface modification layer 50 is provided on the side of the dam apex wall modification layer 40-3 covering the dam apex wall in the defining dam 31 away from the first base, and the second surface modification films on the groove bottom wall modification layer 40-1 covering the groove bottom wall of the groove 60 and the groove side wall modification layer 40-2 covering the groove side wall of the groove 60 are removed.

In an exemplary embodiment, the material of the second surface modification layer 50 may be an inorganic material or an organic material with hydrophobic characteristics, such as silicon nitride or resin, so as to ensure that the surface of the second surface modification layer 50 on the side away from the first substrate has hydrophobic characteristics, and the hydrophobic characteristics may include hydrophobic and oleophilic characteristics. In an exemplary embodiment, the inorganic material may be silicon nitride (SiNx), and the organic material may be photoresist.

In an exemplary embodiment, the thickness of the second surface modification layer 50 may be about 250nm to 350 nm. For example, the thickness of the second surface modification layer 50 may be about 300 nm.

In an exemplary embodiment, the second modifying structure 51 may be located on a surface of the second surface modifying layer 50 on a side away from the first substrate, the second modifying structure 51 being configured to increase the hydrophobic property of the second surface modifying layer 50.

In an exemplary embodiment, the second modifying structure 51 may include a plurality of second protrusions formed on a side surface of the second surface modifying layer 50 away from the second substrate, and the plurality of second protrusions may be sequentially disposed along the second direction and the second direction, respectively. For example, the second direction and the second direction may be perpendicular to each other, and the plurality of second protrusions are arranged in an array.

In an exemplary embodiment, the plurality of second protrusions form micro-nano structures in the shape of a cylinder, a truncated cone, a prism, and the like on the surface of the second surface modification layer 50 away from the second substrate, so that the roughness of the second surface modification layer 50 is increased, the hydrophobic property of the second surface modification layer 50 is increased, and the hydrophobic property outside the micro-reaction chamber is increased.

In an exemplary embodiment, the shape of the second protrusion in a plane parallel to the first substrate may include any one or more of: triangular, square, rectangular, pentagonal, hexagonal, polygonal, circular, and elliptical.

In an exemplary embodiment, the second characteristic length L2 of the second protrusion may be about 0.5 μm to 2 μm in a plane parallel to the first substrate. In the present disclosure, the second characteristic length L2 may be the largest dimension of the second convex shape. For example, when the second protrusions are respectively circular in shape, the diameter of the circle may be about 0.5 μm to 2 μm. For another example, when the second protrusion has a rectangular shape, the long side of the rectangular shape may be about 0.5 μm to 2 μm. For another example, when the second protrusions have an elliptical shape, the major axis of the elliptical shape may be about 0.5 μm to 2 μm.

In an exemplary embodiment, the second protrusion may have a circular shape, and the diameter of the circular shape may be about 1 μm.

In an exemplary embodiment, the cross-sectional shape of the second protrusion in a plane perpendicular to the first substrate may include any one or more of: rectangular, trapezoidal and polygonal, and the sidewalls of the second protrusion may be straight, polygonal or curved.

In an exemplary embodiment, the second height H2 of the second protrusion may be about 0.1 μm to 20 μm in a plane perpendicular to the second substrate. For example, the second height H2 of the second bump may be about 1 μm.

In an exemplary embodiment, the spacing M2 between adjacent second protrusions may be about 0.5 to 2.0 times the second characteristic length L2. For example, the second protrusions may have a circular shape, the diameter of the circular shape may be about 1 μm, and the pitch between adjacent second protrusions may be about 1.5 μm.

From the Wenzel model, it is known that as the roughness of the hydrophobic surface increases, the contact angle increases, i.e., the hydrophobicity increases. The second modification structure comprising the second protrusions is formed at the top of the limiting dam, the roughness of the outer surface of the micro-reaction cavity is increased, the hydrophobic characteristic of the outer surface of the micro-reaction cavity is further increased, under the condition that no driving force is applied to the outside, reaction liquid can automatically enter each micro-reaction cavity based on the hydrophobic characteristic and the capillary phenomenon of the outer surface, automatic sample introduction can be achieved, the reaction liquid can easily enter the micro-reaction cavity with the hydrophilic characteristic, the sample introduction speed is increased, the sample introduction performance of the digital micro-fluidic chip is improved, and based on the hydrophilic and hydrophobic characteristics inside and outside the micro-reaction cavity, the phenomena such as crosstalk and the like cannot occur after the reaction liquid enters the micro-reaction cavity, the stability and effectiveness of an oil seal are improved, and the sample introduction and oil seal performance of the digital micro-fluidic chip is improved.

In an exemplary embodiment, the roughness of the external surface of the micro reaction cavity is increased, so that the specific surface area of the external film layer of the micro reaction cavity is increased, the heat dissipation performance of the film layer is improved, the stress of the film layer is released, the mass production process quality is improved, and the product quality and the service life are improved.

To this end, the first substrate 100 is prepared. In an exemplary embodiment, the first substrate 100 may include a first substrate 10, a control electrode 11 disposed on the first substrate 10, a first insulating layer 12 covering the control electrode 11, a heating electrode 13 disposed on the first insulating layer 12, a second insulating layer 14 disposed on the heating electrode 13, a microcavity defining layer 30 disposed on the second insulating layer 14, a first surface modification layer 40 disposed on the microcavity defining layer 30, and a second surface modification layer 50 disposed on the first surface modification layer 40, wherein a first modification structure 41 for improving hydrophilic characteristics is formed on the first surface modification layer 40, and a second modification structure 51 for improving hydrophobic characteristics is formed on the second surface modification layer 50.

Second part, second substrate preparation

In an exemplary embodiment, the second substrate preparation may include the following operations.

(21) Forming a third surface modification layer pattern. In an exemplary embodiment, forming the third surface modification layer may include: a third surface modification film is deposited or coated on the second substrate, and is patterned through a patterning process, so as to form a third surface modification layer 80 on the second substrate 20, and a third modification structure 81 is formed on the third surface modification layer 80, as shown in fig. 9.

In an exemplary embodiment, the material of the third surface modification layer 80 may be an inorganic material or an organic material having a hydrophobic property, such as silicon nitride or a resin, so as to ensure that the surface of the third surface modification layer 80 on the side away from the second substrate has a hydrophobic property, and the hydrophobic property may include a hydrophobic oleophilic property. In an exemplary embodiment, the material of the third surface modification layer 80 may employ a photoresist of an inorganic material.

In an exemplary embodiment, the thickness of the third surface modification layer 80 may be about 250nm to 350 nm. For example, the thickness of the third surface modification layer 80 may be about 300 nm.

In an exemplary embodiment, the third modification structure 81 may be located on a surface of the third surface modification layer 80 on a side away from the second substrate, and the third modification structure 81 is configured to increase the hydrophobic property of the third surface modification layer 80.

In an exemplary embodiment, the third modification structure 81 may include a plurality of third protrusions formed on a side surface of the third surface modification layer 80 facing away from the second substrate, and the plurality of third protrusions may be sequentially disposed in the third direction and the third direction, respectively. For example, the third direction and the third direction may be perpendicular to each other, and the plurality of third protrusions are arranged in an array.

In an exemplary embodiment, the third protrusions form micro-nano structures in the shape of a cylinder, a truncated cone, a prism, etc. on the surface of the third surface modification layer 80 away from the third substrate, so that the roughness of the third surface modification layer 80 is increased, the hydrophobic property of the third surface modification layer 80 is increased, and thus the hydrophobic property of the outside of the micro-reaction chamber is increased.

In an exemplary embodiment, the shape of the third protrusion in a plane parallel to the second substrate may include any one or more of: triangular, square, rectangular, pentagonal, hexagonal, polygonal, circular, and elliptical.

In an exemplary embodiment, the third characteristic length L3 of the third protrusion may be about 0.5 μm to 2 μm in a plane parallel to the second substrate. In the present disclosure, the third characteristic length L3 may be the largest dimension of the third convex shape. For example, when the shape of the third protrusions is a circle, the diameter of the circle may be about 0.5 μm to 2 μm. For another example, when the third protrusion has a rectangular shape, the long side of the rectangle may be about 0.5 μm to 2 μm. For another example, when the shape of the third protrusion is an ellipse, the major axis of the ellipse may be about 0.5 μm to 2 μm.

In an exemplary embodiment, the third protrusion may have a circular shape, and the diameter of the circular shape may be about 1 μm.

In an exemplary embodiment, the cross-sectional shape of the third protrusion in a plane perpendicular to the second substrate may include any one or more of: rectangular, trapezoidal and polygonal, the side walls of the third protrusion may be straight, broken or curved.

In an exemplary embodiment, the third height H3 of the third protrusion may be about 0.1 μm to 20 μm in a plane perpendicular to the second substrate. For example, the third height H3 of the third bump may be about 1 μm.

In an exemplary embodiment, the spacing M3 between adjacent third protrusions may be about 0.5 to 2.0 times the third characteristic length L3. For example, the third protrusions may have a circular shape, the diameter of the circular shape may be about 1 μm, and the pitch between adjacent third protrusions may be about 1.5 μm.

In an exemplary embodiment, the third height H3 of the third protrusions in the third modified structure 81 may be greater than the second height H2 of the second protrusions in the second modified structure 51, and/or the third pitch M3 of the third protrusions in the third modified structure 81 may be greater than the second pitch M2 of the second protrusions in the second modified structure 51, so that the roughness of the region where the third modified structure 81 is located is greater than that of the region where the second modified structure 51 is located, and the hydrophilic property of the region where the third modified structure 81 is located is greater than that of the region where the second modified structure 51 is located, thereby further improving the sample injection efficiency.

From the Wenzel model, it is known that as the roughness of the hydrophobic surface increases, the contact angle increases, i.e., the hydrophobicity increases. According to the digital micro-fluidic chip, the third modification structure comprising the third protrusions is formed on the second substrate, the roughness of the outer surface of the micro-reaction cavity is increased, the hydrophobic characteristic of the outer surface of the micro-reaction cavity is further increased, the reaction liquid can automatically enter each micro-reaction cavity based on the hydrophobic characteristic and the capillary phenomenon of the outer surface under the condition that no driving force is applied to the outside, automatic sample introduction can be achieved, the reaction liquid can easily enter the micro-reaction cavity with the hydrophilic characteristic, the sample introduction speed is increased, the sample introduction performance of the digital micro-fluidic chip is improved, and the phenomena such as crosstalk and the like cannot occur after the reaction liquid enters the micro-reaction cavity based on the hydrophilic and hydrophobic characteristics inside and outside the micro-reaction cavity, the stability and the effectiveness of an oil seal are improved, and the sample introduction performance and the oil seal performance of the digital micro-fluidic chip are improved.

At this point, the second substrate 200 is prepared. In an exemplary embodiment, the second substrate 200 may include a second substrate 20, a third surface modification layer 80 disposed on the second substrate 20, and a third modification structure 81 formed on the third surface modification layer 80 to improve hydrophobic characteristics.

Third, packaging Process

In exemplary embodiments, the encapsulation process may employ ultraviolet light curing, or may employ thermal curing.

In an exemplary embodiment, the ultraviolet light curing may include the following operations. Placing ultraviolet curing glue (UV glue) doped with a spacer (spacer) with the diameter of about 100 mu m in a glue dispensing machine, setting parameters such as packaging appearance, glue dispensing speed and the like, moving a second substrate through a sucking disc after glue dispensing is performed on a first substrate, aligning and then attaching the second substrate and the first substrate, and performing ultraviolet curing irradiation to complete box-to-box packaging of the first substrate and the second substrate.

In an exemplary embodiment, the thermal curing may include the following operations. And die cutting or laser cutting the thermosetting adhesive film material with the diameter of about 100 microns into single pieces according to the appearance of the second substrate, attaching the single pieces onto the second substrate through a clamp after removing the hard release film, heating to 120 ℃ to enable the thermosetting adhesive to generate viscosity to be adhered to the second substrate, cooling, taking out and removing the soft release film. The second substrate is moved through the sucker and attached to the first substrate after being aligned, and the first substrate and the second substrate are bonded together through the thermosetting adhesive through a heating process, so that box-aligning packaging of the first substrate and the second substrate is completed.

To this end, the box-to-box packaging of the first substrate and the second substrate is completed, as shown in fig. 1. In an exemplary embodiment, the first substrate 100 and the second substrate 200 are connected together by the sealant 90, a side of the first substrate 100 on which the microcavity defining layer is disposed faces the second substrate 200, a side of the second substrate 200 on which the third surface modification layer is disposed faces the first substrate 100, and the first substrate 100, the second substrate 200, and the sealant 90 form a closed reaction chamber.

In an exemplary embodiment, the second substrate 20 and the third surface modification layer 80 of the second substrate are provided with a liquid inlet 82, and the liquid inlet 82 is a through hole penetrating through the second substrate 20 and the third surface modification layer 80. During detection, the reaction solution is injected into the reaction chamber from the solution inlet 82.

When the digital polymerase chain reaction microfluidic device is actually applied, reaction liquid is prepared, diluted reaction liquid is injected into a plurality of micro reaction cavities through a liquid inlet by sample injection operation until the reaction liquid is filled in a plurality of micro reaction cavities (grooves), then mineral oil or perfluoroalkane oil phase is filled above the reaction liquid, and thermal cycle PCR reaction is carried out after oil seal is completed. And an excitation light source is adopted to enter from one side of the first substrate far away from the second substrate or from one side of the second substrate far away from the first substrate, fluorescence is detected, and the number of target molecules in the reaction liquid in each micro-reaction cavity is judged by carrying out data analysis on the collected fluorescence image. Because the target molecules (i.e. DNA templates) in the reaction solution are fully diluted, after the reaction solution enters each micro-reaction cavity, the target molecules in each micro-reaction cavity are less than or equal to 1, i.e. each micro-reaction cavity only comprises one target molecule or does not comprise the target molecules, and thus an accurate detection result can be obtained.

According to the structure and the preparation process of the digital polymerase chain reaction microfluidic device disclosed by the exemplary embodiment of the disclosure, the hydrophilic structure and the hydrophobic structure are respectively designed inside and outside the micro-reaction cavity, so that the defects of incomplete filling of reaction liquid or crosstalk after filling in the sample introduction and amplification processes are effectively avoided, and the sample introduction and oil sealing performance of the self-priming liquid is improved. This is disclosed through limiting the first surface modification layer that the layer formed at the microcavity sets up the recess that has hydrophilic characteristic, and forms first modification structure on the surface of first surface modification layer, has increased the roughness on little reaction chamber internal surface, and then has increased the hydrophilic characteristic of little reaction chamber inside for reaction liquid gets into little reaction chamber more easily, and little reaction chamber is easily filled completely to reaction liquid, has improved from inhaling the liquid sampling performance. This is disclosed through limiting the second surface modification layer that sets up the hydrophobic property on the dam that limits that the layer formed at the microcavity, and form the second on the surface of second surface modification layer and decorate the structure, the hydrophobic property on little reaction chamber external surface has been increased, not only make reaction liquid get into and fill little reaction chamber completely more easily, and based on the hydrophilic and hydrophobic property inside and outside little reaction chamber, phenomenon such as the drunkenness can not appear after reaction liquid gets into little reaction chamber, the stability and the validity of oil blanket have been improved, improved from the liquid of inhaling and advance kind and oil blanket performance. This is disclosed through forming the third surface modification layer that has hydrophobic characteristic on the second base plate, and forms the third on the surface of third surface modification layer and decorate the structure, has increased the outside hydrophobic characteristic of little reaction chamber, has further improved from inhaling the liquid and has advanced kind and oil blanket performance.

The utility model discloses through the hydrophilic characteristic of increasing the inside of little reaction chamber simultaneously and increase the hydrophobic characteristic of little reaction chamber outside, the contact angle of liquid drop in the hydrophilic characteristic of little reaction chamber inside and the hydrophobic characteristic of little reaction chamber outside can be adjusted jointly to the reaction liquid, make the reaction liquid from the inside infiltration of little reaction chamber outside to little reaction chamber, realized high-efficient appearance and advanced a kind, the reaction liquid gets into every little reaction chamber more easily, the reaction liquid easily fills little reaction chamber completely and is difficult for appearing the drunkenness after filling, effectively solved the existing structure and had the reaction liquid in advance a kind and the amplification process and not fill completely or appear the drunkenness scheduling problem after filling, furthest has improved digital micro-fluidic chip from inhaling liquid appearance and oil blanket performance.

This is disclosed through the roughness that increases the surface modification layer for the rete specific surface area increases, has not only improved the heat dispersion of rete, and is favorable to releasing the stress of rete, has improved volume production technology quality, has improved volume production technology stability, has improved validity, stability and the accuracy of chip encapsulation, has promoted product quality and life.

Compared with a reaction system adopting silicon-based processing and preparation, the digital polymerase chain reaction microfluidic control device disclosed by the invention adopts a micromachining mode combining a glass substrate and a semiconductor process, can realize large-scale batch production, can greatly reduce the preparation cost, has a simple preparation process, can effectively utilize process equipment of the semiconductor process, and has the advantages of small process improvement, strong compatibility, simple process realization, high production efficiency, low production cost and high yield. The digital polymerase chain reaction microfluidic control device can detect nucleic acid molecules extracted from body fluids such as blood, urine and the like more simply, conveniently, stably, sensitively and atraumaticly, and can realize auxiliary diagnosis and treatment in the fields of single cell analysis, cancer diagnosis, virus analysis, prenatal diagnosis and the like.

Fig. 10a is a schematic structural view of another dPCR microfluidic device according to an exemplary embodiment of the present disclosure, and fig. 10b is an enlarged view of one of the grooves in fig. 10 a. In an exemplary embodiment, the digital pcr microfluidic device of the present exemplary embodiment has a structure substantially similar to that of the embodiment shown in fig. 1, and includes a first substrate 100 and a second substrate 200 oppositely disposed, the first substrate 100 includes a first substrate 10, and a heating structure layer and a microcavity defining layer 30 sequentially disposed on the first substrate 10, the first surface modification layer 40 and the second surface modification layer 50, the second substrate 200 includes a second substrate 20 and a third surface modification layer 80 disposed on the second substrate 20, the microcavity defining layer 30 may include a plurality of grooves 60 and a defining dam 31 disposed between adjacent grooves 60, a first modification structure 41 for improving hydrophilic property is formed on the first surface modification layer 40, a second modification structure 51 for improving hydrophobic property is formed on the second surface modification layer 50, and a third modification structure 81 for improving hydrophobic property is formed on the third surface modification layer 80. As shown in fig. 10a and 10b, unlike the previous embodiment, a flow guide structure 70 is further disposed on the microcavity defining layer 30, and the flow guide structure 70 is configured to improve the efficiency of the reaction liquid entering the micro-reaction cavity.

In an exemplary embodiment, the flow guiding structure 70 may be disposed on a surface of the first surface modification layer 40 on a side away from the first substrate, and the flow guiding structure 70 and the first modification structure 41 may be simultaneously formed through the same patterning process.

In an exemplary embodiment, the first surface modification layer 40 may include: a groove bottom wall modification layer 40-1 covering the groove bottom wall of the groove 60, a groove side wall modification layer 40-2 covering the groove side wall of the groove 60, and a dam crest wall modification layer 40-3 covering the dam crest wall in the limiting dam 31. The first modification structure 41 may be located on a surface of the bottom wall modification layer 40-1 on a side away from the first substrate, the first modification structure 41 being configured to increase the hydrophilic property inside the micro reaction chamber, the first modification structure 41 being similar to the foregoing exemplary embodiment. The flow guiding structure 70 may be located on a surface of the dam top wall modification layer 40-3 on a side away from the first substrate, and the flow guiding structure 70 is configured to improve efficiency of the reaction liquid entering the micro-reaction chamber.

In an exemplary embodiment, the flow guiding structure 70 may be located at an edge of the dam top wall modification layer 40-3 near one side of the groove 60, and an annular flow guiding structure is formed at the periphery of the micro reaction chamber, and the annular flow guiding structure surrounds the micro reaction chamber, so as to improve the efficiency of the reaction solution entering the micro reaction chamber.

In an exemplary embodiment, a second surface modification layer 50 is disposed on a side of the dam crest wall modification layer 40-3 of the first surface modification layer 40, which is far from the first substrate, the second surface modification layer 50 may be located in a region of the dam crest wall modification layer 40-3, which is far from the groove 60, the second surface modification layer 50 is spaced from the flow guide structure 70, the flow guide structure 70 is located in a peripheral region of the dam crest wall modification layer 40-3, which is near to the groove 60, the second surface modification layer 50 is located in a middle region of the dam crest wall modification layer 40-3, and an orthographic projection of the second surface modification layer 50 on the first substrate does not overlap with an orthographic projection of the flow guide structure 70 on the first substrate.

In the exemplary embodiment, the second surface modification layer 50 has the second modification structure 51 formed thereon, and the second modification structure 51 is similar to the foregoing exemplary embodiment.

In an exemplary embodiment, the flow guiding structure 70 may include a plurality of flow guiding columns formed on a surface of the dam crest wall modification layer 40-3 on a side away from the first substrate, where the plurality of flow guiding columns construct micro-nano structures in shapes of cylinders, truncated cones, prisms, and the like on a surface of the dam crest wall modification layer 40-3 on a side away from the first substrate, so as to increase roughness of a region of the dam crest wall modification layer 40-3 close to the groove 60, and thus increase a hydrophilic characteristic of a region around the groove 60.

In an exemplary embodiment, the shape of the flow guide column in a plane parallel to the first substrate may include any one or more of: triangular, square, rectangular, pentagonal, hexagonal, polygonal, circular, and elliptical.

In an exemplary embodiment, the fourth characteristic length L4 of the flow guide pillar may be about 0.5 μm to 2 μm in a plane parallel to the first substrate. In the present disclosure, the fourth characteristic length L4 may be the largest dimension of the flow guide pillar shape. For example, where the flow post is circular in shape, the diameter of the circle may be about 0.5 μm to 2 μm. For another example, when the guide pillar is rectangular, the long side of the rectangle may be about 0.5 μm to 2 μm. For another example, when the guide pillar has an elliptical shape, the major axis of the elliptical shape may be about 0.5 μm to about 2 μm.

In an exemplary embodiment, the cross-sectional shape of the flow guide pillar in a plane perpendicular to the first substrate may include any one or more of: rectangular, trapezoidal and polygonal, the lateral wall of guide post can be straight line, broken line or camber line.

In an exemplary embodiment, the fourth height H4 of the flow guide pillar may be about 0.1 μm to 20 μm in a plane perpendicular to the first substrate.

In an exemplary embodiment, the fourth spacing M4 between adjacent flow guide columns may be about 0.5 to 2.0 times the fourth characteristic length L4. For example, the flow guide pillars may be circular in shape, the diameter of the circle may be about 1 μm, and the spacing between adjacent flow guide pillars may be about 1 μm.

In an exemplary embodiment, the fourth height H4 of the flow guiding structure 70 may be greater than the first height H1 of the first protrusion in the first modified structure 41, and/or the fourth distance M4 of the flow guiding structure 70 may be greater than the first distance M1 of the first protrusion in the first modified structure 41, so that the roughness of the region where the flow guiding structure 70 is located is greater than the roughness of the region where the first modified structure 41 is located, and the hydrophilic property of the region where the flow guiding structure 70 is located is greater than the hydrophilic property of the region where the first modified structure 41 is located, thereby improving the efficiency of the reaction solution entering the micro-reaction chamber.

The preparation process of the dPCR microfluidic device according to the exemplary embodiment of the present disclosure is substantially similar to that of the foregoing embodiment, except that, in the process of forming the first surface modification layer pattern, not only the first modification structure is formed at the bottom of the groove, but also the flow guide structure is formed in the vicinity area outside the groove; in the process of forming the second surface modification layer pattern, the second modification structure is formed only in the region outside the flow guide structure.

The digital polymerase chain reaction microfluidic device disclosed by the exemplary embodiment of the disclosure not only has the technical effects of the foregoing embodiments, effectively avoids the disadvantages of incomplete filling of the reaction liquid or occurrence of crosstalk after filling in the sample introduction and amplification processes, and improves the sample introduction and oil sealing performance of the self-priming liquid, but also improves the efficiency of the reaction liquid entering the micro-reaction chamber by arranging the flow guide structure at the periphery of the micro-reaction chamber, the reaction liquid easily completely fills the micro-reaction chamber and the crosstalk after filling is not easy to occur, and the sample introduction and oil sealing performance of the self-priming liquid of the digital microfluidic chip is improved to the maximum extent.

Fig. 11a is a schematic structural view of a dPCR microfluidic device according to an exemplary embodiment of the present disclosure, and fig. 11b is an enlarged view of one of the grooves in fig. 11 a. In an exemplary embodiment, the digital pcr microfluidic device of the present exemplary embodiment has a structure substantially similar to that of the embodiment shown in fig. 1, and includes a first substrate 100 and a second substrate 200, which are oppositely disposed, the first substrate 100 includes a first substrate 10, and a heating structure layer, a microcavity defining layer, and a surface modification layer sequentially disposed on the first substrate 10, the second substrate 200 includes a second substrate 20 and a third surface modification layer 80 disposed on the second substrate 20, and the microcavity defining layer 30 may include a plurality of grooves 60 and defining dams 31 between adjacent grooves 60. As shown in fig. 11a and 11b, unlike the previous embodiment, the first surface modification layer 40 is disposed only in the region of the groove 60, and the second modification structure is disposed on the side of the limiting dam 31 away from the first substrate.

In an exemplary embodiment, the first surface modification layer 40 may include: the groove bottom wall modification layer 40-1 covering the groove bottom wall of the groove 60 and the groove side wall modification layer 40-2 covering the groove side wall of the groove 60, that is, the dam top wall in the limiting dam 31 is not provided with the first surface modification layer 40. The first modification structure 41 may be located on a surface of the bottom wall modification layer 40-1 on a side away from the first substrate, the first modification structure 41 being configured to increase the hydrophilic property inside the micro reaction chamber, the first modification structure 41 being similar to the foregoing exemplary embodiment.

In an exemplary embodiment, the microcavity defining layer may be made of a photoresist or the like having a hydrophobic property, the second modification structure 51 is formed on a surface of the side of the dam top wall of the defining dam 31 away from the first substrate, the second modification structure 51 is configured to increase the hydrophobic property outside the micro-reaction chamber, and the second modification structure 51 may include a plurality of second protrusions formed on a surface of the side of the dam top wall of the defining dam 31 away from the first substrate, and the structure of the second protrusions is similar to that of the foregoing exemplary embodiment.

The fabrication process of the digital pcr microfluidic device according to the exemplary embodiment of the present disclosure is substantially similar to that of the foregoing embodiment, except that, in the process of forming the microcavity defining layer pattern, not only the plurality of grooves but also the second modification structure is formed on the surface of the defining dam on the side away from the first substrate. In the process of forming the first surface modification layer pattern, the first surface modification layer and the first modification structure are formed only in the area where the groove is located, and the process of forming the second surface modification layer pattern is not required.

In an exemplary embodiment, the process of forming the microcavity defining layer pattern may employ a patterning process of a Half Tone Mask (Half Tone Mask), a Single Slit diffraction Mask (Single Slit Mask), or a Gray Tone Mask (Gray Tone Mask), and the disclosure is not limited herein.

The digital polymerase chain reaction microfluidic device of the exemplary embodiment of the disclosure can achieve the technical effects of the foregoing embodiments, effectively avoids the disadvantages of incomplete filling of the reaction solution or occurrence of crosstalk after filling in the sample introduction and amplification processes, and improves the sample introduction and oil sealing performance of the self-priming solution.

FIG. 12 is a schematic plan view of a reaction zone according to an exemplary embodiment of the present disclosure. In an exemplary embodiment, along the flowing direction a of the reaction liquid, the reaction region may include a liquid inlet region 110-1, a liquid inlet transition region 110-2, a reaction region 110-3, a liquid outlet transition region 110-4, and a liquid outlet region 110-5, wherein the reaction liquid enters the reaction region from the liquid inlet region 110-1, enters the reaction region 110-3 through the liquid inlet transition region 110-2, and is injected into a plurality of micro reaction chambers, and the reaction liquid after obtaining the detection result flows out from the reaction region 110-3, and flows out to the liquid outlet region 110-5 through the liquid outlet transition region 110-4 to be discharged.

In an exemplary embodiment, the reaction zone 110-3 may include a plurality of micro reaction chambers in which the first trim structure 41 is disposed and a defining dam disposed between adjacent micro reaction chambers in which the second trim structure 51 is disposed. The liquid inlet region 110-1, the liquid inlet transition region 110-2, the liquid outlet transition region 110-4 and the liquid outlet region 110-5 can all comprise a micro-cavity limiting layer, and the micro-cavity limiting layer is provided with a second modification structure 51. That is, the reaction regions other than the plurality of micro reaction chambers have a hydrophilic characteristic, and the reaction regions other than the plurality of micro reaction chambers have a hydrophobic characteristic.

In an exemplary embodiment, the hydrophobic properties of the liquid inlet transition region 110-2, the region outside the plurality of micro reaction chambers in the reaction region 110-3, and the liquid outlet transition region 110-4 may be the same, i.e., the second modification structures 51 of the liquid inlet transition region 110-2, the reaction region 110-3, and the liquid outlet transition region 110-4 may be the same.

In exemplary embodiments, the second modified structure 51 of the inlet region 110-1 and/or the outlet region 110-5 may be different from the second modified structure 51 of the other regions. Taking the example that the second modification structures 51 of the liquid inlet region 110-1 and the liquid inlet transition region 110-2 both include a plurality of second protrusions, the second height of the second protrusions in the liquid inlet region 110-1 may be smaller than the second height of the second protrusions in the liquid inlet transition region 110-2, and/or the second pitch of the second protrusions in the liquid inlet region 110-1 may be smaller than the second pitch of the second protrusions in the liquid inlet transition region 110-2, so that the roughness of the liquid inlet transition region 110-2 is greater than that of the liquid inlet region 110-1, and further, the hydrophobic property of the liquid inlet transition region 110-2 is greater than that of the liquid inlet region 110-1. The sample injection efficiency is further improved by increasing the hydrophobic characteristic of the periphery of the reaction area.

In an exemplary embodiment, the transition length of the liquid-in transition region 110-2 and/or the liquid-out transition region 110-4 may be about 800 μm to 1200 μm along the flow direction a of the reaction liquid. For example, the transition length of the entry transition zone 110-2 and/or the exit transition zone 110-4 may be about 1000 μm or so.

It should be noted that the structure shown in the embodiments of the present disclosure and the preparation process thereof are only an exemplary illustration. In an exemplary embodiment, the corresponding structure may be changed and the patterning process may be added or reduced according to actual needs. For example, the structure of the dPCR microfluidic device may be such that the flow directing structure is disposed on the microcavity defining layer, but the second modification structure is disposed on a side of the defining dam remote from the first substrate. For another example, the dPCR microfluidic device may have a structure in which the first surface modification layer is disposed in a region where the groove is located, the second surface modification layer is disposed in a region other than the groove, the first surface modification layer is provided with the first modification structure, and the second surface modification layer is provided with the second modification structure. For another example, the dPCR microfluidic device may also be provided with other electrodes, leads, and structural film layers, and the disclosure is not limited thereto.

The disclosed exemplary embodiments also provide a method for preparing a digital polymerase chain reaction microfluidic device. In an exemplary embodiment, a method of manufacturing a digital polymerase chain reaction microfluidic device may include:

In an exemplary embodiment, preparing the first substrate may include:

In an exemplary embodiment, preparing the second substrate may include:

According to the digital polymerase chain reaction microfluidic device and the preparation method thereof, the hydrophilic structure and the hydrophobic structure are respectively designed inside and outside the micro reaction cavity, so that the defects of incomplete filling or crosstalk after filling of reaction liquid in the sample introduction and amplification processes are effectively avoided, and the sample introduction and oil sealing performances of the self-priming liquid are improved. This is disclosed through limiting at the microcavity and setting up first surface modification layer in the recess that the layer formed, and at the first surface modification layer surface formation first modification structure, has increased the roughness on little reaction chamber internal surface, and then has increased the hydrophilic characteristic of little reaction chamber inside for reaction liquid gets into little reaction chamber more easily, and little reaction chamber is easily filled completely to reaction liquid, has improved from inhaling liquid sampling performance. This is disclosed through limiting at the microcavity and setting up second surface modification layer on the dam that limits that the layer formed, and form second modification structure on the surface of second surface modification layer, the hydrophobic characteristic on little reaction chamber outside surface has been increased, not only make reaction liquid get into and fill little reaction chamber completely more easily, and based on the hydrophilic and hydrophobic characteristic inside and outside little reaction chamber, phenomenon such as drunkenness can not appear after reaction liquid gets into little reaction chamber, the stability and the validity of oil blanket have been improved, improved from the liquid sampling of inhaling and oil blanket performance. This is disclosed through forming third surface modification layer on the second base plate, and forms third modification structure on the surface of third surface modification layer, has increased the outside hydrophobic characteristic of little reaction chamber, has further improved from inhaling liquid and has advanced kind and oil blanket performance. The utility model discloses through the hydrophilic characteristic of increasing the inside of little reaction chamber simultaneously and increase the hydrophobic characteristic of little reaction chamber outside, the contact angle of liquid drop in the hydrophilic characteristic of little reaction chamber inside and the hydrophobic characteristic of little reaction chamber outside can be adjusted jointly to the reaction liquid, make the reaction liquid from the inside infiltration of little reaction chamber outside to little reaction chamber, realized high-efficient appearance and advanced a kind, the reaction liquid gets into every little reaction chamber more easily, the reaction liquid easily fills little reaction chamber completely and is difficult for appearing the drunkenness after filling, effectively solved the existing structure and had the reaction liquid in advance a kind and the amplification process and not fill completely or appear the drunkenness scheduling problem after filling, furthest has improved digital micro-fluidic chip from inhaling liquid appearance and oil blanket performance. This is disclosed through the roughness that increases the surface modification layer for rete specific surface area increases, has not only improved the heat dispersion of rete, and is favorable to releasing the stress of rete, has improved volume production technology quality, has promoted product quality and life. Compared with a reaction system adopting silicon-based processing and preparation, the digital polymerase chain reaction microfluidic control device disclosed by the invention adopts a micromachining mode combining a glass substrate and a semiconductor process, can realize large-scale batch production, can greatly reduce the preparation cost, has a simple preparation process, can effectively utilize process equipment of the semiconductor process, and has the advantages of small process improvement, strong compatibility, simple process realization, high production efficiency, low production cost and high yield. The digital polymerase chain reaction microfluidic control device can detect nucleic acid molecules extracted from body fluids such as blood, urine and the like more simply, conveniently, stably, sensitively and atraumaticly, and can realize auxiliary diagnosis and treatment in the fields of single cell analysis, cancer diagnosis, virus analysis, prenatal diagnosis and the like.

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 of the present disclosure 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 present disclosure is to be limited only by the terms of the appended claims.

Claims

1. The digital polymerase chain reaction microfluidic device is characterized by comprising a first substrate and a second substrate which are oppositely arranged; the first substrate comprises a first substrate, a microcavity limiting layer arranged on one side of the first substrate, which faces the second substrate, and a first surface modification layer arranged on one side of the microcavity limiting layer, which is far away from the first substrate; the microcavity defining layer includes a plurality of grooves as micro-reaction chambers and defining dams between adjacent grooves, and the first surface modification layer in at least one of the grooves is provided with a first modification structure configured to increase hydrophilic properties inside the micro-reaction chambers.

2. The apparatus of claim 1, wherein the first surface modification layer comprises a bottom wall modification layer covering a bottom wall of the trench and a sidewall modification layer covering a sidewall of the trench, the first modification structure being disposed on a surface of the bottom wall modification layer on a side remote from the first substrate.

3. The apparatus of claim 2, wherein the first modification comprises at least one first protrusion disposed on a surface of the channel bottom wall modification layer on a side thereof remote from the first substrate.

4. The device of claim 3, wherein, in a plane parallel to the first substrate, the first protrusions have a first characteristic length of 0.5 μm to 2 μm, and a first spacing between adjacent first protrusions is 0.5 times to 2.0 times the first characteristic length, the first characteristic length being a maximum dimension of the first protrusions; the first protrusions have a first height of 0.1 to 20 μm in a plane perpendicular to the first substrate.

5. The apparatus of any one of claims 1 to 4, wherein a side of the confining dam remote from the first substrate is provided with a second modification configured to increase hydrophobic properties outside the micro reaction chamber.

6. The apparatus of claim 5, wherein the first surface modification layer comprises a groove bottom wall modification layer covering the groove-in-groove bottom wall, a groove side wall modification layer covering the groove-in-groove side wall, and a dam top wall modification layer covering the dam-in-dam top wall; and a second surface modification layer is arranged on one side, far away from the first substrate, of the at least one dam top wall modification layer, and the second modification structure is arranged on the surface of one side, far away from the first substrate, of the second surface modification layer.

7. The apparatus of claim 5, wherein the first surface modification layer comprises a bottom wall modification layer covering a bottom wall of the trench in the trench and a sidewall modification layer covering a sidewall of the trench in the trench; the second modifying structure is disposed on a surface of a side of the dam apex wall away from the first base in the defining dam.

8. The apparatus of claim 5, wherein the second modifying structure comprises at least one second protrusion provided on a surface of a second surface modifying layer or defining dam on a side of the dam apex wall remote from the first substrate.

9. The device of claim 8, wherein, in a plane parallel to the first substrate, the second protrusions have a second characteristic length of 0.5 μ ι η to 2 μ ι η, and a second spacing between adjacent second protrusions is 0.5 times to 2.0 times the first characteristic length, the second characteristic length being a maximum dimension of the second protrusions; the second protrusions have a second height of 0.1 to 20 μm in a plane perpendicular to the first substrate.

10. The apparatus of any one of claims 1 to 4, wherein the first surface modification layer comprises a groove bottom wall modification layer covering the groove bottom wall in the groove, a groove side wall modification layer covering the groove side wall in the groove, and a dam top wall modification layer covering the dam top wall in the defined dam; and a flow guide structure is arranged on the surface of one side, far away from the first substrate, of at least one dam top wall modification layer, the flow guide structure is positioned on one side, close to the groove, of the dam top wall modification layer, and the flow guide structure is configured to improve the efficiency of reaction liquid entering the micro-reaction cavity.

11. The apparatus of claim 10, wherein the flow directing structure comprises at least one flow directing post disposed on a surface of the dam apex wall modification remote from the first substrate.

12. The apparatus of claim 11, wherein the flow guide posts have a flow guide characteristic length of 0.5 μ ι η to 2 μ ι η and a flow guide spacing between adjacent flow guide posts is 0.5 times to 2.0 times the flow guide characteristic length in a plane parallel to the first substrate, the flow guide characteristic length being the largest dimension of the flow guide post; the flow guide height of the flow guide column in a plane perpendicular to the first substrate is 0.1-20 μm.

13. The apparatus of claim 11, wherein the flow guiding height of the flow guiding pillars in the flow guiding structure is greater than the first height of the first protrusions in the first modifying structure, and/or the flow guiding pitch of the flow guiding pillars in the flow guiding structure is greater than the first pitch of the first protrusions in the first modifying structure.

14. The apparatus of any one of claims 1 to 4, wherein the second substrate comprises a second substrate and a third surface modification layer disposed on a side of the second substrate facing the first substrate, wherein a surface of the third surface modification layer facing the first substrate has a third modification structure disposed thereon, and the third modification structure is configured to increase a hydrophobic property of an exterior of the micro reaction chamber.

15. The apparatus of claim 14, wherein the third modifying structure comprises at least one third protrusion disposed on a surface of the third surface modifying layer facing the first substrate.

16. The device of claim 15, wherein, in a plane parallel to the first substrate, a third feature length of the third protrusions is 0.5 μ ι η to 2 μ ι η, a second pitch between adjacent third protrusions is 0.5 times to 2.0 times the third feature length, the third feature length being a maximum dimension of the third protrusions; a third height of the third protrusion in a plane perpendicular to the first substrate is 0.1 to 20 μm.

17. The apparatus of claim 16, wherein the third height of the third protrusions in the third trim structure is greater than the second height of the second protrusions in the second trim structure, and/or wherein the third pitch of the third protrusions in the third trim structure is greater than the second pitch of the second protrusions in the second trim structure.

18. A method for preparing a microfluidic device for digital polymerase chain reaction, comprising:

19. The method of claim 18, wherein preparing the first substrate comprises:

20. The method of claim 18, wherein preparing the second substrate comprises: