CN113814014B - Digital polymerase chain reaction microfluidic device and preparation method thereof - Google Patents

Abstract

Exemplary embodiments of the present disclosure provide a digital polymerase chain reaction microfluidic device and a preparation method thereof. The digital polymerase chain reaction microfluidic device includes a first substrate and a second substrate oppositely arranged; the first substrate includes a first substrate, a microcavity arranged on the side of the first substrate facing the second substrate A confining layer and a first surface modification layer disposed on the side of the microcavity confining layer away from the first substrate; the microcavity confining layer includes a plurality of grooves as microreaction chambers and is located between 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 chamber. The disclosure improves the self-absorbing liquid sampling and oil sealing performance by respectively designing a hydrophilic structure and a hydrophobic structure inside and outside the micro-reaction chamber.

Description

Digital polymerase chain reaction microfluidic device and preparation method thereof

technical field

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

Background technique

Digital Polymerase Chain Reaction (Digital Polymerase Chain Reaction, referred to as dPCR), dPCR is a quantitative analysis method that provides digital DNA quantification information. The mixture of reagents is dispersed in each miniature micro-reaction chamber in the chip, and the target molecule in each chamber is subjected to independent PCR amplification.

The inventors of the present application have found that the existing digital polymerase chain reaction microfluidic devices have problems such as incomplete filling of the reaction solution during sample injection and amplification or crosstalk after filling.

public content

The following is an overview of the topics described in detail in this article. 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 preparation method thereof, so as to solve the problems of the existing structure that the reaction liquid is not completely filled or cross-talk occurs after filling.

In order to solve the above technical problems, an exemplary embodiment of the present disclosure provides a digital polymerase chain reaction microfluidic device, including a first substrate and a second substrate oppositely arranged; the first substrate includes a first substrate, a set A microcavity-defining layer on the side of the first substrate facing the second substrate and a first surface modification layer disposed on the side of the microcavity-defining layer away from the first substrate; the microcavity-defining layer includes A plurality of grooves as micro-reaction chambers and defined dams between 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 as Increase the hydrophilic properties inside the micro-reaction chamber.

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 walls in the groove, the first surface modification layer A modification structure is disposed on the surface of the groove bottom wall modification layer away from the first base.

In an exemplary embodiment, the first modification structure includes at least one first protrusion disposed on a 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 characteristic length of the first protrusion is 0.5 μm to 2 μm, and the first distance between adjacent first protrusions is 0.5 μm. times to 2.0 times the first characteristic length, the first characteristic length being the maximum dimension of the first protrusion; in a plane perpendicular to the first base, the first height of the first protrusion is 0.1μm to 20μm.

In an exemplary embodiment, the side of the limiting dam away from the first substrate is provided with a second modification structure, and the second modification structure is configured to increase the hydrophobicity of the exterior of the micro-reaction chamber.

In an exemplary embodiment, the first surface modification layer includes 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 groove side wall modification layer covering the groove. A dam crest finish layer defining the dam crest wall in the dam; at least one dam crest finish layer is provided with a second surface finish layer on a side away from the first base, and the second finish structure is set on the second surface The modification layer is on the surface of the side away from the first base.

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 walls in the groove; the second The second modification structure is arranged on the surface of the top wall of the defined dam on the side away from the first base.

In an exemplary embodiment, the second modification structure includes at least one second protrusion disposed on the surface of the second surface modification layer or the top wall of the defined dam on a side away from the first base.

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

In an exemplary embodiment, the first surface modification layer includes 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 groove side wall modification layer covering the groove. A dam crest finishing layer defining the dam crest in the dam; at least one dam crest finishing layer is provided with a flow guide structure on the surface of the side away from the first base, and the flow guiding structure is located in the dam crest finishing layer Near the side of the groove, the flow guide structure is configured to improve the efficiency of the reaction liquid entering the micro-reaction chamber.

In an exemplary embodiment, the diversion structure includes at least one diversion column disposed on a surface of the top wall finishing layer away from the first base.

In an exemplary embodiment, in a plane parallel to the first substrate, the guide column has a guide feature length of 0.5 μm to 2 μm, and the guide distance between adjacent guide posts is 0.5 times to 2 μm. 2.0 times the diversion characteristic length, where the diversion characteristic length is the maximum dimension of the diversion column; in a plane perpendicular to the first substrate, the diversion height of the diversion column is 0.1 μm to 20 μm .

In an exemplary embodiment, the guide height of the guide post in the guide structure is greater than the first height of the first protrusion in the first modification structure, and/or, the guide post in the guide structure The diversion distance is greater than the first distance between the first protrusions in the first modified structure.

In an exemplary embodiment, the second substrate includes a second base and a third surface modification layer disposed on a side of the second base facing the first substrate, and the third surface modification layer faces the first substrate. A third modification structure is arranged on the surface of one side of the substrate, and the third modification structure is configured to increase the hydrophobicity of the exterior of the micro-reaction chamber.

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

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

In an exemplary embodiment, the third height of the third protrusion in the third modified structure is greater than the second height of the second protrusion in the second modified structure, and/or, the third height of the third protrusion in the third modified structure The third pitch of the protrusions is greater than the second pitch of the second protrusions in the second modified structure.

Exemplary embodiments of the present disclosure also provide a method for preparing a digital polymerase chain reaction microfluidic device, including:

Prepare a first substrate and a second substrate respectively; the first substrate includes a first substrate, a microcavity defining layer disposed on the first substrate facing the second substrate, and a microcavity defining layer disposed on a side away from the microcavity defining layer The first surface modification layer on one side of the first substrate; the microcavity-defining layer includes a plurality of grooves as micro-reaction chambers and a limit dam between adjacent grooves, the at least one groove in the groove The first surface modification layer is provided with a first modification structure, and the first modification structure is configured to increase the hydrophilic property inside the micro-reaction chamber;

The first substrate and the second substrate are packaged into 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 comprising a plurality of grooves serving as microreaction chambers and limiting dams between adjacent grooves;

Forming a first surface modification layer, the first surface modification layer includes 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 groove side wall modification layer covering the groove The dam top wall modification layer on the side away from the first base is defined, and at least one groove bottom wall modification layer has a first modification structure formed on the surface of the side away from the first base, and the first modification structure includes at least a first bump;

forming a second surface modification layer, the second surface modification layer is formed on the side of at least one dam top wall modification layer away from the first base, and the second surface modification layer is on the surface of the side away from the first base A second modification structure is formed on the top, and the second modification structure includes at least one second protrusion.

In an exemplary embodiment, preparing the second substrate includes:

A third surface modification layer is formed on the side of the second substrate facing the first substrate, a third modification structure is formed on the surface of the third surface modification layer facing the first substrate, and the third modification The structure includes at least one third protrusion.

Exemplary embodiments of the present disclosure provide a digital polymerase chain reaction microfluidic device and a preparation method thereof. By designing a hydrophilic structure and a hydrophobic structure on the inside and outside of the micro-reaction chamber, it effectively avoids sample injection and During the amplification process, the reaction liquid is not completely filled or cross-talk occurs after filling, which improves the performance of self-absorbing liquid sampling and oil sealing.

Of course, implementing any product or method of the present disclosure does not necessarily need to achieve all the advantages described above at the same time. Additional features and advantages of the disclosure will be set forth in the description which follows, and, in part, will be apparent from the description, or can be learned by practice of the disclosure. The objectives and other advantages of the exemplary embodiments of the present disclosure may be realized and attained by the structure particularly pointed out in the written description, claims hereof as well as the appended drawings.

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

Description of drawings

The accompanying drawings are used to provide a further understanding of the technical solutions of the present disclosure, and constitute a part of the specification, and are used together with the embodiments of the present disclosure to explain the technical solutions of the present disclosure, and do not constitute limitations to the technical solutions of the present disclosure. The shapes and sizes of the various components in the drawings do not reflect true scale, but are only 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;

2 is a schematic structural view of an embodiment of the present disclosure after forming a first conductive layer pattern;

3 is a schematic structural view of an embodiment of the present disclosure after forming a first insulating layer pattern;

4 is a schematic structural view of an embodiment of the present disclosure after forming a second conductive layer pattern;

5 is a schematic structural view of an embodiment of the present disclosure after forming a second insulating layer pattern;

Fig. 6a and Fig. 6b are structural schematic diagrams of the microcavity-defining layer pattern formed in the embodiment of the present disclosure;

Fig. 7a and Fig. 7b are structural schematic diagrams of the present disclosure after the formation of the first surface modification layer pattern;

Fig. 8a and Fig. 8b are structural schematic diagrams of the present disclosure after forming the pattern of the second surface modification layer;

9 is a schematic structural view of an embodiment of the present disclosure after forming a pattern of a third surface modification layer;

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

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

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

Explanation of reference signs:

10—the first substrate; 11—the control electrode; 12—the first insulating layer;

13—heating electrode; 14—second insulating layer; 20—second substrate;

30—microcavity confinement layer; 31—limited dam; 32—dam crest wall;

33—dam side wall; 40—the first surface modification layer; 41—the first modification structure;

50—the second surface modification layer; 51—the second modification structure; 60—the groove;

61—the bottom wall of the groove; 62—the side wall of the groove; 70—the diversion structure;

80—the third surface modification layer; 81—the third modification structure; 82—liquid inlet;

90—sealant; 100—first substrate; 110—reaction area;

120—peripheral area; 200—second substrate.

Detailed ways

In order to make the purpose, technical solution and advantages of the present disclosure clearer, the embodiments of the present disclosure will be described in detail below in conjunction with the accompanying drawings. Note that an embodiment may be embodied in many different forms. Those skilled in the art can easily understand the fact that the means and contents can be changed into various forms without departing from the gist and scope of the present disclosure. Therefore, the present disclosure should not be interpreted as being limited only to the contents described in the following embodiments. In the case of no conflict, the embodiments in the present disclosure and the features in the embodiments can be combined arbitrarily with each other.

The proportions of the drawings in the present disclosure can be used as a reference in the actual process, but are not limited thereto. The drawings described in the present disclosure are only structural schematic diagrams, and an aspect of the present disclosure is not limited to the shapes or numerical values shown in the drawings.

Ordinal numerals such as "first", "second", and "third" in this specification are provided to avoid confusion of constituent elements, and are not intended to limit the number.

In this specification, for convenience, "middle", "upper", "lower", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner" are used , "external" and other words indicating the orientation or positional relationship are used to illustrate the positional relationship of the constituent elements with reference to the drawings, which are only for the convenience of describing this specification and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation , are constructed and operate in a particular orientation and therefore are not to be construed as limitations on the present disclosure. The positional relationship of the constituent elements changes appropriately according to the direction in which each constituent element is described. Therefore, it is not limited to the words and phrases described in the specification, and may be appropriately replaced according to circumstances.

In this specification, unless otherwise specified and limited, the terms "installation", "connection" and "connection" should be interpreted in a broad sense. For example, it may be a fixed connection, or a detachable connection, or an integral connection; it may be a mechanical connection, or an electrical connection; it may be a direct connection, or an indirect connection through an intermediate piece, or an internal communication between two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present disclosure in specific situations.

In the present specification, "parallel" refers to a state where the angle formed by two straight lines is -10° to 10°, and therefore includes a state where the angle is -5° to 5°. In addition, "perpendicular" means a state in which the angle formed by two straight lines is 80° to 100°, and therefore also includes an angle of 85° to 95°.

The triangle, rectangle, trapezoid, pentagon, or hexagon in this specification are not strictly defined, and may be approximate triangles, rectangles, trapezoids, pentagons, or hexagons, etc., and there may be some small deformations caused by tolerances. There can be chamfers, arc edges, deformations, etc.

"About" in the present disclosure refers to a numerical value that is not strictly limited, and is within the range of process and measurement errors.

Digital polymerase chain reaction dPCR is a quantitative analysis method that provides digital DNA quantification information, and is an important technology for molecular biology detection and analysis. Its accurate quantitative DNA copy number is one of the important applications of modern molecular biology and medicine. In recent years, with the rapid development of microfluidics technology, the dPCR microfluidics device combined with microfluidics technology has greatly improved the sensitivity and accuracy. The dPCR microfluidic device adopts a divide-and-conquer detection strategy, using microfluidic technology to disperse the mixture of samples and PCR reagents in the micro-reaction chambers of the chip, and independently PCR amplifies the target molecules in each micro-reaction chamber. Usually, dPCR uses secondary injection for oil sealing, that is, the sample and dPCR reagents are located in the micro-reaction chamber, and the micro-reaction chamber is filled with the oil-phase sample after the hydrophilic and hydrophobic properties and capillary action of the micro-channel, so as to realize micro-reaction. The sample liquid in the cavity is divided. The inventors of the present application found that this method of oil phase liquid seal requires hydrophilic and hydrophobic design inside and outside the micro-reaction chamber, so as to ensure a better secondary injection effect and ensure that the oil phase completely fills the micro-reaction chamber and flows out. However, the existing devices usually only have a hydrophilic design for the interior of the micro-reaction chamber, which leads to problems such as incomplete filling of the reaction solution during sample injection and amplification or cross-talk after filling in the existing structure.

In order to solve the problems of the existing structure that the reaction liquid is not completely filled or disturbed after filling during the sample injection and amplification process, an exemplary embodiment of the present 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 oppositely arranged, and the first substrate may include a first substrate, a substrate disposed on a side of the first substrate facing the second substrate The microcavity-defining layer and the first surface modification layer arranged on the 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 adjacent grooves Defining dams between grooves, the first surface modification layer within at least one groove may be provided with a first modification structure configured to increase the hydrophilic properties of the interior of the microreaction chamber.

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

In an exemplary embodiment, the second substrate may include a second base and a third surface modification layer disposed on a side of the second base facing the first substrate, and the third surface modification layer faces the A third modification structure is arranged on the surface of one side of the first substrate, and the third modification structure is configured to increase the hydrophobicity of the 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 an exemplary embodiment, the digital polymerase chain reaction microfluidic device of the present disclosure can be used to perform a digital polymerase chain reaction, and can be used for a detection process after the reaction. As shown in FIG. 1 , the main body structure of the digital polymerase chain reaction microfluidic device may include a first substrate 100 and a second substrate 200 oppositely arranged, and the first substrate 100 may include a first substrate 10 and be arranged on the first substrate. 10 the microcavity defining layer 30 on the side close to the second substrate 200 and the first surface modification layer 40 disposed on the side of the microcavity defining layer 30 close to the second substrate 200 . The second substrate 200 may include a second base 20 and a third surface modification layer 80 disposed on a side of the second base 20 close to the first base 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 through the sealant 90 .

In an exemplary embodiment, the microcavity-defining layer 30 may include a plurality of grooves 60 and confinement dams 31 between adjacent grooves 60, the confinement dams 31 define a plurality of grooves 60, and the plurality of grooves 60 may be As a micro-reaction chamber for digital polymerase chain reaction.

In an exemplary embodiment, the first surface modification layer 40 has a hydrophilic feature, and the first surface modification layer 40 in at least one groove 60 can be provided with a first modification structure 41, and the first modification structure 41 is configured to increase microscopic Hydrophilic properties of the interior of the reaction chamber.

In an exemplary embodiment, the groove 60 may include a groove bottom wall near a side of the first substrate and a groove side wall located at a periphery of the groove bottom wall. In at least one groove 60, the first surface modification layer 40 may include a groove bottom wall modification layer covering the groove bottom wall in the groove 60 and a groove side wall modification layer covering the groove side walls in the groove 60, the first modification structure 41 It can be arranged on the surface of the groove bottom wall modification layer away from the first base.

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

In an exemplary embodiment, the defining dam 31 may include a crest wall on a side away from the first base and a side wall of the dam on the periphery. The first surface modification layer 40 can also include a dam crest wall modification layer covering at least one of the dam crest walls that define the dam 31, and a second surface modification layer 50 is provided on the side of the dam top wall modification layer away from the first base, and the second surface The modification layer 50 may be provided with a second modification structure 51 configured to increase the hydrophobicity of the exterior of 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 the surface of the second surface modification layer 50 away from the first substrate.

In an exemplary embodiment, the second modification structure 51 may include at least one second protrusion provided on the surface of the second surface modification layer 50 away from the first substrate, and the at least one second protrusion is configured to increase the second The roughness of the surface of the surface modification layer 50 away from the first substrate.

In an exemplary embodiment, the third surface modification layer 80 may be provided with a third modification structure 81 configured to increase the hydrophobic property of the exterior of 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 the 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 provided on the surface of the third surface modification layer 80 away from the second substrate, the at least one third protrusion is configured to increase the third The roughness of the surface of the surface modification layer 80 away from the second substrate.

In an exemplary embodiment, the first substrate 10 may include a reaction area 110 and a peripheral area 120 , and the peripheral area 120 may surround the reaction area 110 , that is, the peripheral area 120 is located outside the reaction area 110 . The second substrate 20 can be arranged opposite to the reaction region 110 on the first substrate 10, and form a closed reaction chamber with the reaction region 110 on the first substrate 10 through the sealant 90, and a plurality of grooves as micro reaction chambers Located in a closed 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 heating electrode 13 disposed on the first substrate 10. The second insulating layer 14 on the heating electrode 13 , the heating electrode 13 may be connected to the control electrode 11 through a connection via hole, and the microcavity defining layer 30 may be disposed on the second insulating layer 14 .

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

In an exemplary embodiment, glass may be used for the first substrate and the second substrate, the shapes of the first substrate and the second substrate may be rectangular, and the size of the second substrate may be smaller than that of the first substrate. For example, the size of the first base may be about 3.2 cm*4.5 cm, and the size of the second base may be about 3.2 cm*3.0 cm.

In an exemplary embodiment, in a plane parallel to the first substrate, the shapes of the plurality of grooves 60 may include any one or more of the following: triangle, square, rectangle, pentagon, hexagon, polygon, Round and oval. In a plane perpendicular to the first substrate, the cross-sectional shapes of the plurality of grooves may include any one or more of the following: rectangle, trapezoid and polygon, and the groove sidewalls of the grooves may be straight lines, broken lines or arcs.

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

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

In an exemplary embodiment, the number of the plurality of grooves 60 serving as micro-reaction chambers may be about 2,000 to 1,000,000. For example, the number of grooves 60 may be about 40,000 to 100,000.

In an exemplary embodiment, in a plane parallel to the first base, the shapes of the plurality of first protrusions, second protrusions and third protrusions may include any one or more of the following: triangle, square, rectangle , pentagon, hexagon, polygon, circle and ellipse. In a plane perpendicular to the first base, the cross-sectional shapes of the plurality of first protrusions, second protrusions and third protrusions may include any one or more of the following: rectangle, trapezoid and polygon, the side walls of the protrusions Can be straight line, polyline or arc.

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

In an exemplary embodiment, the plurality of first protrusions, second protrusions, 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 polymerase chain reaction microfluidic device may further include a temperature sensing device and a program control device, the temperature sensing device may be arranged on the side of the first substrate away from the second substrate, and the program control device may be respectively It is connected with the temperature sensing device, the control electrode and the heating electrode, which is not limited in this disclosure.

The following is an exemplary illustration through the preparation process of the digital polymerase chain reaction microfluidic device. The "patterning process" mentioned in this disclosure includes coating photoresist, mask exposure, development, etching, stripping photoresist and other treatments for metal materials, inorganic materials or transparent conductive materials, and for organic materials, including Coating of organic materials, mask exposure and development, etc. Deposition can use any one or more of sputtering, evaporation, chemical vapor deposition, coating can use any one or more of spray coating, spin coating and inkjet printing, etching can use dry etching and wet Any one or more of the engravings is not limited in the present disclosure. "Thin film" refers to a layer of thin film made of a certain material on a substrate by deposition, coating or other processes. If the "thin film" does not require a patterning process during the entire manufacturing process, the "thin film" can also be called a "layer". If the "thin film" requires a patterning process during the entire production process, it is called a "film" before the patterning process, and it is called a "layer" after the patterning process. The "layer" after the patterning process includes at least one "pattern". "A and B are arranged in the same layer" in this disclosure means that A and B are formed simultaneously through 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 an exemplary embodiment of the present disclosure, "the orthographic projection of B is within the range of the orthographic projection of A" or "the orthographic projection of A includes the orthographic projection of B" means that the boundary of the orthographic projection of B falls within the orthographic projection of A , or the boundary of A's orthographic projection overlaps the boundary of B's orthographic projection.

In an exemplary embodiment, the preparation of the digital polymerase chain reaction microfluidic device may include three parts, which are first substrate preparation, second substrate preparation and encapsulation treatment. Wherein, the preparation of the first substrate and the preparation of the second substrate have no order requirement and can be carried out at the same time, while the encapsulation process needs to be carried out after the preparation of the first substrate and the second substrate is completed. The preparation process of the three parts is described respectively below.

The first part, the first substrate preparation

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

(11) Forming a first conductive layer pattern. In an exemplary embodiment, forming the first conductive layer pattern may include: depositing a first conductive film on the first substrate, patterning the first conductive film through a patterning process, and forming a first conductive film disposed on the first substrate 10. The conductive layer pattern, the first conductive layer pattern may include at least one control electrode 11 , as shown in FIG. 2 .

In exemplary embodiments, at least one control electrode 11 may be located in the peripheral area 120 . The control electrode 11 is configured to apply an electrical signal (such as a voltage signal) to the 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 be a metal material, such as any one or more of silver (Ag), copper (Cu), aluminum (Al), titanium (Ti) and molybdenum (Mo). , or alloy materials of the above metals, may be a single-layer structure, or a multi-layer composite structure. For example, the first conductive layer can be a multi-layer composite structure, including the stacked first sublayer, second sublayer and third sublayer, the material of the first sublayer and the third sublayer can be metal molybdenum, the second The material of the sub-layer can be metal aluminum to form a multi-layer composite structure of Mo/Al/Mo. In an exemplary embodiment, the first sublayer may have a thickness of about 10nm to 30nm, the second sublayer may have a thickness of about 200nm to 400nm, and the third sublayer may have a thickness of about 70nm to 90nm. For example, the thickness of the first sublayer may be about 20 nm, the thickness of the second sublayer may be about 300 nm, and the thickness of the third sublayer may be about 80 nm.

In exemplary embodiments, the temperature for depositing the first conductive film may be about 100°C to 150°C. For example, the temperature for depositing the first conductive film may be about 125°C.

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

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

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

In exemplary embodiments, the temperature for depositing the first insulating film may be about 150°C to 250°C. For example, the temperature for depositing the first insulating film may be about 200°C.

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

In an exemplary embodiment, the heating electrode 13 may be located in the reaction area 110 and the peripheral area 120, and the heating electrode 13 is configured to heat a plurality of micro reaction chambers formed subsequently. Since the heating electrode 13 is connected to the control electrode 11 through the via hole, the heating electrode 13 can receive the 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 subsequent micro-reactions cavity.

In an exemplary embodiment, the present disclosure can effectively realize the heating of the micro-reaction chamber by integrating the heating electrode on the first substrate, and then realize the temperature control of the micro-reaction chamber, without the need for external heating equipment, and has the characteristics of high integration . In addition, compared with some digital polymerase chain reaction microfluidic devices that need to drive droplets to move and sequentially pass through multiple temperature regions, the digital polymerase chain reaction microfluidic device of the present disclosure does not need to drive droplets to operate. The temperature cycle can be realized, the operation is simple, and the production cost is low.

In an exemplary embodiment, the second conductive layer may use a conductive material with a relatively high resistivity, so that the heating electrode generates a large amount of heat when a small electrical signal is provided, so as to improve the energy conversion rate.

In an exemplary embodiment, the second conductive layer may use a transparent conductive material, such as indium tin oxide (ITO) or indium zinc oxide (IZO). The use of a transparent conductive material for the second conductive layer facilitates the incident of laser light into the micro reaction cavity from the side of the first substrate away from the second conductive layer.

In an exemplary embodiment, the second conductive layer may be a metal material, such as any one or more of silver (Ag), copper (Cu), aluminum (Al), titanium (Ti) and molybdenum (Mo) . At this time, the laser light can be incident into the micro-reaction cavity from the 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, so that the multiple micro-reaction chambers in the reaction area 110 are evenly heated.

(14) Forming a second insulating layer pattern. In an exemplary embodiment, forming the second insulating layer pattern may include: depositing a second insulating film on the first substrate on which the foregoing pattern is formed, patterning the second insulating film through a patterning process, and forming the second insulating film on the second conductive layer pattern. A second insulating layer 14 is formed on it, as shown in FIG. 5 .

In an exemplary embodiment, the second insulating layer 14 may be located in the reaction region 110, the second insulating layer 14 is configured to protect the heating electrode 13, and has an insulating effect, which can prevent the liquid from corroding the heating electrode 13 and slow down the heating electrode 13. Aging, and can play a role in planarization.

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

In an exemplary embodiment, the second insulating layer may have a multi-layer structure, and the second insulating layer may include a stacked first insulating sublayer and a second insulating sublayer, the first insulating sublayer may be made of silicon dioxide (SiO ₂ ), and the second insulating layer Silicon nitride (SiNx) can be used for the second insulator layer.

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

(15) Forming a microcavity defining layer pattern. In an exemplary embodiment, forming the pattern of the microcavity-defining layer may include: coating the microcavity-defining film on the first substrate on which the aforementioned pattern is formed, patterning the microcavity-defining film through a patterning process, and forming the microcavity-defining film on the second insulating layer Form the pattern of microcavity-defining layer 30 on 14, and microcavity-defining layer 30 can comprise a plurality of grooves 60 and be positioned at the limit dam 31 between adjacent grooves 60, and a plurality of grooves 60 can be used as carrying out polymerase chain reaction. The micro reaction chamber is shown in Figure 6a and Figure 6b, and Figure 6b is an enlarged view of a groove in Figure 6a.

In an exemplary embodiment, the pattern of the microcavity-defining layer 30 may be located in the reaction region 110, and the number of grooves 60 formed on the microcavity-defining layer 30 may be about 2,000 to 1,000,000. For example, the number of grooves 60 may be about 40,000 to 100,000.

In an exemplary embodiment, the orthographic projections of the plurality of grooves 60 on the first substrate can be within the range of the orthographic projections of the heating electrodes 13 on the first substrate, so as to ensure that the heating electrodes 13 can control all the micro reaction chambers. for heating.

In an exemplary embodiment, a plurality of grooves 60 serving as micro-reaction chambers may be arranged sequentially along a first direction and a second direction respectively, and the first direction and the second direction intersect. For example, the first direction and the second direction can be perpendicular to each other, and a plurality of grooves 60 are arranged in an array. This arrangement can make the images obtained in the subsequent detection stage more regular and orderly, so as to obtain detection results quickly and accurately. .

In an exemplary embodiment, the groove 60 may include a groove bottom wall 61 near the side of the first substrate and a groove side wall 62 located around the groove bottom wall 61 . In a possible implementation manner, the groove 60 may be a through hole penetrating through the microcavity defining layer 30, the entire thickness of the microcavity defining film in the groove 60 is removed, and the groove 60 exposes the surface of the second insulating layer 14 , that is, the groove bottom wall 61 is the surface of the second insulating layer 14 , and the groove sidewall 62 is the surface of the microcavity-defining layer 30 . In another possible implementation, the groove 60 can be a blind hole opened on the microcavity defining layer 30, the microcavity defining film of part thickness in the groove 60 is removed, and the groove 60 exposes the microcavity defining layer The surface of the microcavity-defining layer 30 , that is, the groove bottom wall 61 and the groove sidewall 62 are both surfaces of the microcavity-defining layer 30 .

In an exemplary embodiment, in a plane parallel to the first substrate, the shape of the groove 60 may include any one or more of the following: triangle, square, rectangle, pentagon, hexagon, polygon, circle and oval.

In an exemplary embodiment, the groove 60 may have a characteristic length L of 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, when the shape of the groove 60 is rectangular, the long side of the rectangle may be about 1 μm to 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 a circle, and the diameter of the circle may be about 8 μm.

In an exemplary embodiment, in a plane perpendicular to the first substrate, the cross-sectional shape of the groove 60 may include any one or more of the following: rectangle, trapezoid and polygon, and the groove side wall 62 of the groove 60 may be a straight line , polyline or arc.

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 groove 60 may be about 9.8 μm.

In an exemplary embodiment, it can be understood that the microcavity defining layer 30 includes a plurality of defining dams 31, and the plurality of defining dams 31 can respectively extend along the first direction and along the second direction, and the ones extending along the first direction The plurality of limiting dams 31 intersect with the plurality of limiting dams 31 extending along the second direction to define a plurality of grooves 60 . The limiting dam 31 may include a crest wall 32 away from the first base and a sidewall 33 located around the crest wall 32 , and the sidewall 33 facing the groove 60 also serves as a groove sidewall 62 of the groove. It should be noted that the limiting dam includes not only the area between adjacent grooves, but also the area on the side of the edge groove away from the inner groove, and the limiting dam can not only surround the inner groove, but also surround the edge groove. groove.

In an exemplary embodiment, the microcavity-defining layer may use an inorganic material, such as photoresist. The photoresist can be formed by spin coating, and the thickness is relatively large. For example, the thickness of the microcavity-defining layer can be about 5 μm to 100 μm, for example, the thickness of the microcavity-defining layer can be about 9.8 μm.

(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 forming the aforementioned pattern, and patterning the first surface modification film through a patterning process to form a covering microcavity The first surface modification layer 40 defining the dam 31 and the groove 60 in the limiting layer, the first surface modification layer 40 is formed with a first modification structure 41, as shown in Figure 7a and Figure 7b, Figure 7b is a concave in Figure 7a Enlarged view of the slot.

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

In an exemplary embodiment, the material of the first surface modification layer 40 can be an inorganic material or an organic material with hydrophilic properties, such as silicon oxide, so as to ensure that the first surface modification layer 40 is away from the surface of the first substrate. Having hydrophilic properties, which may include hydrophilic and oleophobic properties. In an exemplary embodiment, the material of the first surface modification layer 40 may be silicon dioxide (SiO ₂ ).

In an exemplary embodiment, the first surface modification layer 40 may have a thickness of about 250 nm 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°C to 250°C. For example, the temperature for depositing the first surface modification film may be about 200°C.

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 40-1 covering the groove side wall of the groove 60 2, and the dam top wall decoration layer 40-3 that covers the dam top wall that defines the dam 31, the groove bottom wall decoration layer 40-1, the groove side wall decoration layer 40-2 and the dam top wall decoration layer 40-3 are interconnected integrated structure.

In an exemplary embodiment, the first modification structure 41 may be located on the surface of the groove bottom wall modification layer 40-1 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 implementations, the first modification structure 41 may be located on the groove side wall modification layer 40-2. In some other possible exemplary embodiments, the first modification structure 41 may be located on the groove bottom wall modification layer 40-1 and the groove side wall modification layer 40-2 at the same time.

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

In an exemplary embodiment, a plurality of first protrusions construct a micro-nano structure (micro-nano structure) in the shape of a cylinder, a circular cone, a prism, etc. on the surface of the groove bottom wall modification layer 40-1 away from the first substrate, increasing The roughness of the groove bottom wall modification layer 40-1 is improved, thereby increasing the hydrophilic property inside the micro-reaction chamber.

In an exemplary embodiment, in a plane parallel to the first base, the shape of the first protrusion may include any one or more of the following: triangle, square, rectangle, pentagon, hexagon, polygon, circle shape and ellipse.

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 protrusion is a circle, the diameter of the circle may be about 0.5 μm to 2 μm. As another example, when the shape of the first protrusion is a rectangle, the long side of the rectangle may be about 0.5 μm to 2 μm. For another example, when the shape of the first protrusion is an ellipse, the major axis of the ellipse may be about 0.5 μm to 2 μm.

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

In an exemplary embodiment, in a plane perpendicular to the first base, the cross-sectional shape of the first protrusion may include any one or more of the following: rectangle, trapezoid and polygon, and the sidewall of the first protrusion may be a straight line , polyline or arc.

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 protrusion may be about 1 μm.

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

The contact angle is a direct criterion used to describe the wetting behavior of a solid surface. For a flat surface, the size of the contact angle is determined by the surface tension of the solid-liquid-gas three-phase equilibrium point of a liquid drop on the solid surface. Usually the size of the contact angle θ is expressed by Young's equation:

where γ _sol-gas , γ _sol-liq and γ _gas-liq are the surface tension coefficients between solid-gas, solid-liquid and gas-liquid, respectively. Based on Young's equation, lyophilicity means that the contact angle of a droplet on a solid surface is less than 90°, while lyophobicity means that the contact angle of a droplet on a solid surface is greater than 90°. If a liquid spreads evenly over a surface without forming droplets, it is considered that such a surface tends to be hydrophilic in nature, allowing water to disperse. Conversely, where water forms droplets on a lyophobic surface, it is assumed that such surfaces tend to be hydrophobic in nature. For a rough surface, the actual contact area of solid-liquid is larger than that of a flat surface, and the size of the contact angle is expressed by the Wenzel model: cosθ _W = rcosθ _Y , θ _W is the apparent contact angle of the rough surface, and θ _Y is the intrinsic contact angle of the smooth surface The characteristic contact angle, r is the roughness factor, which represents the ratio of the actual solid/liquid contact area to the apparent solid/liquid contact area of the rough surface, that is, the ratio of the actual surface area to the surface projected area, and r is always greater than 1.

According to the Wenzel model, as the roughness of the hydrophilic surface increases, the contact angle decreases, that is, the hydrophilicity increases. The present disclosure increases the roughness of the bottom surface of the micro-reaction chamber by forming a first modification structure including a plurality of first protrusions at the bottom of the groove, thereby increasing the hydrophilic property of the inner surface of the micro-reaction chamber, without external drive Under the condition of strong force, the reaction liquid can automatically enter the micro-reaction chamber based on the capillary phenomenon, not only can realize automatic sampling, but also the reaction liquid can enter the micro-reaction chamber more easily, which improves the sampling speed and improves the performance of the digital microfluidic chip. Self-aspiration liquid sampling performance.

In an exemplary embodiment, by increasing the roughness of the inner surface of the micro-reaction chamber, the specific surface area of the film layer in the micro-reaction chamber is increased, which not only improves the heat dissipation performance of the film layer, but also facilitates the release of the stress of the film layer, improving the Mass production process quality improves product quality and service life.

(17) 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 forming the aforementioned pattern, patterning the second surface modification film through a patterning process, and forming a second surface modification film on the first surface A second surface modification layer 50 is formed on the side of the dam crest modification layer 40-3 away from the first substrate in the modification layer 40, and a second modification structure 51 is formed on the second surface modification layer 50, as shown in FIGS. 8a and 8b , Figure 8b is an enlarged view of a groove in Figure 8a.

In an exemplary embodiment, the second surface modification layer 50 is disposed on the side away from the first base covering the crest wall modification layer 40-3 defining the crest wall of the dam 31, and covers the bottom wall of the groove 60. The second surface modification film on the groove bottom wall modification layer 40-1 and the groove side wall modification layer 40-2 covering the groove side walls of the groove 60 is removed.

In an exemplary embodiment, the material of the second surface modification layer 50 can be an inorganic material or an organic material with hydrophobic properties, such as silicon nitride or resin, so as to ensure that the second surface modification layer 50 is away from the surface of the first substrate. Having hydrophobic properties, which may include hydrophobic and lipophilic properties. In an exemplary embodiment, silicon nitride (SiNx) may be used as the inorganic material, and photoresist may be used as the organic material.

In an exemplary embodiment, the second surface modification layer 50 may have a thickness of about 250 nm 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 modification structure 51 may be located on the surface of the second surface modification layer 50 away from the first substrate, and the second modification structure 51 is configured to increase the hydrophobicity of the second surface modification layer 50 .

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

In an exemplary embodiment, a plurality of second protrusions construct micro-nano structures in the shape of cylinders, truncated cones, prisms, etc. on the surface of the second surface modification layer 50 away from the second substrate, increasing the thickness of the second surface modification layer 50. The roughness increases the hydrophobicity of the second surface modification layer 50, thus increasing the hydrophobicity of the outside of the micro-reaction chamber.

In an exemplary embodiment, in a plane parallel to the first base, the shape of the second protrusion may include any one or more of the following: triangle, square, rectangle, pentagon, hexagon, polygon, circle shape and ellipse.

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 shapes of the second protrusions are circular, the diameter of the circular shape may be about 0.5 μm to 2 μm. For another example, when the shape of the second protrusion is a rectangle, the long side of the rectangle may be about 0.5 μm to 2 μm. For another example, when the shape of the second protrusion is an ellipse, the major axis of the ellipse may be about 0.5 μm to 2 μm.

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

In an exemplary embodiment, in a plane perpendicular to the first base, the cross-sectional shape of the second protrusion may include any one or more of the following: rectangle, trapezoid and polygon, and the side wall of the second protrusion may be a straight line , polyline or arc.

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

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

According to the Wenzel model, as the roughness of the hydrophobic surface increases, the contact angle increases, that is, the hydrophobicity increases. The present disclosure increases the roughness of the external surface of the micro-reaction chamber by forming a second modification structure including a plurality of second protrusions on the top of the dam, thereby increasing the hydrophobicity of the external surface of the micro-reaction chamber, without external driving Under the condition of strong force, the reaction solution can automatically enter each micro-reaction chamber based on the hydrophobic characteristics and capillary phenomenon of the external surface, not only can realize automatic sample injection, but also the reaction solution can enter the micro-reaction chamber with hydrophilic characteristics more easily, which improves the process efficiency. The sampling speed improves the self-absorbing liquid sampling performance of the digital microfluidic chip, and based on the hydrophilic and hydrophobic characteristics inside and outside the micro-reaction chamber, there will be no crosstalk after the reaction liquid enters the micro-reaction chamber, which improves the stability and stability of the oil seal. The effectiveness improves the self-absorbing liquid sampling and oil sealing performance of the digital microfluidic chip.

In an exemplary embodiment, by increasing the roughness of the external surface of the micro-reaction chamber, the specific surface area of the film layer outside the micro-reaction chamber is increased, which not only improves the heat dissipation performance of the film layer, but also facilitates the release of the stress of the film layer, improving the Mass production process quality improves product quality and service life.

So far, the preparation of the first substrate 100 is completed. 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 , and a first insulating layer 12 disposed on the first insulating layer 12 . The heating electrode 13, the second insulating layer 14 arranged on the heating electrode 13, the microcavity defining layer 30 arranged on the second insulating layer 14, the first surface modification layer 40 arranged on the microcavity defining layer 30, arranged on The second surface modification layer 50 on the first surface modification layer 40, the first surface modification layer 40 is formed with the first modification structure 41 that improves the hydrophilic property, and the second surface modification layer 50 is formed with the second surface modification layer 50 that improves the hydrophobic property. Modify structure 51.

The second part, the 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: depositing or coating a third surface modification film on the second substrate, patterning the third surface modification film through a patterning process, and forming the third surface modification film on the second substrate 20 A third surface modification layer 80 is formed on the third surface modification layer 80 , 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 can be an inorganic material or an organic material with hydrophobic properties, such as silicon nitride or resin, so as to ensure that the third surface modification layer 80 is away from the surface of the second substrate. Having hydrophobic properties, which may include hydrophobic and lipophilic properties. In an exemplary embodiment, the material of the third surface modification layer 80 may be photoresist of inorganic material.

In an exemplary embodiment, the third surface modification layer 80 may have a thickness of about 250 nm 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 the surface of the third surface modification layer 80 away from the second substrate, and the third modification structure 81 is configured to increase the hydrophobicity 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 the surface of the third surface modification layer 80 away from the second substrate, and the plurality of third protrusions may be respectively along the third direction and the third direction are set in sequence. For example, the third direction and the third direction may be perpendicular to each other, and a plurality of third protrusions are arranged in an array.

In an exemplary embodiment, a plurality of third protrusions construct micro-nano structures in the shape of cylinders, truncated cones, prisms, etc. on the surface of the third surface modification layer 80 away from the third substrate, increasing the thickness of the third surface modification layer 80. The roughness increases the hydrophobicity of the third surface modification layer 80, thus increasing the hydrophobicity of the outside of the micro-reaction chamber.

In an exemplary embodiment, in a plane parallel to the second base, the shape of the third protrusion may include any one or more of the following: triangle, square, rectangle, pentagon, hexagon, polygon, circle shape and ellipse.

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 protrusion is a circle, the diameter of the circle may be about 0.5 μm to 2 μm. As another example, when the shape of the third protrusion is a rectangle, 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 shape of the third protrusion may be a circle, and the diameter of the circle may be about 1 μm.

In an exemplary embodiment, in a plane perpendicular to the second base, the cross-sectional shape of the third protrusion may include any one or more of the following: rectangle, trapezoid and polygon, and the side wall of the third protrusion may be a straight line , polyline or arc.

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 protrusion may be about 1 μm.

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

In an exemplary embodiment, the third height H3 of the third protrusion in the third modification structure 81 may be greater than the second height H2 of the second protrusion in the second modification structure 51, and/or, the third height H3 of the third protrusion in the third modification structure 81 The third spacing M3 of the third protrusions may be greater than the second spacing M2 of the second protrusions in the second modification structure 51 , so that the roughness of the area where the third modification structure 81 is located is greater than the roughness of the area where the second modification structure 51 is located. , so that the hydrophilic property of the area where the third modification structure 81 is located is greater than that of the area where the second modification structure 51 is located, thereby further improving the sampling efficiency.

According to the Wenzel model, as the roughness of the hydrophobic surface increases, the contact angle increases, that is, the hydrophobicity increases. The present disclosure increases the roughness of the external surface of the micro-reaction chamber by forming a third modified structure including a plurality of third protrusions on the second substrate, thereby increasing the hydrophobicity of the external surface of the micro-reaction chamber, without external drive Under the condition of strong force, the reaction solution can automatically enter each micro-reaction chamber based on the hydrophobic characteristics and capillary phenomenon of the external surface, not only can realize automatic sample injection, but also the reaction solution can enter the micro-reaction chamber with hydrophilic characteristics more easily, which improves the process efficiency. The sampling speed improves the self-absorbing liquid sampling performance of the digital microfluidic chip, and based on the hydrophilic and hydrophobic characteristics inside and outside the micro-reaction chamber, there will be no crosstalk after the reaction liquid enters the micro-reaction chamber, which improves the stability and stability of the oil seal. The effectiveness improves the self-absorbing liquid sampling and oil sealing performance of the digital microfluidic chip.

So far, the preparation of the second substrate 200 is completed. 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 that improves hydrophobic properties is formed on the third surface modification layer 80. 81.

The third part, packaging processing

In an exemplary embodiment, the encapsulation process may employ UV curing, or thermal curing.

In an exemplary embodiment, ultraviolet light curing may include the following operations. Place the ultraviolet curable glue (UV glue) doped with a spacer (spacer) with a diameter of about 100 μm in the dispenser, set the package shape, dispensing speed and other parameters, and implement dispensing on the first substrate After the completion, the second substrate is moved by the suction cup, the second substrate is aligned with the first substrate and then bonded, and the box-to-box packaging of the first substrate and the second substrate is completed through ultraviolet curing irradiation.

In an exemplary embodiment, thermal curing may include the following operations. The thermosetting adhesive film material with a diameter of about 100 μm is die-cut or laser-cut into a single piece according to the shape of the second substrate. The thermosetting adhesive is viscous and adheres to the second substrate. After cooling down, take it out and peel off the soft release film. The second substrate is moved by the suction cup, and the second substrate is aligned with the first substrate and bonded together. Through the heating process, the first substrate and the second substrate are bonded together by thermosetting glue, and the first substrate and the second substrate are completed. Packaged in a box.

So far, the 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 a sealant 90 , the side of the first substrate 100 provided with the microcavity defining layer faces the second substrate 200 , and the second substrate 200 The side provided with the third surface modification layer 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 base 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 the second base 20 and the third surface modification layer 80 . During detection, the reaction liquid is injected into the reaction chamber from the liquid inlet 82 .

In the actual application of the digital polymerase chain reaction microfluidic device, the reaction solution is prepared first, and the diluted reaction solution is injected into multiple micro-reaction chambers through the liquid inlet through the sample injection operation until the reaction solution is full of multiple micro-reaction chambers. Micro-reaction chamber (groove), then fill the upper part of the reaction solution with mineral oil or perfluoroalkane oil phase, after completing the oil seal, perform thermal cycle PCR reaction. The excitation light source is incident from the side of the first substrate away from the second substrate or from the side of the second substrate away from the first substrate, and the fluorescence is detected, and the collected fluorescence images are analyzed to determine whether each micro-reaction chamber The number of target molecules in the reaction solution. Since the target molecule (i.e. DNA template) in the reaction solution is fully diluted, when the reaction solution enters each micro-reaction chamber, the target molecule in each micro-reaction chamber is less than or equal to 1, that is, only one micro-reaction chamber is included in each micro-reaction chamber. The target molecule may or may not be included so that accurate detection results can be obtained.

Through the structure and preparation process of the digital polymerase chain reaction microfluidic device in the exemplary embodiment of the present disclosure, it can be seen that the present disclosure effectively avoids the In the process of sample injection and amplification, the reaction liquid is not completely filled or cross-talk occurs after filling, which improves the performance of self-absorbing liquid sampling and oil seal. In the present disclosure, a first surface modification layer with hydrophilic properties is arranged in the groove formed by the microcavity defining layer, and a first modification structure is formed on the surface of the first surface modification layer, thereby increasing the roughness of the inner surface of the microreaction chamber The degree increases the hydrophilic property inside the micro-reaction chamber, making it easier for the reaction liquid to enter the micro-reaction chamber, and the reaction liquid is easy to completely fill the micro-reaction chamber, which improves the performance of self-absorbing liquid sampling. The present disclosure increases the hydrophobicity of the external surface of the micro-reaction chamber by setting a second surface modification layer with hydrophobic properties on the dam formed by the micro-cavity definition layer, and forming a second modification structure on the surface of the second surface modification layer. , not only makes it easier for the reaction liquid to enter and completely fill the micro-reaction chamber, but also based on the hydrophilic and hydrophobic characteristics inside and outside the micro-reaction chamber, there will be no crosstalk after the reaction liquid enters the micro-reaction chamber, which improves the stability and effectiveness of the oil seal. Improved self-priming liquid sampling and oil seal performance. In the present disclosure, a third surface modification layer with hydrophobic properties is formed on the second substrate, and a third modification structure is formed on the surface of the third surface modification layer, thereby increasing the hydrophobic properties outside the micro-reaction chamber, and further improving the self-absorbing liquid. Injection and oil seal performance.

The present disclosure simultaneously increases the hydrophilic property inside the micro-reaction chamber and increases the hydrophobic property outside the micro-reaction chamber, the hydrophilic property inside the micro-reaction chamber and the hydrophobic property outside the micro-reaction chamber can jointly adjust the contact angle of the droplet in the reaction solution , so that the reaction liquid infiltrates from the outside of the micro-reaction chamber to the inside of the micro-reaction chamber, realizing efficient sample injection, the reaction liquid is easier to enter each micro-reaction chamber, the reaction liquid is easy to completely fill the micro-reaction chamber and it is not easy to cross-talk after filling, effectively solving the problem It solves the problems of the existing structure that the reaction liquid is not completely filled during the sample injection and amplification process or crosstalk occurs after filling, and maximizes the self-absorbing liquid sample injection and oil seal performance of the digital microfluidic chip.

In the present disclosure, by increasing the roughness of the surface modification layer, the specific surface area of the film layer is increased, which not only improves the heat dissipation performance of the film layer, but also helps release the stress of the film layer, improves the mass production process quality, and improves the mass production process. Stability, which improves the effectiveness, stability and accuracy of chip packaging, and improves product quality and service life.

Compared with the reaction system prepared by silicon-based processing, the digital polymerase chain reaction microfluidic device of the present disclosure adopts the micro-processing method of glass-based and semiconductor technology, which can realize large-scale batch production, not only can greatly reduce the preparation cost, but also The preparation process is simple, the process equipment of the semiconductor process can be effectively utilized, the process improvement is small, the compatibility is strong, the process is simple to realize, the production efficiency is high, the production cost is low, and the yield rate is high. The digital polymerase chain reaction microfluidic device of the present disclosure can detect nucleic acid molecules extracted from blood, urine and other body fluids more conveniently, more stably, more sensitively and non-invasively, and realize single cell analysis, cancer diagnosis, virus analysis and production. Auxiliary diagnosis and treatment in the fields of pre-diagnosis and other fields.

Fig. 10a is a schematic structural diagram of another dPCR microfluidic device according to an exemplary embodiment of the present disclosure, and Fig. 10b is an enlarged view of a groove in Fig. 10a. In an exemplary embodiment, the main structure of the digital polymerase chain reaction microfluidic device of this exemplary embodiment is basically similar to that of the embodiment shown in FIG. 1 , including a first substrate 100 and a second substrate 200 disposed opposite , the first substrate 100 includes a first base 10 and a heating structure layer, a microcavity defining layer 30 , a first surface modification layer 40 and a second surface modification layer 50 sequentially arranged on the first base 10 , and the second substrate 200 includes a first Two substrates 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 limiting dams 31 between adjacent grooves 60, the first surface modification layer 40 is formed with a first modification structure 41 that improves hydrophilic properties, the second surface modification layer 50 is formed with a second modification structure 51 that improves hydrophobic properties, and the third surface modification layer 80 is formed with a third modification that improves hydrophobic properties. Structure 81. As shown in Fig. 10a and Fig. 10b, different from the previous embodiments, the microcavity defining layer 30 is further provided with a flow guide structure 70, and the flow guide structure 70 is configured to improve the efficiency of the reaction liquid entering the micro reaction chamber.

In an exemplary embodiment, the flow guide structure 70 may be disposed on the surface of the first surface modification layer 40 away from the first substrate, and the flow guide structure 70 and the first modification structure 41 may be formed simultaneously 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 40-1 covering the groove side wall of the groove 60 2, and the crest wall finishing layer 40-3 covering the crest wall defining the dam 31. The first modification structure 41 can be located on the surface of the groove bottom wall modification layer 40-1 away from the first substrate. The first modification structure 41 is configured to increase the hydrophilic property inside the micro-reaction chamber. The first modification structure 41 is the same as the aforementioned Exemplary embodiments are similar. The flow guide structure 70 may be located on the surface of the dam top wall modification layer 40 - 3 away from the first substrate, and the flow guide structure 70 is configured to improve the efficiency of the reaction liquid entering the micro reaction chamber.

In an exemplary embodiment, the flow guide structure 70 can be located on the edge of the top wall modification layer 40-3 near the groove 60, and an annular flow guide structure is formed on the periphery of the micro-reaction chamber, and the annular flow guide structure surrounds the micro-reaction cavity. The reaction chamber improves the efficiency of the reaction liquid entering the micro reaction chamber.

In an exemplary embodiment, in the first surface modification layer 40, the side of the top wall modification layer 40-3 away from the first substrate is provided with a second surface modification layer 50, and the second surface modification layer 50 can be positioned on the top wall modification layer. The layer 40-3 is far away from the region of the groove 60, the second surface modification layer 50 is spaced apart from the diversion structure 70, and the diversion structure 70 is located in the peripheral region of the dam top wall modification layer 40-3 close to the groove 60, the second surface modification The layer 50 is located in the middle region of the dam crest wall modification layer 40-3, and the orthographic projection of the second surface modification layer 50 on the first substrate does not overlap with the orthographic projection of the flow guide structure 70 on the first substrate.

In an exemplary embodiment, a second modification structure 51 is formed on the second surface modification layer 50 , and the second modification structure 51 is similar to the foregoing exemplary embodiments.

In an exemplary embodiment, the diversion structure 70 may include a plurality of diversion pillars formed on the surface of the top wall finishing layer 40 - 3 away from the first base, and the plurality of diversion pillars are formed on the surface of the crest decoration layer 40 - 3 . -3 Micro-nano structures in the shape of cylinders, truncated cones, and prisms are constructed on the surface away from the first base, increasing the roughness of the top wall modification layer 40-3 near the groove 60, thus increasing the surrounding area of the groove 60 hydrophilic properties.

In an exemplary embodiment, in a plane parallel to the first base, the shape of the guide post may include any one or more of the following: triangle, square, rectangle, pentagon, hexagon, polygon, circle and oval.

In an exemplary embodiment, the fourth characteristic length L4 of the guide post 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 shape of the guide post. For example, when the shape of the guide column is a circle, the diameter of the circle may be about 0.5 μm to 2 μm. For another example, when the shape of the deflector column is a rectangle, the long side of the rectangle may be about 0.5 μm to 2 μm. For another example, when the shape of the guide column is ellipse, the major axis of the ellipse may be about 0.5 μm to 2 μm.

In an exemplary embodiment, in a plane perpendicular to the first base, the cross-sectional shape of the guide post may include any one or more of the following: rectangle, trapezoid and polygon, and the side wall of the guide post may be a straight line or a broken line. Or arcs.

In an exemplary embodiment, the fourth height H4 of the guide post 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 guide posts may be about 0.5 times to 2.0 times the fourth characteristic length L4. For example, the shape of the guide posts may be circular, the diameter of the circle may be about 1 μm, and the distance between adjacent guide posts may be about 1 μm.

In an exemplary embodiment, the fourth height H4 of the guide post in the guide structure 70 may be greater than the first height H1 of the first protrusion in the first modification structure 41, and/or, the guide post in the guide structure 70 The fourth spacing M4 of the first modification structure 41 may be greater than the first spacing M1 of the first protrusions in the first modification structure 41, so that the roughness of the area where the flow guide structure 70 is located is greater than the roughness of the area where the first modification structure 41 is located, thereby making the flow guide The hydrophilic property of the area where the structure 70 is located is greater than that of the area where the first modified structure 41 is located, which improves the efficiency of the reaction solution entering the micro-reaction chamber.

The manufacturing process of the dPCR microfluidic device in the exemplary embodiment of the present disclosure is basically similar to the foregoing embodiments, except that in the process of forming the pattern of the first surface modification layer, not only the first modification structure is formed at the bottom of the groove, A flow guiding structure is also formed in a region adjacent to the outside of the groove; in the process of forming the pattern of the second surface modification layer, the second modification structure is only formed in the area outside the flow guiding structure.

The digital polymerase chain reaction microfluidic device in the exemplary embodiment of the present disclosure not only has the technical effects of the foregoing embodiments, but also effectively avoids defects such as incomplete filling of the reaction solution or interference after filling during the process of sample injection and amplification, and improves the Self-absorbing liquid sampling and oil seal performance, and by setting a diversion structure around the micro-reaction chamber, the diversion structure improves the efficiency of the reaction liquid entering the micro-reaction chamber, the reaction liquid is easy to completely fill the micro-reaction chamber and is not prone to cross-talk after filling , to maximize the self-aspiration liquid injection and oil seal performance of the digital microfluidic chip.

Fig. 11a is a schematic structural diagram of another dPCR microfluidic device according to an exemplary embodiment of the present disclosure, and Fig. 11b is an enlarged view of a groove in Fig. 11a. In an exemplary embodiment, the main structure of the digital polymerase chain reaction microfluidic device of this exemplary embodiment is basically similar to that of the embodiment shown in FIG. 1 , including a first substrate 100 and a second substrate 200 disposed opposite , the first substrate 100 includes a first substrate 10 and a heating structure layer, a microcavity defining layer, and a surface modification layer sequentially arranged on the first substrate 10, and the second substrate 200 includes a second substrate 20 and a layer arranged on the second substrate 20. The third surface modification layer 80 of 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 FIG. 11b , different from the previous embodiments, the first surface modification layer 40 is only provided on the area where the groove 60 is located, and the second modification structure is provided 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: a groove bottom wall modification layer 40-1 covering the groove bottom wall of the groove 60 and a groove side wall modification layer 40-1 covering the groove side wall of the groove 60. 2, that is, the top wall of the dam 31 is defined as not having the first surface modification layer 40 . The first modification structure 41 can be located on the surface of the groove bottom wall modification layer 40-1 away from the first substrate. The first modification structure 41 is configured to increase the hydrophilic property inside the micro-reaction chamber. The first modification structure 41 is the same as the aforementioned Exemplary embodiments are similar.

In an exemplary embodiment, the microcavity-defining layer can be made of materials such as photoresist with hydrophobic properties, and a second modification structure 51 is formed on the surface of the dam top wall away from the first substrate in the defined dam 31, and the second modification The structure 51 is configured to increase the hydrophobicity of the outside of the microreaction chamber, and the second modified structure 51 may include a plurality of second protrusions formed on the surface of the dam top wall away from the first base in the defined dam 31, the second protrusions The starting structure is similar to the foregoing exemplary embodiment.

The manufacturing process of the digital polymerase chain reaction microfluidic device in the exemplary embodiment of the present disclosure is basically similar to that of the foregoing embodiments, except that in the process of forming the pattern of the microcavity-defining layer, not only a plurality of grooves are formed, A second modification structure is also formed on the surface defining the side of the dam remote from the first substrate. In the process of forming the pattern of the first surface modification layer, the first surface modification layer and the first modification structure are only formed in the region where the groove is located, and the process of forming the pattern of the second surface modification layer is not required.

In an exemplary embodiment, the process of forming the pattern of the microcavity-defining layer may be patterned using a half-tone mask (HalfTone Mask), a single-slit diffraction mask (Single Slit Mask) or a gray-tone mask (Gray Tone Mask) process, the present disclosure is not limited here.

The digital polymerase chain reaction microfluidic device in the exemplary embodiment of the present disclosure can achieve the technical effects of the foregoing embodiments, effectively avoiding defects such as incomplete filling of the reaction solution or interference after filling during the process of sample introduction and amplification, and improving the Self-priming liquid sampling and oil seal performance.

Fig. 12 is a schematic plan view of a reaction area according to an exemplary embodiment of the present disclosure. In an exemplary embodiment, along the flow direction A of the reaction liquid, the reaction zone may include a liquid inlet zone 110-1, a liquid inlet transition zone 110-2, a reaction zone 110-3, a liquid outlet transition zone 110-4, and a liquid outlet transition zone 110-4. Liquid zone 110-5, the reaction liquid enters the reaction zone from the liquid inlet zone 110-1, enters the reaction zone 110-3 after passing through the liquid inlet transition zone 110-2 and injects it into multiple micro-reaction chambers, and the reaction after the test results are obtained The liquid flows out from the reaction zone 110-3, passes through the liquid outlet transition zone 110-4, and then flows to the liquid outlet zone 110-5 to be discharged.

In an exemplary embodiment, the reaction zone 110-3 may include a plurality of micro-reaction chambers and limiting dams between adjacent micro-reaction chambers, the first modification structure 41 is arranged in the plurality of micro-reaction chambers, and the micro-reaction chambers The outer limiting dam is provided with a second finishing structure 51 . 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 may all include a microcavity defining layer, and the microcavity defining layer is provided with a second modification structure 51 . That is to say, except that the multiple micro-reaction chambers have hydrophilic properties, other reaction regions other than the multiple micro-reaction chambers all have hydrophobic properties.

In an exemplary embodiment, the hydrophobic characteristics of the liquid-entry transition zone 110-2, the regions outside the multiple micro-reaction chambers in the reaction zone 110-3, and the liquid-flow transition zone 110-4 may be the same, that is, the liquid-entry transition zone 110- 2. The second modification structure 51 of the reaction zone 110-3 and the liquid outlet transition zone 110-4 may be the same.

In an exemplary embodiment, the second modified structure 51 of the liquid inlet region 110-1 and/or the liquid outlet region 110-5 may be different from the second modified structure 51 of other regions. Taking the second modification structure 51 of the liquid entry zone 110-1 and the liquid entry transition zone 110-2 both including a plurality of second protrusions as an example, the second height of the second protrusions in the liquid entry zone 110-1 may be smaller than that of the entrance The second height of the second protrusions in the liquid transition area 110-2, and/or, the second spacing of the second protrusions in the liquid entry area 110-1 may be smaller than that of the second protrusions in the liquid entry transition area 110-2. The second distance is such that the roughness of the liquid-entry transition zone 110-2 is greater than that of the liquid-entrance zone 110-1, so that the hydrophobicity of the liquid-entry transition zone 110-2 is greater than that of the liquid-entry zone 110-1. The present disclosure further improves the sampling efficiency by increasing the hydrophobicity around the reaction zone.

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

It should be noted that the structures shown in the embodiments of the present disclosure and their preparation processes are merely illustrative. In exemplary embodiments, the corresponding structure may be changed and the patterning process may be increased or decreased according to actual needs. For example, the structure of the dPCR microfluidic device may be that the microcavity defining layer is provided with a flow guiding structure, but the second modifying structure is provided on the side of the defining dam away from the first substrate. For another example, the structure of the dPCR microfluidic device can be that the first surface modification layer is arranged in the area where the groove is located, the second surface modification layer is arranged in the area outside the groove, and the first surface modification layer is provided with the first modification structure, The second surface modification layer is provided with a second modification structure. For another example, the dPCR microfluidic device can also be provided with other electrodes, leads and structural membrane layers, which are not limited in this disclosure.

Exemplary embodiments of the present disclosure also provide a method for preparing a digital polymerase chain reaction microfluidic device. In an exemplary embodiment, a method for preparing a digital polymerase chain reaction microfluidic device may include:

Prepare a first substrate and a second substrate respectively; the first substrate includes a first substrate, a microcavity defining layer disposed on the first substrate facing the second substrate, and a microcavity defining layer disposed on a side away from the microcavity defining layer The first surface modification layer on one side of the first substrate; the microcavity-defining layer includes a plurality of grooves as micro-reaction chambers and a limit dam between adjacent grooves, the at least one groove in the groove The first surface modification layer is provided with a first modification structure, and the first modification structure is configured to increase the hydrophilic property inside the micro-reaction chamber;

The first substrate and the second substrate are packaged into a box through a packaging process.

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

Forming a microcavity-defining layer on the first substrate, the microcavity-defining layer comprising a plurality of grooves serving as microreaction chambers and limiting dams between adjacent grooves;

Forming a first surface modification layer, the first surface modification layer includes 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 groove side wall modification layer covering the groove The dam top wall modification layer on the side away from the first base is defined, and at least one groove bottom wall modification layer has a first modification structure formed on the surface of the side away from the first base, and the first modification structure includes at least a first bump;

forming a second surface modification layer, the second surface modification layer is formed on the side of at least one dam top wall modification layer away from the first base, and the second surface modification layer is on the surface of the side away from the first base A second modification structure is formed on the top, and the second modification structure includes at least one second protrusion.

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

A third surface modification layer is formed on the side of the second substrate facing the first substrate, a third modification structure is formed on the surface of the third surface modification layer facing the first substrate, and the third modification The structure includes at least one third protrusion.

Through the preparation process of the digital polymerase chain reaction microfluidic device of the present disclosure, it can be seen that the present disclosure effectively avoids the process of sample injection and amplification by designing the hydrophilic structure and the hydrophobic structure inside and outside the micro-reaction chamber respectively. The reaction solution in the middle is not completely filled or cross-talk occurs after filling, which improves the performance of self-absorbing liquid sampling and oil sealing. In the present disclosure, the first surface modification layer is arranged in the groove formed by the microcavity defining layer, and the first modification structure is formed on the surface of the first surface modification layer, thereby increasing the roughness of the inner surface of the microreaction chamber, thereby increasing the The hydrophilic property inside the micro-reaction chamber makes it easier for the reaction liquid to enter the micro-reaction chamber, and the reaction liquid is easy to completely fill the micro-reaction chamber, which improves the self-absorbing liquid sampling performance. In the present disclosure, a second surface modification layer is arranged on the limiting dam formed by the microcavity limiting layer, and a second modification structure is formed on the surface of the second surface modification layer, which increases the hydrophobicity of the external surface of the micro-reaction chamber, not only makes the reaction It is easier for the liquid to enter and completely fill the micro-reaction chamber, and based on the hydrophilic and hydrophobic characteristics inside and outside the micro-reaction chamber, there will be no disturbance after the reaction liquid enters the micro-reaction chamber, which improves the stability and effectiveness of the oil seal and improves the self-priming Liquid sampling and oil seal performance. In the present disclosure, the third surface modification layer is formed on the second substrate, and the third modification structure is formed on the surface of the third surface modification layer, which increases the hydrophobicity of the micro-reaction chamber, and further improves the self-absorption liquid injection and oil seal. performance. The present disclosure simultaneously increases the hydrophilic property inside the micro-reaction chamber and increases the hydrophobic property outside the micro-reaction chamber, the hydrophilic property inside the micro-reaction chamber and the hydrophobic property outside the micro-reaction chamber can jointly adjust the contact angle of the droplet in the reaction solution , so that the reaction liquid infiltrates from the outside of the micro-reaction chamber to the inside of the micro-reaction chamber, realizing efficient sample injection, the reaction liquid is easier to enter each micro-reaction chamber, the reaction liquid is easy to completely fill the micro-reaction chamber and it is not easy to cross-talk after filling, effectively solving the problem It solves the problems of the existing structure that the reaction liquid is not completely filled during the sample injection and amplification process or crosstalk occurs after filling, and maximizes the self-absorbing liquid sample injection and oil seal performance of the digital microfluidic chip. In the present disclosure, by increasing the roughness of the surface modification layer, the specific surface area of the film layer is increased, which not only improves the heat dissipation performance of the film layer, but also helps release the stress of the film layer, improves the mass production process quality, and improves product quality and service life. Compared with the reaction system prepared by silicon-based processing, the digital polymerase chain reaction microfluidic device of the present disclosure adopts the micro-processing method of glass-based and semiconductor technology, which can realize large-scale batch production, not only can greatly reduce the preparation cost, but also The preparation process is simple, the process equipment of the semiconductor process can be effectively utilized, the process improvement is small, the compatibility is strong, the process is simple to realize, the production efficiency is high, the production cost is low, and the yield rate is high. The digital polymerase chain reaction microfluidic device of the present disclosure can detect nucleic acid molecules extracted from blood, urine and other body fluids more conveniently, more stably, more sensitively and non-invasively, and realize single cell analysis, cancer diagnosis, virus analysis and production. Auxiliary diagnosis and treatment in the fields of pre-diagnosis and other fields.

Although the embodiments disclosed in the present disclosure are as above, the content described is only the embodiments adopted to facilitate understanding of the present disclosure, and is not intended to limit the present disclosure. Anyone skilled in the field of this disclosure can make any modifications and changes in the form and details of implementation without departing from the spirit and scope disclosed in this disclosure, but the patent protection scope of this application is still subject to The scope defined by the appended claims shall prevail.

Claims (19)

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 comprises a plurality of grooves serving as micro-reaction chambers and defining 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 chambers;

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 limiting 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.

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 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.

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 flow-directing structure comprises at least one flow-directing post disposed on a surface of the top dam wall finish distal from the first substrate.

11. The apparatus of claim 10, 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.

12. The apparatus of claim 10, 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.

13. 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.

14. The apparatus of claim 13, 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.

15. The device of claim 14, 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.

16. The apparatus of claim 15, wherein a third height of the third protrusions in the third trim structure is greater than a second height of the second protrusions in the second trim structure, and/or wherein a third pitch of the third protrusions in the third trim structure is greater than a second pitch of the second protrusions in the second trim structure.

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

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; 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 limiting dam; 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;

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

18. The method of claim 17, wherein preparing the first substrate comprises:

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.

19. The method of claim 17, wherein preparing the second substrate comprises:

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.

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