Microfluidic substrate, microfluidic chip, method for manufacturing a chip, and method for using a chip

Abstract

A microfluidic substrate, a microfluidic chip, a preparation method and a use method of the microfluidic chip. The microfluidic substrate (100) comprises a substrate (10), the substrate (10) comprises a plurality of microcavity regions (R) arranged in an array, each of the plurality of microcavity regions (R) comprises a first part (R1) and a second part (R2) stacked with each other, the depth of the first part (R1) is x, and the first part (R1) comprises a top opening (1012) which is circular in shape and has a diameter D, the diameter D of the top opening (1012) and the depth x are approximately in the relationship D=2x+y, x ranges from 20 microns to 400 microns, and y ranges from 5 microns to 30 microns.

Description

[0001] Related applications

[0002] This application claims priority to PCT application No. PCT / CN2021 / 127002, filed on October 28, 2021, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biomedical detection, and in particular to a microfluidic substrate, a microfluidic chip including the microfluidic substrate, a method for preparing the microfluidic chip, and a method for using the microfluidic chip. Background Technology

[0004] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, replicating minute amounts of deoxyribonucleic acid (DNA) to a large quantity, significantly increasing its number. Digital polymerase chain reaction (dPCR) is a quantitative analysis technique developed from PCR, providing digital DNA quantitative information. Its combination with microfluidic technology greatly improves sensitivity and accuracy. In dPCR, the nucleic acid sample is sufficiently diluted so that the number of target molecules (i.e., DNA templates) in each reaction unit is less than or equal to one. PCR amplification of the target molecule is performed separately in each reaction unit. After amplification, the fluorescence signals of each reaction unit are statistically analyzed, thereby achieving absolute quantitative detection of single DNA molecules. Due to its advantages of high sensitivity, high specificity, high throughput, and accurate quantification, dPCR is widely used in clinical diagnostics, gene instability analysis, single-cell gene expression, environmental microbial detection, and prenatal diagnosis. Summary of the Invention

[0005] According to one aspect of this disclosure, a microfluidic substrate is provided, comprising a substrate including a plurality of microcavity regions arranged in an array. Each of the plurality of microcavity regions includes a first portion and a second portion stacked on top of each other, the first portion having a depth of x, and the first portion including a top opening of circular shape with a diameter D, the diameter D of the top opening being approximately related to the depth x by D = 2x + y, where x ranges from 20 micrometers to 400 micrometers and y ranges from 5 micrometers to 30 micrometers.

[0006] In some embodiments, the first portion is a blind via and the first portion and the second portion do not penetrate each other, the first portion forming a microcavity of the microfluidic substrate.

[0007] In some embodiments, the substrate further includes an unetched third portion located between any two adjacent microcavity regions among the plurality of microcavity regions. Within each microcavity region, the etched portion of the substrate constitutes the first portion, and the unetched portion of the substrate constitutes the second portion. The orthographic projection of the first portion onto the microfluidic substrate overlaps with the orthographic projection of the second portion onto the microfluidic substrate. The second portion and the third portion are integrally formed.

[0008] In some embodiments, x ranges from 20 micrometers to 100 micrometers.

[0009] In some embodiments, the first portion is a curved body, the first portion includes the top opening, the bottom, and a sidewall connecting the top opening and the bottom, the bottom of the first portion being circular with a diameter of approximately x micrometers.

[0010] In some embodiments, the tangent plane at at least some points on the sidewall is at a non-perpendicular angle to the reference plane on which the microfluidic substrate is located.

[0011] In some embodiments, the second portion includes a bottom opening on a side away from the first portion, the first portion and the second portion penetrate each other to form a through hole, and the through hole forms a microcavity of the microfluidic substrate.

[0012] In some embodiments, the depth of the second portion is x and the shape of the bottom opening of the second portion is circular, and the relationship between the diameter D of the bottom opening and the depth x is approximately D = 2x + y.

[0013] In some embodiments, the first portion and the second portion have the same shape and are axially symmetrical about an axis of symmetry, which is parallel to the reference plane in which the microfluidic substrate is located.

[0014] In some embodiments, y equals 10 micrometers.

[0015] In some embodiments, the substrate includes a first substrate, the first substrate including the plurality of first portions and the plurality of second portions.

[0016] In some embodiments, the substrate includes a first substrate and a defining layer located on the first substrate, the defining layer including the plurality of first portions and the plurality of second portions.

[0017] In some embodiments, the microfluidic substrate further includes a shielding layer. The shielding layer includes a plurality of first openings, each corresponding to one of the plurality of microcavity regions, and the orthographic projection of each of the plurality of microcavity regions onto the microfluidic substrate at least partially overlaps with the orthographic projection of a corresponding first opening onto the microfluidic substrate. The orthographic projection of the shielding layer onto the microfluidic substrate at least partially overlaps with the orthographic projection of the defining layer onto the microfluidic substrate.

[0018] In some embodiments, the microfluidic substrate further includes a spacer region located between any two adjacent microcavity regions among the plurality of microcavity regions and a hydrophobic layer disposed within the spacer region. The hydrophobic layer includes a plurality of second openings, each of the plurality of microcavity regions corresponding to one of the plurality of second openings, and the orthographic projection of each microcavity region on the microfluidic substrate falls within the orthographic projection of a corresponding second opening on the microfluidic substrate.

[0019] In some embodiments, the second opening is circular in shape, and the diameter of the second opening is 5 to 20 micrometers larger than the diameter of the top opening.

[0020] In some embodiments, the microfluidic substrate further includes a hydrophilic layer. The hydrophilic layer is located at least within the plurality of microcavity regions, and the orthographic projection of a portion of the hydrophilic layer within each microcavity region onto the microfluidic substrate falls within the orthographic projection of a second opening corresponding to that microcavity region onto the microfluidic substrate.

[0021] According to another aspect of this disclosure, a microfluidic chip is provided, comprising: a first substrate; a second substrate opposite to the first substrate; a microfluidic substrate according to any of the preceding embodiments, located between the first substrate and the second substrate; and a sealing frame located between the first substrate and the second substrate, wherein the orthographic projection of the microfluidic substrate on the first substrate falls within the orthographic projection of the sealing frame on the first substrate.

[0022] In some embodiments, the sealing frame includes a first side and a second side arranged along a first direction and opposite to each other, and a third side and a fourth side arranged along a second direction different from the first direction and opposite to each other, wherein the first side and the second side are arc-shaped.

[0023] In some embodiments, the microfluidic substrate includes a first edge and a second edge arranged along the second direction and opposite to each other, the distance between the orthographic projection of the third side of the sealing frame on the first substrate and the orthographic projection of the first edge of the microfluidic substrate on the first substrate is 2 mm to 6 mm, and the distance between the orthographic projection of the fourth side of the sealing frame on the first substrate and the orthographic projection of the second edge of the microfluidic substrate on the first substrate is 2 mm to 6 mm.

[0024] In some embodiments, the distance between the microfluidic substrate and the second substrate is 0.1 mm to 0.3 mm.

[0025] In some embodiments, the second substrate includes an inlet port and an outlet port, and the orthographic projections of the inlet port and the outlet port on the first substrate fall within the orthographic projection of the sealing frame on the first substrate.

[0026] In some embodiments, the first substrate includes a second substrate.

[0027] In some embodiments, the first substrate includes: a second substrate; and a heating electrode located between the second substrate and the microfluidic substrate. The orthographic projection of a plurality of microcavity regions of the microfluidic substrate onto the second substrate falls within the orthographic projection of the heating electrode onto the second substrate.

[0028] In some embodiments, the orthographic projection of the sealing frame onto the second substrate falls within the orthographic projection of the heating electrode onto the second substrate.

[0029] In some embodiments, the first substrate further includes: a first dielectric layer located between the second substrate and the heating electrode; and a second dielectric layer located between the heating electrode and the microfluidic substrate.

[0030] In some embodiments, the first substrate further includes a conductive layer located between the second substrate and the first dielectric layer, the conductive layer being electrically connected to the heating electrode via a via in the first dielectric layer.

[0031] According to another aspect of this disclosure, a method for fabricating a microfluidic chip is provided, comprising: providing a first substrate; fabricating a microfluidic substrate as described in any of the preceding embodiments; fixing a sealing frame and the microfluidic substrate onto the first substrate such that the orthographic projection of the microfluidic substrate onto the first substrate falls within the orthographic projection of the sealing frame onto the first substrate; placing a second substrate on the side of the sealing frame and the microfluidic substrate away from the first substrate; and performing an encapsulation process.

[0032] In some embodiments, the step of preparing the microfluidic substrate described in any of the preceding embodiments includes: providing a first substrate and patterning the first substrate to form the plurality of microcavity regions.

[0033] In some embodiments, the step of fabricating the microfluidic substrate described in any of the preceding embodiments includes: providing a first substrate having a thickness of H; forming a hydrophobic layer on the first substrate; forming a mask pattern including a plurality of exposure holes on the side of the hydrophobic layer away from the first substrate, each of the plurality of exposure holes being circular in shape and having a diameter of y, where y ranges from 5 micrometers to 30 micrometers; etching the portion of the first substrate exposed by the plurality of exposure holes to a depth x to form the plurality of microcavity regions, the etched portion in each microcavity region of the first substrate constituting the first portion, and the unetched portion in each microcavity region of the first substrate constituting the second portion, where x ranges from 20 micrometers to 100 micrometers and x is less than H; and removing the mask pattern.

[0034] In some embodiments, the step of preparing the microfluidic substrate described in any of the preceding embodiments includes: providing a first substrate; applying a defining film on the first substrate and patterning the defining film to form the plurality of microcavity regions.

[0035] According to another aspect of this disclosure, a method of using a microfluidic chip is provided, comprising: adding a sample solution into a plurality of microcavities of the microfluidic chip described in any of the preceding embodiments; heating the microfluidic chip to cause the sample solution in the plurality of microcavities to react; and detecting optical signals emitted by the reacted sample solution in the plurality of microcavities using an optical device.

[0036] In some embodiments, the first substrate includes a second substrate, and the step of heating the microfluidic chip includes placing the microfluidic chip in a flatbed thermal cycler.

[0037] In some embodiments, the first substrate includes a second substrate and a heating electrode located between the second substrate and the microfluidic substrate, wherein the orthographic projection of a plurality of microcavities of the microfluidic substrate onto the second substrate falls within the orthographic projection of the heating electrode onto the second substrate. The step of heating the microfluidic chip includes: applying an electrical signal to the microfluidic chip to drive the heating electrode to heat the plurality of microcavities, and using a temperature sensor to detect the temperature of the plurality of microcavity regions to adjust the current flowing through the heating electrode in real time. Attached Figure Description

[0038] To more clearly describe the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1A A plan view of a microfluidic substrate according to an embodiment of the present disclosure is shown schematically;

[0040] Figure 1B Schematic illustration along Figure 1A A cross-sectional view taken from line AA';

[0041] Figure 2A The shape of a microcavity is schematically shown;

[0042] Figure 2B The relationship between the opening diameter and depth of the microcavity is schematically shown;

[0043] Figure 3A This schematically illustrates another shape of the microcavity;

[0044] Figure 3B This schematically illustrates another shape of the microcavity;

[0045] Figure 3C This schematically illustrates another shape of the microcavity;

[0046] Figure 4A A partial cross-sectional view of a microfluidic substrate according to an embodiment of the present disclosure is schematically shown;

[0047] Figure 4B A partial cross-sectional view of a microfluidic substrate according to an embodiment of the present disclosure is schematically shown;

[0048] Figure 5 A schematic plan view of a microfluidic chip according to an embodiment of the present disclosure is shown;

[0049] Figure 6 schematically shown Figure 5 A plan view of the sealing frame of the microfluidic chip in the image;

[0050] Figure 7 schematically shown Figure 5 A plan view of the second substrate of the microfluidic chip in the image;

[0051] Figure 8A schematically shown Figure 5 A partial cross-sectional view of the first substrate of the microfluidic chip in the image;

[0052] Figure 8Bschematically shown Figure 5 A partial cross-sectional view of the first substrate of the microfluidic chip in the image;

[0053] Figure 9A A partial cross-sectional view of a microfluidic chip according to an embodiment of the present disclosure is schematically shown;

[0054] Figure 9B A partial cross-sectional view of another microfluidic chip according to an embodiment of the present disclosure is schematically shown;

[0055] Figure 9C A partial cross-sectional view of another microfluidic chip according to an embodiment of the present disclosure is schematically shown;

[0056] Figure 9D A partial cross-sectional view of another microfluidic chip according to an embodiment of the present disclosure is schematically shown;

[0057] Figure 9E A partial cross-sectional view of another microfluidic chip according to an embodiment of the present disclosure is schematically shown;

[0058] Figure 9F A partial cross-sectional view of another microfluidic chip according to an embodiment of the present disclosure is schematically shown;

[0059] Figure 10 A flowchart illustrating a method for fabricating a microfluidic chip according to an embodiment of the present disclosure is shown schematically.

[0060] Figure 11 The mask pattern used in the fabrication of microfluidic chips is schematically shown.

[0061] Figure 12 A flowchart illustrating a method of using a microfluidic chip according to an embodiment of the present disclosure is shown schematically; and

[0062] Figure 13 A fluorescence image of a microfluidic chip according to an embodiment of the present disclosure after being illuminated by a light source is shown. Detailed Implementation

[0063] The technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0064] Digital polymerase chain reaction (dPCR) is a quantitative analysis method that provides digital DNA information and has demonstrated significant advantages in multiple fields since its inception. With the emergence and rapid development of microfluidic technology in recent years, combining microfluidics with dPCR has significantly improved the sensitivity and accuracy of detection. Digital microfluidic chips based on dPCR technology, due to their numerous advantages such as high sensitivity, high integration, high automation, and high-throughput detection, have shown tremendous technological advantages and commercial potential in research areas such as single-cell analysis, early cancer diagnosis, and prenatal diagnosis.

[0065] However, the inventors of this application have discovered that there are still some problems with current digital microfluidic chips based on microcavity structures, such as how to ensure that the sample solution can be distributed to each microcavity of the microfluidic chip as much as possible, how to reduce the bubbles generated during the sample injection process, how to improve the stability of the microfluidic chip during the heating process, and how to achieve precise control of the fluid.

[0066] In view of this, the embodiments of this disclosure provide a microfluidic substrate, a microfluidic chip including the microfluidic substrate, a method for fabricating the microfluidic chip, and a method for using the microfluidic chip, in order to overcome the many problems mentioned above.

[0067] Figure 1A A plan view of a microfluidic substrate 100 according to an embodiment of the present disclosure is shown. Figure 1B It shows along Figure 1A A partial cross-sectional view taken by line AA' in the image. (See image for details.) Figure 1A and Figure 1B As shown, the microfluidic substrate 100 includes a substrate 10, and the substrate 10 includes a plurality of microcavity regions R arranged in an array. Figure 1B The example only shows three microcavity regions R), each of which includes a first part R1 and a second part R2 stacked on top of each other. The first part R1 has a depth of x and includes a top opening 1012 that is circular in shape (centered at O1) and has a diameter of D. The diameter D of the top opening 1012 is approximately related to the depth x by D = 2x + y, where x ranges from 20 micrometers to 400 micrometers, for example, 20 micrometers, 50 micrometers, 100 micrometers, 150 micrometers, 210 micrometers, 250 micrometers, 300 micrometers, 350 micrometers, 400 micrometers, etc.; and y ranges from 5 micrometers to 30 micrometers, for example, 5 micrometers, 10 micrometers, 15 micrometers, 20 micrometers, 25 micrometers, 30 micrometers, etc.

[0068] The first part R1 and the second part R2 determine the structure and shape of the microcavities in each microcavity region R of the microfluidic substrate 100. The microcavities are reaction chambers of the microfluidic substrate 100, used to contain sample solutions and provide space for the reaction of the sample solutions (e.g., PCR reaction). The top opening 1012 of the first part R1 refers to the top opening of the microcavity, through which the sample solution enters the microcavity.

[0069] In the formula D=2x+y, x is the depth of the first part R1, and y is the diameter of the exposed aperture of the mask pattern used in the fabrication of the microfluidic substrate 100. The smaller the value of y, the higher the precision required for the manufacturing process, and therefore the greater the difficulty. The exposed aperture of the mask pattern is circular with a diameter of y micrometers. Microcavities are typically formed by etching a substrate (e.g., a substrate or a defining layer on top of the substrate). Due to the isotropic nature of the etching process, when etching to form a microcavity, the diameter D of the top opening 1012 of the microcavity is approximately 2x+y micrometers when the etching depth is x micrometers. This formula roughly expresses the influence of the mask pattern's exposed aperture size design on the diameter of the top opening 1012 of the microcavity in isotropic wet etching. In the formula D=2x+y, the coefficient "2" indicates that the lateral etching rate of the substrate is twice the longitudinal etching rate during the etching process. This coefficient is related to factors such as the substrate material and the choice of etching solution. Therefore, this coefficient will change accordingly when the substrate material and / or the composition of the etching solution are changed. For example, when the substrate material and / or the composition of the etching solution are changed, the formula D=2x+y can also become D=3x+y, D=4x+y, D=5x+y, D=6x+y, etc. The mask pattern will be described in more detail later, and will not be elaborated upon here.

[0070] As previously described, the first portion R1 and the second portion R2 determine the structure and shape of the microcavities within each microcavity region R of the microfluidic substrate 100. When the first portion R1 is a blind via and the first portion R1 and the second portion R2 do not penetrate each other, the first portion R1 constitutes a microcavity of the microfluidic substrate 100; in this case, the microcavity is a blind via. That is, during the etching process of the substrate to form the microcavity, the portion etched away within the microcavity region R is the first portion R1 of the microcavity region R, which constitutes a blind via microcavity of the microfluidic substrate 100, while the portion not etched away within the microcavity region R is the second portion R2 of the microcavity region R. When the second portion R2 includes a bottom opening away from the first portion R1 and the first portion R1 and the second portion R2 penetrate each other, the first portion R1 and the second portion R2 are formed as through-holes, which constitute a microcavity of the microfluidic substrate 100; in this case, the microcavity is a through-hole. In other words, during the process of etching the substrate to form a microcavity, the entire thickness of the substrate is etched away within the microcavity region R to form a via. The first part R1 within the microcavity region R constitutes the upper half of the via, and the second part R2 within the microcavity region R constitutes the lower half of the via.

[0071] Figure 2A As an example, a microcavity 101 of a certain shape is shown. This microcavity 101 is a blind via, in which case the first portion R1 is the microcavity 101. The substrate 10 also includes an unetched third portion R3 located between any two adjacent microcavity regions R among a plurality of microcavity regions R. Within each microcavity region R, the etched portion of the substrate 10 constitutes the blind via-type first portion R1, and the unetched portion of the substrate 10 constitutes the second portion R2. The orthographic projection of the first portion R1 onto the microfluidic substrate 100 overlaps with the orthographic projection of the second portion R2 onto the microfluidic substrate 100, and the second portion R2 and the third portion R3 are an integral structure. In other words, in the fabrication Figure 2AIn the illustrated microcavity, within each microcavity region R, a portion of the substrate 10 is etched away to form a first portion R1, which constitutes a blind-hole microcavity of the microfluidic substrate 100. The second portion R2, located directly below the first portion R1, and the third portion R3, located between adjacent microcavities, are not etched, thus making the second portion R2 and the third portion R3 a single structure made of the same material. When the microcavity 101 is a blind hole, it includes a top opening 1012, a bottom 1013, and sidewalls 1011, which connect the top opening 1012 and the bottom 1013. The sidewalls 1011, together with the top opening 1012 and the bottom 1013, constitute the reaction chamber of the microcavity 101 to contain the sample solution. It should be noted that, in this application, the term "sidewall of a microcavity" refers to all the walls surrounding the interior of the microcavity. Any point on the sidewall 1011 of the microcavity 101 forms an angle α with the reference plane containing the microfluidic substrate 100, where α is not equal to 90 degrees. As shown in the figure, the microcavity 101 includes a sidewall 1011, a top opening 1012, and a bottom 1013, with the sidewall 1011 connecting the top opening 1012 and the bottom 1013. The blind-via microcavity 101 can have various suitable shapes, including but not limited to curved surfaces, regular truncated pyramids, etc. For example, in one example, the blind-via microcavity 101 can be approximately "bowl-shaped".

[0072] By designing the microcavity 101 as a blind hole, the sample solution can be stably kept inside the cavity during detection and is not easily carried out of the cavity. In addition, if bubbles are generated during the process of the sample solution entering the microcavity 101, the microcavity 101 can adsorb these bubbles on the sidewall 1011 to prevent the bubbles from being mixed in with the sample solution inside the cavity, thereby avoiding affecting the subsequent fluorescence detection of the sample solution.

[0073] Figure 2BA bowl-shaped microcavity 101 is shown. As shown, the microcavity 101 includes sidewalls 1011, a top opening 1012, and a bottom 1013. The sidewalls 1011 connect the top opening 1012 and the bottom 1013. The top opening 1012 is circular with a center at O1 and a diameter of D. The depth of the microcavity 101 is x. The relationship between the diameter D of the top opening 1012 and the depth x of the microcavity 101 can be roughly considered as D = 2x + y, where y ranges from 5 micrometers to 30 micrometers, for example, 5 micrometers, 10 micrometers, 12 micrometers, 15 micrometers, 20 micrometers, 25 micrometers, 30 micrometers, etc. y is the diameter of the exposed aperture of the mask pattern used in the manufacturing process. The smaller the value of y, the higher the precision required for the manufacturing process, and therefore the greater the difficulty. The exposed apertures of the mask pattern are circular with a diameter of y micrometers. Due to the isotropic nature of the etching process, when etching the substrate to form a blind-hole microcavity, the diameter D of the top opening 1012 of the microcavity 101 is approximately 2x + y micrometers when the etching depth is x micrometers. This formula roughly expresses the influence of the mask pattern's exposed aperture size design on the diameter of the top opening 1012 of the microcavity 101 during isotropic wet etching. As mentioned earlier, in the formula D = 2x + y, the coefficient "2" indicates that the lateral etching rate of the substrate is twice the longitudinal etching rate during the etching process. This coefficient is related to factors such as the substrate material and the choice of etching solution. Therefore, when the substrate material and / or the composition of the etching solution are changed, this coefficient will also change accordingly. For example, when the substrate material and / or the composition of the etching solution are changed, the formula D = 2x + y can also become D = 3x + y, D = 4x + y, D = 5x + y, D = 6x + y, etc. The mask pattern will be described in more detail later, so it will not be elaborated on here. It should be noted that the phrase "the relationship between the diameter D of the top opening 1012 of the microcavity 101 and the depth x of the microcavity 101 can be roughly considered as D = 2x + y" should be understood as meaning that the value of D is basically equal to 2x + y, but a certain numerical deviation due to manufacturing process errors should be allowed. In one example, the diameter D of the top opening 1012 of the microcavity 101 is equal to 2x + y, and y is equal to 10 micrometers.

[0074] In some embodiments, x ranges from 20 micrometers to 100 micrometers, such as 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, etc. In some embodiments, such as... Figure 3B As shown, the bottom 1013 of the microcavity 101 is circular in shape, with its center at O2, and the diameter of the bottom 1013 is approximately x micrometers. In some embodiments, the diameter of the top opening 1012 of the microcavity 101 is 50-200 micrometers, such as 50 micrometers, 80 micrometers, 110 micrometers, 125 micrometers, 150 micrometers, 180 micrometers, 200 micrometers, etc.

[0075] like Figure 2B As shown, the sidewalls 1011 of the microcavity 101 have a certain curvature and are relatively smooth, while the bottom 1013 of the microcavity 101 is relatively flat. With this design, the sample solution can more easily enter the interior of the microcavity 101 along the smooth and curved sidewalls 1011, and air bubbles are less likely to remain at the bottom 1013.

[0076] Microcavities 101 can be blind vias or through-holes, and can have various suitable shapes. The tangent planes at at least some points on the sidewalls 1011 of each microcavity 101 form non-perpendicular angles with the reference plane containing the microfluidic substrate 100. In mathematical textbooks, a "tangent plane" is defined as follows: Under certain conditions, there are infinitely many curves passing through a point M on a surface, and each curve has a tangent line at point M. Under certain conditions, these tangent lines lie in the same plane, which is called the tangent plane of the surface at point M, and point M is called the tangent point. Therefore, the phrase "the tangent plane at at least some points on the sidewall 1011 of each microcavity 101 forms a non-perpendicular angle with the reference plane on which the microfluidic substrate 100 is located" means that at least a portion of the sidewall 1011 of each microcavity 101 is not perpendicular to the reference plane (e.g., a horizontal plane) on which the microfluidic substrate 100 is located. For example, it could mean that all portions of the sidewall 1011 of the microcavity 101 are not perpendicular to the reference plane, or it could mean that one or more portions of the sidewall 1011 of the microcavity 101 are not perpendicular to the reference plane. In other words, at least a portion of the sidewall 1011 of each microcavity 101 has a certain inclination relative to the reference plane, and this inclination angle can be, for example, an acute angle or an obtuse angle. In related technologies, the sidewalls of microcavities are usually perpendicular to the reference plane on which the microfluidic substrate is located. Such steep sidewalls are very unfavorable for sample solution to enter the microcavity, causing the sample solution to enter the microcavity very slowly or even stagnate on the surface of the microfluidic substrate, thereby reducing the sample introduction efficiency and even wasting the already small amount of sample solution. In the embodiments of this disclosure, by making at least a portion of the sidewall 1011 of the microcavity 101 not perpendicular to the reference plane where the microfluidic substrate 100 is located, the slope of the sidewall 1011 of the microcavity 101 relative to the reference plane can be reduced. This facilitates the rapid entry of the sample solution into the interior of each microcavity 101 along the sidewall 1011 without stagnating on the surface of the microfluidic substrate 100, thereby further promoting the distribution of the sample solution into each microcavity 101, improving the injection efficiency, and increasing the utilization rate of the sample solution.

[0077] Figure 3AAs an example, a microcavity 101 of a certain shape is shown, which is a through-hole. When the microcavity 101 is a through-hole, a first portion R1 constitutes the upper half of the through-hole, and a second portion R2 constitutes the lower half of the microcavity. The first portion R1 and the second portion R2 penetrate each other to form a through-hole microcavity. When the microcavity 101 is a through-hole, it includes a top opening 1012, a bottom opening 1014, and a sidewall 1011, which connects the top opening 1012 and the bottom opening 1014. In this application, the term "top opening of the microcavity" refers to the opening through which the sample solution enters the microcavity. The term "bottom opening of the microcavity" refers to the opening opposite the top opening of the microcavity, and is included only when the microcavity is a through-hole. The sidewall 1011 of the microcavity 101 forms an angle α with the reference plane where the microfluidic substrate is located, where α is not equal to 90 degrees.

[0078] Figure 3A The microcavity 101 shown can be considered as consisting of two Figure 2B The illustrated "bowl-shaped" microcavities are combined. The first part R1 is bowl-shaped, and the second part R2 is an inverted bowl-shaped structure. The shapes of the first part R1 and the second part R2 are axially symmetrical about an axis of symmetry parallel to the reference plane on which the microfluidic substrate 100 is located. As shown in the figure, the top opening 1012 of the first part R1 of the microcavity 101 is circular (center O1). The diameter of the top opening 1012 is D, and the depth of the first part R1 is x. The relationship between the diameter D and the depth x of the top opening 1012 is approximately D = 2x + y, where x ranges from 20 micrometers to 400 micrometers, and y ranges from 5 micrometers to 30 micrometers. The bottom opening 1014 of the second part R2 of the microcavity 101 is circular (centered at O1). The diameter of the bottom opening 1014 is D, and the depth of the second part R2 is x. The relationship between the diameter D and the depth x of the bottom opening 1014 is approximately D = 2x + y, where x ranges from 20 micrometers to 400 micrometers, and y ranges from 5 micrometers to 30 micrometers. As mentioned earlier, in the formula D = 2x + y, the coefficient "2" indicates that the lateral etching rate of the substrate is twice the longitudinal etching rate during the etching process. This coefficient is related to factors such as the substrate material and the choice of etching solution. Therefore, when the substrate material and / or the composition of the etching solution are changed, this coefficient will also change accordingly. For example, when the substrate material and / or the composition of the etching solution are changed, the formula D = 2x + y can also become D = 3x + y, D = 4x + y, D = 5x + y, D = 6x + y, etc.

[0079] like Figure 3AAs shown, the first portion R1 and the second portion R2 of the microcavity 101 penetrate each other via a third opening 1016. In some embodiments, the third opening 1016 is circular in shape and has a diameter of approximately x micrometers. The sidewalls 1011 of the microcavity 101 have a certain curvature and are relatively smooth. With this design, the sample solution can more easily enter the interior of the microcavity 101 along the smooth and curved sidewalls 1011.

[0080] Figure 3B As an example, another shape of microcavity 101 is shown, which is a through hole. The microcavity 101 shown can be frustum-shaped or regular frustum-shaped, and the area of ​​the top opening of the microcavity 101 is larger than the area of ​​the bottom opening.

[0081] Figure 3C As an example, another shape of microcavity 101 is shown, which is a through-hole. Microcavity 101 consists of a top first part, a middle second part, and a bottom third part, and is axially symmetric about an axis of symmetry. In one example, the top first part and the bottom third part of microcavity 101 are shaped like a frustum or a regular truncated cone, while the middle second part is shaped like a curved surface. In mathematics textbooks, a "curved surface" is defined as any geometric solid with curved surfaces involved, also called a curved solid. The surface of a curved surface can be entirely composed of curved surfaces, such as a cylinder or a sphere. The surface of a curved surface can also be a combination of curved and flat surfaces.

[0082] Figures 3A to 3C The following examples illustrate several different shapes of the through-hole microcavity 101, but do not exhaust all possible shapes of the through-hole microcavity 101. For example, the shape of the microcavity 101 can be freely combined from one or more of the following: curved surface (e.g., bowl-shaped), frustum, and regular prism.

[0083] In some embodiments, the top opening of the through-hole microcavity 101 is circular, and the diameter of the top opening is 50-200 micrometers, such as 50 micrometers, 80 micrometers, 110 micrometers, 125 micrometers, 150 micrometers, 180 micrometers, 200 micrometers, etc. In some embodiments, the depth of the through-hole microcavity 101 is 300-400 micrometers, such as 300 micrometers, 350 micrometers, 400 micrometers, etc. Due to its greater depth, the through-hole microcavity 101 can accommodate a larger dose of sample solution, allowing a larger dose of sample solution to react simultaneously.

[0084] By designing the microcavity 101 as a through-hole, the sample solution can smoothly enter the interior of the microcavity 101 under capillary action, without stagnating on the surface of the microfluidic substrate 100 and wasting the sample solution. Furthermore, some air bubbles are inevitably generated during the sample injection process. The through-hole design of the microcavity 101 allows these gases to escape from the bottom opening, preventing air bubbles from remaining inside the microcavity 101 and thus not affecting subsequent fluorescence detection of the sample solution.

[0085] like Figure 4AAs shown, the microfluidic substrate 100 may further include a spacer region S located between any two adjacent microcavity regions R in a plurality of microcavity regions R, and a hydrophobic layer 103 disposed within the spacer region S. As shown, the hydrophobic layer 103 includes a plurality of second openings 104, with each of the plurality of microcavities 101 corresponding one-to-one to the plurality of second openings 104 of the hydrophobic layer 103, and the orthographic projection of each microcavity region R onto the microfluidic substrate 100 falls within the orthographic projection of a corresponding second opening 104 onto the microfluidic substrate 100. The microfluidic substrate 100 may further include a hydrophilic layer 102, which is located at least within the plurality of microcavity regions R. For example, when the microcavity 101 is a blind via, the hydrophilic layer 102 at least covers the sidewalls 1011 and bottom 1013 of the microcavity 101. When the microcavity 101 is a through-hole, the hydrophilic layer 102 at least covers the sidewalls 1011 of the microcavity 101. The orthographic projection of the portion of the hydrophilic layer 102 located within each microcavity region R onto the microfluidic substrate 100 falls within the orthographic projection of a second opening 104 corresponding to that microcavity region R onto the microfluidic substrate 100. It should be noted that although the hydrophilic layer 102 is only located within each microcavity region R in the figure, this is merely an example. In some alternative embodiments, the hydrophilic layer 102 may be located not only within each microcavity region R but also within a portion of the spacer region S. By arranging a hydrophobic layer 103 within the spacer region S between two adjacent microcavities 101 of the microfluidic substrate 100, the hydrophobic properties of the outer region of the microcavity 101 can be improved; while by arranging the hydrophilic layer 102 inside the microcavity 101 (at least on the sidewall 1011), the hydrophilic properties of the interior of the microcavity 101 can be improved. Therefore, the hydrophilic layer 102 and the hydrophobic layer 103 can jointly adjust the surface contact angle of the sample solution droplets. Without external driving force applied to the sample solution, the sample solution can automatically enter each microcavity 101 of the microfluidic substrate 100 based on capillary action, thereby improving the uniformity of sample solution distribution and preventing cross-contamination. By arranging microcavity structures within the microfluidic substrate 100, the amount of sample solution flowing into each microcavity 101 can be made substantially the same by designing uniformly sized microcavities 101, thus achieving precise control of the sample solution. The orthographic projection of the portion of the hydrophilic layer 102 located within each microcavity 101 onto the microfluidic substrate 100 falls within the orthographic projection of a second opening 104 corresponding to that microcavity 101 onto the microfluidic substrate 100. In other words, the hydrophilic layer 102 and the hydrophobic layer 103 have a certain boundary distance near each microcavity 101.

[0086] The orthographic projection of each microcavity 101 onto the microfluidic substrate 100 falls within the orthographic projection of a corresponding second opening 104 onto the microfluidic substrate 100. In some embodiments, the top opening of each microcavity 101 is circular, and the shape of the corresponding second opening 104 is also circular. Figure 4A As shown, the diameter of the top opening of the microcavity 101 is D1, and the diameter of the second opening 104 of the hydrophobic layer 103 is D2. In some examples, the diameter D2 of the second opening 104 of the hydrophobic layer 103 is 5 micrometers to 20 micrometers larger than the diameter D1 of the top opening of the microcavity 101.

[0087] like Figure 4A As shown, in some embodiments, the substrate 10 may include a first substrate 105, which includes the aforementioned plurality of microcavities 101, each microcavity 101 being a through-hole or a blind via. In other words, the aforementioned plurality of microcavities 101 are formed by patterning the first substrate 105. In some embodiments, the thickness of the first substrate 105 is 0.3-0.7 mm, for example 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, etc. The first substrate 105 may be formed of various suitable materials, including but not limited to glass, quartz, silicon, etc.

[0088] Figure 4B A partial cross-sectional view of the microfluidic substrate 100' is shown. Except for the arrangement of the microcavities 101 and the shielding layer 107, the structure of the microfluidic substrate 100' in Figure 4 is similar to... Figure 4A The microfluidic substrate 100' has a basically the same structure as the microfluidic substrate 100'. For example, the microfluidic substrate 100' also includes a hydrophilic layer 102 and a hydrophobic layer 103, and Figure 4B The arrangement of the hydrophilic layer 102 and the hydrophobic layer 103 in the middle is the same as Figure 4A The hydrophilic layer 102 and the hydrophobic layer 103 are arranged in the same way. Therefore, Figure 4B The structure and effects of the hydrophilic layer 102 and the hydrophobic layer 103 can be found in the section on Figure 4A The description will not be repeated here. For the sake of brevity, Figure 4B The microfluidic substrate 100' in Figure 4A The similarities between the microfluidic substrate 100 and the microfluidic substrate 100 will not be described again; only the differences will be introduced below.

[0089] like Figure 4B As shown, the substrate 10 includes a first substrate 105 and a defining layer 106. The defining layer 106 is located between the first substrate 105 and the hydrophobic layer 103. The defining layer 106 includes the aforementioned plurality of microcavities 101, each of which can be a through-hole or a blind via. That is, with Figure 4AThe difference is that the microcavity 101 is not formed by patterning the first substrate 105, but by patterning the defining layer 106. The defining layer 106 can be made of various suitable materials, including but not limited to photoresist.

[0090] like Figure 4B As shown, the microfluidic substrate 100' may further include a shielding layer 107. The shielding layer 107 includes a plurality of first openings 108, each of which corresponds one-to-one with a plurality of microcavities 101. The orthographic projection of each microcavity 101 on the microfluidic substrate 100' and the orthographic projection of a corresponding first opening 108 on the microfluidic substrate 100' at least partially overlap. Furthermore, the orthographic projection of the shielding layer 107 on the microfluidic substrate 100' at least partially overlaps with the orthographic projection of the defining layer 106 on the microfluidic substrate 100'. In one example, the orthographic projection of the defining layer 106 on the microfluidic substrate 100' falls entirely within the orthographic projection of the shielding layer 107 on the microfluidic substrate 100'. The shielding layer 107 can be made of any suitable material, as long as the material can block or absorb light. The embodiments of this disclosure do not specifically limit the material of the shielding layer 107. In some embodiments, the material of the shielding layer 107 is an opaque material, such as an opaque metal. In some examples, the material of the shielding layer 107 is a black matrix (BM) commonly used in the display field.

[0091] When the material of the defining layer 106 is photoresist, due to its inherent material properties, the defining layer 106 typically emits unwanted fluorescence after being irradiated by excitation light. This unwanted fluorescence interferes with the fluorescence signal emitted by the sample solution within the microcavity 101. However, in the embodiments of this disclosure, by providing a shielding layer 107 and ensuring that the orthographic projection of the shielding layer 107 on the microfluidic substrate 100' at least partially overlaps with the orthographic projection of the defining layer 106 on the microfluidic substrate 100', when excitation light irradiates the microcavity 101 through the first opening 108 of the shielding layer 107, the shielding layer 107 can at least partially shield the defining layer 106 from being irradiated by the excitation light, thereby preventing the defining layer 106 from generating interfering fluorescence due to excitation light irradiation. Thus, the excitation light can only excite the sample solution within the microcavity 101 through the first opening 108. Therefore, this arrangement reduces or even eliminates fluorescence interference caused by the confinement layer 106, allowing the detector to accurately identify the fluorescence signal emitted by the sample solution within the microcavity 101. This enables more sensitive and accurate interpretation of the reaction signal, improving the fluorescence detection accuracy of the sample solution and providing image data support for subsequent nucleic acid amplification reaction data analysis. Furthermore, this arrangement achieves clearer micro-well array imaging, reduces detection errors caused by false positives, and effectively avoids interference between different channels during multi-channel fluorescence signal detection.

[0092] It should be noted that, although Figure 4B The diagram shows a shielding layer 107 located between the first substrate 105 and the defining layer 106, but this is only an example; the shielding layer 107 can also be located at other locations. In some embodiments, the shielding layer 107 may be located on the side of the first substrate 105 away from the defining layer 106, i.e., on the back side of the first substrate 105. In an alternative embodiment, the shielding layer 107 may be located on the side of the defining layer 106 away from the first substrate 105 and attached to the side surface of the defining layer 106 and the surface away from the first substrate 105. In an alternative embodiment, the shielding layer 104 is not only located between the first substrate 105 and the defining layer 106 and attached to the surface of the defining layer 106 near the first substrate 105, but also located on the side of the defining layer 106 away from the first substrate 105 and attached to the side surface of the defining layer 106 and the surface away from the first substrate 105; that is, the shielding layer 107 surrounds the defining layer 106 from all sides.

[0093] According to another aspect of this disclosure, a microfluidic chip is provided. Figure 5 A planar schematic diagram of the microfluidic chip 200 is shown. (As shown...) Figure 5As shown, the microfluidic chip 200 includes: a first substrate 201; a second substrate 202 opposite to the first substrate 201; a microfluidic substrate 204 located between the first substrate 201 and the second substrate 202, the microfluidic substrate 204 may be the microfluidic substrate 100 or 100' described in any of the preceding embodiments; and a sealing frame 203 located between the first substrate 201 and the second substrate 202, and the orthographic projection of the microfluidic substrate 204 on the first substrate 201 falls within the orthographic projection of the sealing frame 203 on the first substrate 201.

[0094] Figure 6 A plan view of the sealing frame 203 is shown. The sealing frame 203 is configured to maintain an appropriate distance between the first substrate 201 and the second substrate 202 and to keep the microfluidic chip 200 sealed. In some embodiments, the sealing frame 203 is an elastic sealing frame. In some embodiments, the sealing frame 203 is made of silicone, die-cut to a certain shape, and surrounds the periphery of the microfluidic substrate 204. Figure 6 As shown, the sealing frame 203 includes a first side 2031 and a second side 2032 arranged along a first direction D1 and opposite to each other, and a third side 2033 and a fourth side 2034 arranged along a second direction D2 different from the first direction D1 and opposite to each other. The first side 2031 and the second side 2032 are arc-shaped. In one example, the first side 2031 and the second side 2032 of the sealing frame 203 are arc-shaped. This arc or arc-shaped design facilitates the flow and convergence of the sample solution within the microfluidic chip 200 and avoids the generation of air bubbles within the microfluidic chip 200 during sample addition.

[0095] refer to Figure 5 and Figure 6The microfluidic substrate 204 includes a first edge 109 and a second edge 110 arranged along a second direction D2 and opposite to each other. A third side 2033 of the sealing frame 203 is spaced a certain distance from the first edge 109 of the microfluidic substrate 204, and a fourth side 2034 of the sealing frame 203 is spaced a certain distance from the second edge 110 of the microfluidic substrate 204. In some embodiments, the distance between the orthographic projection of the third side 2033 of the sealing frame 203 on the first substrate 201 and the orthographic projection of the first edge 109 of the microfluidic substrate 204 on the first substrate 201 is 2 mm to 6 mm, for example, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm; the distance between the orthographic projection of the fourth side 2034 of the sealing frame 203 on the first substrate 201 and the orthographic projection of the second edge 110 of the microfluidic substrate 204 on the first substrate 201 is 2 mm to 6 mm, for example, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm. In some embodiments, the area of ​​the region where the plurality of microcavities 101 of the microfluidic substrate 204 are located is 15×15mm. 2 The microfluidic substrate 204 has an area of ​​17×17mm. 2 That is, the four edges of the microfluidic substrate 204 are at a certain distance from the area where the microcavity 101 is located. This avoids cutting into the microcavity area during the cutting process of the microfluidic chip 200 and leaves enough space for subsequent packaging.

[0096] In some embodiments, the thickness of the sealing frame 203 is 0.4-0.8 mm, for example, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, etc. The thickness of the sealing frame 203 is greater than the thickness of the microfluidic substrate 204. In some embodiments, the thickness of the sealing frame 203 is 0.1-0.3 mm greater than the thickness of the microfluidic substrate 204, for example, 0.1 mm, 0.2 mm, 0.3 mm greater. In other words, the distance between the microfluidic substrate 204 and the second substrate 202 is 0.1-0.3 mm. By having a small distance between the microfluidic substrate 204 and the second substrate 202, even if bubbles are generated during the heating process of the microfluidic chip 200, under the surface tension and the compression between the microfluidic substrate 204 and the second substrate 202, the bubbles can easily move automatically to the external open area and be discharged from the microfluidic chip 200, thus avoiding the bubbles from circulating with the liquid inside the microfluidic chip 200 and affecting the sample reaction and subsequent fluorescence detection.

[0097] Figure 7 A planar schematic diagram of the second substrate 202 is shown. (As shown) Figure 7As shown, the second substrate 202 includes an inlet port 2021 and an outlet port 2022. The sample solution is injected into the microcavity 101 of the microfluidic chip 200 through the inlet port 2021. The sample solution processed by the microfluidic chip 200 can be transferred to other external devices through the outlet port 2022. The inlet port 2021 and the outlet port 2022 can be circular in shape, with a diameter of approximately 0.5-1.5 mm. (Reference) Figure 5 and Figure 7 The orthographic projections of the sample inlet 2021 and sample outlet 2022 of the second substrate 202 onto the first substrate 201 fall within the orthographic projection of the sealing frame 203 onto the first substrate 201.

[0098] In some embodiments, the second substrate 202 can be cut from a large-size piece of clear glass, and the size of the second substrate 202 can be 40×42mm. 2 In some embodiments, the thickness of the second substrate 202 is 0.3-0.7 mm, for example, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, etc. In some embodiments, the distance between the two side edges of the second substrate 202 along the first direction D1 and the microcavity region is 16 mm, and the distance between the two side edges of the second substrate 202 along the second direction D2 and the microcavity region is 15 mm. This design allows sufficient space for the arrangement of the heating electrodes on the first substrate 201.

[0099] Figure 8A A partial cross-sectional view of a first substrate 201 in one embodiment is shown. As shown, the first substrate 201 includes a second substrate 2011. The second substrate 2011 can be made of various suitable materials, such as glass. When the microfluidic chip 200 includes such a first substrate 201, after the microfluidic chip 200 is packaged, the microcavity 101 of the microfluidic chip 200 can be heated using a flatbed thermal cycler to allow the sample solution within the microcavity 101 to react, for example, in a PCR reaction.

[0100] Figure 8BA partial cross-sectional view of a first substrate 201 in another embodiment is shown. As shown, the first substrate 201 includes a second substrate 2011 and a heating electrode 2012 located between the second substrate 2011 and a microfluidic substrate 204. The orthographic projection of a plurality of microcavities 101 of the microfluidic substrate 204 onto the second substrate 2011 falls within the orthographic projection of the heating electrode 2012 onto the second substrate 2011. The heating electrode 2012 is configured to heat the plurality of microcavities 101. The heating electrode 2012 can receive an electrical signal (e.g., a voltage signal), thereby generating heat when a current flows through the heating electrode 2012, which is conducted into the microcavities 101 for use in a polymerase chain reaction. For example, the heating electrode 2012 can be made of a conductive material with high resistivity, thereby enabling the heating electrode 2012 to generate a large amount of heat with a small electrical signal, thereby improving the energy conversion efficiency. The heating electrode 2012 can be made of a transparent conductive material, such as indium tin oxide (ITO) or tin oxide, or other suitable materials, such as metals. The embodiments disclosed herein are not limited in this regard. By setting the heating electrode 2012 in the first substrate 201 (e.g., integrating the heating electrode 2012 on the second substrate 2011), heating of the microcavity 101 can be achieved without external heating equipment, and the temperature of the microcavity 101 can be controlled in real time. This results in the microfluidic chip 200, including the first substrate 201, having higher integration and more precise temperature control, and improving the stability of the microfluidic chip 200 during the heating process.

[0101] like Figure 8B As shown, the first substrate 201 may further include: a first dielectric layer 2013 located between the second substrate 2011 and the heating electrode 2012, and a second dielectric layer 2014 located between the heating electrode 2012 and the microfluidic substrate 204. The first dielectric layer 2013 and the second dielectric layer 2014 can be made of various suitable materials, and the embodiments of this disclosure do not limit the specific materials of the first dielectric layer 2013 and the second dielectric layer 2014. In one example, the first dielectric layer 2013 and the second dielectric layer 2014 may be made of SiO2. The first substrate 201 may further include a conductive layer 2015 located between the second substrate 2011 and the first dielectric layer 2013, the conductive layer 2015 being electrically connected to the heating electrode 2012 via a via 2016 in the first dielectric layer 2013. The conductive layer 2015 is configured to apply an electrical signal (e.g., a voltage signal) to the heating electrode 2012.

[0102] The microfluidic chip 200 can have essentially the same technical effects as the microfluidic substrate described in the previous embodiments. Therefore, for the sake of brevity, the technical effects of the microfluidic chip 200 will not be described again here.

[0103] Figures 9A-9F Several different microfluidic chips are shown as examples. These microfluidic chips have essentially the same structure, for example, all including a first substrate 201, a second substrate 202, a sealing frame 203, and a microfluidic substrate, and the structures of the second substrate 202 and the sealing frame 203 are similar to those of the microfluidic chips. Figure 5-7 The second substrate 202 and the sealing frame 203 described are identical in structure. The differences between these microfluidic chips lie in the composition of the first substrate 201 and the arrangement of the microcavities 101 within the microfluidic substrate. For simplicity, only the differences between the individual microfluidic chips will be described below.

[0104] Figure 9A A microfluidic chip 200A is shown, which includes a first substrate 201, a second substrate 202, a sealing frame 203, and a microfluidic substrate 204A located between the first substrate 201 and the second substrate 202. The microfluidic substrate 204A includes a first substrate 105, and each microcavity 101 is a blind via formed by patterning the first substrate 105. The specific structure of the microfluidic substrate 204A can be referred to the preceding section on... Figure 1B , Figure 2A , Figure 2B The first substrate 201 is the second substrate 2011. Figure 9A The microfluidic chip 200A shown can be simply referred to as "a blind-hole microcavity formed in a first substrate + a substrate-type first substrate".

[0105] Figure 9B A microfluidic chip 200B is shown, which includes a first substrate 201, a second substrate 202, a sealing frame 203, and a microfluidic substrate 204B located between the first substrate 201 and the second substrate 202. The microfluidic substrate 204B includes a first substrate 105, and each microcavity 101 is a blind via formed by patterning the first substrate 105. The specific structure of the microfluidic substrate 204B can be found in the preceding text. Figure 1B , Figure 2A , Figure 2B The first substrate 201 includes a second substrate 2011, a heating electrode 2012, a first dielectric layer 2013, a second dielectric layer 2014, and a conductive layer 2015. For the specific structure of the first substrate 201, please refer to the description... Figure 8B As shown in the figure, the orthographic projection of the sealing frame 203 on the second substrate 2011 falls within the orthographic projection of the heating electrode 2012 on the second substrate 2011. Figure 9B The microfluidic chip 200B shown can be simply referred to as "a blind-hole microcavity formed in a first substrate + a first substrate with integrated heating electrodes".

[0106] Figure 9CA microfluidic chip 200C is shown, which includes a first substrate 201, a second substrate 202, a sealing frame 203, and a microfluidic substrate 204C located between the first substrate 201 and the second substrate 202. The microfluidic substrate 204C includes a first substrate 105, and each microcavity 101 is a through-hole formed by patterning the first substrate 105. The specific structure of the microfluidic substrate 204C can be referred to the preceding section on... Figure 1B , Figures 3A-3C The first substrate 201 is the second substrate 2011. Figure 9C The microfluidic chip 200C shown can be simply referred to as "a through-hole microcavity formed in a first substrate + a substrate-type first substrate".

[0107] Figure 9D A microfluidic chip 200D is shown, which includes a first substrate 201, a second substrate 202, a sealing frame 203, and a microfluidic substrate 204D located between the first substrate 201 and the second substrate 202. The microfluidic substrate 204D includes a first substrate 105, and each microcavity 101 is a through-hole formed by patterning the first substrate 105. The specific structure of the microfluidic substrate 204D can be referred to the preceding section on... Figure 1B , Figures 3A-3C The first substrate 201 includes a second substrate 2011, a heating electrode 2012, a first dielectric layer 2013, a second dielectric layer 2014, and a conductive layer 2015. For the specific structure of the first substrate 201, please refer to the description... Figure 8B As shown in the figure, the orthographic projection of the sealing frame 203 on the second substrate 2011 falls within the orthographic projection of the heating electrode 2012 on the second substrate 2011. Figure 9D The microfluidic chip 200D shown can be simply referred to as "a through-hole microcavity formed in a first substrate + a first substrate with integrated heating electrodes".

[0108] Figure 9E A microfluidic chip 200E is shown, which includes a first substrate 201, a second substrate 202, a sealing frame 203, and a microfluidic substrate 204E located between the first substrate 201 and the second substrate 202. The microfluidic substrate 204E includes a defining layer 106 and a shielding layer 107. Each microcavity 101 is a through-hole or blind via and is formed by patterning the defining layer 106. The specific structure of the microfluidic substrate 204E can be found in the preceding text. Figure 4B The first substrate 201 is the second substrate 2011. Figure 9E The microfluidic chip 200E shown can be simply referred to as "a microcavity with a through-hole or blind hole formed in a defined layer + a first substrate".

[0109] Figure 9FA microfluidic chip 200F is shown, which includes a first substrate 201, a second substrate 202, a sealing frame 203, and a microfluidic substrate 204F located between the first substrate 201 and the second substrate 202. The microfluidic substrate 204F includes a defining layer 106 and a shielding layer 107. Each microcavity 101 is a through-hole or blind via and is formed by patterning the defining layer 106. For the specific structure of the microfluidic substrate 204F, please refer to the previous section on... Figure 4B The first substrate 201 includes a second substrate 2011, a heating electrode 2012, a first dielectric layer 2013, a second dielectric layer 2014, and a conductive layer 2015. For the specific structure of the first substrate 201, please refer to the description... Figure 8B As shown in the figure, the orthographic projection of the sealing frame 203 on the second substrate 2011 falls within the orthographic projection of the heating electrode 2012 on the second substrate 2011. Figure 9F The microfluidic chip 200F shown can be simply referred to as "a microcavity with a through-hole or blind hole formed in a defined layer + a first substrate with integrated heating electrodes".

[0110] Figure 10 A flowchart of a microfluidic chip fabrication method 1000 is shown. This fabrication method 1000 is applicable to fabricating the microfluidic chips described in any of the preceding embodiments. The steps of the fabrication method 1000 are as follows:

[0111] Step S1001: Provide the first substrate 201;

[0112] Step S1002: Prepare a microfluidic substrate, which can be any of the microfluidic substrates described in the preceding embodiments;

[0113] Step S1003: Fix the sealing frame 203 and the microfluidic substrate prepared above onto the first substrate 201, such that the orthographic projection of the microfluidic substrate onto the first substrate 201 falls within the orthographic projection of the sealing frame 203 onto the first substrate 201.

[0114] Step S1004: Place the second substrate 202 on the side of the sealing frame 203 and the microfluidic substrate away from the first substrate 201; and

[0115] Step S1005: Perform encapsulation processing.

[0116] The following is based on Figure 9A The microfluidic chip 200A shown is used as an example to describe in detail the fabrication method of the microfluidic chip 200A.

[0117] Fabrication steps of microfluidic substrate 204A:

[0118] Step 1101: Provide a first substrate 105 and clean it. The first substrate 105 can be made of any suitable material; in one example, the first substrate 105 is made of glass. The first substrate 105 can have any suitable thickness H; in one example, the thickness H of the first substrate 105 is 300-700 μm.

[0119] Step 1102: A mark is fabricated on the first substrate 105 to provide positioning for subsequent substrate cutting. In one example, the mark formation process is as follows: a Mo film layer with a thickness of approximately 2200 Å is sputtered on the surface of the first substrate 105, and the Mo film layer is exposed, developed, and etched using a photolithography process to form a metal mark.

[0120] Step 1103: Deposit an insulating film layer on the surface of the first substrate 105 with the metal markings formed, and expose, develop, and etch the insulating film layer to form a hydrophobic layer 103. In one example, the process of forming the hydrophobic layer 103 is as follows: Deposit a SiN layer with a thickness of approximately 3000 Å on the surface of the first substrate 105. x The film layer, for this SiN x The film is exposed, developed, and etched to form a hydrophobic layer 103. The hydrophobic layer 103 includes a plurality of second openings 104.

[0121] Step 1104: A mask pattern is formed on the side of the hydrophobic layer 103 away from the first substrate 105. This mask pattern is used to define the shape of the microcavity formed by subsequent etching and to provide isolation and protection to other parts outside the microcavity during the etching process. In one example, the process of forming the mask pattern is as follows: A Mo film layer with a thickness of approximately 2200 Å is sputtered on the side of the hydrophobic layer 103 away from the first substrate 105. The Mo film layer is then exposed, developed, and etched using photolithography to form a metal mask pattern 205. The formed metal mask pattern 205 is as follows: Figure 11 As shown, the metal mask pattern 205 includes a plurality of exposure holes 2051, which correspond to the positions of a plurality of microcavities to be subsequently formed, thereby exposing the areas to be etched to form the microcavities. In some embodiments, each exposure hole of the metal mask pattern 205 is circular in shape and has a diameter of y, where y ranges from 5 micrometers to 30 micrometers. In one example, the value of y is 10 micrometers.

[0122] Step 1105: Forming the microcavity 101 using a wet etching method. Specifically, the steps are as follows: The microfluidic substrate 204A with the metal mask pattern 205 is immersed in an etching solution with a hydrogen fluoride (HF) concentration of approximately 40% and an etching rate of approximately 3.5 μm / min. The surface of the first substrate 105 facing the metal mask pattern 205 is etched. During etching, the etching solution is continuously stirred using blades to ensure more uniform etching of the first substrate 105. The etching time is approximately 60 minutes to form the microcavity 101, which is a blind via. The microcavity 101 is formed using a wet etching method. The exposed aperture 2051 of the metal mask pattern 205 is circular. Due to the isotropic nature of wet etching, the top opening of the microcavity 101 is also typically circular. The diameter of the top opening is approximately 50-200 μm, such as 50 μm, 80 μm, 100 μm, 120 μm, 150 μm, 200 μm, etc. The depth of microcavity 101 is approximately 20-100 μm, such as 20 μm, 40 μm, 60 μm, 80 μm, 100 μm, etc. The specific shape of microcavity 101 can be found in the previous section regarding... Figure 3A and Figure 3B For the sake of brevity, the description will not be repeated here. The blind-hole structure of the microcavity 101 helps to keep the sample solution stably within the cavity during the detection process, making it less likely to be carried out of the microcavity 101. In one example, when the first substrate 105 is etched using the metal mask pattern 205, the relationship between the diameter D of the top opening of the formed blind-hole microcavity 101 and the etching depth x of the microcavity 101 can be roughly considered as D = 2x + y, where x ranges from 20 to 100 μm and y ranges from 10 to 30 μm, for example, 10 μm. The formed microcavity 101 has smooth sidewalls and a flat bottom, which facilitates the flow of the sample solution along the smooth sidewalls into the interior of the microcavity 101 and makes it less likely to be carried out, and can reduce the generation of bubbles. The orthographic projection of each microcavity 101 on the first substrate 105 falls within the orthographic projection of a corresponding second opening 104 on the first substrate 105. In some embodiments, the second opening 104 of the hydrophobic layer 103 is circular in shape, the top opening of the microcavity 101 is circular in shape, and the diameter of the second opening 104 of the hydrophobic layer 103 is 5-20 μm larger than the diameter of the top opening of the microcavity 101.

[0123] Step 1106: After etching the microcavity 101, remove the metal mask pattern 205.

[0124] Step 1107: An insulating film layer is deposited on the surface of the first substrate 105. The insulating film layer is then exposed, developed, and etched to form a hydrophilic layer 102. The hydrophilic layer 102 is located only on the sidewalls and bottom of each microcavity 101. In one example, the process of forming the hydrophilic layer 102 is as follows: A SiO2 film layer is deposited on the surface of the first substrate 105. The SiO2 film layer is then exposed, developed, and etched to form the hydrophilic layer 102.

[0125] Step 1108: The first substrate 105, on which the microcavity 101 is formed, is diced into small pieces to form a microfluidic substrate 204A. The area of ​​the microfluidic substrate 204A is approximately 17 × 17 mm. 2 The area of ​​the region where the multiple microcavities 101 are located is approximately 15×15mm. 2 The area where the microcavity 101 is located is at a certain distance from the edge of the microfluidic substrate 204, which can avoid damage to the microcavity area during the cutting process and leave enough space for subsequent packaging.

[0126] Step 1109: Fabrication of the second substrate 202

[0127] The large-sized substrate is cut to obtain the second substrate 202. In one example, the second substrate 202 has dimensions of 40 × 42 mm. 2 The second substrate 202 includes an inlet hole 2021 and an outlet hole 2022, both of which are circular in shape and have a diameter of approximately 0.5-1.5 mm. The second substrate 202 can be made of various suitable materials, such as glass.

[0128] Step 1110: Fabrication of the first substrate 201

[0129] A large-sized substrate is cut to obtain a first substrate 201. The first substrate 201 can be made of various suitable materials, such as glass.

[0130] Step 1111: Fixing the microfluidic substrate 204A. Place the prepared microfluidic substrate 204A onto the first substrate 201. After aligning the positioning marks on the first substrate 201, fix the four corners of the microfluidic substrate 204A using UV adhesive. Then, place the sealing frame 203 onto the first substrate 201, surrounding the microfluidic substrate 204A. The sealing frame 203 can be an elastic sealing frame. In one example, the elastic sealing frame 203 and the first substrate 201 can be first subjected to plasma activation treatment, and then the treated sealing frame 203 can be placed on the first substrate 201, so that the sealing frame 203 surrounds the microfluidic substrate 204A. The thickness of the sealing frame 203 is approximately 0.1-0.3 mm thicker than the microfluidic substrate 204A; that is, with the first substrate 201 as a reference surface, the height of the sealing frame 203 is approximately 0.1-0.3 mm higher than the height of the microfluidic substrate 204A. The sealing frame 203 is configured to maintain an appropriate distance between the first substrate 201 and the second substrate 202 and to keep the microfluidic chip 200A sealed. In some embodiments, the sealing frame 203 is made of silicone and has a certain shape by die-cutting. The sealing frame 203 includes a first side 2031 and a second side 2032 arranged along a first direction D1 and opposite to each other, and a third side 2033 and a fourth side 2034 arranged along a second direction D2 different from the first direction D1 and opposite to each other. The first side 2031 and the second side 2032 are arc-shaped. In one example, the first side 2031 and the second side 2032 of the sealing frame 203 are arc-shaped. This arc or arc-shaped design facilitates the flow and convergence of the sample solution within the microfluidic chip 200A and avoids the generation of air bubbles within the microfluidic chip 200A during sample addition.

[0131] Step 1112: Sample injection. The mixed sample solution is scraped across the surface of the microcavity 101 with a glass scraper to allow the sample solution to flow into the microcavity 101. Then, a few drops of fluorinated oil are dropped on the area above the microcavity 101 using a pipette. The fluorinated oil can be FC-40 or other mineral oil. After the fluorinated oil is spread out, the second substrate 202 is covered.

[0132] Step 1113: Sealing. Encapsulation can be achieved by plasma treatment of the first substrate 201 and the sealing frame 203. Alternatively, a certain amount of UV adhesive can be injected around the sealing frame 203 using a syringe to seal the outer perimeter and prevent leakage. Fluorinated oil or mineral oil is injected through the inlet 2021 of the second substrate 202 using a pipette. After the fluorinated oil or mineral oil fills the internal cavity of the microfluidic chip 200A, the inlet 2021 and outlet 2022 of the second substrate 202 are sealed with a sealing film or UV adhesive.

[0133] Figure 9BThe manufacturing method of the microfluidic chip 200B shown is similar to... Figure 9A The manufacturing method of the microfluidic chip 200A shown is basically the same, with differences only in a few steps. The same steps can be found in the description of the manufacturing method of microfluidic chip 200A; the following only describes the differences in the manufacturing method of microfluidic chip 200B.

[0134] The microfluidic substrate 204B is prepared using the same method steps and manufacturing sequence as steps 1101-1108, and the second substrate 202 is prepared using the same method steps as step 1109.

[0135] The fabrication method of the first substrate 201 of the microfluidic chip 200B differs from that of the first substrate 201 of the microfluidic chip 200A. The fabrication method of the first substrate 201 of the microfluidic chip 200B is roughly as follows:

[0136] Step A: Provide a second substrate 2011. The second substrate 2011 can be made of any suitable material; in one example, the second substrate 2011 is made of glass.

[0137] Step B: A conductive film layer is formed on the second substrate 2011 at approximately 240°C. In one example, a molybdenum (Mo) layer with a thickness of 200 Å, an aluminum neodymium (AlNd) layer with a thickness of 3000 Å, and a molybdenum (Mo) layer with a thickness of 800 Å are sequentially deposited on the second substrate 2011 to form a conductive film layer. This conductive film layer is patterned, for example, by exposure, development, etching, etc., to form a conductive layer 2015.

[0138] Step C: At approximately 200°C, a first insulating film layer is deposited on the conductive layer 2015, and the first insulating film layer is patterned to form a first dielectric layer 2013 covering the conductive layer 2015. In one example, the first dielectric layer 2013 is a SiO2 layer with a thickness of approximately 3000 Å.

[0139] Step D: Pattern the first dielectric layer 2013 to form at least one via 2016 through the first dielectric layer 2013, the at least one via 2016 exposing a portion of the conductive layer 2015.

[0140] Step E: A conductive film is deposited on the side of the first dielectric layer 2013 away from the second substrate 2011. Then, the conductive film is subjected to exposure, development, etching, and stripping processes to form a patterned heating electrode 2012. In one example, the material of the heating electrode 2012 is ITO.

[0141] Step F: A second insulating film layer is deposited on the side of the heating electrode 2012 away from the second substrate 2011, and the second insulating film layer is patterned to form a second dielectric layer 2014 that at least partially covers the heating electrode 2012. In one example, the second dielectric layer 2014 comprises a SiO2 layer with a thickness of approximately 1000 Å and a SiN layer with a thickness of approximately 2000 Å, stacked sequentially. x layer.

[0142] Then, using the same preparation method and operation sequence as steps 1111-1113, the microfluidic substrate 204B is fixed, sampled, and sealed to form the microfluidic chip 200B.

[0143] Figure 9C The manufacturing method of the microfluidic chip 200C shown is similar to... Figure 9A The manufacturing method of the microfluidic chip 200A shown is basically the same, with differences only in a few steps. The same steps can be found in the description of the manufacturing method of the microfluidic chip 200A; the following only describes the differences in the manufacturing method of the microfluidic chip 200C.

[0144] The same method steps and manufacturing sequence as steps 1101-1104 are used to provide the first substrate 105, form a mark, form a hydrophobic layer 103 and form a mask pattern 205, respectively.

[0145] Then, after step 1104, another hydrophobic layer and another metal mask pattern are sequentially formed on the surface of the first substrate 105 opposite to the metal mask pattern 205 (i.e., the back side) by marking alignment. The position of the other hydrophobic layer corresponds exactly to the position of the hydrophobic layer 103, and the position of the other metal mask pattern corresponds exactly to the position of the metal mask pattern 205. The preparation method of the other hydrophobic layer and the other metal mask pattern is exactly the same as steps 1103 and 1104.

[0146] Step 1105': The microfluidic substrate 204C, with metal mask pattern 205 and another metal mask pattern formed on it, is immersed in an etching solution. The concentration of hydrogen fluoride (HF) in the etching solution is approximately 40%, and the etching rate is approximately 3.5 μm / min. Etching is performed on both surfaces of the first substrate 105. During etching, the etching solution is continuously stirred using blades to ensure more uniform etching of the first substrate 105. The etching time is approximately 60 minutes to form a microcavity 101, which is a through-hole. The opening shape of the microcavity 101 can be circular, with a diameter of approximately 50-200 μm, such as 50 μm, 80 μm, 100 μm, 120 μm, 150 μm, 200 μm, etc. The depth of the microcavity 101 is approximately 300-400 μm, such as 300 μm, 350 μm, 400 μm, etc. The specific shape of the microcavity 101 can be referred to the previous section on... Figure 2A and Figure 2B For the sake of brevity, the description will not be repeated here. Due to its greater depth, the through-hole structure of the microcavity 101 can accommodate more sample solution, facilitating the simultaneous reaction of larger doses of sample solution. In some embodiments, the second opening 104 of the hydrophobic layer 103 is circular, the opening of the microcavity 101 is circular, and the diameter of the second opening 104 of the hydrophobic layer 103 is 5-20 μm larger than the diameter of the opening of the microcavity 101.

[0147] Step 1106': After etching the through-hole microcavity 101, remove the metal mask pattern 205 and another metal mask pattern.

[0148] Step 1107': Prepare the hydrophilic layer 102 using the same method as in step 1107. Since the microcavity 101 is a through hole, the hydrophilic layer 102 is located only on the sidewall of each microcavity 101.

[0149] Then, the microfluidic chip 200C is fabricated using essentially the same method steps and manufacturing sequence as steps 1108-1113. However, in step 1111, when fixing the four corners of the microfluidic substrate 204C with UV adhesive, 100μm spacers can be doped into the UV adhesive. The UV adhesive doped with spacers not only provides fixation but also better support.

[0150] Figure 9D The manufacturing method of the microfluidic chip 200D shown is similar to... Figure 9A The manufacturing method of the microfluidic chip 200A shown is basically the same, with differences only in a few steps. The same steps can be found in the description of the manufacturing method of the microfluidic chip 200A; the following only describes the differences in the manufacturing method of the microfluidic chip 200D.

[0151] The microfluidic substrate 204D is prepared using the exact same method steps and manufacturing sequence as the microfluidic substrate 204C of the microfluidic chip 200C, and the second substrate 202 is prepared using the exact same method steps as in step 1109.

[0152] The first substrate 201 of the microfluidic chip 200D is prepared by using the method step AF for preparing the first substrate 201 of the microfluidic chip 200B.

[0153] Then, using the same preparation method and operation sequence as steps 1111-1113, the microfluidic substrate 204D is fixed, sampled, and sealed to form the microfluidic chip 200D.

[0154] Figure 9E The manufacturing method of the microfluidic chip 200E shown is roughly as follows.

[0155] Fabrication steps of the 204E microfluidic substrate:

[0156] Step I: Provide a first substrate 105 and clean it. The first substrate 105 can be made of any suitable material; in one example, the first substrate 105 is made of glass. The first substrate 105 can have any suitable thickness; in one example, the thickness of the first substrate 105 is 300-700 μm.

[0157] Step II: A masking film layer is coated on the first substrate 105 and patterned to form a masking layer 107 defining the first opening 108. In one example, the specific steps for forming the masking layer 107 may include: spin-coating the masking film layer on the first substrate 105 at a pressure of 30 kPa, a spin-coating speed of approximately 380 rpm, and a spin-coating time of approximately 7 seconds. Then, the spin-coated masking film layer is pre-cured at 90°C for 120 seconds. Next, the masking film layer is exposed, developed, and etched through a photomask for approximately 75 seconds. Finally, the etched masking film layer is post-cured at 230°C for approximately 20 minutes to form the masking layer 107 defining the first opening 108. In one example, the material forming the masking layer 107 includes chromium, chromium oxide, and black resin.

[0158] Step III: A defining film layer is coated on the side of the shielding layer 107 away from the first substrate 105, and the defining film layer is patterned to form a defining layer 106 defining a plurality of microcavities 101. Each microcavity 101 can be a through-hole or a blind via. In one example, the process of forming the defining layer 106 is described as follows: First, at a pressure of 30 kPa, optical adhesive is spin-coated on the surface of the shielding layer 107 away from the first substrate 105 at a speed of 300 rpm for approximately 10 seconds, and then the optical adhesive is cured at a temperature of 90°C for 120 seconds. The above process is repeated twice to obtain the defining film layer. Next, the defining film layer is exposed through a photomask, and then developed with a developer for 100 seconds, followed by etching. The etched defining film layer is cured at a temperature of 230°C for 30 minutes, finally obtaining the defining layer 106 defining the plurality of microcavities 101. The material of the defining layer 106 includes photoresist. Each first opening 108 of the shielding layer 107 has its orthographic projection on the first substrate 105 at least partially overlapping with the orthographic projection of a corresponding microcavity 101 of the defining layer 106 on the first substrate 105, and the orthographic projection of the shielding layer 107 on the first substrate 105 at least partially overlapping with the orthographic projection of the defining layer 106 on the first substrate 105.

[0159] Step IV: At 200°C, an insulating film layer is deposited on the surface of the defining layer 106 away from the first substrate 105. This insulating film layer is then exposed, developed, and etched to form a patterned layer. The patterned layer is treated with a 0.4% KOH solution for approximately 15 minutes to hydrophilically modify it, thereby forming a hydrophilic layer 102, which is located only inside the microcavity 101. For example, when the microcavity 101 is a blind via, the hydrophilic layer 102 covers the sidewalls and bottom of the microcavity 101. As another example, when the microcavity 101 is a through-hole, the hydrophilic layer 102 covers the sidewalls of the microcavity 101. In one example, the hydrophilic layer 102 is a SiO2 layer with a thickness of approximately 3000 Å.

[0160] Step V: An insulating film layer is deposited on the surface of the defining layer 106 away from the first substrate 105. This insulating film layer is then exposed, developed, and etched to form a hydrophobic layer 103. In one example, the process of forming the hydrophobic layer 103 is as follows: A SiN layer with a thickness of approximately 1000 Å is deposited on the surface of the defining layer 106 away from the first substrate 105. x The film layer, for this SiN xThe film layer is exposed, developed, and etched to form a hydrophobic layer 103 including a plurality of second openings 104. The orthographic projection of each microcavity 101 on the first substrate 105 falls within the orthographic projection of a corresponding second opening 104 on the first substrate 105. In some embodiments, the second openings 104 of the hydrophobic layer 103 are circular in shape, the top opening of the microcavity 101 is circular in shape, and the diameter of the second openings 104 of the hydrophobic layer 103 is 5-20 μm larger than the diameter of the top opening of the microcavity 101.

[0161] Step VI: The first substrate 105, on which the microcavities 101 are formed, is diced into small pieces to form a microfluidic substrate 204E. The area of ​​the microfluidic substrate 204E is approximately 17 × 17 mm. 2 The area of ​​the region where the multiple microcavities 101 are located is approximately 15×15mm. 2 .

[0162] Then, the microfluidic chip 200E is formed using the exact same preparation method and operation sequence as steps 1109-1113.

[0163] Figure 9F The manufacturing method of the microfluidic chip 200F shown is roughly as follows.

[0164] The microfluidic substrate 204F was prepared using the same method as steps I-VI of the microfluidic chip 200E.

[0165] The second substrate 202 is prepared using the same method as in step 1109.

[0166] The first substrate 201 of the microfluidic chip 200F is prepared by using the method step AF for preparing the first substrate 201 of the microfluidic chip 200B.

[0167] Then, using the same preparation method and operation sequence as steps 1111-1113, the microfluidic substrate 204F is fixed, sampled, and sealed to form the microfluidic chip 200F.

[0168] For other technical effects of the microfluidic chip fabrication method, please refer to the previous description of the technical effects of microfluidic substrates and microfluidic chips. For the sake of brevity, they will not be repeated here.

[0169] According to another aspect of this disclosure, a method of using a microfluidic chip is provided, which may be the microfluidic chip described in any of the preceding embodiments. Figure 12 A flowchart of the usage method 1200 is shown, which includes the following steps:

[0170] Step S1201: Add the sample solution into multiple microcavities of the microfluidic chip;

[0171] Step S1202: Heating the microfluidic chip to cause the sample solution within the multiple microcavities to react; and

[0172] Step S1203: Use optical equipment to detect the optical signals emitted by the sample solution after the reaction in multiple microcavities.

[0173] When the microfluidic chip is Figure 9A , Figure 9C , Figure 9E When the microfluidic chip is shown, i.e. the first substrate 201 includes the second substrate 2011 but does not include the heating electrode, the step of heating the microfluidic chip in step S1202 may include: placing the sealed microfluidic chip in a flat thermal cycler.

[0174] When the microfluidic chip is Figure 9B , Figure 9D , Figure 9F When the microfluidic chip shown is implemented, the first substrate 201 includes a second substrate 2011 and a heating electrode 2012. The step of heating the microfluidic chip in step S1202 may include: applying an electrical signal to the microfluidic chip to drive the heating electrode 2012 to heat multiple microcavities 101, and using a temperature sensor to detect the temperature of the area where the multiple microcavities 101 are located to adjust the current flowing through the heating electrode 2012 in real time. By integrating the heating electrode 2012 into the first substrate 201, heating of the microcavities 101 can be achieved without external heating equipment, and the temperature of the microcavities 101 can be controlled in real time. This results in the microfluidic chip including the first substrate 201 having higher integration and more precise temperature control, improving the stability of the microfluidic chip during the heating process.

[0175] Figure 13 A fluorescence image of a microfluidic chip provided according to an embodiment of the present disclosure is shown, where each dot represents a microcavity 101 containing positive cells. After the sample solution within the microfluidic chip completes the PCR reaction, the sample solution containing positive cells in the microcavity emits fluorescence under excitation light, while the sample solution containing negative cells does not. Therefore, by placing the microfluidic chip under an observation lens, the number of bright and dark areas in the microcavities can be observed. Figure 13As can be seen, the colors of each microcavity 101 and its surrounding area differ significantly, with a very obvious contrast. Each microcavity 101 displays a brighter color, while the surrounding area appears black. Therefore, the microfluidic chip provided in this embodiment can provide high resolution and clarity for fluorescence detection of sample solutions, enabling the fluorescence signal emitted by the sample solution within the microcavity 101 to be accurately identified by the detector. This allows for more sensitive and accurate reading of the reaction signal, improving the accuracy of fluorescence detection of sample solutions and providing image data support for subsequent nucleic acid amplification reaction data analysis.

[0176] It will be understood that although the terms first, second, third, etc., may be used herein to describe various elements, components, areas, layers, and / or parts, these elements, components, areas, layers, and / or parts should not be limited by these terms. These terms are used only to distinguish one element, component, area, layer, or part from another. Therefore, the first element, component, area, layer, or part discussed above may be referred to as a second element, component, area, layer, or part without departing from the teachings of this disclosure.

[0177] Spatial relative terms such as “row,” “column,” “below,” “above,” “left,” “right,” etc., may be used herein for ease of description to describe the relationship between one element or feature illustrated in the figures and another element(s). It will be understood that these spatial relative terms are intended to cover different orientations of the device in use or operation, in addition to those depicted in the figures. For example, if the device in the figure is flipped, then an element described as “below other elements or features” will be oriented “above other elements or features.” Thus, the exemplary term “below” can cover both orientations above and below. Devices may be oriented in other ways (rotated 90 degrees or otherwise) and the spatial relative descriptors used herein will be interpreted accordingly. Additionally, it will be understood that when a layer is referred to as “between two layers,” it may be the only layer between those two layers, or there may be one or more intermediate layers.

[0178] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprising” and / or “including”, when used in this specification, specify the presence of the stated features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. In the description of this specification, references to the terms “one embodiment,” “another embodiment,” etc., mean that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. The illustrative expressions of the foregoing terms in this specification do not necessarily refer to the same embodiments or examples. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, those skilled in the art can combine and integrate the different embodiments or examples described herein, as well as the features of the different embodiments or examples, without contradiction.

[0179] It will be understood that when a component or layer is referred to as "on another component or layer," "connected to another component or layer," "coupled to another component or layer," or "adjacent to another component or layer," it may be directly on another component or layer, directly connected to another component or layer, directly coupled to another component or layer, or directly adjacent to another component or layer, or there may be intermediate components or layers. Conversely, when a component is referred to as "directly on another component or layer," "directly connected to another component or layer," "directly coupled to another component or layer," or "directly adjacent to another component or layer," no intermediate components or layers exist. However, in any case, "on" or "directly on" should not be interpreted as requiring a layer to completely cover the layer below.

[0180] Embodiments of this disclosure are described herein with reference to illustrative illustrations (and intermediate structures) of idealized embodiments. Therefore, variations in the illustrated shapes should be expected, for example, as a result of manufacturing techniques and / or tolerances. Consequently, embodiments of this disclosure should not be construed as limited to the specific shapes of the regions illustrated herein, but should include, for example, shape deviations due to manufacturing processes. Thus, the regions illustrated are schematic in nature, and their shapes are not intended to illustrate the actual shape of regions of a device and are not intended to limit the scope of this disclosure.

[0181] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with their meanings in the relevant field and / or the context of this specification, and will not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0182] As those skilled in the art will understand, although the steps of the methods in this disclosure are described in a specific order in the accompanying drawings, this does not require or imply that the steps must be performed in that specific order unless the context clearly indicates otherwise. Additional or alternatively, multiple steps may be combined into a single step, and / or a single step may be broken down into multiple steps. Furthermore, other method steps may be inserted between steps. Inserted steps may represent improvements to the method described herein, or may be unrelated to the method. Moreover, a given step may not be fully completed before the next step begins.

[0183] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A microfluidic substrate, comprising a substrate, the substrate including a plurality of microcavity regions arranged in an array, wherein, Each of the plurality of microcavity regions includes a first portion and a second portion stacked on top of each other. The first portion has a depth of x and includes a top opening of circular shape with a diameter D. The diameter D of the top opening is related to the depth x by the formula D = 2x + y, where y represents the diameter of the exposure hole of the mask pattern used in the etching process of the microfluidic substrate, x ranges from 20 micrometers to 400 micrometers, and y ranges from 5 micrometers to 30 micrometers. in, The first portion is a blind via and the first portion and the second portion do not penetrate each other; the first portion constitutes a microcavity of the microfluidic substrate; or; The second part includes a bottom opening on the side away from the first part. The first part and the second part penetrate each other to form a through hole. The through hole forms a microcavity of the microfluidic substrate. The depth of the second part is x1 and the shape of the bottom opening of the second part is circular. The relationship between the diameter D1 of the bottom opening and the depth x1 is D1 = 2x1 + y.

2. The microfluidic substrate according to claim 1, wherein, The first portion is a blind via, and the substrate further includes an unetched third portion located between any two adjacent microcavity regions among the plurality of microcavity regions. Within each microcavity region, the etched portion of the substrate constitutes the first portion, and the unetched portion of the substrate constitutes the second portion. The orthographic projection of the first portion onto the microfluidic substrate overlaps with the orthographic projection of the second portion onto the microfluidic substrate. The second part and the third part are an integral structure.

3. The microfluidic substrate according to claim 2, wherein, The range of x is from 20 micrometers to 100 micrometers.

4. The microfluidic substrate according to claim 1, wherein, The first part is a blind hole, and the shape of the first part is a curved body. The first part includes the top opening, the bottom, and the sidewall connecting the top opening and the bottom. The bottom of the first part is circular in shape and has a diameter of x micrometers.

5. The microfluidic substrate according to claim 4, wherein, The tangent planes at at least some points on the sidewall are at a non-perpendicular angle to the reference plane on which the microfluidic substrate is located.

6. The microfluidic substrate according to claim 1, wherein, The first part and the second part penetrate each other to form a through hole. The first part and the second part have the same shape and are symmetrical about an axis of symmetry, which is parallel to the reference plane where the microfluidic substrate is located.

7. The microfluidic substrate according to any one of claims 1-6, wherein, y equals 10 micrometers.

8. The microfluidic substrate according to any one of claims 1-6, wherein, The substrate includes a first substrate, which includes the plurality of first portions and the plurality of second portions.

9. The microfluidic substrate according to any one of claims 1-6, wherein, The substrate includes a first substrate and a defining layer located on the first substrate, the defining layer including the plurality of first portions and the plurality of second portions.

10. The microfluidic substrate according to claim 9, further comprising a shielding layer, in, The shielding layer includes a plurality of first openings, each of which corresponds one-to-one with a plurality of microcavity regions. The orthographic projection of each of the plurality of microcavity regions onto the microfluidic substrate and the orthographic projection of a corresponding first opening onto the microfluidic substrate at least partially overlap. Wherein, the orthographic projection of the shielding layer on the microfluidic substrate and the orthographic projection of the defining layer on the microfluidic substrate at least partially overlap.

11. The microfluidic substrate according to any one of claims 1-6, further comprising a spacer region located between any two adjacent microcavity regions in the plurality of microcavity regions and a hydrophobic layer disposed in the spacer region. in, The hydrophobic layer includes a plurality of second openings, and the plurality of microcavity regions correspond one-to-one with the plurality of second openings. The orthographic projection of each microcavity region on the microfluidic substrate falls within the orthographic projection of a second opening corresponding to the microcavity region on the microfluidic substrate.

12. The microfluidic substrate according to claim 11, wherein, The second opening is circular in shape, and the diameter of the second opening is 5 to 20 micrometers larger than the diameter of the top opening.

13. The microfluidic substrate according to claim 11, further comprising a hydrophilic layer, wherein, The hydrophilic layer is located at least within the plurality of microcavity regions, and the orthographic projection of the portion of the hydrophilic layer within each microcavity region onto the microfluidic substrate falls within the orthographic projection of a second opening corresponding to that microcavity region onto the microfluidic substrate.

14. A microfluidic chip, comprising: First substrate; The second substrate is opposite to the first substrate. ； The microfluidic substrate according to any one of claims 1-13 is located between the first substrate and the second substrate; as well as A sealing frame is located between the first substrate and the second substrate, and the orthographic projection of the microfluidic substrate on the first substrate falls within the orthographic projection of the sealing frame on the first substrate.

15. The microfluidic chip according to claim 14, wherein, The sealing frame includes a first side and a second side arranged along a first direction and opposite to each other, and a third side and a fourth side arranged along a second direction different from the first direction and opposite to each other, wherein the first side and the second side are arc-shaped.

16. The microfluidic chip according to claim 15, wherein, The microfluidic substrate includes a first edge and a second edge arranged along the second direction and opposite to each other. The distance between the orthographic projection of the third side of the sealing frame on the first substrate and the orthographic projection of the first edge of the microfluidic substrate on the first substrate is 2 mm to 6 mm. The distance between the orthographic projection of the fourth side of the sealing frame on the first substrate and the orthographic projection of the second edge of the microfluidic substrate on the first substrate is 2 mm to 6 mm.

17. The microfluidic chip according to claim 14, wherein, The distance between the microfluidic substrate and the second substrate is 0.1 mm to 0.3 mm.

18. The microfluidic chip according to claim 14, wherein, The second substrate includes an inlet port and an outlet port, and the orthographic projections of the inlet port and the outlet port on the first substrate fall within the orthographic projection of the sealing frame on the first substrate.

19. The microfluidic chip according to claim 14, wherein, The first substrate includes a second substrate.

20. The microfluidic chip according to any one of claims 14-18, wherein, The first substrate includes: Second substrate; and The heating electrode is located between the second substrate and the microfluidic substrate. In this embodiment, the orthographic projection of the multiple microcavity regions of the microfluidic substrate onto the second substrate falls within the orthographic projection of the heating electrode onto the second substrate.

21. The microfluidic chip according to claim 20, wherein, The orthographic projection of the sealing frame onto the second substrate falls within the orthographic projection of the heating electrode onto the second substrate.

22. The microfluidic chip according to claim 20, wherein, The first substrate further includes: A first dielectric layer is located between the second substrate and the heating electrode; and The second dielectric layer is located between the heating electrode and the microfluidic substrate.

23. The microfluidic chip according to claim 22, wherein, The first substrate further includes a conductive layer located between the second substrate and the first dielectric layer, wherein the conductive layer is electrically connected to the heating electrode via a via in the first dielectric layer.

24. A method for fabricating a microfluidic chip, comprising: Provide a first substrate; Prepare a microfluidic substrate according to any one of claims 1-13; The sealing frame and the microfluidic substrate are fixed on the first substrate such that the orthographic projection of the microfluidic substrate on the first substrate falls within the orthographic projection of the sealing frame on the first substrate. The second substrate is placed on the side of the sealing frame and the microfluidic substrate away from the first substrate; as well as Perform encapsulation processing.

25. The preparation method according to claim 24, wherein, The step of preparing the microfluidic substrate according to any one of claims 1-13 includes: A first substrate is provided and the first substrate is patterned to form the plurality of microcavity regions.

26. The preparation method according to claim 25, wherein, The step of preparing the microfluidic substrate according to any one of claims 1-13 includes: The first substrate is provided, and the thickness of the first substrate is H; A hydrophobic layer is formed on the first substrate; A mask pattern comprising a plurality of exposure holes is formed on the side of the hydrophobic layer away from the first substrate. Each of the plurality of exposure holes is circular in shape and has a diameter of y, where y ranges from 5 micrometers to 30 micrometers. The portion of the first substrate exposed by the plurality of exposed vias is etched to a depth x to form the plurality of microcavity regions, wherein the etched portion within each microcavity region of the first substrate constitutes the first portion, and the unetched portion within each microcavity region of the first substrate constitutes the second portion, wherein x ranges from 20 micrometers to 100 micrometers and x is less than H; and Remove the mask pattern.

27. The preparation method according to claim 26, wherein, The step of preparing the microfluidic substrate according to any one of claims 1-13 includes: Provide a first substrate; A defining film is applied to the first substrate and the defining film is patterned to form the plurality of microcavity regions.

28. A method of using a microfluidic chip as described in any one of claims 14-18, comprising: The sample solution is added into multiple microcavities of the microfluidic chip; The microfluidic chip is heated to cause the sample solution within the multiple microcavities to react; as well as Optical signals emitted by the reacted sample solution within the multiple microcavities are detected using optical equipment.

29. The method of use according to claim 28, wherein, The first substrate includes a second substrate, and the step of heating the microfluidic chip includes: The microfluidic chip was placed in a flat-panel thermal cycler.

30. The method of use according to claim 28, wherein, The first substrate includes a second substrate and a heating electrode located between the second substrate and the microfluidic substrate. The orthographic projection of a plurality of microcavities of the microfluidic substrate onto the second substrate falls within the orthographic projection of the heating electrode onto the second substrate. The step of heating the microfluidic chip includes: An electrical signal is applied to the microfluidic chip to drive the heating electrode to heat the multiple microcavities, and a temperature sensor is used to detect the temperature of the multiple microcavity regions to adjust the current flowing through the heating electrode in real time.

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