Microfluidic substrate and microfluidic chip

Abstract

The present disclosure provides a microfluidic substrate and a microfluidic chip comprising the same. The microfluidic substrate comprises a plurality of microcavities arranged in an array, at least some of the plurality of microcavities being through-holes, and a tangent plane at at least some points on a sidewall of each microcavity forms a non-perpendicular angle with a reference plane on which the microfluidic substrate lies.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomedical detection, and in particular, to a microfluidic substrate and a microfluidic chip comprising the same. BACKGROUND

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments, which can replicate a small amount of deoxyribonucleic acid (DNA) to a large amount, greatly increasing the quantity. Digital Polymerase Chain Reaction (dPCR) technology is a quantitative analysis technology that can provide digital DNA quantification information developed on the basis of PCR, which, in combination with microfluidic technology, greatly improves sensitivity and accuracy. In the dPCR technology, nucleic acid samples are fully diluted so that the number of target molecules (i.e., DNA templates) in each reaction unit is less than or equal to 1. PCR amplification is performed on the target molecules in each reaction unit, and after the amplification, statistical analysis is performed on the fluorescence signals of each reaction unit, thereby realizing absolute quantitative detection of single molecule DNA. Due to the high sensitivity, strong specificity, high detection throughput, accurate quantification and other advantages, dPCR is widely used in clinical diagnosis, gene instability analysis, single-cell gene expression, environmental microbial detection, prenatal diagnosis and other fields. SUMMARY

[0003] According to an aspect of the present disclosure, a microfluidic substrate is provided, which includes a plurality of microcavities arranged in an array. At least some of the plurality of microcavities are through holes, and a tangent plane at at least some points on the sidewall of each microcavity forms a non-perpendicular angle with a reference plane on which the microfluidic substrate is located.

[0004] In some embodiments, the sidewall of each microcavity includes at least one of a curved surface and an inclined surface, and the inclined surface is not perpendicular to the reference plane.

[0005] In some embodiments, each of the plurality of microcavities is a through hole, and each microcavity includes a top opening and a bottom opening.

[0006] In some embodiments, the shape of each microcavity is a circular truncated pyramid or a regular truncated pyramid, and the area of the top opening of each microcavity in the orthographic projection on the reference plane is greater than the area of the bottom opening in the orthographic projection on the reference plane.

[0007] In some embodiments, the angle between the normal line of any point on the sidewall of each microcavity and a reference line perpendicular to the reference plane is 82°-85°.

[0008] In some embodiments, the microfluidic substrate further comprises a hydrophobic layer. The hydrophobic layer is located on opposite first and second surfaces of the microfluidic substrate, a portion of the hydrophobic layer located on the first surface comprises a plurality of first through-holes, and a portion of the hydrophobic layer located on the second surface comprises a plurality of second through-holes. The plurality of first through-holes and the plurality of second through-holes respectively correspond one-to-one to the plurality of microcavities, and a top opening of each of the plurality of microcavities has a footprint on the reference plane that is within a footprint on the reference plane of a first through-hole corresponding to the microcavity, and a bottom opening of each of the plurality of microcavities has a footprint on the reference plane that overlaps a footprint on the reference plane of a second through-hole corresponding to the microcavity.

[0009] In some embodiments, each microcavity is axially symmetric about a symmetry axis that is parallel to the reference plane.

[0010] In some embodiments, each microcavity comprises a first portion and a second portion that are stacked and penetrated each other, the first portion and the second portion are axially symmetric about the symmetry axis, and the first portion and the second portion are one of a circular truncated cone shape and a regular truncated prism shape. An area of a top first opening of the first portion on the reference plane is greater than an area of a bottom second opening of the first portion on the reference plane, and an area of a top third opening of the second portion on the reference plane is less than an area of a bottom fourth opening of the second portion on the reference plane.

[0011] In some embodiments, each microcavity further comprises a third portion between and connecting the first portion and the second portion, the bottom second opening of the first portion is a top fifth opening of the third portion, the top third opening of the second portion is a bottom sixth opening of the third portion, and the third portion is axially symmetric about the symmetry axis.

[0012] In some embodiments, the first portion and the second portion are a circular truncated cone shape, and the third portion is a cylindrical shape; or, the first portion and the second portion are a regular quadrangular truncated prism shape, and the third portion is a cuboid shape.

[0013] In some embodiments, the first portion and the second portion are a circular truncated cone shape, and the third portion is a curved surface body, a vertical distance from any point on a side wall of the third portion to a reference line is greater than a radius of the top fifth opening of the third portion, the reference line passes through centers of the top fifth opening and the bottom sixth opening of the third portion and is perpendicular to the reference plane.

[0014] In some embodiments, the top opening of each microcavity is circular in shape, and the diameter of the circle is 110-130 μm.

[0015] In some embodiments, each microcavity comprises a fourth portion and a fifth portion stacked with each other and penetrating through, the fourth portion and the fifth portion being axisymmetric about the axis of symmetry. The fourth portion and the fifth portion are curved in shape, the top opening and the bottom opening of each microcavity are circular in shape, and the perpendicular distance from any point on the sidewall of each microcavity to the reference line is greater than the radius of the top opening, the reference line passing through the center of the top opening and the bottom opening and being perpendicular to the reference plane.

[0016] In some embodiments, the diameter of the top opening is 210-230 μm.

[0017] In some embodiments, the depth of each microcavity is 300 μm.

[0018] In some embodiments, some of the plurality of microcavities are blind holes.

[0019] In some embodiments, the blind hole is curved in shape, the blind hole comprising an opening, a sidewall, and a bottom, the opening of the blind hole being the top opening of the microcavity and being circular in shape, and the perpendicular distance from any point on the sidewall of the blind hole to the reference line being greater than the radius of the top opening, the reference line passing through the center of the top opening and being perpendicular to the reference plane.

[0020] In some embodiments, the depth of the blind hole is 50-100 μm, and the diameter of the opening of the blind hole is 110-130 μm.

[0021] In some embodiments, the ratio of the maximum value of the perpendicular distance to the radius of the top opening is 1.2:1.

[0022] In some embodiments, the spacing between two adjacent microcavities of the plurality of microcavities is 20-50 μm.

[0023] In some embodiments, the plurality of microcavities are disposed in a glass substrate of the microfluidic substrate.

[0024] In some embodiments, the microfluidic substrate further comprises a heating electrode. The heating electrode is located in the region between two adjacent microcavities on at least one of the first surface and the second surface of the microfluidic substrate.

[0025] In some embodiments, the microfluidic substrate further comprises a hydrophobic layer. The heating electrode is located at a region between two adjacent microcavities on opposite first and second surfaces of the microfluidic substrate, and the hydrophobic layer is located on a side of the heating electrode away from the first surface and a side of the heating electrode away from the second surface.

[0026] In some embodiments, the microfluidic substrate further comprises: a first dielectric layer located on a side of the heating electrode close to the first surface and a side of the heating electrode close to the second surface; a second dielectric layer located on a side of the first dielectric layer away from the first surface and a side of the first dielectric layer away from the second surface; and a conductive layer located between the first and second dielectric layers and arranged at a peripheral edge of the microfluidic substrate, the conductive layer being electrically connected to the heating electrode via a via in the second dielectric layer.

[0027] According to another aspect of the present disclosure, there is provided a microfluidic chip comprising the microfluidic substrate described in any one of the preceding embodiments.

[0028] In some embodiments, the microfluidic chip further comprises a counter substrate opposite to the microfluidic substrate and an encapsulation glue located between the microfluidic substrate and the counter substrate. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly describe the technical solutions in the embodiments of the present disclosure, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0030] Figure 1 A plurality of microcavities of a microfluidic substrate according to an embodiment of the present disclosure are shown;

[0031] Figure 2A A cross-sectional view of a partial structure of a microfluidic substrate according to an embodiment of the present disclosure is shown;

[0032] Figure 2B A structural schematic diagram of a microcavity in Figure 2A is shown;

[0033] Figure 2C Another structural schematic diagram of a microcavity in Figure 2A is shown;

[0034] Figure 3A A cross-sectional view of a partial structure of a microfluidic substrate according to an embodiment of the present disclosure is shown;

[0035] Figure 3B A structural schematic diagram of a microcavity in Figure 3Aa structural diagram of a microcavity in the microfluidic substrate 1000;

[0036] Figure 3C a cross-sectional view of a part of the microfluidic substrate 1000 is shown; Figure 3A a structural diagram of a microcavity in the microfluidic substrate 1000;

[0037] Figure 4A a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0038] Figure 4B a cross-sectional view of a part of the microfluidic substrate 1000 is shown; Figure 4A a structural diagram of a microcavity in the microfluidic substrate 1000;

[0039] Figure 4C a cross-sectional view of a part of the microfluidic substrate 1000 is shown; Figure 4A a structural diagram of a microcavity in the microfluidic substrate 1000;

[0040] Figure 5A a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0041] Figure 5B a cross-sectional view of a part of the microfluidic substrate 1000 is shown; Figure 5A a structural diagram of a microcavity in the microfluidic substrate 1000;

[0042] Figure 6A a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0043] Figure 6B a structural diagram of a microcavity in the microfluidic substrate 1000; Figure 6A a structural diagram of a microcavity in the microfluidic substrate 1000;

[0044] Figure 7A a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0045] Figure 7B a cross-sectional view of a part of the microfluidic substrate 1000 is shown; Figure 7A a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0046] Figure 8A a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0047] Figure 8B a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0048] Figure 9 a cross-sectional view of a part of the microfluidic substrate 1000 is shown;

[0049] Figure 10 a structural diagram of a hydrophobic layer in the microfluidic substrate 1000 is shown; and Figure 9 a structural diagram of a hydrophobic layer in the microfluidic substrate 1000 is shown; and

[0050] Figure 11A structural schematic diagram of a microfluidic chip according to an embodiment of the present disclosure is shown. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by a person of ordinary skill in the art without creative labor are within the protection scope of the present disclosure.

[0052] dPCR is widely used in clinical diagnosis, single-cell analysis, early cancer diagnosis, gene instability analysis, environmental microorganism detection, and prenatal diagnosis, etc. due to its high sensitivity, strong specificity, high detection throughput, and accurate quantification. The principle of dPCR technology, which is an absolute quantification technology for nucleic acid molecules, can be roughly described as follows: a sample solution containing target nucleic acid molecules is sufficiently diluted, and then the diluted sample solution is distributed into a large number of microcavities of a microfluidic chip, so that only one or zero nucleic acid molecules are contained in each microcavity. Then, single-molecule PCR amplification is performed in each microcavity to form a solution to be detected. Then, the fluorescence intensity of the solution to be detected in each microcavity is detected using a fluorescence microscope or a flow cytometer, and finally the number (or concentration) of target nucleic acid molecules in the original sample can be calculated through the number of positive microcavities and Poisson distribution statistics, thereby realizing absolute quantification.

[0053] The microfluidic chip includes a plurality of microcavities with small sizes. At present, there are still many challenges in the application of the microfluidic chip based on the microcavity structure. For example, the abundance of circulating tumor DNA (ctDNA) in blood is usually very low, and the ctDNA usually needs to be enriched when the microfluidic chip is used for ctDNA detection. To reduce this operation, the total reaction volume of the microcavities of the microfluidic chip can be selected to be increased to improve the minimum detection limit of the microfluidic chip. The total reaction volume of the microcavities can improve the minimum detection limit of the microfluidic chip because, according to the Poisson distribution, it is generally considered that c = -ln(b / n) / v, wherein c is the concentration of the sample detection target (unit: copy per microliter), b is the number of microcavities that are negative, n is the total number of microcavities, and v is the volume of a single microcavity (unit: microliter). As can be seen, the greater v is, the lower the lower limit of the detectable target concentration is. However, the volume of a single microcavity is too large, which affects the stability of the fixing effect of the sample solution in the microcavity. Because of the large volume of the microcavity, on the one hand, the ratio of the opening diameter of the microcavity to the depth of the microcavity can be too large, so that the packaging oil is easy to flush the sample solution in the microcavity out of the microcavity or into another adjacent microcavity, causing waste or crosstalk of the sample solution; on the other hand, a larger volume of the microcavity can accommodate more doses of sample solution, but too much sample solution is easy to flow out of the bottom opening of the microcavity under the influence of its own gravity, thereby failing to be stably retained in the microcavity. In addition, in the related art, the sidewall of the microcavity is usually a vertical wall, that is, the sidewall of the microcavity is perpendicular to the surface of the microfluidic chip. The steep sidewall is very unfavorable for the 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 chip, thereby reducing the sample efficiency or even causing waste of the sample solution which is already in a small amount.

[0054] To solve the problems in the related art, the embodiments of the present disclosure provide a microfluidic substrate. The microfluidic substrate provided by the embodiments of the present disclosure can not only perform dPCR detection and analysis on nucleic acids extracted from tumor tissue cells, peripheral blood samples and the like, but also can be applied to digital isothermal amplification, single molecule immunization and other digital analysis biological detection, thereby providing a new choice for popular medical fields such as single cell analysis, early diagnosis of cancer and prenatal diagnosis.

[0055] Figure 1A planar top view of the microfluidic substrate 01 is shown, which includes a plurality of microcavities 02 arranged in an array, at least some of the plurality of microcavities 02 are through holes, and a tangent plane at at least some points on the sidewall of each microcavity 02 forms a non-perpendicular angle with a reference plane on which the microfluidic substrate 01 lies. The plurality of microcavities 02 can all be through holes, or only a part of them are through holes. The inner wall of the microcavity 02 usually has a hydrophilic effect due to the selection of the material (for example, glass), and the microfluidic substrate 01 is usually provided with a hydrophobic layer on the surface which has a hydrophobic effect. Under the action of hydrophilic and hydrophobic and capillary, and because the volume of a single microcavity is very small (on the order of microliters), the sample solution in liquid form can be kept in the microcavity 02 of the through hole structure. It should be noted that in the specification of the present application, the term "sidewall of the microcavity" refers to all the walls around the inside of the microcavity. The microcavity 02 includes a top opening, a bottom opening, and a sidewall connecting the top opening and the bottom opening, and the sidewall of the microcavity 02, together with the top opening and the bottom opening, forms a reaction chamber of the microcavity 02 to accommodate the sample solution. The definition of "tangent plane" in mathematical textbooks is that under certain conditions, there are countless curves passing through a certain point M on the curved surface, and each curve has a tangent line at the point M. Under certain conditions, these tangent lines lie in the same plane, which is called the tangent plane of the curved surface at the point M, and the point M is called the tangent point. Therefore, the phrase "a tangent plane at at least some points on the sidewall of each microcavity 02 forms a non-perpendicular angle with a reference plane on which the microfluidic substrate 01 lies" means that at least a part of the sidewall of each microcavity 02 is not perpendicular to the reference plane (for example, the horizontal plane) on which the microfluidic substrate 01 lies, for example, it can be that all parts of the sidewall of the microcavity 02 are not perpendicular to the reference plane, or it can be that one or more parts of the sidewall of the microcavity 02 are not perpendicular to the reference plane. In other words, at least a part of the sidewall of each microcavity 02 has a certain inclination relative to the reference plane, which inclination angle may, for example, be an acute angle (greater than 0° and less than 90°) or an obtuse angle (greater than 90° and less than 180°).

[0056] By designing the microcavity 02 as a through hole, it is beneficial to make the sample solution enter the inside of the microcavity 02 smoothly under the action of capillary, without stagnating on the surface of the microfluidic substrate 01, causing waste of the sample solution. In addition, some bubbles will inevitably be produced in the sample solution during sampling. By using the through hole design of the microcavity 02, the gas can be discharged from the bottom opening of the microcavity 02, avoiding the bubbles remaining in the microcavity 02, so as not to affect the subsequent fluorescence detection of the sample solution. By making at least a part of the sidewall of the microcavity 02 not perpendicular to the reference plane on which the microfluidic substrate 01 lies, the slope of the sidewall of the microcavity 02 relative to the reference plane can be reduced, which is beneficial to make the sample solution enter the inside of the microcavity 02 quickly along the sidewall without stagnating on the surface of the microfluidic substrate 01, thereby improving the sampling efficiency and improving the utilization rate of the sample solution.

[0057] In some embodiments, the sidewall of each microcavity 02 includes at least one of a curved surface and an inclined surface, and the inclined surface is not perpendicular to the reference plane. The curved surface can be a curved surface with arbitrary curvature (e.g., varying curvature), such as an arc surface, a spherical surface, etc., which is not perpendicular to the reference plane. The inclined surface can be an inclined plane with a certain inclination angle to the reference plane.

[0058] In the following, several embodiments are described respectively to illustrate several different shapes of the microcavity 02 and the arrangement of other film layers. It should be noted that the following description is only illustrative of several different shapes of the microcavity 02, but is not exhaustive of all possible shapes of the microcavity 02. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present disclosure.

[0059] Figure 2A A cross-sectional view of a partial structure of a microfluidic substrate 100 including a plurality of microcavities 101 is shown. In this embodiment, each microcavity 101 of the microfluidic substrate 100 is a through-hole. As shown, the microfluidic substrate 100 includes a substrate 10, which can be any suitable material, including but not limited to glass, silicon, silicon oxide, etc. In one example, the substrate 10 is a glass substrate, and the microcavities 101 are formed in the glass substrate and form through-holes by penetrating through the glass substrate. Or in other words, the glass substrate is etched to form a plurality of through-holes, thereby forming a plurality of microcavities 101. The microcavity 101 includes a top opening 103, a bottom opening 104, and a sidewall 102, which is inclined at a certain inclination angle with respect to the reference plane on which the microfluidic substrate 100 is located. By making the sidewall 102 have a certain inclination angle with respect to the reference plane, it is beneficial to make the sample solution fully fill each microcavity 101 when the sample solution flows.

[0060] Figure 2A The shape of the microcavity 101 can be a circular truncated pyramid or a regular truncated pyramid, which can be a regular quadrangular truncated pyramid, a regular pentagonal truncated pyramid, or a regular polygonal truncated pyramid.

[0061] Figure 2BAs an example, a microcavity 101 with a frustum shape is shown. The microcavity 101 includes a top opening 103, a bottom opening 104, and a sidewall 102. The top opening 103 and the bottom opening 104 are both circular, the top opening 103 has a center O, the bottom opening 104 has a center O', and the area of the top opening 103 in the normal projection on the reference plane is greater than the area of the bottom opening 104 in the normal projection on the reference plane. The sidewall 102 has a certain inclination angle with respect to the reference plane. As shown, any point P on the sidewall 102 of the microcavity 101 has a normal AA' which forms a right triangle with a first reference line BB' and a second reference line CC', wherein the first reference line BB' is perpendicular to the reference plane (also perpendicular to the plane on which the top opening 103 and the bottom opening 104 lie), and the second reference line CC' is parallel to the generatrix of the frustum. The angle between the normal AA' and the first reference line BB' is a, and the angle between the first reference line BB' and the second reference line CC' is β, a and β are complementary angles, a can be an acute angle with any appropriate value, and similarly, β can also be an acute angle with any appropriate value, as long as their sum is 90 degrees. In an example, the angle a between the normal AA' and the first reference line BB' is 82°-85°, that is, the angle β between the first reference line BB' and the second reference line CC' is 5°-8°, so it can be roughly considered that the slope angle of the sidewall of the microcavity 101 is 5°-8°. As mentioned before, by making the sidewall 102 have a certain inclination angle with respect to the reference plane, it is beneficial to make the sample solution flow into the microcavity 101 along the inclined sidewall 102 of the microcavity 101, so that the sample solution can fully fill each microcavity 101.

[0062] Figure 2CAs another example, a microcavity 101 with a shape of a regular quadrangular frustum is shown. The microcavity 101 includes a top opening 103, a bottom opening 104, and a sidewall 102. The top opening 103 and the bottom opening 104 are both squares, and the area of the orthographic projection of the top opening 103 on a reference plane is greater than the area of the orthographic projection of the bottom opening 104 on the reference plane. The sidewall 102 includes four sides, which are congruent isosceles trapezoids. The sidewall 102 has a certain angle of inclination with respect to the reference plane. As shown, any point P on the sidewall 102 of the microcavity 101 has a normal AA' that forms a right triangle with a first reference line BB' and a second reference line CC', where the first reference line BB' is perpendicular to the reference plane (also perpendicular to the plane on which the top opening 103 and the bottom opening 104 lie), and the second reference line CC' is parallel to the side edge of the regular quadrangular frustum. The angle between the normal AA' and the first reference line BB' is a, and the angle between the first reference line BB' and the second reference line CC' is b, a and b are complementary angles, a can be an acute angle with any appropriate value, and similarly, b can also be an acute angle with any appropriate value, as long as their sum is 90 degrees. In one example, the angle a between the normal AA' and the first reference line BB' is 82-85°, that is, the angle b between the first reference line BB' and the second reference line CC' is 5-8°, and thus, it can be considered that the slope angle of each side of the sidewall 102 of the microcavity 101 is 5-8°. As described above, by making the sidewall 102 have a certain angle of inclination with respect to the reference plane, it is beneficial to make the sample solution flow into the microcavity 101 along the inclined sidewall 102 of the microcavity 101, so that the sample solution can fully fill each microcavity 101.

[0063] The thickness of the substrate 10 is about 300 pm, and thus the depth of the microcavity 101 is about 300 pm. In some embodiments, the density of the microcavities 101 of the microfluidic substrate 100 is about 9000 per cm 2 In embodiments in which the top opening 103 of the microcavity 101 is circular, the diameter of the top opening 103 is 110-130 pm, for example, 110 pm, 120 pm, or 130 pm. The size of the microfluidic substrate 100 can be any appropriate size, and the number of the microcavities 101 can be any appropriate number, and embodiments of the present disclosure do not specifically limit the size of the microfluidic substrate 100 and the number of the microcavities 101. In one example, the size of the microfluidic substrate 100 is 5 cm*5 cm, and the number of the microcavities 101 is 100*100.

[0064] Referring back to Figure 2AThe microfluidic substrate 100 can further include a hydrophobic layer 105. The microfluidic substrate 100 includes opposite first and second surfaces 108 and 109, and the hydrophobic layer 105 is located on the opposite first and second surfaces 108 and 109 of the microfluidic substrate 100. The hydrophobic layer 105 has a hydrophobic and oleophilic property, and the material of the hydrophobic layer 105 can be resin or silicon nitride. Since the sample solution is an aqueous liquid, the hydrophobic layer 105 provided on the first and second surfaces 108 and 109 of the microfluidic substrate 100 can prevent the aqueous sample solution from staying on the surface of the microfluidic substrate 100 and promote its entry into the microcavities 101. The part of the hydrophobic layer 105 located on the first surface 108 includes a plurality of first through holes 106, and the part of the hydrophobic layer 105 located on the second surface 109 includes a plurality of second through holes 107. The plurality of first through holes 106 and the plurality of second through holes 107 respectively correspond to the plurality of microcavities 101 one by one, that is, the number of first through holes 106 is the same as the number of microcavities 101, and the number of second through holes 107 is also the same as the number of microcavities 101. The shapes of the first and second through holes 106 and 107 are adapted to the shapes of the top and bottom openings 103 and 104 of the microcavities 101, for example, when the shapes of the top and bottom openings 103 and 104 of the microcavities 101 are circular respectively, the shapes of the first and second through holes 106 and 107 are also circular respectively; when the shapes of the top and bottom openings 103 and 104 of the microcavities 101 are regular polygons respectively, the shapes of the first and second through holes 106 and 107 are also regular polygons respectively. The orthographic projection of the top opening 103 of each microcavity 101 on a reference plane is within the orthographic projection of the first through hole 106 corresponding to the microcavity 101 on the reference plane, and the orthographic projection of the bottom opening 104 of each microcavity 101 on the reference plane overlaps with the orthographic projection of the second through hole 107 corresponding to the microcavity 101 on the reference plane, for example, the orthographic projection of the bottom opening 104 of each microcavity 101 on the reference plane can completely overlap with the orthographic projection of the second through hole 107 corresponding to the microcavity 101 on the reference plane. In other words, the edges of the first through holes 106 of the hydrophobic layer 105 are horizontally apart from the edges of the top openings 103 of the microcavities 101 by a certain distance, and the edges of the second through holes 107 of the hydrophobic layer 105 substantially coincide with the edges of the bottom openings 104 of the microcavities 101 in the vertical direction. By designing the first and second through holes 106 and 107 of the hydrophobic layer 105 in this way, the sample solution can be promoted to enter the microcavities 101 along the side walls 102 of the microcavities 101 and be difficult to flow out of the microcavities 101.

[0065] Figure 3A A cross-sectional view of a part of the structure of the microfluidic substrate 200 is shown, and the microfluidic substrate 200 includes a plurality of microcavities 201. In this embodiment, each microcavity 201 of the microfluidic substrate 200 is a through hole. Unlike the microfluidic substrate 100 in Figure 2A The difference between the microfluidic substrate 200 in Figure 3A The shapes of the microcavities 201 of the microfluidic substrate 200 inFigure 2A the shape of the microcavity 101 of the microfluidic substrate 100 in Figure 3A the arrangement of the hydrophobic layer 105 of the microfluidic substrate 200 in Figure 2A the arrangement of the hydrophobic layer 105 of the microfluidic substrate 100 in

[0066] As shown in Figure 3A each microcavity 201 comprises a first portion 1011 and a second portion 1012 stacked with each other and penetrating through, the first portion 1011 and the second portion 1012 are axially symmetric about a symmetry axis QQ’ parallel to the reference plane. That is, the first portion 1011 is completely overlapped with the second portion 1012 after being flipped 180 degrees about the symmetry axis QQ’. The shape of the first portion 1011 and the second portion 1012 can be one of a circular truncated cone and a regular truncated pyramid, and the regular truncated pyramid can be a regular quadrangular truncated pyramid, a regular pentagonal truncated pyramid or any regular polygonal truncated pyramid.

[0067] Figure 3B The shape of the microcavity 201 is shown as an example, wherein the first portion 1011 and the second portion 1012 of the microcavity 201 are both circular truncated cones. The first portion 1011 comprises a top first opening 110 and a bottom second opening 111, and the second portion 1012 comprises a top third opening 112 and a bottom fourth opening 113, the bottom second opening 111 of the first portion 1011 and the top third opening 112 of the second portion 1012 are the same opening, that is, they are completely overlapped. The top first opening 110 and the bottom second opening 111 of the first portion 1011 and the top third opening 112 and the bottom fourth opening 113 of the second portion 1012 are all circular. The top first opening 110 of the first portion 1011 is the top opening 103 of the microcavity 201, and the bottom fourth opening 113 of the second portion 1012 is the bottom opening 104 of the microcavity 201.

[0068] Figure 3CAs another example, the shape of the microcavity 201 is shown, in which the first portion 1011 and the second portion 1012 of the microcavity 201 are both regular quadrangular pyramids. The first portion 1011 includes a top first opening 110 and a bottom second opening 111, and the second portion 1012 includes a top third opening 112 and a bottom fourth opening 113. The bottom second opening 111 of the first portion 1011 and the top third opening 112 of the second portion 1012 are the same opening, i.e., they completely overlap. The top first opening 110 and the bottom second opening 111 of the first portion 1011 and the top third opening 112 and the bottom fourth opening 113 of the second portion 1012 are all squares. The sidewall 102 of the microcavity 201 includes eight sides, and the eight sides are all congruent isosceles trapezoids. The top first opening 110 of the first portion 1011 is the top opening 103 of the microcavity 201, and the bottom fourth opening 113 of the second portion 1012 is the bottom opening 104 of the microcavity 201.

[0069] In some embodiments, the microcavity 201 has a first portion 1011 and a second portion 1012. The first portion 1011 includes a top first opening 110 and a bottom second opening 111, and the second portion 1012 includes a top third opening 112 and a bottom fourth opening 113. The bottom second opening 111 of the first portion 1011 and the top third opening 112 of the second portion 1012 are the same opening, i.e., they completely overlap. The top first opening 110 and the bottom second opening 111 of the first portion 1011 and the top third opening 112 and the bottom fourth opening 113 of the second portion 1012 are all squares. The sidewall 102 of the microcavity 201 includes eight sides, and the eight sides are all congruent isosceles trapezoids. The top first opening 110 of the first portion 1011 is the top opening 103 of the microcavity 201, and the bottom fourth opening 113 of the second portion 1012 is the bottom opening 104 of the microcavity 201. Figure 3B or Figure 3C In some embodiments, the microcavity 201 has a first portion 1011 and a second portion 1012. The first portion 1011 includes a top first opening 110 and a bottom second opening 111, and the second portion 1012 includes a top third opening 112 and a bottom fourth opening 113. The bottom second opening 111 of the first portion 1011 and the top third opening 112 of the second portion 1012 are the same opening, i.e., they completely overlap. The top first opening 110 and the bottom second opening 111 of the first portion 1011 and the top third opening 112 and the bottom fourth opening 113 of the second portion 1012 are all squares. The sidewall 102 of the microcavity 201 includes eight sides, and the eight sides are all congruent isosceles trapezoids. The top first opening 110 of the first portion 1011 is the top opening 103 of the microcavity 201, and the bottom fourth opening 113 of the second portion 1012 is the bottom opening 104 of the microcavity 201.

[0070] The thickness of the substrate 10 is about 300 μm, so the depth of the microcavity 201 is about 300 μm. In some embodiments, the density of the microcavity 201 of the microfluidic substrate 200 is about 9000 per cm 2 In embodiments in which the top opening 103 of the microcavity 201 is circular, the diameter of the top opening 103 is 110-130 μm, for example, 110 μm, 120 μm, or 130 μm. The size of the microfluidic substrate 200 can be any appropriate size, and the number of microcavities 201 can be any appropriate number, and embodiments of the present disclosure do not specifically limit the size of the microfluidic substrate 200 and the number of microcavities 201. In one example, the size of the microfluidic substrate 200 is 5 cm*5 cm, and the number of microcavities 201 is 100*100.

[0071] Similar to microcavity 101, the sidewall 102 of microcavity 201 has a certain slope angle relative to the reference plane. In one example, the slope angle of the sidewall 102 of microcavity 201 is 5°-8°. By making the sidewall 102 have a certain tilt angle relative to the reference plane, it is beneficial to allow the sample solution to flow into the interior of microcavity 201 along the tilted sidewall 102, so that the sample solution can fully fill each microcavity 201.

[0072] Regarding the hydrophobic layer 105 Figure 3A The arrangement of the hydrophobic layer 105 of the microfluidic substrate 200 shown is similar to... Figure 2A The arrangement of the hydrophobic layer 105 of the microfluidic substrate 100 shown is basically the same. The only difference is that the orthographic projection of the top opening 103 of each microcavity 201 on the reference plane overlaps with the orthographic projection of a first via 106 corresponding to the microcavity 201 on the reference plane, and the orthographic projection of the bottom opening 104 of each microcavity 201 on the reference plane overlaps with the orthographic projection of a second via 107 corresponding to the microcavity 201 on the reference plane.

[0073] Figure 4A A cross-sectional view of a portion of the structure of a microfluidic substrate 300 is shown. The microfluidic substrate 300 includes a plurality of microcavities 301. Figure 4B and 4C Two shapes of the microcavity 301 are shown as examples. Figure 4A-4C The microfluidic substrate 300 shown has the same characteristics as in... Figure 3A-3C The microfluidic substrate 200 shown in the figure has a substantially the same structure, and therefore the same reference numerals are used to refer to the same components. Therefore, Figure 4A-4C It has with Figure 3A-3C For detailed descriptions of the functions and uses of components with the same reference numerals in the accompanying drawings, please refer to the [reference needed]. Figure 3A-3C The explanation will not be repeated here; the differences will only be introduced below.

[0074] like Figure 4A-4C As shown, each microcavity 301 also includes a third portion 1013 located between the first portion 1011 and the second portion 1012 and connecting the first portion 1011 and the second portion 1012, the third portion 1013 being axially symmetric about the axis of symmetry QQ'. Figure 4A-4C Part 1011 and Part 1012 can be referenced. Figure 3A-3C The description of the first part 1011 and the second part 1012 is as follows. Since the first part 1011 and the second part 1012 are also axially symmetric about the axis of symmetry QQ', the microcavity 301 composed of the first part 1011, the second part 1012 and the third part 1013 is axially symmetric about the axis of symmetry QQ'.

[0075] likeFigure 4B As shown, when the shapes of the first part 1011 and the second part 1012 are circular frustum, the shape of the third part 1013 can be a cylinder, and the side wall of the cylinder is perpendicular to the reference plane. As shown in Figure 4C As shown, when the shapes of the first part 1011 and the second part 1012 are right quadrangular frustum, the shape of the third part 1013 can be a cuboid, and the side wall of the cuboid is perpendicular to the reference plane. The third part 1013 includes a top fifth opening 114 and a bottom sixth opening 115, and the top fifth opening 114 and the bottom sixth opening 115 are both square in Figure 4C In the embodiment, the top fifth opening 114 and the bottom sixth opening 115 are both square. The cuboid includes a square cuboid and a rectangular cuboid, and when the height of the cuboid (i.e. the side edge of the third part 1013 between the first part 1011 and the second part 1012) is equal to the side length of the top fifth opening 114 and the bottom sixth opening 115, the cuboid is a square cuboid; and when the height of the cuboid is not equal to the side length of the top fifth opening 114 and the bottom sixth opening 115, the cuboid is a rectangular cuboid.

[0076] As shown in Figure 4B and 4C As shown, the bottom second opening 111 of the first part 1011 of the microcavity 301 is the top fifth opening 114 of the third part 1013, and the top third opening 112 of the second part 1012 of the microcavity 301 is the bottom sixth opening 115 of the third part 1013.

[0077] Similar to the microcavity 201 in Figure 3A , the side wall 102 of the microcavity 301 in Figure 4A has a certain slope angle with respect to the reference plane. In an example, the slope angle of the side wall 102 of the microcavity 301 is 5°-8°. By making the side wall 102 have a certain slope angle with respect to the reference plane, it is beneficial to make the sample solution flow into the microcavity 301 along the inclined side wall 102 of the microcavity 301, so that the sample solution can fully fill each microcavity 301. In addition, compared with the microcavity 201 in Figure 3A , the microcavity 301 in Figure 4A has a middle part 1013, and the existence of the middle part 1013 can increase the cavity volume, so that each microcavity 301 can contain more sample solution, and more doses of the to-be-tested reagent can be obtained after the PCR amplification reaction, so as to improve the minimum detection limit of the microfluidic substrate 300; on the other hand, the side wall of the third part 1013 is perpendicular to the reference plane, so that the sample solution not only can smoothly enter the microcavity 301 along the inclined side wall of the first part 1011 of the microcavity 301 and fully fill each microcavity 301, but also can be stably kept in the microcavity 301 due to the existence of the vertical side wall of the third part 1013, and is not easy to flow out of the cavity.

[0078] The thickness of the substrate 10 is about 300 μm, i.e. the depth of the microcavity 301 is about 300 μm. In some embodiments, the density of the microcavities 301 of the microfluidic substrate 300 is about 9000 per cm 2 In the embodiments in which the top opening 103 of the microcavity 301 is circular, the diameter of the top opening 103 is 110-130 μm, e.g. 110 μm, 120 μm, 130 μm. The size of the microfluidic substrate 300 can be any suitable size, and the number of the microcavities 301 can be any suitable number, and the embodiments of the present disclosure do not make specific limitation on the size of the microfluidic substrate 300 and the number of the microcavities 301. In one example, the size of the microfluidic substrate 300 is 5 cm*5 cm, and the number of the microcavities 301 is 100*100.

[0079] Figure 5A A cross-sectional view showing part of the structure of a microfluidic substrate 400 including a plurality of microcavities 401 is shown. Figure 5B An example shape of the microcavity 401 is shown. In Figure 5A-5B The microfluidic substrate 400 shown in Figure 3A-3C has substantially the same configuration as the microfluidic substrate 200 shown in Figure 5A-5B , and thus the same reference numerals are used to refer to the same components. Thus, Figure 3A-3C the detailed roles and functions of the components having the same reference numerals in Figure 3A-3C are referred to the descriptions of the components having the same reference numerals in , which will not be repeated here, and only the differences will be introduced below.

[0080] As shown in Figure 5A , each microcavity 401 further includes a third portion 1013 located between the first portion 1011 and the second portion 1012 and connecting the first portion 1011 and the second portion 1012, the third portion 1013 being axially symmetrical about the axis of symmetry QQ'. Figure 5A The first portion 1011 and the second portion 1012 in Figure 3A-3C may refer to the descriptions of the first portion 1011 and the second portion 1012 in . Since the first portion 1011 and the second portion 1012 are also axially symmetrical about the axis of symmetry QQ', the microcavity 401 composed of the first portion 1011, the second portion 1012 and the third portion 1013 is axially symmetrical about the axis of symmetry QQ'.

[0081] Figure 5BAs shown, as an example, when the first part 1011 and the second part 1012 are truncated cones, the third part 1013 is a curved solid. The third part 1013 includes a fifth opening 114 at the top and a sixth opening 115 at the bottom, both of which are circular. The term "curved solid" refers to any curved geometric shape in which curved surfaces are involved, also known as a curved solid. The surface of a curved solid can be entirely composed of curved surfaces, such as cylinders and spheres. The surface of a curved solid can also be a combination of curved and flat surfaces. As shown in the figure, the second opening 111 at the bottom of the first part 1011 of the microcavity 401 is the fifth opening 114 at the top of the third part 1013, and the third opening 112 at the top of the second part 1012 of the microcavity 401 is the sixth opening 115 at the bottom of the third part 1013.

[0082] like Figure 5B As shown, the sidewall of the third part 1013 is an arc-shaped surface with a certain curvature, which protrudes outward relative to the fifth opening 114 at the top of the third part 1013. The perpendicular distance S from any point on the sidewall of the third part 1013 to the reference line BB' is greater than the radius R of the fifth opening 114 at the top of the third part 1013. The reference line BB' passes through the center O of the fifth opening 114 at the top of the third part 1013 and the center O' of the sixth opening 115 at the bottom, and is perpendicular to the reference plane. In one example, the maximum value of this perpendicular distance S (e.g., the perpendicular distance from the intersection of the sidewall of the third part 1013 and the axis of symmetry QQ' to the reference line BB') is 1.2:1 to the radius R of the fifth opening 114 at the top. In a conventional microcavity with vertical sidewalls (e.g., a cylindrical microcavity), the perpendicular distance S from any point on the sidewall of the microcavity to the reference line BB' is always equal to the radius R of the microcavity opening. In the microcavity 401 provided in this embodiment, by specially designing the shape of the microcavity 401, the ratio of the vertical distance S from a point on the sidewall of the microcavity to the reference line BB' to the radius R of the microcavity opening varies with the position of the point. This shape design makes it easier for the sample solution flowing on the hydrophobic layer 105 to enter the microcavity 401 and remain stably in the cavity.

[0083] and Figure 4A Similar to the microcavity 301 in the middle, Figure 5A The sidewall 102 of the microcavity 401 has a certain slope angle relative to the reference plane. By making the sidewall 102 tilted relative to the reference plane, it is beneficial for the sample solution to flow into the interior of the microcavity 401 along the tilted sidewall 102, so that the sample solution can fully fill each microcavity 401. In addition, Figure 5AThe microcavity 401 has a central portion 1013. The presence of this central portion 1013 increases the internal volume of the cavity, allowing each microcavity 401 to hold more sample solution. This results in a larger dose of the detection reagent after PCR amplification, thereby improving the minimum detection limit of the microfluidic substrate 400. Furthermore, the sidewalls of the third portion 1013 are outwardly convex arc-shaped surfaces, making it more difficult for sample solution entering the microcavity 401 to flow out along these sidewalls. Therefore, the structural design of the microcavity 401 not only facilitates the entry of sample solution into the microcavity 401 and ensures complete filling of each microcavity 401, but also ensures that the sample solution entering the microcavity 401 remains stably within the microcavity 401 during the detection process, preventing it from being carried out of the microcavity 401.

[0084] The substrate 10 has a thickness of approximately 300 μm, meaning the depth of the microcavity 401 is approximately 300 μm. In some embodiments, the density of the microcavities 401 in the microfluidic substrate 400 is approximately 9000 per cm³. 2 In embodiments where the top opening 103 of the microcavity 401 is circular, the diameter of the top opening 103 is 110-130 μm, for example, 110 μm, 120 μm, or 130 μm. The size of the microfluidic substrate 400 can be any suitable size, and the number of microcavities 401 can be any suitable quantity. The embodiments of this disclosure do not impose specific limitations on the size of the microfluidic substrate 400 or the number of microcavities 401. In one example, the size of the microfluidic substrate 400 is 5 cm * 5 cm, and the number of microcavities 401 is 100 * 100.

[0085] Figure 6A A cross-sectional view of a portion of the structure of a microfluidic substrate 500 is shown, which includes a plurality of microcavities 501. Figure 6B One shape of the microcavity 501 is shown as an example. Figure 6A-6B The microfluidic substrate 500 shown has the same characteristics as in... Figure 3A-3C The microfluidic substrate 200 shown in the figure has a substantially the same structure, and therefore the same reference numerals are used to refer to the same components. Therefore, Figure 6A-6B It has with Figure 3A-3C For detailed descriptions of the functions and uses of components with the same reference numerals in the accompanying drawings, please refer to the [reference needed]. Figure 3A-3C The explanation will not be repeated here; the differences will only be introduced below.

[0086] like Figure 6A-6BAs shown, each microcavity 501 comprises a fourth portion 1014 and a fifth portion 1015 stacked with each other and penetrating through, the fourth portion 1014 and the fifth portion 1015 are axially symmetric about the symmetry axis QQ', so that the microcavity 501 is an axially symmetric pattern. The fourth portion 1014 and the fifth portion 1015 are shaped as curved surface bodies, the shape of the curved surface body of the fourth portion 1014 is substantially the same as that of the third portion 1013. The fourth portion 1014 comprises a top seventh opening 116 and a bottom eighth opening 117, the fifth portion 1015 comprises a top ninth opening 118 and a bottom tenth opening 119, the bottom eighth opening 117 and the top ninth opening 118 are the same opening. The top seventh opening 116 of the fourth portion 1014 is the top opening 103 of the microcavity 501, and the bottom tenth opening 119 of the fifth portion 1015 is the bottom opening 104 of the microcavity 501. The top seventh opening 116 and the bottom eighth opening 117 of the fourth portion 1014 and the top ninth opening 118 and the bottom tenth opening 119 of the fifth portion 1015 are all circular. Figure 5B

[0087] Similarly, Figure 5B Figure 6B In the fourth portion 1014 and the fifth portion 1015, the side wall is an arc surface with a certain curvature, which is more outwardly convex relative to the top seventh opening 116 of the fourth portion 1014. The vertical distance S from any point on the side wall of the fourth portion 1014 or the fifth portion 1015 to the reference line BB' is greater than the radius R of the top seventh opening 116 of the fourth portion 1014, the reference line BB' passes through the center O of the top seventh opening 116 of the fourth portion 1014 and the center O' of the bottom tenth opening 119 of the fifth portion 1015 and is perpendicular to the reference plane. In an example, the ratio of the maximum value of the vertical distance S to the radius R of the top seventh opening 116 is 1.2:1. In a conventional microcavity with a vertical side wall (for example, a cylindrical microcavity), the vertical distance S from any point on the side wall of the microcavity to the reference line BB' is always equal to the radius R of the microcavity opening. In the microcavity 501 provided by the embodiment of the present disclosure, by specially designing the shape of the microcavity 501, the ratio of the vertical distance S from a point on the side wall of the microcavity to the reference line BB' to the radius R of the microcavity opening changes with the position of the point. Such a structural design can make the sample solution flowing on the hydrophobic layer 105 more easily enter the microcavity 501 and stably remain in the cavity.

[0088] ​​The fourth part 1014 and the fifth part 1015 of the microcavity 501 are both curved surfaces, so that the sidewall of the microcavity 501 is an outwardly convex arc surface. This shape design makes it more difficult for the sample solution entering the inside of the microcavity 501 to flow out of the microcavity 501 along the sidewall. Therefore, the structural design of the microcavity 501 can make the sample solution entering the inside of the microcavity 501 stably remain in the microcavity 501 during the detection process and not easily taken out of the microcavity 501.

[0089] The thickness of the substrate 10 is about 300 μm, that is, the depth of the microcavity 501 is about 300 μm. In some embodiments, the density of the microcavity 501 of the microfluidic substrate 500 is about 3500 per cm 2 The diameter of the top opening 103 of the microcavity 501 is 210-230 μm, for example, 210 μm, 220 μm, or 230 μm.

[0090] The above various embodiments introduce that the microfluidic substrate includes a plurality of microcavities, and each microcavity in the plurality of microcavities is a through hole. In alternative embodiments, part of the plurality of microcavities of the microfluidic substrate can be through holes, and the remaining part can be blind holes.

[0091] Figure 7A A plan view of a microfluidic substrate 600 is shown, which includes a plurality of microcavities, part of which are through holes, and the microcavities with through hole shapes can be the microcavities 101, 201, 301, 401, 501 introduced in the previous embodiments; another part of the plurality of microcavities are blind holes 601. Although Figure 7A The microcavities with through hole shapes are arranged adjacent to each other, and the microcavities with blind hole shapes are arranged adjacent to each other, but this is only an example, and the arrangement of the through hole type microcavities and the blind hole type microcavities can be flexibly selected according to actual needs. For example, in alternative embodiments, the through hole type microcavities and the blind hole type microcavities can be alternately arranged.

[0092] Figure 7B A cross-sectional view taken along the II' line in Figure 7A is shown, in which only one blind hole type microcavity 601 is shown. The shape of the blind hole type microcavity 601 is a curved surface, and the shape of the curved surface can be referred to Figure 5B and Figure 6B for description. Unlike the curved surface of Figure 5B and Figure 6B , in Figure 5B and Figure 6B , since the microcavities 401 and 501 are both through holes, the curved surface includes a top opening, a bottom opening, and a sidewall connecting the top opening and the bottom opening. In Figure 7BIn this design, since the microcavity 601 is a blind aperture, the curved surface includes an opening 126, a sidewall 127, and a bottom 128. The sidewall 127 and the bottom 128 together form the reaction chamber of the microcavity 601 to contain the sample solution. The opening 126 of the curved surface is the top opening 103 of the microcavity 601 and is circular in shape, with a center O. Figure 7B In the diagram, the part where the microcavity 601 intersects with the dashed segments DD' and EE' is the sidewall 127 of the microcavity 601, and the remaining bottom part is the bottom 128 of the microcavity 601.

[0093] like Figure 7B As shown, the sidewall 127 of the microcavity 601 is an arc-shaped surface with a certain curvature, which protrudes outward relative to the top opening 103 of the microcavity 601. The vertical distance S from any point on the sidewall 127 of the microcavity 601 to the reference line BB' is greater than the radius R of the top opening 103 of the microcavity 601. The reference line BB' passes through the center O of the top opening 103 of the microcavity 601 and is perpendicular to the reference plane. In one example, the maximum value of this vertical distance S (e.g., the vertical distance from the maximum curvature of the sidewall 127 to the reference line BB') is 1.2:1 to the radius R of the top opening 103. In conventional microcavities with vertical sidewalls (e.g., cylindrical microcavities), the vertical distance S from any point on the sidewall of the microcavity to the reference line BB' is always equal to the radius R of the microcavity opening. In the microcavity 601 provided in this embodiment, by specially designing the shape of the microcavity 601, the ratio of the vertical distance S from a point on the sidewall of the microcavity to the reference line BB' to the radius R of the microcavity opening varies with the position of the point. This structural design makes it easier for the sample solution to enter the microcavity 601 and remain stably in the cavity.

[0094] Because the microcavity 601 is a blind cavity with a certain curvature on its sidewalls, the sample solution can be stably held inside the cavity during detection and is not easily carried out of the cavity after flowing into it. Furthermore, if bubbles are generated during the flow of the sample solution into the microcavity 601, the microcavity 601 can adsorb these bubbles on its sidewall 127, preventing them from mixing with the sample solution inside the cavity and thus avoiding interference with subsequent fluorescence detection of the sample solution.

[0095] In some embodiments, the depth of the microcavity 601 is 50-100 μm, for example, 50 μm, 75 μm, or 100 μm. In some embodiments, the diameter of the top opening 103 of the microcavity 601 is 110-130 μm, for example, 110 μm, 120 μm, or 130 μm.

[0096] like Figure 7BAs shown, the microfluidic substrate 600 further comprises a hydrophobic layer 105, which is only disposed on the first surface 108 of the microfluidic substrate 600. The shape of the first via hole 106 of the hydrophobic layer 105 is adapted to the shape of the top opening 103 of the microcavity 601, and the orthographic projection of the top opening 103 of each microcavity 601 on the reference plane and the orthographic projection of one first via hole 106 corresponding to the microcavity 601 on the reference plane overlap, for example, can completely overlap.

[0097] In Figure 7A and 7B In the embodiment shown, the microfluidic substrate 600 comprises a plurality of microcavities 601, part of which are through holes, and the through hole type microcavity can be the microcavity 101, 201, 301, 401, 501 described in any one of the preceding embodiments, and the other part of the plurality of microcavities 601 are blind holes, i.e. the microcavity 601. Therefore, the microfluidic substrate 600 combines all the advantages of through hole microcavities and blind hole microcavities, which not only facilitates the rapid entry of the sample solution into the microcavity, but also can make the sample solution stable in the cavity and not easily taken out of the cavity.

[0098] Figure 8A An arrangement of a plurality of microcavities on a microfluidic substrate is shown, which can be the microcavity 101, 201, 301, 401, 501, 601 described in any one of the preceding embodiments or any combination thereof, and in this figure, the openings of the microcavities are taken as an example of a regular hexagon. As shown, the plurality of microcavities are arranged in a two-dimensional hexagonal close-packed manner on the microfluidic substrate, and the spacing between any two adjacent microcavities in the plurality of microcavities is 20-50um, for example, 20um, 30um, 40um, 50um. The term "two-dimensional hexagonal close-packed" means that the plurality of microcavities are arranged in a honeycomb-like manner on the microfluidic substrate to maximize the use of space area, but it is necessary to ensure that there is a suitable spacing between the microcavities to avoid mutual interference between the microcavities. As Figure 8A As shown by the dashed box in the figure, the two-dimensional hexagonal close-packed arrangement makes the line connecting the centers of the six adjacent microcavities form a regular hexagon, and another microcavity is arranged at the center of the regular hexagon, and the center of the microcavity at the center of the regular hexagon coincides with the center of the regular hexagon.

[0099] Figure 8BAnother arrangement of a plurality of microcavities on a microfluidic substrate is shown, which can be the microcavity 101, 201, 301, 401, 501, 601 or any combination thereof described in any of the previous embodiments. In this figure, the opening of the microcavity is taken as an example of a circle. As shown, the plurality of microcavities are arranged in a two-dimensional square lattice on the microfluidic substrate, and the spacing between any two adjacent microcavities in the plurality of microcavities is 20-50um, for example 20um, 30um, 40um, 50um. The term "two-dimensional square lattice" means that the plurality of microcavities are regularly arranged on the microfluidic substrate, and the intersection of two adjacent rows of microcavities and two adjacent columns of microcavities is four microcavities, and the line connecting the centers of the bottoms of the four microcavities forms a square. This arrangement of microcavities can maximize the use of space while ensuring appropriate spacing between microcavities to avoid interference between microcavities.

[0100] In the dPCR reaction, the double-stranded structure of the DNA fragment is denatured into a single-stranded structure at high temperature (e.g. 90°C), the primer binds to the single strand according to the base complementary pairing principle at low temperature (e.g. 65°C), and the base binding and extension is realized at the optimum temperature of DNA polymerase (e.g. 72°C). The above process is the temperature cycle process of denaturation, annealing and extension. Through multiple temperature cycles of denaturation, annealing and extension, the DNA fragment can be replicated in large quantities. In order to realize the above temperature cycle process, in the related art, a series of external devices are required to heat and cool the microfluidic device, which makes the device bulky, the operation complex, the integration of the microfluidic device low, and the cost expensive.

[0101] To solve the above problems, as shown in Figure 9 An embodiment of the present disclosure provides a microfluidic substrate 700, which includes a microcavity and a heating electrode 121. The microcavity can be any one of the microcavities 101, 201, 301, 401, 501, 601 described in the previous embodiments or any combination thereof, and has the corresponding advantages. Figure 9 Taking the microfluidic substrate 700 including the microcavity 401 as an example.

[0102] The heating electrode 121 can be located on at least one of the opposite first surface 108 and the second surface 109 of the substrate 10, and located in the region between the adjacent two microcavities 401. For example, the heating electrode 121 can be located only on the first surface 108 of the substrate 10, only on the second surface 109 of the substrate 10, or on both the opposite first surface 108 and the second surface 109 of the substrate 10. The heating electrode 121 is configured to heat the microcavity 401 to provide a suitable temperature for the reaction of the sample solution in the microcavity 401. Figure 9The diagram shows heating electrodes 121 located on opposing first surfaces 108 and second surfaces 109 of substrate 10, in the region between two adjacent microcavities 401. Arranging heating electrodes 121 on both sides of substrate 10 provides superior heating performance. Heating electrodes 121 can receive electrical signals (e.g., voltage signals), thereby generating heat when current flows through them. This heat can be conducted to adjacent microcavities 401 for use in polymerase chain reactions. For example, heating electrodes 121 can be fabricated using conductive materials with high resistivity, allowing them to generate significant heat with relatively small electrical signals, thus improving energy conversion efficiency. Heating electrodes 121 can be fabricated, for example, using transparent conductive materials such as indium tin oxide (ITO), tin oxide, etc., or other suitable materials such as metals; the embodiments disclosed herein are not limited to these methods.

[0103] In the embodiments of this disclosure, by integrating the heating electrode 121 in the microfluidic substrate 700, the microcavity 401 of the microfluidic substrate 700 can be effectively heated, thereby achieving temperature control of the microcavity 401 without the need for external heating equipment. Therefore, it has high integration, small size, simple operation, and can reduce costs.

[0104] like Figure 9 As shown, the microfluidic substrate 700 may further include a hydrophobic layer 122, wherein the hydrophobic layer 122 is located on the side of the heating electrode 121 away from the first surface 108 and on the side away from the second surface 109. The hydrophobic layer 122 has hydrophobic and oleophilic properties, which facilitates the flow of aqueous sample solution flowing on it into the microcavity 401. Figure 10 A schematic diagram of one structure of the hydrophobic layer 122 is shown, wherein the circular opening is a microcavity 401, and the hydrophobic layer 122 is disposed in the region between two adjacent microcavities 401. The material of the hydrophobic layer 122 can be resin or silicon nitride. In one example, the material of the hydrophobic layer 122 is SiN.

[0105] The microfluidic substrate 700 can further include a conductive layer 125 located on the side of the heating electrode 121 close to the first surface 108 and the second surface 109, and arranged around the four edges of the microfluidic substrate 700. The conductive layer 125 is electrically connected to the heating electrode 121. The conductive layer 125 is configured to apply an electrical signal (e.g., a voltage signal) to the heating electrode 121, and the heating electrode 121 can generate heat under the action of the electrical signal after receiving the electrical signal, so as to heat the microcavity 401. The resistance value of the heating electrode 121 can be greater than the resistance value of the conductive layer 125, so that the heating electrode 121 generates more heat and the conductive layer 125 generates less heat under the action of the same electrical signal, thereby reducing energy loss. For example, the conductive layer 125 can be made of a material with a small resistivity, thereby reducing energy loss on the conductive layer 125. The conductive layer 125 can be made of a metal material, for example, molybdenum (Mo), copper or copper alloy, aluminum or aluminum alloy, etc., which can be a single metal layer or a composite metal layer. The embodiments of the present disclosure do not limit this.

[0106] The microfluidic substrate 700 can further include a first dielectric layer 123 and a second dielectric layer 124. The first dielectric layer 123 is located on the side of the heating electrode 121 close to the first surface 108 and the second surface 109; the second dielectric layer 124 is located on the side of the first dielectric layer 123 away from the first surface 108 and the second surface 109. The conductive layer 125 is located between the first dielectric layer 123 and the second dielectric layer 124, and can be electrically connected to the heating electrode 121 via a via in the second dielectric layer 124. The first dielectric layer 123 and the second dielectric layer 124 can be any appropriate material, and the embodiments of the present disclosure do not limit this. In one example, the material of the first dielectric layer 123 is SiN, and the material of the second dielectric layer 124 is SiO.

[0107] According to another aspect of the present disclosure, a microfluidic chip is provided. Figure 11 A structural schematic diagram of a microfluidic chip 800 is shown, which includes a microfluidic substrate 801, which can be any one of the microfluidic substrates 100, 200, 300, 400, 500, 600, 700 described in the foregoing embodiments.

[0108] The microfluidic chip 800 can further include an opposing substrate 802 opposite to the microfluidic substrate 801, and an encapsulating glue 803 between the microfluidic substrate 801 and the opposing substrate 802.

[0109] In one example, the microfluidic substrate 801 and the counter substrate 802 each comprises a glass substrate. The microfluidic substrate 801 and the counter substrate 802 are oppositely arranged to provide protection, support, isolation, etc. The microfluidic chip 800 is manufactured by using a micro-processing method combining semiconductor technology and glass substrate, so that mass production can be realized and the production cost can be greatly reduced.

[0110] The encapsulation glue 803 is configured to seal the microfluidic substrate 801 and the counter substrate 802, and configured to maintain a proper interval between the microfluidic substrate 801 and the counter substrate 802 to provide sufficient space for the flow of sample solution.

[0111] The microfluidic chip 800 can have substantially the same technical effects as the microfluidic substrates described in the above various embodiments, and therefore, for the purpose of brevity, the technical effects of the microfluidic chip 800 will not be described again.

[0112] Still another aspect of the present disclosure provides a method 900 for manufacturing a microfluidic substrate. The different microfluidic substrates described in the above various embodiments have substantially the same manufacturing steps, except for some differences in the details of certain steps. The method steps will be described below by taking the microfluidic substrate 100 shown in the figures as an example. Figure 2A-2C

[0113] Step 901: providing a substrate 10 and cleaning it. The substrate 10 can be made of any suitable material, and in one example, the substrate 10 is made of glass. The substrate 10 can have any suitable thickness, and in one example, the thickness of the substrate 10 is 300 μm.

[0114] Step 902: preparing a mark on the substrate 10 to provide positioning function for subsequent microcavity etching and cutting of the substrate. In one example, the mark is formed as follows: under the conditions of a sputtering chamber temperature of about 230°C, a volume flow rate of Ar of about 100 sccm (standard cubic centimeter per minute), a pressure of about 0.3 Pa, a power of about 12 KW, and a scanning frequency of about 15 scans, a metal Mo film layer with a thickness of about 2200 Å is sputtered on the surface of the substrate 10, and the Mo film layer is exposed, developed, and etched by using a photolithography process to form a metal mark.

[0115] ​Step 903: depositing an insulating film layer on the first surface 108 of the substrate 10, and exposing, developing and etching the insulating film layer to form the hydrophobic layer 105. In an example, the hydrophobic layer 105 is formed as follows: in a plasma enhanced chemical vapor deposition (PECVD) device, at a temperature of about 390°C, a power of about 600 W, a pressure of about 1200 mtorr, and a distance between a target material in the PECVD device and a sample to be deposited of about 1000 mils, SiH4 (volume flow rate of about 140 sccm), NH3 (volume flow rate of about 700 sccm) and N2 (volume flow rate of about 2260 sccm for about 225 seconds) are introduced into the reaction chamber to deposit a SiN x film layer on the first surface 108 of the substrate 10 with a thickness of about 3000 Å, and exposing, developing and etching the SiN x film layer to form the hydrophobic layer 105.

[0116] Step 904: forming a first metal mask on the side of the hydrophobic layer 105 away from the first surface 108, which is used to provide isolation protection for other parts of the microfluidic substrate except the microcavity in subsequent etching of the microcavity. In an example, the first metal mask is formed as follows: under the conditions of a sputtering chamber temperature of about 230°C, a volume flow rate of Ar of about 100 sccm, a pressure of about 0.3 Pa, a power of about 12 KW, and a scanning frequency of about 15 scans, a metal Mo film layer with a thickness of about 2200 Å is sputtered on the side of the hydrophobic layer 105 away from the first surface 108, and a photolithography process is used to expose, develop and etch the Mo film layer to form the first metal mask. The first metal mask includes a plurality of vias corresponding to the positions of the plurality of microcavities to be formed later and having the same shape to expose the areas where the microcavities are to be etched and formed later.

[0117] Step 905: sequentially forming a hydrophobic layer 105 and a second metal mask on the second surface 109 of the substrate 10 by mark alignment, the position of the hydrophobic layer 105 on the second surface 109 completely corresponds to the position of the hydrophobic layer 105 on the first surface 108, and the position of the second metal mask on the second surface 109 completely corresponds to the position of the first metal mask on the first surface 108. The preparation methods of the hydrophobic layer 105 and the second metal mask on the second surface 109 are the same as those in steps 903 and 904.

[0118] Step 906: The substrate 10 is etched using dry etching to form multiple microcavities 101, all of which are through-holes. In one example, the process of forming multiple microcavities 101 using dry etching is as follows: Using inductively coupled plasma etching (ICP), the substrate 10 is etched for approximately 375 minutes under the following conditions: power approximately 2500 W, temperature approximately 20 °C, pressure approximately 0.6 Pa, C4F8 flow rate approximately 60 ml / min, Ar flow rate approximately 120 ml / min, and etching rate approximately 0.8 μm / min, to form multiple microcavities 101. The top opening 103 of the microcavities 101 formed by dry etching can be circular or a regular polygon. When the top opening 103 of the microcavities 101 is circular, the diameter of the top opening 103 is approximately 110-130 μm, for example, 120 μm, and the density of microcavities 101 is approximately 9000 / cm³. 2 The depth of microcavity 101 is 300 μm, and the spacing between two adjacent microcavities 101 is 20-50 μm. The sidewalls 102 of microcavity 101 have a certain tilt angle, which facilitates the full filling of microcavity 101 by the sample solution during injection flow. For the specific technical effects of microcavity 101, please refer to the previous section on... Figure 2A-2C The description will not be repeated here.

[0119] Step 907: After etching the microcavity 101, remove the first metal mask and the second metal mask.

[0120] Step 908: Place the etched microfluidic substrate 100 on the base, and use a specific tool (e.g., a scraper) to slide the sample solution in the same direction to fill the microcavity 101. After filling, drop mineral oil onto the top opening 103 of the microcavity 101. At this time, fix the encapsulating adhesive to the base, and simultaneously fix the opposing substrate to the encapsulating adhesive to form a microfluidic device. Then, fill the sample inlet of the microfluidic device with mineral oil and seal the sample inlet to achieve encapsulation of the microfluidic device.

[0121] Figure 3A-3C The manufacturing method of the microfluidic substrate 200 shown is similar to... Figure 2A-2C The manufacturing methods of the microfluidic substrate 100 shown are basically the same, with differences only in a few steps. The same method steps can be found in the description of the manufacturing method of the microfluidic substrate 100; the differences in the manufacturing method of the microfluidic substrate 200 will be described below.

[0122] The microfluidic substrate 200 is prepared using the exact same method steps and manufacturing sequence as steps 901-905.

[0123] Then, in step 906A, the etching method for the microcavities 201 of the microfluidic substrate 200 is slightly different from the etching method for the microcavities 101 of the microfluidic substrate 100. The process of etching the substrate 10 using dry etching to form multiple microcavities 201 is as follows: Using the ICP method, under the conditions of approximately 2500W power, approximately 20°C temperature, approximately 0.6Pa pressure, approximately 60ml / min C4F8 flow rate, approximately 120ml / min Ar flow rate, and approximately 0.8µm / min within the reaction chamber, one side of the substrate 10 is first etched for approximately 188 minutes to form the first portion 1011 of the microcavities 201 in the substrate 10; then the other side of the substrate 10 is etched for approximately 188 minutes to form the second portion 1012 of the microcavities 201 in the substrate 10, thereby forming multiple microcavities 201. The top opening 103 of the microcavity 201 formed by dry etching can be circular or a regular polygon. When the top opening 103 of the microcavity 201 is circular, the diameter of the top opening 103 is approximately 110-130 μm, for example, 120 μm, and the density of the microcavities 201 is approximately 9000 / cm³. 2 The depth of microcavity 201 is 300 μm, and the spacing between two adjacent microcavities 201 is 20-50 μm. The sidewall 102 of microcavity 201 has a certain tilt angle, which is beneficial for the sample solution to fully fill the microcavity 201 during injection flow. For the specific technical effects of microcavity 201, please refer to the previous section on... Figure 3A-3C The description will not be repeated here.

[0124] Then, the microfluidic substrate 200 is prepared using the exact same method steps and manufacturing sequence as steps 907-908 to complete the encapsulation.

[0125] Figure 4A-4C The manufacturing method of the microfluidic substrate 300 shown is similar to... Figure 2A-2C The manufacturing methods of the microfluidic substrate 100 shown are basically the same, with differences only in a few steps. The same method steps can be found in the description of the manufacturing steps of the microfluidic substrate 100; the differences in the manufacturing method of the microfluidic substrate 300 will be described below.

[0126] The microfluidic substrate 300 is prepared using the exact same method steps and manufacturing sequence as steps 901-905.

[0127] Then, in step 906B, the etching method for the microcavities 301 of the microfluidic substrate 300 is slightly different from the etching method for the microcavities 101 of the microfluidic substrate 100. Similarly, the process of etching the substrate 10 using dry etching to form multiple microcavities 301 is as follows: Using the ICP method, under the conditions of approximately 2500W power, approximately 20°C temperature, approximately 0.6Pa pressure, approximately 60ml / min C4F8 flow rate, approximately 120ml / min Ar flow rate, and approximately 0.8µm / min within the reaction chamber, one side of the substrate 10 is first etched for approximately 125 minutes to form the first portion 1011 of the microcavities 301 in the substrate 10; then the other side of the substrate 10 is etched for approximately 125 minutes to form the second portion 1012 of the microcavities 301 in the substrate 10. Then, using laser etching, a suitable laser spot is selected and positioned at the center of each microcavity for ablation to form the third part 1013, thereby forming multiple microcavities 301. The top opening 103 of the microcavities 301 formed by dry etching can be circular or a regular polygon. When the top opening 103 of the microcavities 301 is circular, the diameter of the top opening 103 is approximately 110-130 μm, for example, 120 μm, and the density of the microcavities 301 is approximately 9000 / cm³. 2 The depth of microcavity 301 is 300 μm, and the spacing between two adjacent microcavities 301 is 20-50 μm. The sidewall 102 of microcavity 301 has a certain tilt angle, which is beneficial for the sample solution to fully fill the microcavity 301 during injection flow. For the specific technical effects of microcavity 301, please refer to the previous section on... Figure 4A-4C The description will not be repeated here.

[0128] Then, the microfluidic substrate 300 is prepared using the exact same method steps and manufacturing sequence as steps 907-908 to complete the encapsulation.

[0129] Figure 5A-5B The manufacturing method of the microfluidic substrate 400 shown is similar to... Figure 2A-2C The manufacturing methods of the microfluidic substrate 100 shown are basically the same, with differences only in a few steps. The same method steps can be found in the description of the manufacturing steps of the microfluidic substrate 100; the differences in the manufacturing method of the microfluidic substrate 400 will be described below.

[0130] The microfluidic substrate 400 is prepared using the exact same method steps and manufacturing sequence as steps 901-905.

[0131] Then at step 906C, the etching method of the microcavities 401 of the microfluidic substrate 400 is slightly different from the etching method of the microcavities 101 of the microfluidic substrate 100. The process of forming the plurality of microcavities 401 is as follows: first, using the ICP method, under the conditions that the power in the reaction chamber is about 2500W, the temperature is about 20°C, the pressure is about 0.6Pa, the flow rate of C4F8 is about 60ml / min, the flow rate of Ar is about 120ml / min, and the etching speed is about 0.8um / min, the first side of the substrate 10 is etched first to form the first part 1011 of the microcavity 401 in the substrate 10, and the etching time is about 60 minutes; then the other side of the substrate 10 is etched to form the second part 1012 of the microcavity 401 in the substrate 10, and the etching time is about 60 minutes. Then, a third metal mask is formed on the microfluidic substrate 400, which is used to provide isolation protection for the first and second parts of the microcavity 401 that have been formed and other parts of the microfluidic substrate 400 during the subsequent etching of the third part of the microcavity. In one example, the process of forming the third metal mask is as follows: sputtering a Mo film layer with a thickness of about 2200Å on the microfluidic substrate 400 under the conditions that the sputtering chamber temperature is about 230°C, the volume flow rate of Ar is about 100sccm, the pressure is about 0.3Pa, the power is about 12KW, and the scanning frequency is about 15scan, and then using photolithography process to expose, develop and etch the Mo film layer to form the third metal mask. The third metal mask covers the areas that need to be protected, while exposing the third part of the microcavity that needs to be etched subsequently. Then, the third part 1013 of the microcavity 401 is formed by wet etching. The specific steps can be described as follows: immerse the microfluidic substrate 400 in the etching solution, the concentration of hydrogen fluoride (HF) in the etching solution is about 40%, the etching speed is about 3.5um / min, and the etching solution is continuously stirred by a blade during etching to make the etching solution more uniformly etch the substrate 10 of the microfluidic substrate 400. The etching time needs about 30 minutes to etch and form the third part 1013 of the microcavity 401, thereby forming the microcavity 401. The microcavity 401 is formed by combining dry etching and wet etching. Due to the isotropic property of wet etching, the shape of the top opening 103 of the microcavity 401 is usually circular, the diameter of the top opening 103 is about 110-130μm, for example, 120um, the density of the microcavities 401 is about 9000 / cm 2 , and the depth of the microcavities 401 is 300μm. The spacing between two adjacent microcavities 401 is 20-50μm. The sidewall 102 of the microcavity 401 has a certain inclination angle, which is beneficial to fully fill the microcavity 401 with the sample solution when the sample solution flows, and the sample solution is easy to stably remain during the detection process and is not easy to be taken out of the microcavity 401. The specific technical effects of the microcavity 401 can be referred to the description of the microcavity 101 in the foregoing Figure 5A-5B , which will not be described here again.

[0132] Then in step 907C, after the etching of the microcavity 401 is completed, the first metal mask, the second metal mask and the third metal mask are removed.

[0133] Finally, the microfluidic substrate 400 is prepared by using the same method steps as step 908 to complete the packaging.

[0134] Figure 6A-6B The manufacturing method of the microfluidic substrate 500 shown is basically the same as the manufacturing method of the microfluidic substrate 100, and only differs in individual steps. The same method steps can refer to the description of the manufacturing steps of the microfluidic substrate 100, and only the differences in the manufacturing method of the microfluidic substrate 500 are introduced below. Figure 2A-2C The manufacturing method of the microfluidic substrate 100 shown is basically the same as the manufacturing method of the microfluidic substrate 100, and only differs in individual steps. The same method steps can refer to the description of the manufacturing steps of the microfluidic substrate 100, and only the differences in the manufacturing method of the microfluidic substrate 500 are introduced below.

[0135] The microfluidic substrate 500 is prepared by using the same method steps and manufacturing sequence as steps 901-905.

[0136] Then in step 906D, the etching method of the microcavity 501 of the microfluidic substrate 500 is slightly different from the etching method of the microcavity 101 of the microfluidic substrate 100. The process of forming a plurality of microcavities 501 is as follows: the microcavities 501 are etched by using a wet etching method. The specific steps can be described as follows: the microfluidic substrate 500 is immersed in an etching solution, the concentration of hydrogen fluoride (HF) in the etching solution is about 40%, the etching speed is about 3.5 um / min, and the first surface 108 and the second surface 109 of the substrate 10 are etched at the same time. The etching solution is continuously stirred by using a blade during etching to make the etching solution more uniformly etch the substrate 10 of the microfluidic substrate 500. The etching time needs about 60 minutes to etch the fourth part 1014 and the fifth part 1015 of the microcavity 501 to form the microcavity 501. The microcavity 501 is formed by using a wet etching method, and due to the isotropic properties of wet etching, the shape of the top opening 103 of the microcavity 501 is usually circular, the diameter of the top opening 103 is about 210-230 μm, for example, 220 um, and the density of the microcavity 501 is about 3500 / cm 2 The depth of the microcavity 501 is 300 μm, and the spacing between two adjacent microcavities 501 is 20-50 μm. The structure of the microcavity 501 is beneficial to stably keep the sample solution in the cavity during the detection process and is not easy to be taken out of the microcavity 501. The specific technical effects of the microcavity 501 can refer to the description of the microcavity 101 in the foregoing, which will not be described here. Figure 6A-6B

[0137] Finally, the microfluidic substrate 500 is prepared by using the same method steps and manufacturing sequence as steps 907-908 to complete the packaging.

[0138] Figure 7A-7B ​The manufacturing method of the microfluidic substrate 600 shown is substantially the same as that of the microfluidic substrate 100 shown, with only differences in individual steps. Since the microfluidic substrate 600 includes a plurality of microcavities, some of which are through-holes and some of which are blind holes. The manufacturing method of the through-hole microcavity can refer to the description above, which will not be repeated here. Only the manufacturing method of the blind hole microcavity 601 will be introduced below. Figure 2A-2C The manufacturing method of the microfluidic substrate 100 shown is substantially the same, with only differences in individual steps. Since the microfluidic substrate 600 includes a plurality of microcavities, some of which are through-holes and some of which are blind holes. The manufacturing method of the through-hole microcavity can refer to the description above, which will not be repeated here. Only the manufacturing method of the blind hole microcavity 601 will be introduced below.

[0139] The microfluidic substrate 600 is prepared using the same method steps and manufacturing sequence as steps 901-905.

[0140] Then at step 906E, the etching method of the microcavity 601 of the microfluidic substrate 600 is slightly different from that of the microcavity 101 of the microfluidic substrate 100. The process of forming a plurality of microcavities 601 is as follows: etching is used to form microcavities 601. The specific steps can be described as follows: the microfluidic substrate 600 is immersed in an etching solution, the concentration of hydrogen fluoride (HF) in the etching solution is about 40%, the etching speed is about 3.5um / min, and the first surface 108 of the substrate 10 is etched. The etching solution is continuously stirred using a blade during etching to make the etching solution more uniformly etch the substrate 10 of the microfluidic substrate 600. The etching time needs about 30 minutes to form the microcavity 601, which is a blind hole. This microcavity 601 is formed by using wet etching, and due to the isotropic properties of wet etching, the shape of the top opening 103 of the microcavity 601 is usually circular, the diameter of the top opening 103 is about 110-130μm, for example 120um, and the density of the microcavity 601 is about 9000 / cm 2 The depth of the microcavity 601 is about 50-100μm, and the spacing between two adjacent microcavities 601 is about 20-50μm. The blind hole structure of the microcavity 601 is beneficial to keep the sample solution stable in the cavity during the detection process and not easily taken out of the microcavity 601. The specific technical effects of the microcavity 601 can refer to the description above, which will not be repeated here. Figure 7A-7B

[0141] Finally, the microfluidic substrate 600 can be prepared using the same method steps and manufacturing sequence as steps 907-908 to complete the packaging. In alternative embodiments, the packaging method of step 908 can also be the following process: a frame is formed around the periphery of the etched microfluidic substrate 600 using UV glue mixed with spacers or oil-resistant adhesive film, and then the opposing substrate is bonded to the microfluidic substrate 600 to form a microfluidic device. After the sample solution is added through the sample inlet hole on the opposing substrate, mineral oil is then added from the sample inlet hole, and after the internal space is completely filled, the sample inlet hole and the sample outlet hole of the microfluidic device are closed.

[0142] Figure 9 ​The method of fabricating the microfluidic substrate 700 is substantially the same as Figure 5A-5B The method of fabricating the microfluidic substrate 400 is substantially the same, with only differences in individual steps. The same method steps can be referred to the description of the method steps of fabricating the microfluidic substrate 400, and only the differences of the method of fabricating the microfluidic substrate 700 are introduced below.

[0143] First, the microfluidic substrate 700 is fabricated using the same method steps and fabrication sequence as steps 901-905. It is noted that the hydrophobic layer 105 formed in steps 903 and 905 does not serve as the hydrophobic layer of the microfluidic substrate 700, but is used as the first dielectric layer 123, except that the method and material of forming the first dielectric layer 123 are the same as the method and material of forming the hydrophobic layer 105 in steps 903 and 905.

[0144] Then, the microcavities 401 of the microfluidic substrate 700 are fabricated using the same method as steps 906C and 907C of the microfluidic substrate 400.

[0145] After step 907C is completed, a metal layer is deposited on the side of the first dielectric layer 123 away from the first surface 108 and the second surface 109, and the metal layer is patterned to form the conductive layer 125, which is arranged around the four peripheral edges of the microfluidic substrate 700. In one example, the conductive layer 125 is a Mo-AlNd-Mo stack structure, and the corresponding film thicknesses are 200Å, 3000Å and 800Å, respectively.

[0146] Then, an insulating layer is deposited on the side of the first dielectric layer 123 away from the first surface 108 and the second surface 109, and the insulating layer is patterned to form the second dielectric layer 124. The second dielectric layer 124 can be any suitable material, and in one example, the material of the second dielectric layer 124 is SiOx. In one example, the thickness of the second dielectric layer 124 is 3000Å.

[0147] Then, a conductive film layer is deposited on the side of the second dielectric layer 124 away from the first surface 108 and the second surface 109, and the conductive film layer is patterned to form the heating electrode 121. The heating electrode 121 is located in the region between two adjacent microcavities. The heating electrode 121 can be any suitable material, and in one example, the material of the heating electrode 121 is indium tin oxide (ITO). In one example, the thickness of the heating electrode 121 is 1350Å.

[0148] Then, an insulating film layer is deposited on the side of the heating electrode 121 away from the first surface 108 and the side of the heating electrode 121 away from the second surface 109. The insulating film layer is exposed, developed, and etched to form the hydrophobic layer 122. In one example, the hydrophobic layer 122 is formed as follows: in a plasma enhanced chemical vapor deposition (PECVD) device, at a temperature of about 390 °C, a power of about 600 W, a pressure of about 1200 mtorr, and a distance between a target and a sample to be deposited in the PECVD device of about 1000 mils, SiH4 (volume flow rate of about 140 seem), NH3 (volume flow rate of about 700 seem), and N2 (volume flow rate of about 2260 seem for about 225 seconds) are introduced into the reaction chamber to deposit a SiN x film layer on the side of the heating electrode 121 away from the first surface 108 and the side of the heating electrode 121 away from the second surface 109 with a thickness of about 3000 A, and the SiN x film layer is exposed, developed, and etched to form the hydrophobic layer 122.

[0149] Finally, the microfluidic substrate 700 is prepared using the same method steps as step 908 to complete the package.

[0150] It will be understood that, although the terms first, second, third, etc. can be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Thus, a first element, component, region, layer or section discussed above could be termed a second element, component, region, layer or section without departing from the teachings of the present disclosure.

[0151] Spatially relative terms such as "row," "column," "below," "above," "left," "right," and the like can be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientations depicted in the figures. For example, if a device in the figures is turned over, elements described as "below" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "below" can encompass both an orientation of above and below. The device can be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. In addition, it will also be understood that the term "between" when used in relation to two layers means that there can be only those two layers, or there can also be one or more intervening layers.

[0152] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the 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 "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, 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 the specification, the description of the terms "one embodiment," "another embodiment," and the like means that a particular feature, structure, material, or characteristic being described is included in at least one embodiment of the disclosure. The illustrative appearances of the above-mentioned terms in the description are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples. Additionally, the disclosure can include a variety of different embodiments or examples that are not necessarily mutually exclusive. Different embodiments or examples described herein can be combined and combined in different ways to form additional embodiments or examples that are not expressly mentioned.

[0153] It will be understood that when an element or layer is referred to as being "on" or "connected to" or "coupled to" another element or layer, it can be directly on or connected to or coupled to the other element or layer or intervening elements or layers can be present. In contrast, when an element is referred to as being "directly on" or "directly connected to" or "directly coupled to" another element or layer, there are no intervening elements or layers present. It will also be understood that when an element is referred to as being "on" or "connected to" or "coupled to" another element or layer, it can be directly on or connected to or coupled to the other element or layer, or intervening elements or layers can be present. In contrast, when an element is referred to as being "directly on" or "directly connected to" or "directly coupled to" another element or layer, there are no intervening elements or layers present. It will be understood that, in any of the claims, the expression "on" or "connected to" or "coupled to" or "directly on" or "directly connected to" or "directly coupled to" as used herein is used in the sense of contact or connection or coupling of one element to another element. However, "on" or "connected to" or "coupled to" or "directly on" or "directly connected to" or "directly coupled to" should not be construed as requiring an element to directly contact or be directly connected to or be directly coupled to another element.

[0154] Embodiments of the disclosure are described herein with reference to schematic illustrations of idealized embodiments (and intermediate structures) of the disclosure. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and / or tolerances, are to be expected. Thus, embodiments of the disclosure should not be construed as limited to the particular shapes of regions illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. The regions illustrated in the figures are schematic in nature and their shapes are not intended to illustrate the actual shape of a region of a device and are not intended to limit the scope of the disclosure.

[0155] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and / or the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0156] The above description is only specific embodiments of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present disclosure, which should be covered by the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.

Claims

1. A microfluidic substrate comprising a plurality of microcavities arranged in an array, wherein at least some of the plurality of microcavities are through-holes, and a tangent plane at at least some points on a sidewall of each microcavity forms a non-perpendicular angle with a reference plane on which the microfluidic substrate lies, wherein each through-hole comprises a first portion and a second portion stacked with each other and penetrating through, and a third portion between the first portion and the second portion and connecting the first portion and the second portion, the third portion comprises a top opening and a bottom opening, and a shape of the third portion is selected from one of the following: a cylinder shape; a cuboid shape; a curved surface body, a sidewall of the curved surface body is an arc surface, the arc surface protrudes outward relative to the top opening, wherein a shape of the first portion and the second portion is a circular truncated cone shape, and a shape of the third portion is the cylinder shape or the curved surface body; or a shape of the first portion and the second portion is a regular quadrangular truncated cone shape, and a shape of the third portion is the cuboid shape, wherein an area of a normal projection of a top first opening of the first portion on the reference plane is greater than an area of a normal projection of a bottom second opening of the first portion on the reference plane, an area of a normal projection of a top third opening of the second portion on the reference plane is less than an area of a normal projection of a bottom fourth opening of the second portion on the reference plane, and wherein the bottom second opening of the first portion is the top opening of the third portion, the top third opening of the second portion is the bottom opening of the third portion, the first portion and the second portion are axially symmetrical about a symmetry axis, the third portion is axially symmetrical about the symmetry axis, and the symmetry axis is parallel to the reference plane.

2. The microfluidic substrate of claim 1, wherein, Each of the plurality of microcavities is a through-hole.

3. The microfluidic substrate of claim 1, wherein, A normal of any point on a sidewall of the first portion or the second portion forms an angle of 82°-85° with a reference line, the reference line is perpendicular to the reference plane. 4.The microfluidic substrate of claim 1, further comprising a hydrophobic layer, wherein the hydrophobic layer is located on opposite first and second surfaces of the microfluidic substrate, a portion of the hydrophobic layer located on the first surface comprises a plurality of first vias, a portion of the hydrophobic layer located on the second surface comprises a plurality of second vias, and wherein the plurality of first vias and the plurality of second vias respectively correspond one-to-one to the plurality of through-holes, a normal projection of the top first opening on the reference plane and a normal projection of one first via corresponding to the through-hole on the reference plane overlap, and a normal projection of the bottom fourth opening on the reference plane and a normal projection of one second via corresponding to the through-hole on the reference plane overlap.

5. The microfluidic substrate of claim 1, wherein, A perpendicular distance from any point on a sidewall of the curved surface body to a reference line is greater than a radius of the top opening of the third portion, the reference line passes through centers of the top opening and the bottom opening of the third portion and is perpendicular to the reference plane.

6. The microfluidic substrate of any one of claims 1-5, wherein, A shape of the top first opening is a circle, and a diameter of the circle is 110-130 μm.

7. The microfluidic substrate of any one of claims 1-5, wherein, A depth of each through-hole is 300 μm.

8. The microfluidic substrate of claim 1, wherein, Some of the plurality of microcavities are blind holes.

9. The microfluidic substrate of claim 8, wherein, The blind hole has a curved shape, and includes an opening, a sidewall, and a bottom. The opening of the blind hole has a circular shape. A vertical distance from any point on the sidewall of the blind hole to a reference line is greater than a radius of the opening. The reference line passes through a center of the opening and is perpendicular to the reference plane.

10. The microfluidic substrate of claim 9, wherein, The blind hole has a depth of 50-100 μm, and the opening of the blind hole has a diameter of 110-130 μm.

11. The microfluidic substrate of claim 5 or 9, wherein, A ratio of a maximum value of the vertical distance to the radius is 1.2:

1.

12. The microfluidic substrate of any one of claims 1-5, wherein, A spacing between two adjacent microcavities of the plurality of microcavities is 20-50 μm.

13. The microfluidic substrate of any one of claims 1-5, wherein, The plurality of microcavities are disposed in a glass substrate of the microfluidic substrate.

14. The microfluidic substrate of claim 1, further comprising a heating electrode, wherein, The heating electrode is located on at least one of opposite first and second surfaces of the microfluidic substrate in a region between two adjacent microcavities.

15. The microfluidic substrate of claim 14, further comprising a hydrophobic layer, wherein, The heating electrode is located on opposite first and second surfaces of the microfluidic substrate in a region between two adjacent microcavities, and the hydrophobic layer is located on a side of the heating electrode away from the first surface and a side away from the second surface.

16. The microfluidic substrate of claim 15, further comprising: a first dielectric layer located on a side of the heating electrode close to the first surface and a side close to the second surface; a second dielectric layer located on a side of the first dielectric layer away from the first surface and a side away from the second surface; and a conductive layer located between the first and second dielectric layers and arranged around a perimeter edge of the microfluidic substrate, the conductive layer being electrically connected to the heating electrode via a via in the second dielectric layer.

17. A microfluidic chip comprising the microfluidic substrate of any one of claims 1-16.

18. The microfluidic chip of claim 17, further comprising an opposing substrate that is bonded to the microfluidic substrate, and an encapsulation glue located between the microfluidic substrate and the opposing substrate. ​

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