CN114308160A

CN114308160A - Digital PCR microcavity chip and preparation method thereof

Info

Publication number: CN114308160A
Application number: CN202111645217.4A
Authority: CN
Inventors: 殷敏; 李睿文
Original assignee: Zhenzhun Bio Technology Co ltd
Current assignee: Zhenzhun Bio Technology Co ltd
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2022-04-12
Anticipated expiration: 2041-12-29
Also published as: CN114308160B

Abstract

The application discloses a digital PCR microcavity chip and a preparation method thereof, wherein the digital PCR microcavity chip comprises: the front surface of the substrate is provided with a plurality of microcavity structures, the back surface of the substrate is provided with a flow channel communicated with the microcavity structures, the first bonding layer is bonded with the front surface of the substrate, and the second bonding layer is bonded with the back surface of the substrate. The preparation method comprises the following steps: carrying out micro-cavity structure pattern photoetching on the front side of the substrate; etching a micro-cavity structure; carrying out flow channel pattern photoetching on the back of the substrate; etching a flow channel; and respectively bonding the first bonding layer and the second bonding layer on the front surface and the back surface of the substrate. The microcavity structure and the flow channel are respectively arranged on the two sides of the substrate, so that the advantages of microcavity chip type are kept, liquid drop sampling is convenient, and the development of a high-flux automatic digital PCR system is facilitated; the double-sided etching process provided by the application can ensure the preparation precision of the flow channel and the microcavity structure, and the preparation method is simple and easy to realize.

Description

Digital PCR microcavity chip and preparation method thereof

Technical Field

The application belongs to the technical field of digital PCR, and particularly relates to a digital PCR microcavity chip and a preparation method thereof.

Background

The digital PCR (Polymerase Chain Reaction) is the latest generation of trace nucleic acid absolute quantification tool, and the principle is that the Reaction solution is averagely dispersed into a pico-liter to nano-liter trace Reaction system, and after the PCR circulation Reaction, the original DNA concentration is judged according to the proportion of the trace Reaction system which emits a fluorescent signal, so that the absolute quantification of nucleic acid molecules is realized.

Providing reliable, uniform, high quality, high density reaction units is the core of dPCR (digital PCR). The current technical route of droplet segmentation is classified according to droplet segmentation methods and fluorescence detection methods, and mainly comprises droplet chip type, droplet chip type and microcavity chip type. The micro-cavity chip type is characterized in that different micro reaction units are pre-divided in a physical dividing mode, then fluorescence detection is carried out in a photographing mode, and the stability and uniformity of the system size can be guaranteed not to change along with the change of a reaction system by a solid phase dividing method. The micro-cavity chip type has higher compatibility to reagents, is convenient for in-vitro diagnostic enterprises to develop own application kits on the system, and creates a completely open digital PCR platform. In the microcavity chip-type technical route, the modes of microcavity construction, surface treatment and liquid entering into the microcavity structure determine whether the sample can be efficiently subjected to average division and the PCR reaction can be smoothly performed.

Disclosure of Invention

In view of the above disadvantages or shortcomings of the prior art, the present application provides a digital PCR microcavity chip and a method for manufacturing the same.

The method is realized through the following technical scheme:

the application provides a digital PCR microcavity chip, including: the front surface of the substrate is provided with a plurality of microcavity structures, and the back surface of the substrate is provided with a runner communicated with the microcavity structures, wherein the first bonding layer is bonded with the front surface of the substrate, and the second bonding layer is bonded with the back surface of the substrate.

Optionally, in the digital PCR microcavity chip, a depth of the flow channel is smaller than a depth of the microcavity structure.

Optionally, in the digital PCR microcavity chip, a sum of the depth of the flow channel and the depth of the microcavity structure is equal to a thickness of the substrate.

Optionally, in the digital PCR microcavity chip, the first bonding layer is made of a transparent material.

Optionally, in the digital PCR microcavity chip, the flow channel includes a main flow channel and a plurality of branch flow channels communicated with the main flow channel, and the branch flow channels are communicated with the microcavity structure disposed correspondingly thereto.

Optionally, in the digital PCR microcavity chip, a width of the main flow channel is greater than a width of the branch flow channel.

Optionally, in the digital PCR microcavity chip, the microcavity structure is etched on the front surface of the substrate by etching.

Optionally, in the digital PCR microcavity chip, the flow channel is etched on the back surface of the substrate by etching.

Optionally, in the digital PCR microcavity chip, the substrate is a silicon substrate.

The application also provides a preparation method of the digital PCR microcavity chip, and the preparation method comprises the following steps:

carrying out micro-cavity structure pattern photoetching on the front side of the substrate;

etching the micro-cavity structure;

carrying out flow channel pattern photoetching on the back of the substrate;

etching the flow channel;

and respectively bonding the first bonding layer and the second bonding layer on the front surface and the back surface of the substrate.

Compared with the prior art, the method has the following technical effects:

the microcavity structure and the flow channel are respectively arranged on the two sides of the substrate, so that the advantages of microcavity chip type are kept, liquid drop sampling is convenient, and the development of a high-flux automatic digital PCR system is facilitated; the double-sided etching process provided by the application can ensure the preparation precision of the flow channel and the microcavity structure, and the preparation method is simple and easy to realize.

In the application, liquid flows into the microcavity structure through the flow channel under certain external pressure, and the inside surface treatment of the microcavity structure can ensure that the PCR reaction is carried out efficiently.

The application relates to substrate double-sided etching, firstly etching a microcavity structure on the front side, then etching a runner on the back side, or firstly etching a runner on the front side, then etching a microcavity structure on the back side, intersecting the runner with the microcavity structure, and packaging the digital PCR microcavity chip after completing the structure preparation; when the digital PCR microcavity chip is used, reaction liquid is added to the inlet of the chip, proper external force is applied to the inlet, and the liquid flows into the microcavity structures uniformly to complete liquid division.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:

FIG. 1: the packaging structure diagram of the digital PCR microcavity chip in one embodiment of the application is I;

FIG. 2: a second packaging structure diagram of the digital PCR microcavity chip in the first embodiment of the application;

FIG. 3: schematic diagram of a microcavity structure in an embodiment of the present application;

FIG. 4: a schematic structural diagram of a flow channel in an embodiment of the present application;

FIG. 5: in one embodiment of the present application, a front side plan view of a substrate.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

As shown in fig. 1 and 2, in one embodiment of the present application, a digital PCR microcavity chip includes: the micro-cavity structure comprises a substrate 1, a first bonding layer 5 and a second bonding layer 6, wherein a plurality of micro-cavity structures 2 are arranged on the front surface of the substrate 1, a runner communicated with the micro-cavity structures 2 is arranged on the back surface of the substrate 1, the first bonding layer 5 is bonded with the front surface of the substrate 1, and the second bonding layer 6 is bonded with the back surface of the substrate 1. In the embodiment, the microcavity structure 2 and the flow channel are respectively arranged on the two sides of the substrate 1, so that the advantages of microcavity chip type are kept, the liquid drop sample separation is convenient, and the development of a high-throughput automatic digital PCR system is facilitated.

In the present embodiment, the substrate 1 is preferably a silicon substrate 1.

Further, the micro-cavity structure 2 is etched on the front surface of the substrate 1 in an etching manner, and the runner is etched on the back surface of the substrate 1 in an etching manner. In the embodiment, the microcavity structure 2 with a uniform volume can be conveniently formed on the substrate 1 by using a semiconductor etching method, and the microcavity structure 2 can be used as a reaction chamber for digital PCR. Meanwhile, a runner with a certain depth can be etched beside each microcavity structure 2, and the PCR reaction liquid can be drained into the microcavity structure 2 through the runners by external force, so that uniform sample division of the digital PCR reaction liquid can be realized.

In this embodiment, the setting depth of the flow channel is smaller than the setting depth of the microcavity structure 2, and the above structure can increase the capillary force of the microcavity structure 2, so that the PCR reaction solution can be more easily guided into the microcavity structure 2.

For example, in one embodiment, the substrate 1 is disposed to a thickness of 300 μm, the flow channel may be disposed to a depth of 100 μm, and the microcavity structure 2 may be disposed to a depth of 200 μm.

In this embodiment, preferably, the sum of the installation depth of the flow channel and the installation depth of the microcavity structure 2 is equal to the thickness of the substrate 1, so that the flow channel can be ensured to be communicated with the microcavity structure 2, and a PCR reaction solution can be guided into the microcavity structure 2.

As shown in fig. 4 and 5, the flow channel includes a main flow channel 3 and a plurality of branch flow channels 4 communicated with the main flow channel 3, and the branch flow channels 4 are communicated with the micro-cavity structures 2 correspondingly disposed therein. In this embodiment, the number of the branch flow channels 4 is not limited, and the number of the branch flow channels 4 is the same as the number of the microcavity structures 2. The micro-cavity structures 2 may be arranged in a rectangular array, as shown in fig. 3, and the arrangement is merely an example, and does not limit the protection scope of the present application.

Further, the width of the main flow channel 3 is greater than the width of the branch flow channels 4. For example, in one embodiment, the etching width of the main channel 3 may be 100 μm, and the etching width of the branch channel 4 may be 20 μm.

Wherein, the first bonding layer 5 is made of transparent material, such as transparent glass. The first bonding layer 5 is arranged to ensure that the fluorescent observation of the microcavity structure 2 is carried out smoothly after the PCR reaction.

The material of the second bonding layer 6 is not limited in this embodiment, and it is desirable that the second bonding layer 6 can be bonded to the substrate 1, wherein the second bonding layer 6 may be made of, for example, glass, plastic, or the like.

In another aspect, this embodiment further provides a method for preparing the digital PCR microcavity chip, where the method includes the following steps:

carrying out pattern photoetching on the microcavity structure 2 on the front surface of the substrate 1;

etching the micro-cavity structure 2;

carrying out flow channel pattern photoetching on the back of the substrate 1;

etching the flow channel;

and respectively bonding the first bonding layer 5 and the second bonding layer 6 on the front surface and the back surface of the substrate 1.

In the semiconductor etching process, the following steps are roughly carried out: coating photoresist, photoetching, HM etching, removing the photoresist, cleaning and the like; in the digital PCR microcavity chip, if the flow channels and the microcavity structure 2 with different depths are to be constructed, the construction needs to be carried out in two steps. Firstly, etching a channel, and then performing secondary etching on the microcavity structure 2 on the channel pattern through the steps of coating glue again, photoetching, etching and the like; or firstly etching the micro-cavity structure 2 to obtain the micro-cavity structure 2 with large density, and then performing secondary etching on the flow channel on the pattern of the micro-cavity structure 2 through the steps of coating glue again, photoetching, etching and the like. However, this method has some disadvantages: 1) the pattern etched for the second time needs to be strictly aligned with the pattern etched for the first time; 2) during secondary etching, the existing pattern (the microcavity structure 2 or the runner) can influence the spin coating uniformity of the photoresist and influence the precision and uniformity of the secondary etching pattern; 3) during the secondary etching, the photoresist still needs to be coated in a spinning mode, and the photoresist enters the flow channel or the micro-cavity structure 2, so that the subsequent cleaning is troublesome. The present embodiment adopts a double-sided etching method to avoid the above technical defects. Specifically, firstly, a high-density microcavity structure 2 is etched on a substrate 1 through the steps of gluing, photoetching, etching and the like, so as to avoid the influence of the existing pattern on the subsequent secondary etching; and etching the flow channel on the back, and realizing the construction of the flow channel through the steps of gluing, photoetching, etching and the like, so as to ensure that the sum of the depth of the flow channel and the depth of the microcavity structure 2 is equal to the thickness of the substrate 1. Thus, the runner can be connected with the micro-cavity structure 2, and the reaction liquid can be guided into the micro-cavity structure 2. After the etching of the runner and the microcavity structure 2 is realized, transparent plastic or glass is used for carrying out double-sided packaging on the silicon wafer, so that the fluorescent observation of the microcavity structure 2 can be smoothly carried out after the PCR reaction is ensured.

The order of the double-sided etching is not limited in this embodiment, and the front-side etching may be performed first, or the back-side etching may be performed first. The above preparation process is described in detail below by way of two examples.

Example 1

In this embodiment, a digital PCR microcavity chip is prepared by a double-sided etching process. Specifically, the method comprises the following steps:

step one, taking a silicon wafer substrate 1 with the thickness of 300 microns, and cleaning the silicon wafer substrate by using solvents such as IPA (isopropyl alcohol);

performing pattern photoetching of the microcavity structure 2 on the front surface of the substrate 1, and performing steps of gluing, exposure, development and the like;

step three, deep silicon etching: etching the micro-cavity structure 2 shown in the figures 1 and 3, wherein the depth is about 200 μm, and the diameter of the micro-cavity structure 2 is about 100 μm;

cleaning and removing the photoresist by a dry method and a wet method;

fifthly, carrying out flow channel pattern photoetching on the back surface of the substrate 1, and carrying out steps of gluing, exposing, developing and the like;

sixthly, etching the flow channel shown in the figures 2 and 5, wherein the depth is about 100 microns, the flow channel can be intersected with the microcavity structure 2, the width of the main flow channel 3 is about 100 microns, and the width of the branch flow channel is about 20 microns;

step seven, after the double-sided etching is finished, carrying out hydrophilic pattern layer modification on the chip;

and step eight, bonding the substrate 1 by adopting transparent glass and glass.

Step nine, after bonding, conveying the digital PCR reaction liquid into the flow channel and the microcavity structure 2 through an external force through the flow channel surface;

step ten, after PCR amplification, the optical system acquires a fluorescence image through the microcavity surface for subsequent analysis.

Example 2

step two, carrying out flow channel pattern photoetching on the back surface of the substrate 1, and carrying out steps of gluing, exposing, developing and the like;

etching the flow channel shown in the figures 2 and 5, wherein the depth is about 100 μm, the width of the main flow channel 32 is about 100 μm, and the width of the branch flow channel is about 20 μm;

cleaning and removing the photoresist by a dry method and a wet method;

fifthly, carrying out pattern photoetching of the micro-cavity structure 2 on the front surface of the substrate 1, and carrying out steps of gluing, exposure, development and the like;

step six, deep silicon etching: etching the micro-cavity structure 2 shown in fig. 1 and fig. 3, intersecting with the flow channel, wherein the depth is about 200 μm, and the diameter of the micro-cavity structure 2 is about 100 μm;

The method comprises the steps of respectively etching a microcavity structure 2 and a flow channel on the front side and the back side of a substrate 1 by a double-sided etching method, and packaging the digital PCR microcavity chip; when the digital PCR microcavity chip is used, reaction liquid is added to the inlet of the chip, proper external force is applied to the inlet, and the liquid flows into the microcavity structures 2 uniformly to complete liquid division. And the fluorescent observation of the microcavity structure 2 can be smoothly carried out after the PCR reaction is ensured.

In the description of the present application, unless expressly stated or limited otherwise, the terms "connected," "connected," and "fixed" are to be construed broadly, e.g., as meaning permanently connected, removably connected, or integral to one another; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art as appropriate.

In this application, unless expressly stated or limited otherwise, the first feature "on" or "under" the second feature may comprise direct contact of the first and second features, or may comprise contact of the first and second features not directly but through another feature in between. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.

In the description of the present embodiment, the terms "upper", "lower", "left", "right", and the like are used based on the orientations and positional relationships shown in the drawings only for convenience of description and simplification of operation, and do not indicate or imply that the referred device or element must have a specific orientation, be configured and operated in a specific orientation, and thus, should not be construed as limiting the present application. Furthermore, the terms "first" and "second" are used only for descriptive purposes and are not intended to have a special meaning.

The above embodiments are merely to illustrate the technical solutions of the present application and are not limitative, and the present application is described in detail with reference to preferred embodiments. It will be understood by those skilled in the art that various modifications and equivalent arrangements may be made in the present invention without departing from the spirit and scope of the present invention and shall be covered by the appended claims.

Claims

1. A digital PCR microcavity chip, comprising: the front surface of the substrate is provided with a plurality of microcavity structures, and the back surface of the substrate is provided with a runner communicated with the microcavity structures, wherein the first bonding layer is bonded with the front surface of the substrate, and the second bonding layer is bonded with the back surface of the substrate.

2. The digital PCR microcavity chip of claim 1, wherein the runners are provided at a depth less than that of the microcavity structure.

3. The digital PCR microcavity chip of claim 1, wherein the sum of the depth of the flow channels and the depth of the microcavity structure is equal to the thickness of the substrate.

4. The digital PCR microcavity chip of claim 1, wherein the first bonding layer is made of a transparent material.

5. The digital PCR microcavity chip of claim 1, wherein the flow channel includes a main flow channel and a plurality of branch flow channels connected to the main flow channel, and the branch flow channels are connected to the microcavity structure disposed corresponding to the branch flow channels.

6. The digital PCR microcavity chip of claim 5, wherein the main flow channels have a width greater than the branch flow channels.

7. The digital PCR microcavity chip according to any one of claims 1 to 6, wherein the microcavity structure is etched on the front surface of the substrate by etching.

8. The digital PCR microcavity chip of any one of claims 1-6, wherein the flow channels are etched in the back side of the substrate by etching.

9. The digital PCR microcavity chip of any one of claims 1-6, wherein the substrate is a silicon substrate.

10. A method for preparing the digital PCR microcavity chip according to any one of claims 1 to 9, wherein the method comprises the following steps:

etching the micro-cavity structure;

carrying out flow channel pattern photoetching on the back of the substrate;

etching the flow channel;