CN114308160B

CN114308160B - Digital PCR microcavity chip and preparation method

Info

Publication number: CN114308160B
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: 2023-06-20
Anticipated expiration: 2041-12-29
Also published as: CN114308160A

Abstract

The application discloses a digital PCR microcavity chip and a preparation method thereof, wherein the digital PCR microcavity chip comprises: the substrate, first bonding layer and second bonding layer, the front of substrate is provided with a plurality of microcavity structures, the back of substrate is equipped with the runner with microcavity structure intercommunication, first bonding layer bonds with the front of substrate, second bonding layer bonds with the back of substrate. The preparation method comprises the following steps: carrying out micro-cavity structure pattern photoetching on the front surface of the substrate; etching a microcavity structure; carrying out flow channel pattern photoetching on the back surface of the substrate; etching a runner; the first bonding layer and the second bonding layer are bonded on the front surface and the back surface of the substrate respectively. According to the method, the microcavity structure and the runner are respectively arranged on the two sides of the substrate, so that the advantages of microcavity chip type are reserved, the liquid drop sample separation is convenient, and the development of a high-flux automatic digital PCR system is convenient to realize; 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

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 absolute quantitative tool for trace nucleic acid, and the principle is that reaction liquid is evenly dispersed into a micro-reaction system which is picoliter to nanoliter, after PCR circular reaction, the original DNA concentration is judged according to the proportion occupied by the micro-reaction system which emits fluorescent signals, and 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 routes for droplet splitting are classified according to a droplet splitting method and a fluorescence detection method, and mainly comprise a droplet type, a droplet chip type and a microcavity chip type. The microcavity chip type micro reaction units are segmented in advance in a physical segmentation mode, then fluorescent detection is carried out in a photographing mode, and the stability and uniformity of the system can be guaranteed not to change along with the change of the reaction system by adopting a solid phase segmentation method. The microcavity chip type has higher compatibility to reagents, is convenient for in-vitro diagnosis 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 two main cores determine whether the sample can be divided efficiently and whether the PCR reaction can be carried out smoothly or not in the mode of microcavity construction, surface treatment and liquid entering the microcavity structure.

Disclosure of Invention

Aiming at the defects or shortcomings of the prior art, the technical problem to be solved by the application is to provide a digital PCR microcavity chip and a preparation method.

The application is realized by the following technical scheme:

the application provides a digital PCR microcavity chip, comprising: the substrate, first bonding layer and second bonding layer, the front of substrate is provided with a plurality of microcavity structures, the back of substrate be equipped with microcavity structure intercommunication's runner, wherein, first bonding layer with the front bonding of substrate, the second bonding layer with the back bonding of substrate.

Optionally, the digital PCR microcavity chip is described above, wherein the set depth of the flow channel is less than the set depth of the microcavity structure.

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

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

Optionally, the digital PCR microcavity chip includes a main flow channel and a plurality of branch flow channels that are communicated with the main flow channel, where the branch flow channels are communicated with the microcavity structure that is correspondingly arranged.

Optionally, in the digital PCR microcavity chip, the width of the main flow channel is greater than the 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, the digital PCR microcavity chip is described above, wherein the substrate is a silicon substrate.

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

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

etching the microcavity structure;

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

etching the runner;

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

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

according to the method, the microcavity structure and the runner are respectively arranged on the two sides of the substrate, so that the advantages of microcavity chip type are reserved, the liquid drop sample separation is convenient, and the development of a high-flux automatic digital PCR system is convenient to realize; 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 this application, liquid flows into the microcavity structure through the runner under certain external pressure, and microcavity structure inside is through surface treatment, can guarantee that the PCR reaction is gone on with high efficiency.

The application relates to double-sided etching of a substrate, wherein a microcavity structure is etched on the front side, then a runner is etched on the back side, or the runner is etched on the front side, then a microcavity structure is etched on the back side, the runner is intersected with the microcavity structure, and after the structure preparation is completed, the digital PCR microcavity chip is packaged; when the digital PCR microcavity chip is used, the reaction liquid is added into the inlet of the chip, the inlet is applied with proper external force, and the liquid uniformly flows into each microcavity structure to finish liquid segmentation.

Drawings

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

fig. 1: the first packaging structure diagram of the digital PCR microcavity chip of the embodiment of the application;

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

fig. 3: schematic diagram of microcavity structure in one embodiment of the present application;

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

fig. 5: the front top view of the substrate in one embodiment of the present application.

Detailed Description

The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.

As shown in fig. 1 and 2, in one embodiment of the present application, a digital PCR microcavity chip comprises: the substrate 1, the first bonding layer 5 and the second bonding layer 6, wherein the front surface of the substrate 1 is provided with a plurality of microcavity structures 2, the back surface of the substrate 1 is provided with a runner communicated with the microcavity structures 2, 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. According to the embodiment, the microcavity structure 2 and the runner are respectively arranged on the two sides of the substrate 1, so that the advantages of microcavity chip type are reserved, the liquid drop sample separation is convenient, and the development of the high-flux automatic digital PCR system is convenient to realize.

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

Further, the microcavity structure 2 is etched on the front surface of the substrate 1 by etching, and the runner is etched on the back surface of the substrate 1 by etching. The micro-cavity structure 2 with consistent volume can be conveniently formed on the substrate 1 by using the semiconductor etching method in the embodiment, and the micro-cavity structure 2 can be used as a reaction chamber of 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 structures 2 through the runners by external force, so that uniform sample separation of the digital PCR reaction liquid can be realized.

In this embodiment, the depth of the flow channel is smaller than that 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 is more easily guided into the microcavity structure 2.

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

In this embodiment, preferably, 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, so that the flow channel is ensured to be communicated with the microcavity structure 2, and the 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 microcavity structure 2 correspondingly arranged. In this embodiment, the number of the branched flow channels 4 is not limited, and the number of the branched flow channels 4 is the same as the number of the microcavity structures 2. The arrangement manner of the microcavity structure 2 may be arranged in a rectangular array, as shown in fig. 3, which is only for illustration and does not limit the protection scope of the present application.

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

Wherein the first bonding layer 5 is made of a transparent material, such as transparent glass. The arrangement of the first bonding layer 5 ensures that the fluorescence observation of the microcavity structure 2 is smoothly performed after the PCR reaction.

The material of the second bonding layer 6 is not limited in this embodiment, so long as the second bonding layer 6 can be bonded to the substrate 1, and the second bonding layer 6 may be glass, plastic, or the like.

On the other hand, the embodiment also provides a preparation method of the digital PCR microcavity chip, which comprises the following steps:

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

etching the microcavity structure 2;

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

etching the runner;

the first bonding layer 5 and the second bonding layer 6 are bonded to the front surface and the back surface of the substrate 1, respectively.

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

The sequence 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 method is described in detail by two examples below.

Example 1

The present example uses a double-sided etching process to prepare a digital PCR microcavity chip. Specifically, the method comprises the following steps:

step one, taking a silicon wafer substrate 1 with the thickness of 300 mu m, and cleaning the silicon wafer substrate by solvents such as IPA;

step two, carrying out graphic photoetching of the microcavity structure 2 on the front surface of the substrate 1, and carrying out the steps of gluing, exposure, development and the like;

step three, deep silicon etching: etching the microcavity structure 2 shown in fig. 1 and 3 to a depth of about 200 μm and a diameter of about 100 μm of the microcavity structure 2;

step four, cleaning and removing glue by a dry method and a wet method;

fifth, carrying out flow channel pattern photoetching on the back surface of the substrate 1, and carrying out the steps of gluing, exposure, development and the like;

step six, etching a runner as shown in fig. 2 and 5, wherein the depth is about 100 μm, the runner can intersect with the microcavity structure 2, the width of the main runner 3 is about 100 μm, and the width of the branch runner is about 20 μm;

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

and step eight, bonding the transparent glass and the glass with the substrate 1.

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

and step ten, after PCR amplification, the optical system acquires fluorescent images 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 the steps of gluing, exposure, development and the like;

step three, etching the flow channel shown in fig. 2 and 5 to a depth of about 100 μm, a width of about 100 μm of the main flow channel 32, and a width of about 20 μm of the sub flow channel;

step four, cleaning and removing glue by a dry method and a wet method;

step five, carrying out graphic photoetching of the microcavity structure 2 on the front surface of the substrate 1, and carrying out the steps of gluing, exposure, development and the like;

step six, deep silicon etching: etching a microcavity structure 2 shown in fig. 1 and 3, intersecting the flow channel to a depth of about 200 μm, and a diameter of the microcavity structure 2 of about 100 μm;

The method comprises the steps of respectively etching a microcavity structure 2 and a runner on the front side and the back side of a substrate 1 by adopting a double-sided etching method, and packaging the digital PCR microcavity chip; when the digital PCR microcavity chip is used, the reaction liquid is added into the inlet of the chip, the inlet is applied with proper external force, and the liquid uniformly flows into each microcavity structure 2, so that the liquid segmentation can be completed. After the PCR reaction, the fluorescence observation of the microcavity structure 2 can be smoothly carried out.

In the description of the present application, unless explicitly stated and limited otherwise, the terms "connected," "connected," and "fixed" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the terms in this application will be understood by those of ordinary skill in the art as the case may be.

In this application, unless expressly stated or limited otherwise, a first feature "above" or "below" a second feature may include both the first and second features being in direct contact, and may also include the first and second features not being in direct contact but being in contact with each other by way of additional features therebetween. Moreover, a first feature being "above," "over" and "on" a second feature includes the first feature being directly above and obliquely above the second feature, or simply indicating that the first feature is higher in level than the second feature. The first feature being "under", "below" and "beneath" the second feature includes the first feature being directly under and obliquely below the second feature, or simply means that the first feature is less level than the second feature.

In the description of the present embodiment, the terms "upper", "lower", "left", "right", etc. azimuth or positional relationship are based on the azimuth or positional relationship shown in the drawings, and are merely for convenience of description and simplification of operations, and do not indicate or imply that the apparatus or element referred to must have a specific azimuth, be configured and operated in a specific azimuth, and thus should not be construed as limiting the present application. Furthermore, the terms "first," "second," and the like, are used merely for distinguishing between descriptions and not for distinguishing between them.

The above embodiments are only for illustrating the technical solution of the present application, not for limiting, and the present application is described in detail with reference to the preferred embodiments. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made to the technical solution of the present application without departing from the spirit and scope of the technical solution of the present application, and it is intended to cover within the scope of the claims of the present application.

Claims

1. A digital PCR microcavity chip, comprising: the substrate comprises a substrate, a first bonding layer and a second bonding layer, wherein the front surface of the substrate is provided with a plurality of microcavity structures, the back surface of the substrate is provided with a runner 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 arrangement depth of the flow channel is smaller than that of the microcavity structure.

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

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

4. The digital PCR microcavity chip according to claim 1, wherein the flow channel comprises a main flow channel and a plurality of branch flow channels in communication with the main flow channel, the branch flow channels being in communication with the microcavity structure in which they are disposed.

5. The digital PCR microcavity chip of claim 4, wherein the width of the main flow channel is greater than the width of the branch flow channel.

6. The digital PCR microcavity chip according to any one of claims 1 to 5, characterized in that the microcavity structure is etched on the front side of the substrate by means of etching.

7. The digital PCR microcavity chip according to any one of claims 1 to 5, characterized in that the flow channel is etched in the back side of the substrate by means of etching.

8. The digital PCR microcavity chip according to any one of claims 1 to 5, characterized in that the substrate is a silicon substrate.

9. A method of preparing a digital PCR microcavity chip according to any one of claims 1 to 8, characterized in that the method of preparing comprises the steps of:

etching the microcavity structure;

etching the runner;