阅读说明：本技术 基于通孔结构和微流控的dPCR原位芯片及制作方法 (dPCR in-situ chip based on through hole structure and microfluidics and manufacturing method ) 是由 吴炫烨 关一民 于 2018-09-05 设计创作，主要内容包括：本发明提供一种基于通孔结构和微流控的dPCR原位芯片及制作方法,芯片包括：微流道层,微流道层中形成有微流道；微通孔层,微通孔层中形成有多个微通孔,多个微通孔沿微流道的走向排布,且微通孔与微流道连通；第一封闭层,密封覆盖于微通孔层上,第一封闭层为透气而不透液的封闭层；以及第二封闭层,密封覆盖于微流道层上,第二封闭层中形成有与微流道连通的注入口。本发明通过微流道和微通孔以及透气不透液的封闭层的结合,采用施加液压的方式即可完成dPCR试剂的分散,解决了基于穿孔dPCR技术试剂需要通过刮涂完成液体分散的不便,同时有效解决基于穿孔dPCR技术试剂通过刮涂时样本的损失,可大大降低检验成本。(The invention provides a dPCR in-situ chip based on a through hole structure and microfluidics and a manufacturing method thereof, wherein the chip comprises: a micro channel layer in which a micro channel is formed; the micro-via layer is provided with a plurality of micro-vias which are arranged along the trend of the micro-channel and are communicated with the micro-channel; the first sealing layer is hermetically covered on the micro-via layer, and is a breathable and liquid-tight sealing layer; and a second sealing layer hermetically covering the micro flow channel layer, wherein an injection port communicated with the micro flow channel is formed in the second sealing layer. The invention can complete the dispersion of the dPCR reagent by combining the micro-flow channel, the micro-through hole and the air-permeable liquid-tight sealing layer and adopting a hydraulic mode, solves the problem that the reagent based on the perforation dPCR technology needs to complete the dispersion of liquid by blade coating, effectively solves the problem of sample loss when the reagent based on the perforation dPCR technology is subjected to blade coating, and can greatly reduce the inspection cost.)

1. A dPCR in-situ chip based on a through hole structure and microfluidics is characterized by comprising:

a micro channel layer in which a micro channel is formed;

the micro-through hole layer is formed with a plurality of micro-through holes, the micro-through holes are distributed along the trend of the micro-channel, and the micro-through holes are communicated with the micro-channel;

the first sealing layer is hermetically covered on the micro-via layer, and is a breathable and liquid-tight sealing layer; and

and the second sealing layer is hermetically covered on the micro-flow channel layer, and an injection port communicated with the micro-flow channel is formed in the second sealing layer.

2. The through-hole structure and microfluidic based dPCR in situ chip of claim 1, wherein: and liquid-phase dPCR reagent enters the micro-flow channel through the injection port, and fills each micro-through hole with the liquid-phase dPCR reagent through pressure based on the gas permeability and liquid impermeability of the first sealing layer.

3. The through-hole structure and microfluidic based dPCR in situ chip of claim 1, wherein: the width of the micro flow channel is not less than the aperture of the micro through hole.

4. The through-hole structure and microfluidic based dPCR in situ chip of claim 1, wherein: the micro-channel layer and the micro-via layer are respectively formed on the first surface and the second surface of the rigid substrate, and the micro-via is communicated with the micro-channel.

5. The through-hole structure and microfluidic based dPCR in situ chip of claim 1, wherein:

the micro-via layer comprises a rigid substrate, and the micro-via penetrates through the rigid substrate;

the micro-channel layer comprises a photosensitive material layer, is combined on the surface of the rigid substrate, and penetrates through the photosensitive material layer to be communicated with the micro-through hole.

6. The through-hole structure and microfluidic based dPCR in situ chip of claim 1, wherein: the material of the first sealing layer comprises polydimethylsiloxane PDMS.

7. The through-hole structure and microfluidic based dPCR in situ chip of claim 6, wherein: and chemically bonding the dimethyl siloxane PDMS and the micro-via layer after oxygen plasma treatment.

8. The through-hole structure and microfluidic based dPCR in situ chip of claim 1, wherein: the second sealing layer is made of one of Polydimethylsiloxane (PDMS) and photoetching materials.

9. The through-hole structure and microfluidic based dPCR in situ chip of claim 1, wherein: the micro-channel extends in a zigzag manner in the micro-channel layer.

10. A manufacturing method of a dPCR in-situ chip based on a through hole structure and microfluidics is characterized by comprising the following steps:

1) providing a rigid substrate, wherein the rigid substrate comprises a first surface and a second surface which are opposite;

2) etching a micro channel on the first surface of the rigid substrate;

3) etching a plurality of micro-through holes on the second surface of the rigid substrate, wherein the micro-through holes are distributed along the direction of the micro-channel and are communicated with the micro-channel;

4) and hermetically covering a first sealing layer on the micro-through hole of the rigid substrate, wherein the first sealing layer is a breathable and liquid-tight sealing layer, and hermetically covering a second sealing layer on the micro-channel of the rigid substrate, and an injection port communicated with the micro-channel is formed in the second sealing layer.

11. The method for manufacturing the dPCR in-situ chip based on the through hole structure and the microfluidics according to claim 10, wherein: and liquid-phase dPCR reagent enters the micro-flow channel through the injection port, and fills each micro-through hole with the liquid-phase dPCR reagent through pressure based on the gas permeability and liquid impermeability of the first sealing layer.

12. The method for manufacturing the dPCR in-situ chip based on the through hole structure and the microfluidics according to claim 10, wherein: the material of the first sealing layer comprises polydimethylsiloxane PDMS.

13. The method for manufacturing the dPCR in-situ chip based on the through hole structure and the microfluidics according to claim 12, wherein: the material of the second sealing layer comprises Polydimethylsiloxane (PDMS), and the step 4) comprises the following steps: and simultaneously carrying out oxygen plasma treatment on the first surface of the rigid substrate with the micro flow channel and the second surface of the rigid substrate with the micro through hole, and then chemically bonding the second sealing layer and the first sealing layer with the rigid substrate at the same time.

14. A manufacturing method of a dPCR in-situ chip based on a through hole structure and microfluidics is characterized by comprising the following steps:

1) providing a rigid substrate, wherein the rigid substrate comprises a first surface and a second surface which are opposite, and micro through holes penetrating through the rigid substrate are etched in the rigid substrate;

2) forming a micro-channel material layer on the first surface of the rigid substrate, forming a micro-channel in the micro-channel material layer, wherein the micro-channel penetrates through the micro-channel material layer to be communicated with the micro-through hole;

3) and hermetically covering a first sealing layer on the second surface of the rigid substrate, wherein the first sealing layer is a breathable and liquid-tight sealing layer, and the micro-channel material layer is hermetically covered with a second sealing layer, and an injection port communicated with the micro-channel is formed in the second sealing layer.

15. The method for manufacturing the dPCR in-situ chip based on the through hole structure and the microfluidics according to claim 14, wherein: and liquid-phase dPCR reagent enters the micro-flow channel through the injection port, and fills each micro-through hole with the liquid-phase dPCR reagent through pressure based on the gas permeability and liquid impermeability of the first sealing layer.

16. The method for manufacturing the dPCR in-situ chip based on the through hole structure and the microfluidics according to claim 14, wherein: the material of the first sealing layer comprises polydimethylsiloxane PDMS.

17. The method for manufacturing the dPCR in-situ chip based on the through hole structure and the microfluidics according to claim 16, wherein: and 3) firstly carrying out oxygen plasma treatment on the second surface of the rigid substrate and the first sealing layer, and then chemically bonding the rigid substrate and the first sealing layer.

18. The method for manufacturing the dPCR in-situ chip based on the through hole structure and the microfluidics according to claim 14, wherein: the material of the second sealing layer comprises photoetching material.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a dPCR in-situ chip based on a through hole structure and microfluidics and a manufacturing method thereof.

Background

Polymerase Chain Reaction (PCR) has been proposed for 20 years, during which PCR has developed into a key technology and a conventional technology in the field of molecular biology, and has greatly promoted the development of various fields of life science. Particularly, in the later 90 s, real-time fluorescent quantitative PCR (qPCR) technology and related products proposed by ABI company in America developed PCR from in vitro synthesis and qualitative/semi-quantitative detection technology into a gene analysis technology with high sensitivity, high specificity and accurate quantification.

Although the qPCR technique has been used for diagnosis of all diseases except for trauma and nutritional deficiency through rapid development over a period of ten years, there are many factors affecting the amplification efficiency during PCR amplification, and it cannot be guaranteed that the amplification efficiency remains the same during reaction and that the amplification efficiency is the same between the actual sample and the standard sample as well as between different samples, thereby leading to the basis on which its quantitative analysis depends-the Cycle Threshold (CT) is not constant. Therefore, qPCR is only "relative quantitative", and the accuracy and reproducibility thereof still cannot meet the requirements of molecular biological quantitative analysis.

Vogelstein et al proposed the concept of digital PCR (digital PCR) by dividing a sample into tens to tens of thousands of portions, assigning them to different reaction units, each containing one or more copies of a target molecule (DNA template), performing PCR amplification of the target molecule in each reaction unit, and performing statistical analysis of the fluorescent signals of the reaction units after amplification is complete. Different from qPCR, digital PCR does not depend on CT value, so that the method is not influenced by amplification efficiency, the average concentration (content) of each reaction unit is calculated by direct counting or a Poisson distribution formula after amplification is finished, the error can be controlled within 5%, and absolute quantitative analysis can be realized by digital PCR without reference to a standard sample and a standard curve.

Digital PCR (also known as single molecule PCR) generally involves two parts, PCR amplification and fluorescence signal analysis. In the PCR amplification stage, unlike the conventional art, digital PCR generally requires that a sample be diluted to a single molecule level and equally distributed into several tens to several tens of thousands of units for reaction. Unlike the method of real-time fluorescence measurement for each cycle by qPCR, the digital PCR technique is to collect the fluorescence signal of each reaction unit after amplification is completed. And finally, calculating to obtain the original concentration or content of the sample through direct counting or a Poisson distribution formula.

Since digital PCR is an absolute nucleic acid molecule quantification technique, compared to qPCR, the number of DNA molecules can be directly counted, which is an absolute quantification of the starting sample, and thus it is particularly suitable for application fields that cannot be well resolved depending on CT values, such as copy number variation, mutation detection, gene relative expression studies (e.g., allele imbalance expression), second-generation sequencing result verification, miRNA expression analysis, single-cell gene expression analysis, and the like.

There are three major types of digital PCR technology currently on the market. One is to form droplets by cutting off the PCR solution of the aqueous phase with flowing oil in a specific instrument and then to perform PCR and detection in two other instruments; one is to distribute PCR solution on a hollowed silicon chip, and then carry out PCR in a specific instrument and carry out detection in another instrument; the last one is to inject the liquid into the chamber through a narrow channel on one instrument to form droplets, and to perform PCR, and then to perform detection in another instrument. Due to the manual nature of the doctor blading, the last method tends to have poor hole fill and repeatability on the silicon wafer and to have severe sample loss. In addition, blade coating limits the automation and miniaturization of the overall system. This not only increases the cost of purchase of the instrument, limits the widespread use of digital PCR, but also increases the complexity of the experimental procedures.

Based on the above, the dPCR in-situ chip and the manufacturing method thereof are necessary, wherein the dPCR in-situ chip can replace the manual blade coating of the dPCR of the through hole by utilizing a microfluidic automation method, so that the filling rate of the through hole and the experimental repeatability are improved.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a dPCR in situ chip based on a via structure and microfluidics and a manufacturing method thereof, so as to replace manual blade coating of via dPCR with an automated microfluidic method, thereby improving the filling rate of via holes and the experimental repeatability.

To achieve the above and other related objects, the present invention provides a dPCR in situ chip based on a via structure and microfluidics, comprising: a micro channel layer in which a micro channel is formed; the micro-through hole layer is formed with a plurality of micro-through holes, the micro-through holes are distributed along the trend of the micro-channel, and the micro-through holes are communicated with the micro-channel; the first sealing layer is hermetically covered on the micro-via layer, and is a breathable and liquid-tight sealing layer; and the second sealing layer is hermetically covered on the micro-flow channel layer, and an injection port communicated with the micro-flow channel is formed in the second sealing layer.

Optionally, a liquid-phase dPCR reagent enters the micro flow channel through the injection port, and fills each of the micro through-holes with the liquid-phase dPCR reagent by pressure based on gas permeability and liquid impermeability of the first sealing layer.

Optionally, the width of the micro flow channel is not less than the pore diameter of the micro through hole.

Optionally, the micro channel layer and the micro via layer are formed on the first surface and the second surface of the rigid substrate, respectively, and the micro via is communicated with the micro channel.

Optionally, the microvia layer comprises a rigid substrate through which the microvias extend; the micro-channel layer comprises a photosensitive material layer, is combined on the surface of the rigid substrate, and penetrates through the photosensitive material layer to be communicated with the micro-through hole.

Optionally, the material of the first sealing layer includes polydimethylsiloxane PDMS.

Optionally, the dimethylsiloxane PDMS is chemically bonded to the micro-via layer after oxygen plasma treatment.

Optionally, the material of the second sealing layer includes one of polydimethylsiloxane PDMS and a photolithographic material.

Optionally, the microchannel extends in a zigzag manner in the microchannel layer.

The invention also provides a manufacturing method of the dPCR in-situ chip based on the through hole structure and the microfluidics, which comprises the following steps: 1) providing a rigid substrate, wherein the rigid substrate comprises a first surface and a second surface which are opposite; 2) etching a micro channel on the first surface of the rigid substrate; 3) etching a plurality of micro-through holes on the second surface of the rigid substrate, wherein the micro-through holes are distributed along the direction of the micro-channel and are communicated with the micro-channel; 4) and hermetically covering a first sealing layer on the micro-through hole of the rigid substrate, wherein the first sealing layer is a breathable and liquid-tight sealing layer, and hermetically covering a second sealing layer on the micro-channel of the rigid substrate, and an injection port communicated with the micro-channel is formed in the second sealing layer.

Optionally, a liquid-phase dPCR reagent enters the micro flow channel through the injection port, and fills each of the micro through-holes with the liquid-phase dPCR reagent by pressure based on gas permeability and liquid impermeability of the first sealing layer.

Optionally, the material of the first sealing layer includes polydimethylsiloxane PDMS.

Optionally, the material of the second sealing layer includes polydimethylsiloxane PDMS, and step 4) includes: and simultaneously carrying out oxygen plasma treatment on the first surface of the rigid substrate with the micro flow channel and the second surface of the rigid substrate with the micro through hole, and then chemically bonding the second sealing layer and the first sealing layer with the rigid substrate at the same time.

The invention also provides a manufacturing method of the dPCR in-situ chip based on the through hole structure and the microfluidics, which comprises the following steps: 1) providing a rigid substrate, wherein the rigid substrate comprises a first surface and a second surface which are opposite, and micro through holes penetrating through the rigid substrate are etched in the rigid substrate; 2) forming a micro-channel material layer on the first surface of the rigid substrate, forming a micro-channel in the micro-channel material layer, wherein the micro-channel penetrates through the micro-channel material layer to be communicated with the micro-through hole; and 3) sealing and covering a first sealing layer on the second surface of the rigid substrate, wherein the first sealing layer is a breathable and liquid-tight sealing layer, and a second sealing layer is sealed and covered on the micro-channel material layer, and an injection port communicated with the micro-channel is formed in the second sealing layer.

Optionally, a liquid-phase dPCR reagent enters the micro flow channel through the injection port, and fills each of the micro through-holes with the liquid-phase dPCR reagent by pressure based on gas permeability and liquid impermeability of the first sealing layer.

Optionally, the material of the first sealing layer includes polydimethylsiloxane PDMS.

Optionally, step 3) first performs oxygen plasma treatment on the second surface of the rigid substrate and the first sealing layer, and then chemically bonds the rigid substrate and the first sealing layer.

Optionally, the material of the second sealing layer includes a photolithographic material.

As mentioned above, the dPCR in-situ chip based on the through hole structure and the microfluidics and the manufacturing method thereof have the following beneficial effects:

1) according to the invention, through the combination of the micro-flow channel, the micro-through hole and the air-permeable liquid-tight sealing layer, the dispersion of the dPCR reagent can be completed by applying hydraulic pressure, and the inconvenience that the reagent needs to be coated with blade coating to complete liquid dispersion based on the perforation dPCR technology is solved.

2) The invention can effectively solve the sample loss when the reagent based on the perforation dPCR technology is coated by blade coating, and can greatly reduce the inspection cost.

3) The invention adopts the silicon substrate to manufacture the micro-through holes, reduces the thermal expansion coefficient of the micro-through hole substrate material, and can solve the problem of overlarge difference of a reaction system caused by temperature change during operation, thereby ensuring that dPCR liquid drops in each detection have higher sizes which are basically the same and improving the detection accuracy.

4) The invention ensures the sealing property of the in-situ dPCR chip by using a complete processing mode of a semiconductor process.

Drawings

FIG. 1 shows the overall structure of the dPCR in-situ chip based on the through hole structure and microfluidics.

FIG. 2 is a schematic diagram showing a partial enlarged structure of a dPCR in-situ chip based on a through hole structure and microfluidics.

FIG. 3 shows a schematic cross-sectional structure at A-A' of the dPCR in-situ chip based on the through hole structure and microfluidics in FIG. 2.

FIG. 4 shows a schematic diagram of the principle of the dPCR in situ chip based on the through hole structure and microfluidics of the present invention.

Fig. 5 is a schematic flow chart showing steps of a method for manufacturing a dPCR in-situ chip based on a via structure and microfluidics in embodiment 2 of the present invention, and fig. 6 is a schematic structural diagram presented by the manufacturing method.

Fig. 7 is a schematic flow chart showing steps of a method for manufacturing a dPCR in-situ chip based on a via structure and microfluidics in embodiment 3 of the present invention, and fig. 8 is a schematic structural diagram presented by the manufacturing method.

Description of the element reference numerals

10 micro flow channel layer

101 micro flow channel

11 micro via layer

111 micro via

12 second sealing layer

121 injection port

13 first sealing layer

S11-S14

S21-S23

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Please refer to fig. 1 to 8. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention, and the drawings only show the components related to the present invention rather than being drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of each component in actual implementation may be changed arbitrarily, and the layout of the components may be more complicated.