Preparation method of micro-droplet array chip for dPCR reaction and micro-droplet array chip

Abstract

The invention provides a preparation method of a micro-droplet array chip for dPCR reaction and the micro-droplet array chip. The method comprises the following steps: step 1, forming a silicon nitride layer on the surface of a silicon-based substrate; 2, etching the silicon nitride layer and a part of the silicon-based substrate so as to form a micro-well array on the silicon-based substrate; 3, forming a silicon dioxide layer in the micro-well of the micro-well array; 4, removing the silicon nitride layer to obtain a silicon-based substrate with a micro-well array; and step 5, bonding a glass cover plate on the surface, with the micro-well array, of the silicon-based substrate, wherein a groove corresponding to the micro-well array in position is formed in the surface, used for bonding, of the glass cover plate. According to the preparation method of the micro-droplet array chip for the dPCR reaction and the micro-droplet array chip disclosed by the invention, a scraping cover plate or sample introduction under negative pressure is not needed. The inner cavity of the whole chip belongs to a hydrophilic surface, so that the whole chip can be filled with a water-phase solution under normal pressure, and the water-phase solution forms mutually independent micro-droplet units under the action of interfacial tension of an oil phase subsequently.

Description

Technical Field

[0001] The present invention relates to PCR biotechnology, and particularly to a preparation method of a microdroplet array chip for dPCR reaction and the microdroplet array chip. Background Art

[0002] Current Digital Polymerase Chain Reaction (dPCR) relies on the technology of micro-differentiating an aqueous reaction solution into multiple independent reaction units distributed in an oil phase to form a microdroplet array. Currently, there are mainly two technical routes: based on microdroplet generation and static droplet reaction on a microplate. The microdroplet generation technology involves a droplet generator. This method not only requires a PCR reaction system but also additional auxiliary equipment, increasing the complexity of the design. Moreover, during the thermal cycling reaction of the droplets and the transfer to the reading process, droplet fusion or loss is likely to occur, affecting the accuracy of the results. Most of the dPCR reactions based on microplates are open arrays, and during the sample injection process, the liquid is prone to evaporation in the microholes, thus limiting the volume of the smallest reaction unit. At the same time, during the PCR thermal cycling process, the reaction solution also has a tendency to evaporate, which may lead to series connection between reaction units and affect the accuracy of the results.

[0003] Therefore, there is an urgent need for a droplet dispensing system applicable to dPCR reaction to solve the above various problems. Summary of the Invention

[0004] Embodiments of the present invention provide a preparation method of a microdroplet array chip for dPCR reaction and the microdroplet array chip to at least solve one of the problems existing in the related art. To achieve this purpose, the present invention is realized through the following technical solutions.

[0005] On the one hand, embodiments of the present invention provide a preparation method of a microdroplet array chip for dPCR reaction, including:

[0006] Step 1: Form a silicon nitride layer on the surface of a silicon-based substrate;

[0007] Step 2: Etch the silicon nitride layer and part of the silicon-based substrate to form a micro-well array on the silicon-based substrate;

[0008] Step 3: Form a silicon dioxide layer in the micro-wells of the micro-well array;

[0009] Step 4: Remove the silicon nitride layer to obtain a silicon-based substrate with a micro-well array;

[0010] Step 5: Bond a glass cover plate to the surface of the silicon-based substrate having the micro-well array, and grooves corresponding to the positions of the micro-well array are formed on the surface of the glass cover plate for bonding.

[0011] Further, the thickness of the silicon nitride layer is 10 - 100 angstroms.

[0012] Further, in step 2, a micro-well array is formed on the silicon-based substrate by photolithography or dry etching.

[0013] Further, in step 3, a silicon dioxide layer is formed in the micro-wells of the micro-well array by wet oxidation.

[0014] Further, the depth of the micro-wells of the micro-well array is 10 - 50 microns.

[0015] Further, the thickness of the silicon dioxide layer is 1000 - 1500 angstroms.

[0016] Further, the opening shape of the micro-wells in the micro-well array is circular, quadrilateral or hexagonal.

[0017] On the other hand, an embodiment of the present invention provides a micro-droplet array chip for dPCR reaction, which is prepared according to the above preparation method.

[0018] Further, the aqueous solution for dPCR reaction is filled into the micro-droplet array chip under normal pressure, and then is pinned into each micro-well under the action of the interfacial tension of the oil-phase liquid to form independent micro-droplet units.

[0019] Further, the surface contact angle of the aqueous solution in each micro-well is close to zero degree.

[0020] The embodiment of the present invention has the following beneficial effects:

[0021] (1) By forming a silicon dioxide layer inside the micro-wells, the present invention obtains a super-hydrophilic silicate micro-well array on the silicon-based substrate, making the aqueous solution for dPCR reaction more easily distributed in the micro-wells and separated by the oil phase due to the difference in surface tension, and finally forming independent micro-droplet units.

[0022] (2) Compared with the existing chemical grafting method to improve hydrophilicity, the present invention adopts a method without chemical grafting, and the prepared hydrophilic layer can remain stable under high temperature and high pressure, so that the performance is not affected during the subsequent anodic bonding process of the glass cover plate and is compatible with the MEMS process.

[0023] (3) The preparation method of the microdroplet array chip for dPCR reaction and the microdroplet array chip provided by the present invention do not require a scraping cover plate or sample injection under negative pressure. Since the entire inner cavity of the chip is a hydrophilic surface, the aqueous solution for dPCR reaction can fill the entire chip under normal pressure, and then the subsequent oil phase is distributed into independent reaction units due to the action of interfacial tension, enabling the dispersion of droplets to be completed only relying on the hydrodynamic force depending on the hydrophilic and hydrophobic properties of the chip surface.

[0024] (4) Through the preparation method of the microdroplet array chip for dPCR reaction and the microdroplet array chip provided by the present invention, the volume of the distributed microdroplet units is controllable and the size of the microdroplet units can be smaller than existing mature products.

[0025] (5) By using the microdroplet array chip for dPCR reaction provided by the present invention and in cooperation with the use of a fixture, PCR detection thermal cycling can be carried out under normal pressure, and the stability of static microdroplets can be maintained. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0027] Figure 1 It is a schematic flow chart of the preparation method of the microdroplet array chip for dPCR reaction according to the embodiment of the present invention;

[0028] Figure 2 It is an exploded view of the structure of the microdroplet array chip for dPCR reaction according to the embodiment of the present invention;

[0029] Figure 3 It is the SEM (scanning electron microscope) image and energy dispersive X-ray spectrum analysis result of the microdroplet array chip for dPCR reaction according to the embodiment of the present invention;

[0030] Figure 4 It is a contact angle photo of the bare silicon and the silicon dioxide layer formed on the surface according to the embodiment of the present invention;

[0031] Figure 5 It is a photo of the aqueous solution of 5-carboxyfluorescein dye distributed in the micro-well array with a depth of 50 μm according to the embodiment of the present invention;

[0032] Figure 6 It is the numerical simulation result of the distribution of the aqueous solution in a single micro-well according to the embodiment of the present invention;

[0033] Figure 7 It is a fluorescence image of the dPCR reaction result of the microdroplet array chip according to the embodiment of the present invention;

[0034] Figure 8 It is a fluorescence image of the reaction result of an existing commercial dPCR system. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will elaborate on each implementation manner of the present invention in conjunction with the accompanying drawings. However, those of ordinary skill in the art can understand that in each implementation manner of the present invention, many technical details are presented for the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following implementation manners, the technical solutions claimed in the present application can still be realized. The division of the following embodiments is for convenience of description and should not constitute any limitation to the specific implementation manners of the present invention. The various embodiments can be combined and cross-referenced with each other on the premise of no contradiction.

[0036] One aspect of the embodiments of the present invention provides a method for preparing a microdroplet array chip for dPCR reaction. Figure 1 It is a schematic flowchart of the method for preparing a microdroplet array chip for dPCR reaction according to the embodiments of the present invention. As Figure 1 shown, the preparation method includes the following steps.

[0037] Step 1: Form a silicon nitride layer on the surface of a silicon-based substrate. Specifically, low-pressure chemical vapor deposition is performed on a single-side polished silicon-based substrate to form a silicon nitride layer. In this embodiment, the thickness of the silicon nitride layer is 10 - 100 angstroms.

[0038] Step 2: Etch the silicon nitride layer and part of the silicon-based substrate to form a micro-well array on the silicon-based substrate. Specifically, a lithography method is used. For example, a positive photoresist is used to lithograph a pattern, and deep silicon etching is performed according to actual application requirements to etch a certain depth, that is, the silicon nitride layer and part of the silicon-based substrate layer are etched away. Subsequently, the photoresist is removed by plasma treatment and sulfuric acid cleaning is performed, thereby forming a micro-well array on the silicon-based substrate. In this embodiment, the depth of the micro-wells in the micro-well array is 10 - 50 micrometers.

[0039] Step 3: Form a silicon dioxide layer in the micro-wells of the micro-well array. Specifically, a silicon dioxide layer is formed in the micro-wells of the micro-well array by wet oxidation. Among them, since the unetched area of the silicon-based substrate is protected by the silicon nitride layer, the silicon dioxide layer is only formed in the etched area of the silicon-based substrate (i.e., inside the micro-wells). In this embodiment, the thickness of the silicon dioxide layer is 1000 - 1500 angstroms.

[0040] Step 4: Remove the silicon nitride layer to obtain a silicon-based substrate with a micro-well array. Specifically, immerse the silicon-based substrate in a 14% buffered oxide etchant, and by controlling the reaction time, etch and remove the nitrogen oxides naturally formed on the silicon nitride surface in air. Then, treat it with heated 85 wt% phosphoric acid to remove the silicon nitride layer, and then rinse with deionized water and centrifuge to dry. Finally, a super-hydrophilic silica micro-well array is obtained on the silicon-based substrate. Among them, the opening shape of the micro-wells in the micro-well array can be circular, quadrilateral or hexagonal.

[0041] Step 5: Bond a glass cover plate to the surface of the silicon-based substrate with the micro-well array. Grooves corresponding to the position of the micro-well array are formed on the surface of the glass cover plate for bonding. Specifically, use wet etching and laser processing methods to form grooves, inlets and outlets on the glass cover plate, and then anodically bond the groove surface of the glass cover plate to the micro-well array surface of the silicon-based substrate to obtain a micro-droplet array chip for dPCR reaction.

[0042] Another aspect of the embodiments of the present invention provides a micro-droplet array chip for dPCR reaction prepared according to the above method. Figure 2 It is an exploded view of the structure of the micro-droplet array chip for dPCR reaction according to the embodiments of the present invention. As Figure 2 shown, the micro-droplet array chip includes an upper glass cover plate 1 and a lower silicon-based substrate 2 (left figure). Among them, grooves 3, an inlet 4 and an outlet 5 for reaction reagents are formed on the glass cover plate 1, and a micro-well array 6 is formed on the silicon-based substrate. The right figure is an enlarged view of the micro-well array 6. When the groove surface of the glass cover plate 1 and the micro-well array surface of the silicon-based substrate 2 are bonded, the grooves 3 and the micro-well array 6 are vertically corresponding.

[0043] The preparation method and the micro-droplet array chip for dPCR reaction provided by the present invention do not require a scraping cover plate or sample injection under negative pressure. Since the entire inner cavity of the chip belongs to a hydrophilic surface, the aqueous solution for dPCR reaction can fill the entire chip under normal pressure, and then the subsequent oil phase forms independent micro-droplet units by being pinned into each micro-well under the action of interfacial tension, so that the dispersion of droplets can be completed only by relying on the hydrodynamic force of the hydrophilic and hydrophobic properties of the chip surface.

[0044] To further illustrate the feasibility and the beneficial effects of the preparation method and the micro-droplet array chip for dPCR reaction provided by the present invention, the following conducts performance characterization and performance tests on the micro-droplet array chip for dPCR reaction.

[0045] In this embodiment, the thickness of the deposited silicon nitride layer is After deep silicon etching, Silicon dioxide oxide layer. It was treated with a 14% buffered oxide etchant for 15 seconds and then with 85 wt% phosphoric acid at 160 °C for 30 minutes to remove the silicon nitride layer. After preparation, a silicon dioxide oxide layer of about was formed on the sidewalls and bottom of the micro-wells. The prepared chip was cross-sectioned and observed under a scanning electron microscope, and energy-dispersive X-ray spectroscopy analysis was performed. Figure 3 SEM (scanning electron microscope) images and energy-dispersive X-ray spectroscopy analysis results of the micro-droplet array chip for dPCR reaction according to the embodiments of the present invention are shown. As Figure 3 shown, the depth of the micro-well array pattern (a 50*50 μm square) is about 51 μm, and the energy-dispersive X-ray spectroscopy analysis spectrum proves the existence of a silicon dioxide surface inside the micro-wells.

[0046] Figure 4 Contact angle photos of bare silicon and silicon dioxide layer formed on the surface according to the embodiments of the present invention are shown. Among them, the contact angle of the silicon dioxide oxide layer surface is close to 0°, much lower than the contact angle of 72.69° of the bare silicon surface. Therefore, it shows that a super-hydrophilic micro-well array can be obtained by the preparation method of the micro-droplet array chip for dPCR reaction provided by the embodiments of the present invention.

[0047] Figure 5 Photos of the aqueous solution of 5-carboxyfluorescein dye distributed in the 50 μm deep micro-well array according to the embodiments of the present invention are shown. It can be seen from the photos that the aqueous solution can be distributed into the micro-wells. Further experiments show that droplet partitioning can be achieved in both square and hexagonal micro-arrays with depths of 50 μm and 10 μm, which proves that the preparation method of the micro-droplet array chip for dPCR reaction and the micro-droplet array chip provided by the embodiments of the present invention can be widely applied to various scenarios.

[0048] Figure 6 Numerical simulation results of the distribution of the aqueous solution in a single micro-well according to the embodiments of the present invention are shown. It can be seen from the numerical simulation results that under the condition of controlling the oil phase injection speed, the micro-droplet array chip provided by the embodiments of the present invention can distribute the aqueous solution into the micro-wells, and the filling rate of the aqueous solution in the micro-wells can reach 98.56%.

[0049] Furthermore, a dPCR reaction was carried out on the micro-droplet array chip to detect the DNA copy number concentration of a sample (hepatitis B virus plasmid template), and the fluorescence image of the reaction result is as Figure 7 shown. The number of positive micro-wells accounts for 0.0895 of the total number of micro-wells included in the analysis, and then the DNA concentration of the sample was calculated to be 3409 copies / μL. When the same sample was tested on a commercial dPCR system, the fluorescence image of the reaction result is as Figure 8As shown, the DNA concentration of the sample was then calculated to be 3623 copies / μL, and the difference between the two results was within an acceptable range. It was also found by comparison that there were obvious problems with the commercial chips, such as uneven droplet size, uneven distribution, and droplet fusion, while the microdroplet array chip provided in the embodiments of the present invention did not have these problems.

[0050] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a microdroplet array chip for dPCR reaction, characterized in that: include: Step 1: forming a silicon nitride layer on the surface of a silicon-based substrate; Step 2: etching the silicon nitride layer and a portion of the silicon-based substrate to form a micro-well array on the silicon-based substrate; Step 3: forming a silicon dioxide layer in the micro-wells of the micro-well array; Step 4: removing the silicon nitride layer to obtain a silicon-based substrate with a micro-well array; Step 5: bonding a glass cover plate to the surface of the silicon-based substrate having the micro-well array, wherein the surface of the glass cover plate used for bonding is formed with grooves corresponding to the positions of the micro-well array.

2. The method for preparing a micro-droplet array chip for dPCR reaction according to claim 1, characterized in that: The thickness of the silicon nitride layer is 10-100 angstroms.

3. The method for preparing a micro-droplet array chip for dPCR reaction according to claim 1, characterized in that: Step 2: forming a micro-well array on the silicon-based substrate by photolithography or dry etching.

4. The method for preparing a micro-droplet array chip for dPCR reaction according to claim 1, characterized in that: Step 3: forming a silicon dioxide layer in the micro-wells of the micro-well array by wet oxidation.

5. The method for preparing a micro-droplet array chip for dPCR reaction according to claim 1, characterized in that: The depth of the micro-wells of the micro-well array is 10-50 microns.

6. The method for preparing a micro-droplet array chip for dPCR reaction according to claim 1, characterized in that: The thickness of the silicon dioxide layer is 1000-1500 angstroms.

7. The method for preparing a micro-droplet array chip for dPCR reaction according to claim 1, characterized in that: The opening shape of the micro-wells in the micro-well array is circular, quadrilateral or hexagonal.

8. A microdroplet array chip for dPCR reaction, characterized in that: The micro-droplet array chip is prepared according to the preparation method according to any one of claims 1-7.

9. The micro-droplet array chip for dPCR reaction according to claim 8, characterized in that: The aqueous solution used for dPCR reaction is filled into the micro-droplet array chip under normal pressure, and then pinned into each micro-well under the action of the interfacial tension of the oil phase liquid to form independent micro-droplet units.

10. The micro-droplet array chip for dPCR reaction according to claim 9, characterized in that: The surface contact angle of the aqueous solution in each of the micro-wells is close to zero.

Images