CN113663747A - High-dynamic-range multiple digital PCR chip and preparation method thereof - Google Patents
High-dynamic-range multiple digital PCR chip and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection, and discloses a high dynamic range multiple digital PCR chip and a preparation method thereof, wherein the chip comprises a substrate layer, a cavity layer, a supporting layer and a sealing layer which are sequentially bonded from bottom to top; wherein the chamber layer comprises: a first-stage sample inlet; a plurality of detection regions, the detection regions comprising: one end of the serpentine channel is communicated with the primary sample inlet; one end of the second-stage sample inlet is communicated with the other end of the serpentine channel; one end of the main channel is communicated with the other end of the secondary sample inlet; the reaction unit is at least provided with four chambers with different volumes, and the chambers are connected into the main channel; and the sample outlet is communicated with the other end of the main channel. The multiplex detection capability and the dynamic detection range of the digital PCR chip are improved, the dependence on expensive instruments is avoided, and the method is very suitable for a nucleic acid detection scene.
Description
Technical Field
The application relates to the field of molecular biology, in particular to a high dynamic range multiple digital PCR chip and a preparation method thereof.
Background
Microfluidic technology (Microfluidics), featuring micro-machined structures, typically manipulates fluids on the micrometer scale with high throughput and sensitivity. The micro-fluidic platform has been applied to a plurality of fields such as tissue engineering, microparticle manufacturing, drug delivery, point of care testing (POCT), tumor screening, and the like. With the great progress of micro-fabrication methods, microfluidic platforms are increasingly used in the fields of pathogen detection, disease monitoring, and the like due to their significant advantages of low cost, well-controlled microstructure, reduced sample consumption, rapid fluid processing, satisfactory detection sensitivity, and the like.
Digital PCR (digital PCR, dPCR) is a new method for absolutely quantifying target nucleic acid without a standard curve, and the method has great potential in the detection field. In recent years, many new digital PCR chips have been reported in the literature, mainly in two main categories, one being droplet-based digital PCR chips and the other being microarray-based digital PCR chips. However, most of these chips can only detect one sample at a time, and cannot realize simultaneous detection of multiple samples, which greatly limits the practical application capability of the digital PCR chip. In addition, the digital PCR chip reported at present cannot realize the detection with high dynamic range. However, in actual sample diagnosis and clinical monitoring, it is important to perform multiplex detection with high dynamic range on a sample. Therefore, it is a key issue to be solved at present to improve the dynamic monitoring range and the multiple detection capability of the digital PCR chip. Few studies are reported in this respect. The Chinese patent application with the publication number of CN110575852A develops a multi-digital RPA micro-fluidic chip integrating sample pretreatment, which can only carry out quadruple detection at most, and the cavity of the invention has only one size, and can not realize the detection of a dynamic range.
In the process of implementing the invention, the inventor finds that at least the following problems exist in the prior art:
1. the digital PCR chip has insufficient capability in the aspect of multiplex detection, and 2. the detection dynamic range of the digital PCR chip is limited.
Disclosure of Invention
In view of the above, embodiments of the present application provide a high dynamic range multiple digital PCR chip and a method for manufacturing the same, so as to solve the above problems.
According to a first aspect of the embodiments of the present application, a high dynamic range multiple digital PCR chip is provided, wherein the chip includes a substrate layer, a chamber layer, a supporting layer, and a sealing layer, which are sequentially bonded from bottom to top;
wherein the chamber layer comprises:
a first-stage sample inlet;
a plurality of detection regions, the detection regions comprising:
one end of the serpentine channel is communicated with the primary sample inlet;
one end of the second-stage sample inlet is communicated with the other end of the serpentine channel;
one end of the main channel is communicated with the other end of the secondary sample inlet;
the reaction unit is at least provided with four chambers with different volumes, and the chambers are connected into the main channel; and
and the sample outlet is communicated with the other end of the main channel.
Optionally, the reaction unit has four chambers of different volumes, the four chambers having diameters of 100 μm, 400 μm, 800 μm and 1200 μm, respectively, and a height of 100 μm.
Alternatively, 1656 of the chambers with a diameter of 100 μm, 360 of the chambers with a diameter of 400 μm, 288 of the chambers with a diameter of 800 μm and 360 of the chambers with a diameter of 1200 μm.
Optionally, the sample volume of a single detection region is 10 μ L.
Optionally, each of the detection regions has six sets of reaction units.
Optionally, the main channel is formed by connecting four sub-channels with different widths, and one sub-channel corresponds to one chamber.
Optionally, the four sub-channels with different widths increase sequentially along the sample flow direction.
Optionally, the four sub-channels with different widths are 50 μm, 100 μm, 200 μm, and 300 μm, respectively.
Optionally, the chamber layer and the supporting layer are both made of polydimethylsiloxane.
Optionally, the chamber layer and the substrate layer are bonded by an irreversible plasma.
According to a second aspect of embodiments of the present application, there is provided a method for preparing a high dynamic range multiplex digital PCR chip according to the first aspect, the method comprising the steps of:
(1) preparing a clean 4-inch silicon wafer;
(2) manufacturing a cavity layer mold by adopting a standard soft lithography method;
(3) carrying out spin coating treatment on the die by using a release agent, and carrying out ventilation drying;
(4) preparing A: b is 5:1, pouring the PDMS on a cavity layer mould, and heating and curing to form a cavity layer;
(5) preparing A: b is 10:1, pouring the PDMS on the cavity layer, and curing to form a supporting layer;
(6) heating PDMS in different proportions and then adhering the PDMS together, demolding the cured PDMS and punching the PDMS by using a puncher to form a sample inlet and outlet;
(7) sealing the cavity layer and the substrate through plasma treatment;
(8) and a sealing layer is adhered on the supporting layer to seal the sample inlet and outlet.
The technical scheme provided by the embodiment of the application can have the following beneficial effects:
(1) the high dynamic range multiple digital PCR chip provided by the invention is relatively simple to prepare. Due to the fact that the pre-embedded probe and the secondary sample inlet of the primer are arranged, a detection object can be flexibly changed according to detection requirements. When the chip is applied, the material of the chip is a hydrophobic material with air permeability and certain air storage property, so the sample injection mode is self-absorption liquid-separation sample injection based on negative pressure without applying external force.
(2) Due to the different volume size of the chambers, a high dynamic range of detection can be achieved.
(3) The arrangement of a plurality of detection areas ensures that a plurality of samples can be detected simultaneously only by one sample introduction.
(4) The high dynamic range multiple digital PCR chip provided by the invention has the advantages of small volume, simple operation, no need of large instruments and suitability for field detection.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.
FIG. 1 is a schematic diagram illustrating a cross-sectional structure of a high dynamic range multiple digital PCR chip, according to an exemplary embodiment.
FIG. 2 is a schematic diagram illustrating a structure of a chamber layer, according to an example embodiment.
The reference numerals in the figures are:
100. a base layer; 200. a chamber layer; 300. a support layer; 400. a sealing layer; 210. a first-stage sample inlet; 220. a detection zone; 221. a serpentine channel; 222. a second-stage sample inlet; 223. a main channel; 224. a first chamber; 225. a second chamber; 226. a third chamber; 227. a fourth chamber; 228. and a sample outlet.
Detailed Description
Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
It is to be understood that although the terms first, second, third, etc. may be used herein to describe various information, such information should not be limited to these terms. These terms are only used to distinguish one type of information from another. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of the present application. The word "if" as used herein may be interpreted as "at … …" or "when … …" or "in response to a determination", depending on the context.
Referring to fig. 1 and 2, an embodiment of the present invention provides a high dynamic range multiplex digital PCR chip, wherein the chip comprises a substrate layer 100, a chamber layer 200, a support layer 300, and a sealing layer 400, which are sequentially bonded from bottom to top; wherein the chamber layer 200 comprises: a primary inlet 210 and a plurality of detection zones 220, the detection zones 220 comprising: the device comprises a serpentine channel 221, a secondary sample inlet 222, a main channel 223, a reaction unit and a sample outlet 228, wherein one end of the serpentine channel 221 is communicated with the primary sample inlet 210; one end of the secondary sample inlet 222 is communicated with the other end of the serpentine channel 221; one end of the main channel 223 is communicated with the other end of the secondary sample inlet 222; the reaction unit has at least four chambers of different volumes, which are all connected to the main channel 223; the sample outlet 228 communicates with the other end of the main passage 223.
According to the embodiment, the detection object can be flexibly changed according to the detection requirement due to the pre-embedded probe and the primer injection port. When the chip is applied, the material of the chip is a hydrophobic material with air permeability and certain air storage property, so the sample injection mode is self-absorption liquid-separation sample injection based on negative pressure without applying external force. Due to the different volume size of the chambers, a high dynamic range of detection can be achieved. The arrangement of a plurality of detection areas ensures that a plurality of samples can be detected simultaneously only by one sample introduction. The PCR chip has small volume and simple operation, does not need to use a large instrument, and is very suitable for field detection.
In this embodiment, the dynamic range of detection can be significantly improved by using chambers of four reaction units of different volumes, and detection of six different components can be realized by pre-embedding primers and probes. Specifically, four reaction units with different volumes are respectively marked as a first chamber 224, a second chamber 225, a third chamber 226 and a fourth chamber 227, the four reaction units are all cylindrical chambers with diameters of 100 μm, 400 μm, 800 μm and 1200 μm respectively, and the height is 100 μm, and 4 chambers with different volumes can be used for improving the dynamic range of the chip.
In this example 1656 first chambers 224 with a diameter of 100 μm, 360 second chambers 225 with a diameter of 400 μm, 288 third chambers 226 with a diameter of 800 μm and 360 fourth chambers 227 with a diameter of 1200 μm theoretically allow a dynamic range of 8 orders of magnitude to be achieved.
In this embodiment, the optimal sample volume of a single detection region 220 is 10 μ L, and the optimal sample volume of the whole chip is 60 μ L (including loss), so as to realize accurate digital PCR detection with reduced reagent waste.
In this embodiment, each detection area 220 has six main channels 223, each detection area 220 has six groups of four reaction units with different volumes, and detection of 6 different targets can be realized by one sample injection.
Further, the main channel 223 is formed by connecting four sub-channels with different widths, one sub-channel corresponds to one reaction unit, specifically, the four sub-channels with different widths include a first sub-channel, a second sub-channel, a third sub-channel and a fourth sub-channel which are connected in sequence, the first cavity 224 corresponds to the first sub-channel, the second cavity 225 corresponds to the second sub-channel, the third cavity 226 corresponds to the third sub-channel, and the fourth cavity 227 corresponds to the fourth sub-channel; the four sub-channels with different widths are sequentially increased along the flow direction of the sample, specifically, the four sub-channels with different widths are respectively 50 μm, 100 μm, 200 μm and 300 μm, and the gradient channel design can ensure rapid sample introduction and realize effective segmentation of the sample.
In this embodiment, the substrate layer 100 is made of glass or other materials capable of sealing and bonding with Polydimethylsiloxane (PDMS).
In this embodiment, the chamber layer 200 and the substrate layer 100 are bonded by the irreversible plasma, so as to realize the integral packaging and prevent liquid leakage and air leakage.
In this embodiment, the chamber layer 200 is made of Polydimethylsiloxane (PDMS), which is transparent and easy to observe, and has strong air permeability and air storage property, and hydrophobicity.
In this embodiment, the supporting layer 300 is made of Polydimethylsiloxane (PDMS), has a height of 2mm to 5mm, and is used for supporting the chamber layer 200 and providing a certain negative pressure.
The embodiment of the invention also provides a preparation method of the high dynamic range multiple digital PCR chip, which comprises the following steps:
(1) preparing a clean 4-inch silicon wafer;
(2) manufacturing a cavity layer 200 mould by adopting a standard soft lithography method;
(3) carrying out spin coating treatment on the die by using a release agent, and carrying out ventilation drying;
(4) preparing A: b is 5:1, pouring the PDMS on a cavity layer 200 mould, and heating and curing to form a cavity layer 200;
(5) preparing A: b is 10:1, and pouring the PDMS on the chamber layer 200, and curing to form a support layer 300;
(6) demolding the PDMS layer chip and punching by using a puncher to form a sample inlet and outlet 228;
(7) sealing the chip and the substrate after plasma treatment;
(8) a polypropylene film is attached to the support layer 300 as a sealing layer 400 to seal the inlet/outlet 228.
The following is a detailed description of the preparation process by means of specific examples.
Examples
The high dynamic range multiple digital PCR chip provided by the invention is prepared by the following steps:
(1) preparing a silicon wafer: and (3) baking the clean silicon wafer on a hot plate at 200 ℃ for 4-5 min.
(2) Manufacturing a mould: pouring SU-83025 negative photoresist with the size of one coin in the center of the silicon wafer, and setting the coating procedure of the spin coater as follows: 500rpm, 10 s; 1200rpm, 30s, and a thickness of 50 μm for spin-coated photoresist. Pre-baking: baking at 95 deg.C for 15min, and slowly cooling to room temperature. Exposure: and aligning the channel mask to the silicon wafer coated with the photoresist, and exposing by using a single-side photoetching machine for 6 s. Post-baking: baking at 65 deg.C for 1min, baking at 95 deg.C for 5min, and slowly cooling to room temperature. Coating photoresist for the second time: pouring SU-83025 negative photoresist with the size of one coin in the center of the silicon wafer, and setting the coating procedure of the spin coater as follows: 500rpm, 10 s; 1200rpm, 30s, and a thickness of 50 μm for spin-coated photoresist. And (3) secondary prebaking: baking at 95 deg.C for 15min, and slowly cooling to room temperature. And (3) second exposure: and aligning the cavity mask to the silicon wafer with the exposed channel, and exposing by using a single-side photoetching machine for 11 s. Post-baking: baking at 65 deg.C for 1min, baking at 95 deg.C for 5min, and slowly cooling to room temperature. And (3) developing: the process was carried out in a fume hood. Putting the silicon wafer into a flat dish, pouring a developing solution to enable the silicon wafer to submerge into the developing solution, covering the flat dish and placing the flat dish on a shaking table, taking out the silicon wafer after developing for 15min, firstly washing the silicon wafer with isopropanol, if milky floccules appear on the silicon wafer, putting the silicon wafer into the developing solution to continue developing until the milky sediments can not appear after the silicon wafer is washed with the isopropanol, then washing the silicon wafer with absolute ethyl alcohol, observing the structure of a mold by a microscope, and finally washing the silicon wafer with deionized water if no problem exists, and drying the silicon wafer with air. And placing the developed silicon wafer on a hot plate for hard baking, wherein the set program is baking at 95 ℃ for 5min and baking at 200 ℃ for 30 min. And (3) when the silicon wafer is cooled to room temperature, coating a layer of release agent by using a spin coater to enable PDMS to be released easily.
(3) Fabrication of the chamber layer 200: 6g of PDMS prepolymer with the weight ratio of 5:1 is prepared, and uniformly mixed by a vacuum spin coater and bubbles are discharged. The prepared prepolymer is poured on a prepared die and spin-coated by using a spin coater, and the procedure is as follows: 500rpm, 10 s; 1500rpm, 30s, thickness of about 300 μm, baking at 85 deg.C for 5 min.
(4) Manufacturing the support layer 300: a layer of 10:1 PDMS, approximately 2-5mm thick, was poured over the chamber layer 200 and baked at 85 ℃ for 1 h.
(5) The cured PDMS was next cut from the mold and punched out.
(6) Chip sealing: the plasma processing chamber layer 200 and the surface of the substrate layer 100 are bonded, and then baked for 600min at 90 ℃.
(7) Finally, a transparent adhesive tape is attached to the upper layer of the chip to seal the sample inlet and outlet 228.
The chip provided by the invention is uniquely designed to realize digital PCR detection of 6 different targets by single sample introduction; the detection of 6 targets can be realized by utilizing single-color fluorescence, and the multiple detection capability of the digital PCR chip is improved; the dynamic range of each detection area can reach at least 7 orders of magnitude, which is equivalent to a cavity of a million-level array, so that the multiple detection capability of the digital PCR is improved, and the dynamic detection range of the chip is also improved; by utilizing the self-absorption sample introduction mode of PDMS, no external pump valve is needed, and the sample can be automatically introduced after negative pressure treatment; due to the design of the pre-embedding structure of the chip, liquid cross-talk between different areas can be prevented while the pre-embedding of a specific area is realized.
Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
It will be understood that the present application is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.
Claims (10)
1. A high dynamic range multiple digital PCR chip, wherein, the chip comprises a substrate layer, a chamber layer, a supporting layer and a sealing layer which are adhered in turn from bottom to top;
wherein the chamber layer comprises:
a first-stage sample inlet;
a plurality of detection regions, the detection regions comprising:
one end of the serpentine channel is communicated with the primary sample inlet;
one end of the second-stage sample inlet is communicated with the other end of the serpentine channel;
one end of the main channel is communicated with the other end of the secondary sample inlet;
the reaction unit is at least provided with four chambers with different volumes, and the chambers are connected into the main channel; and
and the sample outlet is communicated with the other end of the main channel.
2. The high dynamic range multiplex digital PCR chip of claim 1, wherein said reaction unit has four chambers with different volumes, the four chambers have diameters of 100 μm, 400 μm, 800 μm and 1200 μm, and the height is 100 μm.
3. A high dynamic range multiplex digital PCR chip as defined in claim 2, 1656 of 100 μm diameter chambers, 360 of 400 μm diameter chambers, 288 of 800 μm diameter chambers, and 360 of 1200 μm diameter chambers.
4. The high dynamic range multiplex digital PCR chip of claim 1, wherein the sample volume of each detection region is 10 μ L.
5. The high dynamic range multiplex digital PCR chip of claim 1, each of said detection regions having six sets of reaction units.
6. The high dynamic range multiple digital PCR chip of claim 1, wherein the main channel is formed by connecting four sub-channels with different widths, and one sub-channel corresponds to one chamber.
7. The high dynamic range multiplex digital PCR chip of claim 1, wherein said four different width sub-channels increase sequentially along the sample flow direction.
8. The high dynamic range multiplex digital PCR chip of claim 7, wherein said four different width sub-channels are 50 μm, 100 μm, 200 μm, 300 μm.
9. The high dynamic range multiplex digital PCR chip of claim 1, wherein said chamber layer and said support layer are made of PDMS, and said chamber layer and said substrate layer are bonded by an irreversible plasma.
10. The method for preparing a high dynamic range multiplex digital PCR chip according to claim 1, comprising the steps of:
(1) preparing a clean 4-inch silicon wafer;
(2) manufacturing a cavity layer mold by adopting a standard soft lithography method;
(3) carrying out spin coating treatment on the die by using a release agent, and carrying out ventilation drying;
(4) preparing A: b is 5:1, pouring the PDMS on a cavity layer mould, and heating and curing to form a cavity layer;
(5) preparing A: b is 10:1, pouring the PDMS on the cavity layer, and curing to form a supporting layer;
(6) heating PDMS in different proportions and then adhering the PDMS together, demolding the cured PDMS and punching the PDMS by using a puncher to form a sample inlet and outlet;
(7) sealing the cavity layer and the substrate through plasma treatment;
(8) and a sealing layer is adhered on the supporting layer to seal the sample inlet and outlet.
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