A micro-chamber digital PCR disk chip connected with a silicon-based microvalve

Abstract

The present invention relates to a micro-cavity digital PCR (digital Polymerase Chain Reaction, dPCR) disk chip connected with a silicon-based microvalve, comprising a disk-shaped disk (1) and a plurality of reaction units (2), wherein the reaction units (2) sequentially comprise a sample area, a silicon-based microvalve and a silicon-based micro-cavity array connected by a connecting pipe from the inside to the outside of the disk. The present invention realizes the whole process of spontaneous and orderly distribution of samples to micro-cavity digital PCR reaction units by centrifugal force and capillary force driving, and then uses a temperature control module and an optical detection system to realize high-throughput, high-sensitivity absolute quantitative detection of low-abundance nucleic acid samples.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic chips, and in particular relates to a micro-chamber digital PCR disc chip connected with a silicon-based microvalve. Background Art

[0002] Digital PCR (digital Polymerase Chain Reaction, dPCR) is a third-generation PCR technology developed on the basis of conventional PCR and fluorescent quantitative PCR. As an in vitro DNA amplification technology, it has become an important detection method in the field of life sciences with its strong specificity, high sensitivity, absolute quantification, simplicity and speed. However, the existing commercial dPCR platform is limited by expensive equipment or complex operations, making it difficult to achieve widespread application. In addition, the current dPCR chip based on polydimethylsiloxane (PDMS) material has problems such as excessive water volatilization and protein adsorption caused by the permeability and hydrophobicity of the material itself, which greatly limits the application of such chips. The above content shows that there are still problems that need to be solved in the application of nucleic acid amplification detection methods in the field of pathogen detection.

[0003] At present, the research on disc chips mainly focuses on the joint inspection of multiple items with one sample addition, but there is still no relevant research published on issues such as excessive evaporation of water. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a micro-chamber digital PCR disc chip connected to a silicon-based microvalve. The disc chip is driven by centrifugal force and capillary force to realize the whole process of spontaneous and orderly distribution of samples. Then, a temperature control module and an optical detection system can be used to achieve high-throughput and highly sensitive absolute quantitative detection of low-abundance nucleic acid samples.

[0005] The present invention provides a micro-chamber digital PCR disc chip connected with a silicon-based microvalve, comprising a disc-shaped disc and a plurality of reaction units, wherein a single reaction unit comprises, from the inside to the outside of the disc, a sample area, a silicon-based microvalve and a silicon-based microcavity array connected by a connecting pipe; wherein the silicon-based microvalve is composed of a first silicon substrate and a plurality of hydrophilic and oleophobic capillaries; and the silicon-based microcavity array is composed of a second silicon substrate and a plurality of microcavities.

[0006] The plurality of reaction units are distributed in a centrally symmetrical manner.

[0007] The silicon-based microvalve is composed of a first silicon substrate and a plurality of mutually parallel hydrophilic and oleophobic capillaries.

[0008] The silicon-based microcavity array is composed of a second silicon substrate and tens of thousands of nanoliter-scale microcavities with hydrophobic surfaces and hydrophilic pore inner walls.

[0009] The silicon-based microcavity array is a digital PCR amplification area.

[0010] The microcavity is a vertically transparent structure.

[0011] The present invention integrates the hydrophilic and oleophobic silicon-based microvalve structure with the fluid control function with the microcavity digital PCR. Driven by centrifugal force and capillary force, the reaction reagents can pass through the microvalve in an orderly manner and be spontaneously distributed to the microcavity reaction array. The change in the magnitude of the centrifugal force is cleverly combined with the hydrophilic and oleophobic characteristics of the silicon-based microvalve, which can realize the flow limiting and switching control functions of the fluid, so that the water phase and the oil phase enter the PCR amplification area in an orderly manner; utilizing the special structure and characteristics of the microcavity array, the aqueous reagents can be spontaneously filled into the cavity by capillary force, and the oil phase covers it. Finally, driven by centrifugal force and capillary force, the whole process of spontaneous and orderly distribution of samples to the microcavity digital PCR reaction unit is realized, and then the temperature control module and the optical detection system are used to realize high-throughput and highly sensitive absolute quantitative detection of low-abundance nucleic acid samples.

[0012] Beneficial Effects

[0013] (1) The present invention adopts a symmetrically distributed disc-shaped disk and uses centrifugal force as the driving force of the liquid, without the need for external pumping to provide pressure; at the same time, the flow rate of the sample fluid can be controlled by changing the rotation speed of the chip.

[0014] (2) The present invention uses a hydrophilic and oleophobic silicon-based microvalve structure to control the orderly entry of the water phase and the oil phase into the PCR amplification area, which can achieve the flow limiting function of the fluid on the chip.

[0015] (3) The silicon-based microcavity digital PCR provided by the present invention includes tens of thousands of nanoliter micro-reaction chambers. Each silicon-based cavity uses capillary force to absorb aqueous reagents into the cavity and is oil-sealed. It has the advantages of high throughput, pump-free and easy automation. At the same time, the microcavity structure realizes absolute quantification of nucleic acid molecules and has excellent sensitivity, specificity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 1 is a structural diagram of the disk-shaped chip of the present invention; 1 is a disk-shaped disc, and 2 is a single reaction unit.

[0017] Figure 2 It is a structural diagram of a reaction unit in the disk chip of the present invention; wherein 201 is a silicon substrate, 202 is a capillary, 203 is an oil phase, 204 is an aqueous phase, 205 is a silicon substrate, and 206 is a microcavity in a microcavity array.

[0018] Figure 3 Schematic diagram of the hydrophilic and oleophobic microvalve structure of the present invention; wherein the left side is a top view and the right side is a side view.

[0019] Figure 4 Schematic diagram of the microcavity structure of the present invention.

[0020] Figure 5 It is a schematic diagram of the three-dimensional structure of the microcavity structure of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0022] Example 1

[0023] Depend on Figure 1 and 2 As shown, this embodiment provides a micro-chamber digital PCR disc chip connected with a silicon-based microvalve, including a disc-shaped disc 1 and a plurality of reaction units 2, wherein a single reaction unit 2 includes a sample area, a silicon-based microvalve and a silicon-based microcavity array connected by a connecting pipe from the inside to the outside of the disc; wherein the silicon-based microvalve is composed of a first silicon substrate 201 and a plurality of mutually parallel hydrophilic and oleophobic capillaries 202; and the silicon-based microcavity array is composed of a second silicon substrate 205 and tens of thousands of nanoliter-scale hydrophobic microcavities 206 with hydrophobic surfaces and hydrophilic inner walls. Driven by centrifugal force, the aqueous phase such as the PCR premix (containing the extracted and purified target nucleic acid) first enters the silicon-based microcavity array through the hydrophilic valve, and as the centrifugal force further increases, the oil phase enters the silicon-based microcavity array and covers the microcavity. Extremely trace nucleic acid samples undergo rapid and quantitative fluorescence amplification reactions under temperature control conditions, and can be quantitatively analyzed.

[0024] Among them, Figure 1 As shown, several reaction units 2 are centrally symmetrically distributed on the disc-shaped chip, so that each reaction unit is subjected to the same centrifugal force. The centrifugal force generated when the chip moves in a circular motion driven by a micromotor is used as the driving force of the liquid flow, without the need for external pumping to provide pressure; the fluid flow limiting and switching control functions can be achieved by changing the chip rotation speed and designing different channel configurations.

[0025] Among them, Figure 2 , 3 As shown in the figure, the silicon-based microvalve uses silicon as the substrate, and a microfluidic channel is etched on its surface through micromachining technology. Then, surface modification and other operations are used to make it hydrophilic and oleophobic, which can control the orderly entry of the water phase and the oil phase into the PCR amplification area, that is, the silicon-based microcavity array. The processed microchannel is open, and in order to make the channel form a capillary, it is usually necessary to seal it with another polymer or glass as a cover plate.

[0026] Among them, Figure 4 , 5 As shown, each silicon-based microcavity array consists of tens of thousands of nanoliter-scale hydrophobic microcavities 206 with hydrophilic inner walls and can hold 20 μL with an area of ​​1 cm×0.5 cm. Each microcavity 206 is a top-to-bottom transparent structure, and the aqueous reagent is sucked into the cavity by capillary force and oil-sealed on top.

[0027] After the PCR premix is ​​obtained through the nucleic acid extraction and purification module on the disc chip, the chip is centrifuged. T1 , f 水通过 <f T1 ), the aqueous reagent will approach the valve due to the centrifugal force. At this time, due to the hydrophilic valve, the aqueous reagent can easily pass through and be spontaneously distributed to the microcavity reaction array under the action of capillary force. When the speed is changed to further increase the centrifugal force (f T2 , f 油通过 <f T2 ), the oil phase enters the PCR amplification area through the valve and covers the microcavity. Under temperature control conditions, trace nucleic acid samples undergo rapid quantitative amplification reactions in the microcavity reaction unit, and after the reaction is completed, the fluorescence signal can be detected under the optical detection system.

Claims

1. A micro-chamber digital PCR disk chip connected with a silicon-based microvalve, comprising a disk-shaped disc (1) and a plurality of reaction units (2), characterized in that: A single reaction unit (2) includes, from the inside to the outside of the disk, a sample area, a silicon-based microvalve, and a silicon-based microcavity array connected by a connecting pipe; wherein the silicon-based microvalve is composed of a first silicon substrate (201) and a plurality of mutually parallel hydrophilic and oleophobic capillaries (202); and the silicon-based microcavity array is composed of a second silicon substrate (205) and tens of thousands of nanoliter-scale microcavities (206) with hydrophobic surfaces and hydrophilic inner walls.

2. The disc-shaped chip according to claim 1, characterized in that: The plurality of reaction units (2) are distributed in a centrally symmetrical manner.

3. The disc-shaped chip according to claim 1, characterized in that: The silicon-based microcavity array is a digital PCR amplification area.

4. The disc-shaped chip according to claim 1, characterized in that: The microcavity (206) is a transparent structure from top to bottom.

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