阅读说明：本技术 一种数字pcr芯片 (digital PCR chip ) 是由 任鲁风 李洁昆 蔡亦梅 高静 范东雨 于 2018-05-28 设计创作，主要内容包括：本发明提供了一种数字PCR芯片,其包括基座、旋转上盖、微阵列反应仓、密封层、密封圈、弹力卡扣、转轴一、转轴二、扭簧。其中微阵列反应仓表面均布400~1亿个微孔,用于独立分隔和承载反应试剂体系；旋转上盖与基座绕转轴一相互转动,实现样品填充；旋转上盖上置有密封层和密封圈,用于隔离和密封微阵列反应仓,避免交叉污染和蒸发；弹力卡扣与基座绕转轴二相互转动,通过转轴二内置的扭簧实现压紧和密封。本发明的数字PCR芯片无需外接驱动装置、节省试剂、操作简单。(the invention provides a digital PCR chip, which comprises a base, a rotary upper cover, a microarray reaction bin, a sealing layer, a sealing ring, an elastic buckle, a first rotating shaft, a second rotating shaft and a torsion spring. Wherein 400-1 hundred million micropores are uniformly distributed on the surface of the microarray reaction bin and are used for independently separating and bearing a reaction reagent system; the rotary upper cover and the base rotate around the first rotating shaft to realize sample filling; the rotary upper cover is provided with a sealing layer and a sealing ring for isolating and sealing the microarray reaction bin to avoid cross contamination and evaporation; the elastic buckle and the base rotate around the second rotating shaft, and the compression and the sealing are realized through a torsion spring arranged in the second rotating shaft. The digital PCR chip of the invention does not need an external driving device, saves reagents and has simple operation.)

1. A digital PCR chip is characterized by comprising a base, a rotary upper cover, a microarray reaction bin, a sealing layer, a sealing ring, an elastic buckle, a first rotating shaft, a second rotating shaft and a torsion spring;

The base is connected with the rotary upper cover through the first rotating shaft;

The second rotating shaft is connected with the base and the elastic buckle through a torsion spring;

A microarray reaction bin is arranged on the base;

A sealing layer is arranged on the rotary upper cover;

The sealing layer is low-melting-point solid straight-chain alkane;

The microarray reaction bin consists of micropores.

2. the digital PCR chip of claim 1, wherein the microarray reaction chamber is made of a material selected from the group consisting of resin, plastic, silicon wafer, glass, quartz, metal, and ceramic.

3. The digital PCR chip of claim 1, wherein the microarray reaction chamber has 400-1 hundred million micropores distributed on the surface thereof, the volume of the micropores is 0.5 pL-1 μ L, and the microarray reaction chamber is divided into 1-100 partitions for performing digital PCR reaction on 1-100 samples simultaneously.

4. The digital PCR chip of claim 1, wherein the shape of the microwells is circular, square, triangular, hexagonal, polygonal, and arranged in horizontal and vertical directions or in close-packed arrangement.

5. The digital PCR chip of claim 3, wherein the micro-via processing method comprises photo-etching, glass ceramic metal etching, high precision mold injection, machining cutting, 3D printing, deposition and bonding.

6. The digital PCR chip of claim 1, wherein the microarray reaction chamber has a functional modification group or surface hydrophilic treatment.

7. The digital PCR chip of claim 1, wherein the solid linear alkane is solid at room temperature, has a melting point of 30 ℃ to 70 ℃, has a density lower than that of the reaction reagent after melting, is immiscible with the reagent, and does not react with the reagent, and is made of paraffin and solid grease.

8. The digital PCR chip of claim 1, wherein the sealing ring is located outside the microarray reaction chamber or outside the sealing layer.

9. The digital PCR chip of claim 1, wherein two ends of the torsion spring are fixed on the rotary upper cover and the base respectively, and the elastic buckle is pressed on the base in a free state.

10. The digital PCR chip of claim 1, wherein the chip is used for separating and supporting each independent reaction system in the digital PCR reaction.

Technical Field

The invention relates to the technical field of digital PCR, in particular to a digital PCR chip.

Background

The Polymerase Chain Reaction (PCR) is a molecular biological technology capable of amplifying DNA fragments in vitro, and the digital PCR (dPCR) is a new technology for absolutely quantifying nucleic acid molecules, does not depend on a standard curve and a reference sample, does not need to be provided with a control, and can directly detect the copy number of target molecules. The principle of dPCR is similar to the 'divide-and-conquer' algorithm in computer science, the reagent with low template is distributed into a large number of micropores, after statistical analysis, most micropores have no or only 1 target molecule, after amplification, counting is carried out through an optical detection module, and the initial template amount is obtained. Compared with the traditional quantitative PCR, the dPCR has higher sensitivity, specificity, high tolerance and accuracy, and the technology is widely applied to the aspects of trace nucleic acid sample detection, CNV analysis, complex sample gene expression detection and the like.

Digital PCR allows for two detection methods.

The method comprises the steps of firstly, detecting the quantity of a template by an emulsion droplet method, forming reaction systems containing nucleic acid molecules into ten thousand microdroplets, wherein the microdroplets are about 1nL in volume, each microdroplet contains 1 nucleic acid molecule at most (except individual difference) according to a statistical rule, using each microdroplet as a PCR reaction container, carrying out cyclic amplification, using a microdroplet analyzer to check whether each microdroplet is amplified one by one, counting the amplified microdroplets, and finally calculating the copy number of the template according to the Poisson distribution principle and the proportion of positive microdroplets. This method has a large error in generating droplets, affects the detection result, and is costly.

The second is to use the micro-fluidic chip to detect, and the micro-fluidic principle is to extrude the reagent into the chip pore channel through the external applied pressure, but the driving structure is complicated, the occupied space is large, the cost is high, the fluid channel resistance is large, gas residue is easy to occur, and the mode generally needs a large amount of surplus of the reagent to fill the chip, thus causing great waste to the reagent.

The material needs to comprehensively consider heat conductivity, heat resistance, biocompatibility and light transmittance, silicon and glass can be selected, the two materials have very good heat conductivity and light transmittance, the traditional micro-processing technology can be used, such as wet etching, dry etching, deep reactive ion etching, optical etching and the like, plastics and polymers can be selected, the material is easier to process and manufacture compared with silicon and glass, and the traditional micro-machining and rapid forming method such as injection molding can be selected.

The invention is a digital PCR chip which does not need an external driving device, avoids cross contamination among micropores, avoids liquid evaporation, reduces reagent consumption and has low cost.

Disclosure of Invention

The invention provides a digital PCR chip.

in some embodiments, the digital PCR chip architecture essentially comprises: the microarray device comprises a base, a rotary upper cover, a microarray reaction bin, a sealing layer, a sealing ring, an elastic buckle, a first rotating shaft, a second rotating shaft and a torsion spring, wherein the base and the rotary upper cover are connected through the first rotating shaft and can rotate mutually; the rotating shaft two is connected with the base and the elastic buckle through the torsion spring, so that the elastic buckle and the base can rotate mutually.

In some embodiments, a microarray reaction chamber is disposed on the base.

In some embodiments, 400-1 hundred million micropores are distributed on the surface of the microarray reaction bin, and the manufacturing materials of the microarray reaction bin comprise resin, plastic, glass, quartz, silicon wafers, metal and ceramic, preferably the silicon wafers, and the microarray reaction bin has the advantages of good heat-conducting property, good biocompatibility and suitability for various processing methods;

In some embodiments, the microwell volume is 0.5pL to 1 uL, preferably 4pL to 1nl, based on statistical analysis, which can be regarded as the number of DNAs in each microwell ≦ 1.

In some embodiments, the microarray reaction chamber is divided into 1-100 partitions for performing digital PCR reaction on 1-100 samples simultaneously.

In some embodiments, the shape of the micropores is circular, square, triangular, hexagonal, polygonal, arranged in transverse and longitudinal directions or in close-packed arrangement.

In some embodiments, the microarray reaction chamber has a groove around it, which is beneficial for storing excess reagents.

in some embodiments, the microarray reaction chamber is fabricated by photolithography, glass ceramic metal etching, high precision mold injection, machining, cutting, 3D printing, deposition, bonding, and the like, preferably photolithography.

In some embodiments, the microarray reaction chamber has a functional modification group or is surface-hydrophilically treated, which advantageously allows the reagents to smoothly enter the microwells, avoiding the formation of bubbles.

In some embodiments, a sealing layer is disposed on the spin-on lid, the sealing layer being a low melting point solid linear alkane. Wherein the low-melting solid straight-chain alkane is solid at normal temperature, the melting point is 30-70 ℃, the density of the melted straight-chain alkane is less than that of a reaction reagent, the straight-chain alkane is not mutually soluble with the reagent and does not react with the reagent, and the straight-chain alkane can be made of paraffin and solid grease.

In some embodiments, the sealing layer or the microarray reaction chamber is provided with a sealing ring at the outer side, and the sealing ring has the beneficial effect that the melted solid linear alkane is sealed above the microarray reaction chamber.

In some embodiments, two ends of the torsion spring are respectively fixed on the rotary upper cover and the base, and the elastic buckle is pressed on the base in a free state.

Compared with the traditional microfluidic chip, the microfluidic chip has the advantages of no need of an external driving device, reagent saving, cross contamination prevention, liquid evaporation prevention and simple operation.

Drawings

fig. 1 is a schematic view showing the overall structure of the embodiment of the present invention.

FIG. 2 is a schematic view showing a structure of a microarray reaction chamber according to an embodiment of the present invention.

FIG. 3 is a diagram illustrating a sample application process according to an embodiment of the present invention.

FIG. 4 is a schematic view showing the completion of sample application according to an embodiment of the present invention.

Detailed Description

The technical solution of the present invention will be specifically described below with reference to the accompanying drawings.

the digital PCR chip of the embodiment is a disposable consumable. The structure comprises a base (11), a rotary upper cover (12), a microarray reaction bin (15), a sealing layer (14), a sealing ring (17), an elastic buckle (18), a first rotating shaft (13), a second rotating shaft (16) and a torsion spring (19). Wherein the base (11) and the rotary upper cover (12) are connected by a first rotating shaft (13) so that the base and the rotary upper cover can rotate mutually; the second rotating shaft (16) is connected with the base (11) and the elastic buckle (18) through a torsion spring (19), so that the elastic buckle (18) and the base (11) can rotate mutually.

In the embodiment, the first rotating shaft (13) is internally provided with the sliding bearing, and the beneficial effect is that the rotating precision of the rotating upper cover and the base is improved.

In this embodiment, a microarray reaction chamber (15) is disposed on the base (11), wherein the microarray reaction chamber is made of silicon wafer, the outer dimension is 25mm × 25mm, 30625 micropores (151) with a diameter of 0.1mm and a depth of 0.05mm and a volume of about 0.4nL are uniformly distributed on the surface of the base with a center of 20mm × 20mm by photolithography. A circle of groove (152) with the width of 0.2mm is arranged around the micropore and used for storing redundant reaction reagents and avoiding the reagent from overflowing.

In this embodiment, the BSA solution is used to perform hydrophilic treatment on the microarray reaction chamber, so that the reagent can smoothly enter the micropores, thereby preventing the generation of bubbles.

The rotary upper cover (12) is provided with a sealing layer (14) which is low-melting-point solid straight-chain alkane. The low-melting-point solid straight-chain alkane is solid at normal temperature, the melting point of the low-melting-point solid straight-chain alkane is 30-70 ℃, the density of the low-melting-point solid straight-chain alkane after melting is smaller than that of a reaction reagent, the low-melting-point solid straight-chain alkane is not mutually soluble with the reagent and does not react with the reagent, the material of the low-melting-point solid straight-chain alkane can be paraffin or solid grease, and the low-melting-point solid alkane in the embodiment is paraffin.

In the embodiment, a sealing ring (17) is arranged on the outer side of the sealing layer (14), and is made of silica gel.

In the embodiment, 3 torsion springs (19) are used, and two ends of each torsion spring are respectively fixed on the rotary upper cover (12) and the base (11).

The sample adding process of this example is: horizontally placing a digital PCR chip (10), dripping a reagent (31) on a microarray reaction bin, rotating an upper cover (12) by hand to gradually reduce an included angle alpha between the upper cover (12) and a base (11), and rotating the upper cover (12) to drive liquid to enter a micropore (151) when a sealing layer (14) is contacted with the reagent (31) and the included angle alpha is reduced; when the included angle alpha is close to 0 degree, the sealing layer (14) can cover the upper part of the micropore area, the elastic buckle (18) is pressed manually, the elastic buckle (18) presses the rotary upper cover (12), and the sealing ring is extruded by the elasticity of the torsion spring (19) to deform to realize sealing. When the digital PCR chip is used for PCR amplification, paraffin is positioned between the upper layer of the microarray reaction bin and the sealing ring after melting along with the temperature rise to 53 ℃, so that the sealing effect is realized, and the cross contamination and the reagent evaporation are avoided.