CN115678758A - Liquid drop type digital PCR chip - Google Patents

Abstract

The invention discloses a liquid drop type PCR digital chip. The chip consists of a substrate and a cover plate; the substrate is provided with a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity; a micro-fluidic channel is arranged between the mixing cavity and the liquid drop cavity, and is provided with an atomizing nozzle with magnetic bead filtering and non-return functions; a liquid drop laying pore passage is arranged between the liquid drop cavity and the detection cavity and is in an hourglass shape. The liquid drop type PCR digital chip provided by the invention not only needs to be bonded once during production, but also does not need to form a vacuum or negative pressure environment in the using process, and can complete the work of processing, liquid drop generation and detection of a nucleic acid sample on a single chip, thereby improving the integration and automation degree of liquid drop type digital PCR detection, and simultaneously reducing the risks of nucleic acid leakage polluting the environment and influencing the body health of detection personnel.

Description

Liquid drop type digital PCR chip

Technical Field

The invention belongs to the field of nucleic acid detection chips, and particularly relates to a liquid drop type digital PCR chip.

Background

Because the quantitative detection does not depend on a standard curve, the quantitative detection is not influenced by the PCR amplification efficiency, and the sensitivity and the accuracy are higher, the liquid drop type digital PCR technology gradually becomes a detection technical means which plays an important role in the fields of clinical medical auxiliary diagnosis such as accurate cancer screening, pathogenic microorganism detection, infectious disease screening and the like. It is known that the detection accuracy of droplet-based digital PCR can be improved and the detection limit of droplet-based digital PCR can be reduced by adding droplets. But the sample processing function of nucleic acid purification is not included in the current digital PCR detection flow in the droplet mode. In other words, in the case of droplet-based digital PCR detection, a nucleic acid tester needs to process a sample outside a chip and then transfer the sample to an additional droplet generation chip to obtain micro-droplets for digital PCR detection. However, the application of droplet-based digital PCR is greatly affected by the inevitable sample loss and sample contamination during sample transfer.

Therefore, it is necessary to develop a droplet-type digital PCR chip capable of realizing "sample in and result out" to improve the integration and automation degree of droplet-type digital PCR detection, and simultaneously reduce the risks of nucleic acid leakage polluting the environment and affecting the health of detection personnel.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art of the droplet-type digital PCR chip, provide a droplet-type digital PCR chip which integrates the whole process of nucleic acid sample cracking, washing, elution, PCR reaction liquid mixing, droplet generation and digital PCR detection and can overcome the problems or partially solve the problems, and provide powerful guarantee for the implementation of high-efficiency and accurate digital PCR detection service in vast PCR laboratories.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a liquid drop type digital PCR chip is composed of a substrate and a cover plate, wherein the substrate comprises a cracking cavity for a nucleic acid sample, a washing cavity, an elution cavity, a PCR reaction liquid mixing cavity (mixing cavity for short), a liquid drop cavity and a digital PCR detection cavity (detection cavity for short), the work of processing the nucleic acid sample, generating liquid drops and detecting the nucleic acid sample can be finished on a single chip, the integration and automation degree of liquid drop type digital PCR detection is improved, and the risk that the nucleic acid leaks to pollute the environment and influence the body health of detection personnel is reduced.

According to the embodiment of the invention, the invention can be further optimized, and the following is the technical scheme formed after optimization:

in one preferred embodiment, the liquid drop PCR digital chip comprises a substrate and a cover plate which are attached to each other.

In one preferred embodiment, the liquid drop PCR digital chip is prepared by packaging a substrate and a cover plate of the chip through thermal compression bonding.

In one preferred embodiment, the liquid drop PCR digital chip is prepared by packaging a substrate and a cover plate of the chip through plasma bonding.

In one preferred embodiment, the cover plate of the liquid drop PCR digital chip is made of a mixed material of PMMA and silicon nitride, and the mass content of the silicon nitride in the mixed material is 1-15%.

In one preferred embodiment, the cover plate of the liquid drop PCR digital chip is made of a mixed material of PDMS and zirconium boride, and the mass content of zirconium boride in the mixed material is 0.5-10%.

In one preferred embodiment, the substrate of the liquid drop PCR digital chip is made of a mixed material of silicon dioxide and PDMS, and the mass content of PDMS in the mixed material is 5% -25%.

In one preferred embodiment, the substrate of the liquid drop PCR digital chip is made of a mixed material of boron nitride and PDMS, and the mass content of PDMS in the mixed material is 20% -40%.

In one preferred embodiment, the liquid drop type PCR digital chip comprises a substrate provided with a lysis cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity.

In some embodiments of the invention, one surface of a cover plate which is not attached to a substrate of the chip is marked with characters of a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity.

In one preferred embodiment, the liquid drop type PCR digital chip is provided with a substrate provided with injection pipelines of a lysis cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity respectively.

In one preferred embodiment, the liquid drop type PCR digital chip is provided with a safety pipeline on a substrate.

In one preferred embodiment, a microfluidic channel is arranged between the lysis cavity of the chip substrate and the washing cavity of the substrate; the microfluidic channel is provided with a check valve.

In one preferred embodiment, a microfluidic channel is arranged between the washing cavity of the chip substrate and the elution cavity of the substrate; the microfluidic channel is provided with a check valve.

In one preferred embodiment, a microfluidic channel is arranged between the elution cavity of the chip substrate and the mixing cavity of the substrate; the microfluidic channel is provided with a check valve.

In one preferred embodiment, a microfluidic channel is arranged between the mixing cavity of the chip substrate and the droplet cavity of the substrate; the micro-fluidic channel is provided with a check valve and an atomizing nozzle with magnetic bead filtering and check functions.

In one preferred embodiment, a droplet tiling hole channel is arranged between the droplet cavity of the chip substrate and the detection cavity of the chip substrate; the liquid drop tiling pore passage is provided with a check valve; the liquid drop tiling pore canal is designed in an hourglass shape.

In one preferred embodiment, the diameters of the injection pipelines and the safety pipelines which are connected with different cavities are consistent or inconsistent.

In one preferred embodiment, the pipe diameter of the injection pipeline and the safety pipeline which are connected with different cavities is 0.5-1.5 mm. In one embodiment, the diameter of the pipe connecting the injection line and the safety line of the different chambers is 0.51 mm. In certain embodiments, the infusion line and the safety line connecting the different lumens have a tube diameter of 0.52 mm, 0.53 mm, 0.54 mm, 0.55 mm, 0.56 mm, 0.57 mm, 0.58 mm, 0.59 mm, 0.6 mm. In certain embodiments, the diameter of the tube connecting the infusion line and the safety line of the different chambers is 0.61 mm to 0.7 mm. In certain embodiments, the injection line and the safety line connecting the different chambers have a tube diameter of 0.71 mm to 0.8 mm. In certain embodiments, the injection line and the safety line connecting the different chambers have a tube diameter of 0.81 mm to 0.9 mm. In certain embodiments, the diameter of the safety line and the infusion line connecting the different lumens has a diameter of 0.91 mm to 1 mm. In certain embodiments, the diameter of the safety line and the infusion line connecting the different lumens has a diameter of 1.1 mm to 1.2 mm. In certain embodiments, the diameter of the tube connecting the infusion line and the safety line of the different lumens is between 1.21 mm and 1.3 mm. In certain embodiments, the injection line and the safety line connecting the different chambers have a tube diameter of 1.31 mm to 1.4 mm. In certain embodiments, the injection line and the safety line connecting the different chambers have a tube diameter of 1.41 mm to 1.5 mm.

In one preferred embodiment, the cross-sectional area of a microfluidic channel, which is formed by respectively connecting a lysis cavity, a washing cavity, an elution cavity, a mixing cavity, a droplet cavity and a detection cavity, of the chip substrate is 1/4-2/3 of the cross-sectional area of a sample inlet channel of the lysis cavity. In one embodiment, the cross-sectional area of the microfluidic channel is 1/3 of the cross-sectional area of the channel of the injection channel of the lysis chamber. In certain embodiments, wherein the microfluidic channel cross-sectional area is 1/3 and 1/2 of the lysis chamber injection channel cross-sectional area.

In one preferred embodiment, the volume of the detection cavity of the droplet PCR digital chip is 0.1 microliter-200 microliter. In one embodiment, wherein the volume of said single detection chamber is 0.2 microliter. In certain embodiments, the volume of the single detection chamber is 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, 1.0 microliters, respectively. In certain embodiments, the volume of a single detection chamber is 1.1-5 microliters. In certain embodiments, the volume of a single detection chamber is 5.1 to 10 microliters. In certain embodiments, the volume of the single detection chamber is 10.1-15 microliters. In certain embodiments, the volume of a single detection chamber is 15.1-20 microliters. In certain embodiments, the volume of the single detection chamber is 20.1-25 microliters. In certain embodiments, the volume of the single detection chamber is 25.1 to 50 microliters. In certain embodiments, the volume of a single detection chamber is 50.1-100 microliters. In certain embodiments, the volume of a single detection chamber is 100.1-150 microliters. In certain embodiments, the volume of a single detection chamber is 150.1-199.9 microliters.

In one preferred embodiment, the micro-droplets formed by the atomizing nozzle have a diameter of 0.05-50 μm.

In one preferred embodiment, the height of the droplet tiling channel is 1-2 times the diameter of the micro-droplets.

In one preferred embodiment, the height of the detection cavity of the droplet-type PCR digital chip is 1.02-1.25 times of the diameter of the micro-droplet. In one embodiment, wherein the height of the detection chamber is 1.1 times the diameter of the micro-droplet. In certain embodiments, wherein the height of the detection chamber is 1.2 times the diameter of the microdroplet.

In one preferred embodiment, the number of the detection cavities is 1-16. In one embodiment, wherein the number of detection chambers is 2. In certain embodiments, wherein the number of detection chambers is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, respectively.

In one preferred embodiment, the liquid drop PCR digital chip is characterized in that a chip substrate and a cover plate are bonded to form a two-layer structure.

In one preferred embodiment, the thickness of the cover plate is 0.1-0.5 mm. In one embodiment, wherein the thickness of the cover plate is 0.11 mm. In certain embodiments, the cover plate has a thickness of 0.12 mm, 0.13 mm, 0.14 mm, 0.15 mm, 0.16 mm, 0.17 mm, 0.18 mm, 0.19 mm, 0.2 mm, respectively. In certain embodiments, wherein the thickness of the cover plate is between 0.21 mm and 0.3 mm. In certain embodiments, wherein the cover plate has a thickness of 0.31 mm to 0.4 mm. In certain embodiments, wherein the thickness of the cover plate is between 0.41 mm and 0.5 mm.

A preparation method of a liquid drop type digital PCR chip comprises the following steps:

firstly, a silicon wafer mould is manufactured by adopting a photoetching technology, and edge lines of a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity and microfluidic channels which are sequentially communicated with the cavities are etched; casting on a silicon wafer mould, forming a chip substrate after mould reversing, perforating pore channels in a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity, and functionalizing; and thirdly, bonding the functionalized chip substrate with one surface of a cover plate which is not marked with characters of a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity by adopting a bonding packaging technology to finish the chip manufacturing.

A method for using a droplet type digital PCR chip comprises the following steps:

the method comprises the following steps: respectively opening injection port protection plugs of a chip cracking cavity, a washing cavity, an elution cavity and a mixing cavity in sequence, feeding oil through a pipettor, enabling the heights of oil phases in the cracking cavity, the washing cavity, the elution cavity and the mixing cavity to be half of the height of the cavity, and then resetting each injection port protection plug; step two: opening an injection port protection plug of the lysis cavity, sequentially adding lysis solution, protease K and magnetic beads through a liquid shifter, and then resetting the injection port protection plug; step three: opening an injection port protective plug of the washing cavity, sequentially adding an ethanol aqueous solution through a pipettor, and then resetting the reagent injection port protective plug; step four: opening a protective plug of an injection port of the elution cavity, and sequentially adding PEG and magnesium chloride solution through a liquid transfer device; step five: opening an injection port protective plug of the mixing cavity, sequentially adding a primer, an enzyme and water of the PCR reaction solution through a liquid shifter, and then resetting the injection port protective plug; step six: opening an injection port protective plug of the chip cracking cavity, adding a sample through a liquid transfer device, and then resetting the injection port protective plug; step seven: placing the chip on a magnetic frame for a period of time to fully and uniformly mix all components in the cracking cavity, and then placing the chip in a thermostat for a period of time to promote the nucleic acid molecules in the sample to be fully combined with the magnetic beads; step eight: opening a microfluidic channel valve between the cracking cavity and the washing cavity, allowing the magnetic beads in the cracking cavity to enter the washing cavity by virtue of a magnetic frame, resetting the microfluidic channel valve, and fully and uniformly mixing the magnetic beads and the washing liquid; step nine: opening a micro-fluidic channel valve between the washing cavity and the elution cavity, allowing the magnetic beads in the washing cavity to enter the elution cavity, resetting the micro-fluidic channel valve, and fully and uniformly mixing the magnetic beads and the eluent; step ten: opening a micro-fluidic channel valve between the elution cavity and the mixing cavity, allowing magnetic beads in the elution cavity to enter the mixing cavity, resetting the micro-fluidic channel valve, fully mixing uniformly, standing for a period of time, and allowing nucleic acid and the magnetic beads to be separated; step eleven: opening a micro-fluidic channel valve between the mixing cavity and the droplet cavity, allowing the PCR reaction solution in the mixing cavity to flow through an atomizing nozzle with a magnetic bead filtering function, leaving the magnetic beads in the elution cavity, and resetting the micro-fluidic channel valve; the mixture separated from the magnetic beads spreads out into a liquid layer while passing through the margin of the nozzle hole of the atomizer having a non-return function, and the liquid layer is broken into a thick and thin cylinder in the shape of an elongated tubular hole and then changed into a liquid droplet due to aerodynamic instability. Liquid is extruded into the nozzle through internal pressure, a quartz blade is arranged in the atomizing nozzle, and the high-speed moving liquid forms independent micro-droplets with uniform size after impact and rebound of a cyclone cavity of the quartz blade; step twelve: opening the micro-droplet tiled pore channel valves of the droplet cavity and the detection cavity, enabling the micro-droplets to enter and be tiled in the detection cavity along the tiled pore channel, resetting the micro-droplet tiled pore channel valve, placing the chip in a PCR instrument for thermal circulation, transferring the chip to a fluorescence microscope for sequential imaging after amplification is completed, counting the proportion of positive (fluorescence) micro-droplets and negative (no fluorescence) micro-droplets through software, obtaining the concentration of sample nucleic acid according to a digital PCR calculation formula, and giving out a detection result. Similarly, the chip can be applied to the treatment of protein samples and realize digital enzyme-linked immunosorbent assay (ELISA) detection according to the similar working mode. If the safety fault problem occurs, the safety outlet plug and the safety valve can be opened, and the safety fault is eliminated through the safety outlet and the safety pipeline, so that the operation safety is ensured.

Compared with the prior art, the invention has the beneficial effects that:

(1) The liquid drop type digital PCR chip only needs to be bonded once during production, and a vacuum or negative pressure environment does not need to be formed in the using process;

(2) The droplet type digital PCR chip integrates the functions of cracking and washing of a nucleic acid sample, mixing of nucleic acid and PCR reaction liquid, droplet generation and PCR reaction detection, and realizes the whole process of 'sample input and result output' of nucleic acid analysis;

(3) The liquid drop type digital PCR chip realizes different functions of a plurality of different cavities connected in series by means of descending design of the different cavities and the microfluidic channel valve with a non-return function among the cavities, and liquid among the cavities can be fully isolated without cross contamination; in addition, the process of generating the micro-droplets by the atomizing nozzle is stable, and the micro-droplets can be independently and stably paved in the detection cavity through the micro-droplet paving pore channel, so that the detection accuracy is improved;

(4) The sample of the droplet type digital PCR chip exists on the chip in the form of droplets in oil, so that the pollution of nucleic acid molecules to the detection environment in the form of aerosol is avoided; and due to the isolation effect of the oil, the nucleic acid sample is not directly contacted with the chip structure in the whole process, so that the condition that nucleic acid molecules are adsorbed on the surfaces of different chip cavities or channels to cause loss or residue is avoided, and the chip can be repeatedly used through proper design.

Drawings

FIG. 1 is a schematic diagram of a droplet PCR digital chip according to an embodiment of the present invention.

Fig. 2 is a schematic view of the structure of the atomizing nozzle 29 in fig. 1.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. For convenience of description, the words "upper", "lower", "left" and "right" in the following description are used only to indicate the correspondence with the upper, lower, left and right directions of the drawings, and do not limit the structure. The embodiments of the present invention are provided only for explaining the present invention and not for limiting the present invention, and the embodiments of the present invention are not limited to the embodiments given in the specification. The examples were prepared under conventional conditions or conditions recommended by the material suppliers without specifying specific experimental conditions or operating conditions. Furthermore, it is to be understood that one or more method steps recited in the present disclosure are not exclusive of other method steps that may also be present before or after the recited combination of steps or that other method steps may also be inserted between the explicitly recited steps, unless otherwise indicated; it is also to be understood that references to combinations of connections in this invention do not preclude the presence or addition of further connections before or after the combination or intervening connections between two of the explicitly mentioned connections, unless expressly stated otherwise. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content. In the following examples, reagents, materials and instruments used are commercially available unless otherwise specified.

The substrate 1, the cover plate 2, the cleavage cavity injection port 3, the washing cavity injection port 4, the elution cavity injection port 5, the mixing cavity injection port 6, the cleavage cavity injection line 7 and the check valve 8, the washing cavity injection line 9 and the check valve 10, the elution cavity injection line 11 and the check valve 12, the mixing cavity injection line 13 and the check valve 14, the cleavage cavity 15, the washing cavity 16, the elution cavity 17, the mixing cavity 18, the droplet cavity 19, the detection cavity 20, the microfluidic channel 21 and the check valve 22 between the cleavage cavity 15 and the washing cavity 16, the microfluidic channel 23 and the check valve 24 between the washing cavity 16 and the elution cavity 17, the microfluidic channel 25 and the check valve 26 between the elution cavity 17 and the mixing cavity 18, the microfluidic channel 27 between the mixing cavity 18 and the droplet cavity 19, the check valve 28 and the atomizing nozzle 29, the droplet tiling channel 30 and the check valve 31 between the droplet cavity 19 and the detection cavity 20, the safety line 32, the safety valve 33, the safety outlet 34, the safety outlet 35, the injection port 36, the plug of the washing cavity, the plug 37, the plug 38 of the elution cavity, and the plug 39 are all known by the conventional technical or technical manual.

Example 1

A liquid drop type PCR digital chip is shown in figure 1 and mainly comprises a substrate 1 and a cover plate 2, wherein a cracking cavity injection port 3, a washing cavity injection port 4, an elution cavity injection port 5, a mixing cavity injection port 6, a cracking cavity injection pipeline 7, a check valve 8, a washing cavity injection pipeline 9, a check valve 10, an elution cavity injection pipeline 11, a check valve 12, a mixing cavity injection pipeline 13, a check valve 14, a cracking cavity 15, a washing cavity 16, an elution cavity 17, a mixing cavity 18, a liquid drop cavity 19 and a detection cavity 20 are arranged on the substrate 1. A micro-fluidic channel 21 and a check valve 22 are arranged between the cracking cavity 15 and the washing cavity 16; a micro-fluidic channel 23 with a non-return function and a non-return valve 24 are arranged between the washing cavity 16 and the elution cavity 17; a micro-fluidic channel 25 and a check valve 26 with a check function are arranged between the elution cavity 17 and the mixing cavity 18; a microfluidic channel 27, a check valve 28 and an atomizing nozzle 29 are arranged between the mixing cavity 18 and the liquid drop cavity 19; a liquid drop laying channel 30 and a check valve 31 are arranged between the liquid drop cavity 19 and the detection cavity 20.

In embodiment 1, a safety pipeline 32, a safety valve 33, a safety outlet 34 and a safety outlet plug 35 are arranged below the liquid drop cavity 19;

in example 1, the lysis chamber injection port is provided with a plug 36, the washing chamber injection port is provided with a plug 37, the elution chamber injection port is provided with a plug 38, and the mixing chamber injection port is provided with a plug 39;

in example 1, the atomizing nozzle 29 is provided with a filter 40 for filtering magnetic beads and a quartz blade 41, as shown in FIG. 2;

in the embodiment 1, the pipe diameters of a cracking cavity injection pipeline 7, a washing cavity injection pipeline 9, an elution cavity injection pipeline 11 and a mixing cavity injection pipeline 13 are all 0.25 mm, and the cross-sectional area of the microfluidic channel is 1/2 of the cross-sectional area of the injection pipeline;

in example 1, the number of detection chambers was 2, and the volume of the single detection chamber was 0.5. Mu.l;

in example 1, the substrate 1 was made of a mixed material of boron nitride and PDMS, and the mass content of PDMS in the mixed material was 40%;

in example 1, the cover plate 2 was made of a mixed material of PDMS and zirconium boride, the mass content of zirconium boride in the mixed material being 2.5%;

in example 1, the size of the cover plate was 0.25 x 45 x 75mm;

in example 1, the inner diameters of the lysis chamber 15, the washing chamber 16, the elution chamber 17 and the mixing chamber 18 are all 5mm;

in example 1, the diameter of the micro-droplets formed by the atomizing nozzle 29 is 0.05 to 50 μm;

in example 1, the height of the droplet tiling channel 30 is 1.2 times the diameter of the micro-droplets;

in example 1, the height of the detection chamber is 1.2 times the diameter of the micro-droplet.

A preparation method of a liquid drop type digital PCR chip comprises the following steps:

firstly, drawing a designed liquid drop type PCR digital chip by using AutoCAD software to manufacture a film mask for photoetching; the method comprises the steps of taking a four-inch monocrystalline silicon wafer as a substrate, manufacturing a silicon wafer mold by adopting a photoetching technology, etching out the cleavage cavity 15, the washing cavity 16, the elution cavity 17, the mixing cavity 18, the edge lines of the liquid drop cavity 15 and the detection cavity 16 and the cleavage cavity injection pipelines 7 respectively communicated with the cavities, the washing cavity injection pipeline 8, the elution cavity injection pipeline 9, the mixing cavity injection pipeline 10, the microfluidic channel 21 between the cleavage cavity 15 and the washing cavity 16, the microfluidic channel 23 between the washing cavity 16 and the elution cavity 17, the microfluidic channel 25 between the elution cavity 17 and the mixing cavity 18, and the liquid drop flat laying channel 30 between the liquid drop cavity 15 and the detection cavity 16 on photoresist;

secondly, casting on a mould, and after mould reversing, functionalizing the cracking cavity 15, the washing cavity 16, the elution cavity 17, the mixing cavity 18, the liquid drop cavity 19 and the detection cavity 20;

thirdly, preparing a cover plate 2 corresponding to the substrate 1, wherein the cover plate 2 which does not face the substrate 1 is provided with marks corresponding to each cavity and each channel of the substrate 1;

and fourthly, bonding the functionalized substrate and the cover plate by adopting a hot-pressing packaging technology to manufacture the chip.

A method for using a droplet type digital PCR chip comprises the following steps:

the method comprises the following steps: respectively opening injection port protective plugs 36, 37, 38 and 39 of the chip cracking cavity 15, the washing cavity 16, the elution cavity 17 and the mixing cavity 18 in sequence, respectively injecting mineral oil through a pipette, enabling the heights of oil phases in the cracking cavity 15, the washing cavity 16, the elution cavity 17 and the mixing cavity 18 to be half of the heights of the cavities, and then resetting the injection port protective plugs; step two: opening an injection port protective plug 36 of the lysis cavity 15, sequentially adding lysis solution, proteinase K and magnetic beads through a pipette, and then resetting the injection port protective plug 36; step three: opening the injection port protective plug 37 of the washing cavity, sequentially adding an ethanol aqueous solution through a pipette, and then resetting the injection port protective plug 37; step four: opening the injection port protective plug 38 of the elution cavity, sequentially adding PEG and magnesium chloride solution through a liquid transfer device, and then resetting the injection port protective plug 38 of the elution cavity; step five: opening an injection port protective plug 39 of the mixing cavity, sequentially adding a primer, an enzyme and water of the PCR reaction solution through a pipettor, and then resetting the injection port protective plug 39; step six: opening an injection port protective plug 36 of the chip cracking cavity, adding a sample through a liquid transfer device, and then resetting the injection port protective plug 36; step seven: placing the chip on a magnetic frame for a period of time to fully and uniformly mix all components in the cracking cavity 15, and then placing the chip in a thermostat for a period of time to promote the nucleic acid molecules in the sample to be fully combined with the magnetic beads; step eight: opening a check valve 22 of a microfluidic channel 21 between the cracking chamber 15 and the washing chamber 16 to enable magnetic beads in the cracking chamber 15 to enter the washing chamber 16, resetting the check valve 22, and fully mixing the magnetic beads and the washing liquid; step nine: opening a check valve 24 of a microfluidic channel 23 between the washing cavity 16 and the elution cavity 17 to allow magnetic beads in the washing cavity 16 to enter the elution cavity 17, resetting the check valve 24, and fully and uniformly mixing washing liquid and eluent; step ten: opening a check valve 25 of a microfluidic channel 24 between the elution cavity 17 and the mixing cavity 18 to allow magnetic beads in the elution cavity 17 to enter the mixing cavity 18, resetting the check valve 25, fully mixing uniformly, standing for a period of time to allow nucleic acid and the magnetic beads to be separated; step eleven: the atomizing nozzle 29 with magnetic bead filtering and non-return functions between the mixing chamber 18 and the droplet chamber 19 is opened to allow the PCR reaction solution in the mixing chamber 18 to enter the droplet chamber 19, the magnetic beads are left in the mixing chamber 18, and the mixed solution separated from the magnetic beads spreads into a liquid layer when flowing through the margin of the hole of the atomizing nozzle 29 with non-return functions, and the liquid layer is broken into a thick and thin cylinder in the shape of an elongated tubular hole due to the instability of aerodynamic force and then becomes a droplet. The liquid is extruded into the nozzle 29 by internal pressure, a quartz blade 41 is arranged in the atomizing nozzle 29, the high-speed moving liquid is impacted and rebounded by a cyclone cavity of the quartz blade 41 to form dispersed micro-droplets with uniform size and diameter of about 0.05-50 microns, and the atomizing nozzle 29 is reset; step twelve: opening a micro-droplet tiled pore channel valve 31 of the droplet cavity and the detection cavity to enable micro-droplets to enter and be tiled in the detection cavity 20, resetting the micro-droplet tiled pore channel valve 31, placing the chip in a PCR instrument for thermal cycle, transferring the chip to a fluorescence microscope to sequentially image the micro-droplets after amplification is finished, counting the proportion of positive (with fluorescence) micro-droplets and negative (without fluorescence) micro-droplets through software, obtaining the concentration of sample nucleic acid according to a digital PCR calculation formula, and giving a detection result. Similarly, the chip can be applied to the treatment of protein samples and realize digital enzyme-linked immunosorbent assay (ELISA) detection according to a similar working mode as the above. If the safety fault problem occurs, the safety outlet plug and the safety valve can be opened, and the safety fault is eliminated through the safety outlet and the safety pipeline, so that the operation safety is ensured.

Example 2

Firstly, sequentially opening injection port protection plugs 36, 37, 38 and 39 of a chip cracking cavity 15, a washing cavity 16, an elution cavity 17 and a mixing cavity 18 respectively, injecting mineral oil through a pipette respectively to ensure that the heights of oil phases in the cracking cavity 15, the washing cavity 16, the elution cavity 17 and the mixing cavity 18 are half of the heights of the cavities, and then resetting the protection plugs of oil inlets;

secondly, opening an injection port protective plug 36 of the lysis cavity 15, adding 10 microliters of lysis system reagents (lysis solution, proteinase K and magnetic beads) through a liquid transfer device, and resetting the injection port protective plug 36; opening the injection port-protecting plug 37 of the washing chamber 16, adding 5. Mu.l of washing solution 1 (60% ethanol aqueous solution) by a pipette, and resetting the injection port-protecting plug 37; opening the injection port-protecting plug 38 of the elution chamber 17, adding 5. Mu.l of washing solution (13% PEG-8000,1mM magnesium chloride solution), and resetting the injection port-protecting plug 38; the injection port-protecting plug 39 of the mixing chamber 18 is opened, 4. Mu.L of the PCR reaction solution (primer, enzyme, water) is added by a pipette, and the injection port-protecting plug 39 is reset; finally, 2.5 microliters of sample (plasma free nucleic acids from cancer patients) is added to the lysis chamber 15, followed by resetting the injection port guard plug 36;

thirdly, placing the chip on a magnetic frame for 2 minutes and uniformly mixing, and then placing the chip in a 50-degree thermostat for 12 minutes to ensure that the nucleic acid molecules are fully combined with the magnetic beads;

fourthly, opening the check valve 22, dragging the magnetic beads from the cracking cavity 15 to the washing cavity 16 along the microfluidic channel 21, resetting the check valve 22, and uniformly mixing the magnetic beads and the washing solution for 1 minute;

fifthly, opening the check valve 24, dragging the magnetic beads from the washing cavity 16 to the elution cavity 17 along the microfluidic channel 23, and uniformly mixing the magnetic beads and the eluent for 1 minute;

sixthly, opening a check valve 26, dragging the magnetic beads from the elution cavity 17 to the mixing cavity 18 along the microfluidic channel 25, uniformly mixing the magnetic beads and the reaction solution for 1 minute, and then standing for 10 minutes;

seventhly, opening a check valve 28, enabling the mixture to pass through an atomizing nozzle 29 with magnetic filtering beads and non-return functions from the mixing cavity 18 along the microfluidic channel 27, separating the magnetic beads from the PCR reaction solution, and enabling the PCR reaction solution to enter the liquid drop cavity in a micro-liquid drop mode;

eighthly, opening a check valve 31 of the liquid drop laying channel 30 to enable the liquid drops to be laid in the detection cavity 20;

and step nine, placing the chip in an in-situ PCR instrument, starting an amplification program, detecting the negative and positive of the liquid drops under a fluorescence microscope after amplification, obtaining the concentration of the sample nucleic acid according to a digital PCR calculation formula, and giving a detection result.

Example 3

The detection result of the droplet type PCR digital chip in the embodiment 2 of the invention on the sample is very consistent with that of a commercial PCR detection chip, but compared with the commercial PCR digital chip, the invention shortens the operation time required by sample detection by 60 minutes, and greatly improves the efficiency of the detection process. In addition, compared with the commercial PCR detection chip, the detection accuracy of the sample is improved by 10 percent and reaches 100 percent.

Example 4

The 13 droplet-type PCR digital chips prepared in example 1 of the present invention perform droplet-type PCR digital detection on samples such as collected wound swabs, plasma, cerebrospinal fluid, bone marrow fluid, aqueous humor, pleural effusion, joint effusion, tissue, urine, abscess aspirate, alveolar lavage fluid, sputum or nasopharyngeal swabs after processing according to the standard operation procedures of clinical examination and detection, and the results show that the detection results of the 13 droplet-type PCR digital chips constructed in example 1 can accurately reflect the actual conditions of the samples. Compared with the commercial PCR digital chip, the 13 droplet PCR digital chips constructed in example 1 not only have the detection accuracy up to 100%, but also have short detection time. According to the cost price calculation, the detection cost of each sample is averagely saved by 37 yuan, so that the hospital medical insurance purchasing and using are facilitated, and a detection expense can be saved for a detector.

The above-described embodiments are set forth so that this disclosure will be thorough and complete, and will not be limited by any theory presented in the preceding claims, which may suggest themselves to those skilled in the art after reading this disclosure and all equivalents thereof that fall within the scope of the invention as defined in the claims appended hereto.

Claims (9)

1. A liquid drop type PCR digital chip is characterized in that the chip is composed of a substrate and a cover plate; the substrate is provided with a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity; a micro-fluidic pore passage is arranged between the mixing cavity and the liquid drop cavity, and is provided with an atomizing nozzle with magnetic filtering beads and non-return functions; a liquid drop laying pore passage is arranged between the liquid drop cavity and the detection cavity and is in an hourglass shape.

2. The liquid drop PCR digital chip of claim 1, wherein the lysis chamber, the washing chamber, the elution chamber and the mixing chamber are respectively provided with an injection pipeline.

3. The digital chip for droplet PCR according to claim 1, wherein the cross-sectional area of the microfluidic channel is 1/4-2/3 of the cross-sectional area of the channel of the injection channel of the lysis chamber.

4. The digital chip for droplet PCR according to claim 1, wherein the volume of the single detection cavity is 0.5-200. Mu.l.

5. The droplet-based PCR digital chip of claim 1, wherein the height of the droplet tiling channel is 1-2 times the diameter of the droplet.

6. The droplet-type PCR digital chip of claim 1, wherein the chip substrate is provided with a safety pipeline.

7. The liquid drop type PCR digital chip of claim 1, wherein the side of the cover plate not attached to the substrate of the chip has the marks of the lysis chamber, the washing chamber, the elution chamber, the mixing chamber, the liquid drop chamber and the detection chamber, and the chip substrate and the cover plate are bonded to form a two-layer structure.

8. The droplet-type PCR digital chip according to claim 1, wherein the method for preparing the droplet-type PCR digital chip comprises the following steps: firstly, a silicon wafer mould is manufactured by adopting a photoetching technology, and edge lines of a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity, and an injection pipeline and a microfluidic channel which are communicated with the cavities in sequence are etched; secondly, casting on a silicon wafer mold, forming a chip substrate after reverse molding, and opening corresponding pipelines and pore channels in a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity and functionalizing the pipelines and the pore channels; and thirdly, bonding the functionalized chip substrate with the back surface of a cover plate marked with a cracking cavity, a washing cavity, an elution cavity, a mixing cavity, a liquid drop cavity and a detection cavity by adopting a bonding packaging technology to finish the chip manufacturing.

9. The droplet-type PCR digital chip according to claim 1, wherein the method for using the droplet-type PCR digital chip comprises the following steps: the method comprises the following steps: respectively opening injection port protection plugs of a chip cracking cavity, a washing cavity, an elution cavity and a mixing cavity in sequence, feeding oil through a pipettor, enabling the heights of oil phases in the cracking cavity, the washing cavity, the elution cavity and the mixing cavity to be half of the height of the cavity, and then resetting each injection port protection plug; step two: opening an injection port protection plug of the lysis cavity, sequentially adding lysis solution, protease K and magnetic beads through a liquid shifter, and then resetting the injection port protection plug; step three: opening an injection port protective plug of the washing cavity, sequentially adding an ethanol aqueous solution through a liquid transfer device, and then resetting the injection port protective plug; step four: opening a filling port protective plug of the elution cavity, and respectively and sequentially adding PEG and magnesium chloride solution through a liquid transfer device; step five: opening an injection port protective plug of the mixing cavity, sequentially adding a primer, an enzyme and water of the PCR reaction solution through a liquid transfer device, and then resetting the injection port protective plug; step six: opening an injection port protective plug of the chip cracking cavity, adding a sample through a liquid transfer device, and then injecting the injection port protective plug; step seven: placing the chip on a magnetic frame for a period of time to fully and uniformly mix all components in the cracking cavity, and then placing the chip in a thermostat for a period of time to promote the nucleic acid molecules in the sample to be fully combined with the magnetic beads; step eight: opening a micro-fluidic channel valve between the cracking cavity and the washing cavity, allowing magnetic beads in the cracking cavity to enter the washing cavity by virtue of a magnetic frame, resetting the micro-fluidic channel valve, and fully mixing the magnetic beads and the washing liquid; step nine: opening a micro-fluidic channel valve between the washing cavity and the elution cavity, allowing the magnetic beads in the washing cavity to enter the elution cavity, resetting the micro-fluidic channel valve, and fully and uniformly mixing the magnetic beads and the eluent; step ten: opening a micro-fluidic channel valve between the elution cavity and the mixing cavity, allowing magnetic beads in the elution cavity to enter the mixing cavity, resetting the micro-fluidic channel valve, fully mixing, and standing for a period of time to separate nucleic acid from the magnetic beads; step eleven: opening a micro-fluidic channel valve between the mixing cavity and the droplet cavity, allowing the PCR reaction solution in the mixing cavity to enter the mixing cavity through an atomizing nozzle with magnetic bead filtering and non-return functions, leaving the magnetic beads in the elution cavity, and then resetting the micro-fluidic channel valve; the mixed liquid separated from the magnetic beads is unfolded into a liquid layer when flowing through the margin of an atomizing nozzle hole with a non-return function, and the liquid layer is broken into a cylinder with the thickness of an elongated tubular hole due to the instability of aerodynamic force and then becomes liquid drops; liquid is extruded into a nozzle through internal pressure, a quartz blade is placed in an atomizing nozzle, and the liquid moving at high speed is impacted and rebounded through a cyclone cavity of the quartz blade to form independent micro-droplets with uniform size; step twelve: opening the micro-droplet tiling pore channel valves of the droplet cavity and the detection cavity to enable micro-droplets to enter and be tiled in the detection cavity, and resetting the micro-droplet tiling pore channel valve; placing the chip in a PCR instrument for thermal cycle, transferring the chip to a fluorescence microscope for imaging micro-droplets in sequence after amplification is finished, counting the proportion of positive (with fluorescence) micro-droplets to negative (without fluorescence) micro-droplets through software, obtaining the concentration of sample nucleic acid according to a digital PCR calculation formula, and giving a detection result; similarly, the chip can be applied to the treatment of protein samples and realize digital enzyme-linked immunosorbent assay according to the similar working mode; if the safety fault problem occurs, the safety outlet plug and the safety valve can be opened, and the safety fault is eliminated through the safety outlet and the safety pipeline, so that the operation safety is ensured.

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