Centrifugal force driving-based disc PCR micro-fluidic chip and control method thereof

Abstract

The application relates to a centrifugal force driving-based disc PCR micro-fluidic chip and a control method thereof. The chip comprises a chip disc; the chip disc comprises a micro-fluid channel layer, a circular cavity is arranged in the center as a central sample inlet area, and a micro-fluid channel is arranged in the interior; the micro-fluid channel is divided into a PCR amplification area micro-fluid channel and a detection area micro-fluid channel from inside to outside along the radial direction of the chip; the PCR amplification area micro-fluid channel comprises a plurality of temperature reaction areas; a bonding layer is used for bonding and connecting the micro-fluid channel layer and a temperature control layer; the temperature control layer is provided with a plurality of heating electrodes corresponding to the temperature reaction areas and a plurality of sensor electrodes corresponding to the heating electrodes; a micro column array is further arranged on the surface of the temperature control layer, and the micro column array extends into the central sample inlet area. The application not only can realize parallelization and high-throughput nucleic acid amplification and detection of a large batch of samples, guarantee high efficiency and economy of amplification, but also can make the chip reusable, does not need frequent replacement, reduces operation complexity, and reduces amplification cost required by a single sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, and in particular to a disc PCR microfluidic chip based on centrifugal force driving and a control method thereof. BACKGROUND

[0002] With the development of microfluidic technology, polymerase chain reaction (PCR) on a microfluidic chip has been widely used. Due to the scale of the PCR reaction unit being only microns, higher heat and mass transfer efficiency is brought, greatly shortening the time required for PCR amplification, while the reagent consumption is less, and the economic effect is greatly improved.

[0003] Disc PCR technology, as a product of the combination of microfluidic technology and PCR technology, uses centrifugal force as the driving force for the sample, without the need for additional fluid driving units, which not only reduces the complexity of operation, but also facilitates the portability and lightness of the device, and therefore has made great progress in the field of microfluidics. Some successful disc PCR application products have also been introduced, such as the ELISA comprehensive sample response system, the centrifugal integrated microfluidic chemical analysis system designed by the University of Freiburg in Germany, and the Piccolo clinical blood analyzer system of Abaxis. However, considering the cross-contamination problem when multiple samples are amplified, and the complexity of the sampling method, the existing disc PCR microfluidic chip can only amplify a single sample or a few samples simultaneously. At the same time, the existing PCR microfluidic chip is disposable, and when facing the demand for large-scale sample amplification, it not only cannot provide efficient and rapid amplification, but also faces the high cost of replacing the chip.

[0004] Therefore, a microfluidic chip for large-scale parallel PCR amplification and detection is proposed, which has the advantages of high amplification efficiency, high integration, good economy, and no cross-contamination between samples, which has high application value and significance for large-scale infectious disease screening and crop breeding. SUMMARY

[0005] The present application aims to provide a disc PCR microfluidic chip based on centrifugal force driving and a control method thereof, which can solve the problems in the prior art, not only can be used for large-scale sample parallelization, high-throughput nucleic acid amplification and detection, ensuring high efficiency and economy of amplification, but also can be reused, without frequent replacement, thereby reducing the complexity of operation and the cost of amplification for a single sample.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In a first aspect of the present application, a centrifugal force driven disc PCR microfluidic chip is disclosed.

[0008] Specifically, the chip comprises a chip disc; the chip is composed of a central sample injection area, a microfluidic channel area and a temperature control area, and the bottom of the chip is connected with a conductive slip ring through a connecting piece, and further connected with a motor shaft.

[0009] The chip disc comprises a microfluidic channel layer, a bonding layer and a temperature control layer arranged in sequence.

[0010] The microfluidic channel layer is centrally provided with a circular cavity as a central sample injection area, and internally arranged with a microfluidic channel; the microfluidic channel is divided into a PCR amplification area microfluidic channel and a detection area microfluidic channel along the radial direction of the chip from inside to outside; the PCR amplification area microfluidic channel comprises a plurality of temperature reaction regions;

[0011] The bonding layer is used for bonding connection of the microfluidic channel layer and the temperature control layer, and a through hole is provided in the middle thereof;

[0012] The temperature control layer is provided with a plurality of heating electrodes corresponding to the temperature reaction regions and a plurality of sensor electrodes corresponding to the heating electrodes; the surface of the temperature control layer is further provided with a micro column array, and the micro column array extends into the central sample injection area after passing through the through hole. The upper surface of the temperature control layer disc is processed with a micro column array with a height of 300 um in a circular area with a center radius of 10 mm, and the micro column diameter is 800 um, which is uniformly arranged on 6 equidistant concentric circles.

[0013] Further, the side surface of the circular cavity has a groove;

[0014] The groove side wall has a collection port for collecting micro droplets;

[0015] The collection port is in a trumpet shape;

[0016] The collection port end is connected with the inlet of the microfluidic channel.

[0017] Further, the first segment of the microfluidic channel is a serpentine microfluidic channel, the second segment is a multi-turn spiral line microfluidic channel, and the third segment is an arc microfluidic channel tangent to the outside of the spiral line;

[0018] The outlet of the microfluidic channel is located on the edge wall surface of the microfluidic channel layer;

[0019] The arc microfluidic channel leads to the outlet of the microfluidic channel.

[0020] Further, the PCR amplification area microfluidic channel comprises three temperature zones, namely a first temperature zone, a second temperature zone and a third temperature zone;

[0021] The first temperature zone and the second temperature zone are located in the lower semicircular sector, each occupying 1 / 4 of the circular ring area, and the third temperature zone is located in the upper semicircular sector, occupying 1 / 2 of the circular ring area, and each temperature zone is spaced apart by 14 mm.

[0022] Further, the upper surface of the temperature control layer is provided with heating electrodes corresponding to the three temperature zones and sensor electrodes corresponding to the heating electrodes, respectively, first heating electrode, second heating electrode, third heating electrode and first sensor electrode, second sensor electrode and third sensor electrode; the arrangement position of the heating electrode and the sensor electrode corresponds to the first temperature zone, the second temperature zone, the third temperature zone in the vertical direction in space.

[0023] The upper surface of the temperature control layer is processed with a heating electrode and a sensor electrode with a nanometer level thickness in a circular ring area with a radius of 25 mm to a radius of 60 mm, the electrode material is platinum, the heating electrode is divided into a first heating electrode, a second heating electrode and a third heating electrode, and is arranged in a snake shape, the arrangement area corresponds to the three temperature zones of the top micro-channel layer; the sensor electrode is divided into a first sensor electrode, a second sensor electrode and a third sensor electrode, and is arranged equidistantly with the first, second and third heating electrodes.

[0024] Further, the end of the heating electrode and the sensor electrode is connected with an electrode sheet; the bottom of the electrode sheet is embedded with a conductive metal; the conductive metal is used to connect the electrode sheet to the electrode terminal arranged on the back of the temperature control layer.

[0025] The two ends of the heating electrode and the sensor electrode are connected with the electrode sheet, the material and thickness of the electrode sheet are the same as the electrode, the shape is a rounded square with a side length of 6 mm, the conductive metal is embedded through the 5 mm thick polyimide substrate vertically at the bottom of each electrode sheet, and is led to the metal electrode terminal with a diameter of 5 mm and a height of 10 mm on the back of the temperature control layer substrate disc, and then is led out to the external circuit through the wires of the conductive slip ring, the number of electrode sheets, electrode terminals and wire lines is 12.

[0026] The orientation of the electrode sheets at the two ends of the first heating electrode is 4 o'clock and 5 o'clock, the orientation of the electrode sheets at the two ends of the second heating electrode is 7 o'clock and 8 o'clock, and the orientation of the electrode sheets at the two ends of the third heating electrode is 11 o'clock and 1 o'clock; the orientation of the electrode sheets at the two ends of the first sensor electrode is 3 o'clock and 6 o'clock, the orientation of the electrode sheets at the two ends of the second sensor electrode is 6 o'clock and 9 o'clock, and the orientation of the electrode sheets at the two ends of the third sensor electrode is 9 o'clock and 3 o'clock.

[0027] Further, the micro-channel layer and the bonding layer are made of PDMS material.

[0028] The temperature control layer is made of polyimide material.

[0029] Further, the temperature control layer is provided with a connecting piece;

[0030] The connecting piece is detachably connected with a conductive slip ring.

[0031] Further, the back surface of the temperature control layer is provided with a threaded counterbore;

[0032] The connecting piece is connected into the threaded counterbore through a screw;

[0033] The connecting piece is provided with a keyway;

[0034] The connecting piece is connected with the conductive slip ring through a flat key matched with the keyway.

[0035] In the second aspect of the application, a control method of the microfluidic chip is disclosed.

[0036] Specifically, the method comprises:

[0037] S1, the chip disc rotates, and mineral oil is continuously injected into the central sample injection area as a continuous phase for isolating sample microdroplets, and a continuously updated rotating oil film is formed to take away sample microdroplet waste liquid generated in a previous impact process on the disc surface of the chip disc to perform sample injection of the next sample;

[0038] S2, a pre-prepared sample droplet is dropped into the central sample injection area of the rotating chip disc, the sample droplet contains a to-be-amplified gene fragment and various reaction substrates required for PCR amplification, and then the sample droplet is broken to generate a plurality of oil-in-microdroplets after impacting the rotating microcolumn array, under the action of centrifugal force, the oil-in-microdroplets with a larger particle size are thrown out of the disc, and the oil-in-microdroplets with a smaller particle size enter the microchannel;

[0039] S3, the oil-in-microdroplets entering the microchannel flow to the outside of the chip along the microchannel due to the action of the centrifugal force radially outward, in the process of flowing through the microchannel, the sample microdroplets perform pre-denaturation, denaturation, annealing and extension to perform a PCR amplification reaction, when all sample microdroplets are amplified and flow into the detection area microchannel, the chip disc stops rotating and oil injection, and all samples in the microchannel are detected after amplification, and then the disc is rotated again, and all sample microdroplets are discharged from the chip disc.

[0040] Compared with the prior art, the application has the following advantages:

[0041] (1) The preparation, injection, driving, PCR amplification and detection of sample microdroplets are integrated on one chip, the chip has simple structure, small size and high integration degree, a large number of samples can be simultaneously amplified in the chip at a time, and the dependence on peripheral additional equipment is very small, which has great advantages for the miniaturization and portability of the equipment.

[0042] (2) The present application adopts the method of oil-coated rotating micro-column breaking micro-droplets by droplet impact on the central sample inlet area. The generated micro-droplets diffuse to the periphery of the chip under the action of centrifugal force, and then the micro-droplets are screened by the combined action of the slope and the trumpet-shaped collection port outside the sample inlet area. The micro-droplets of a specific size enter the micro-channel, and the micro-droplets of an oversized size are thrown out of the chip disc surface. Therefore, the preparation and sampling of micro-droplets of a single sample can be realized within two seconds. This ingenious method not only saves the time of sample-by-sample sampling, but also prevents cross contamination between sample micro-droplets by the presence of continuous phase oil film. Therefore, the chip can perform multiplex PCR amplification and detection, greatly improving the efficiency of parallel operation.

[0043] (3) The present application adopts a micro-droplet driving technology based on centrifugal force. The sample micro-droplets entering the micro-channel move to the outer periphery of the spiral micro-channel under the action of centrifugal force of the rotating chip disc, and then flow through the three set temperature zones one by one, realizing PCR amplification of the sample in the micro-droplets. Based on the relationship between centrifugal force and the movement speed of micro-droplets in the spiral micro-pipe, the driving speed of micro-droplets can be indirectly controlled by adjusting the speed of the chip, thereby replacing the external devices such as syringe pumps used in traditional driving methods, simplifying the overall structure and operation complexity, and reducing the cost and stability of the equipment.

[0044] (4) The present application uses different regions of the spiral micro-channel as PCR amplification reaction zones and detection zones, respectively. By detecting the fluorescence of the stationary sample micro-droplets in the micro-channel after amplification reaction, the process of re-collection and transfer of sample micro-droplets is saved, realizing on-chip multi-sample amplification and in-situ detection, and greatly saving operation time and labor cost.

[0045] (5) The present application uses a spin-coated ultra-thin PDMS film to realize strong bonding between the PDMS micro-channel layer and the polyimide temperature control layer. The thickness of the PDMS film can reach ten microns under high-speed spinning. As an intermediate layer between the electrode and the sample micro-droplets, its thickness is small compared to the size of the micro-droplets. Compared with the traditional bonding method of using heat-conducting double-sided adhesive tape, the heat transfer efficiency is higher, the temperature control is more accurate, and the rapid temperature response of the sample micro-droplets in the PCR amplification process is more rapid. It has great significance for shortening the amplification time and improving the amplification efficiency.

[0046] (6) The temperature control layer in the application is made by making nanoscale thin film electrodes as temperature zone heat sources and nanoscale thin film sensor electrodes as temperature measurement feedback units on the substrate of the polyimide material. Since the sensor electrode is separated from the micro channel by only an ultra-thin PDMS layer, compared with the traditional temperature measurement by pasting temperature sensors or infrared temperature measurement, the temperature measurement result is more accurate, which is conducive to the temperature control and adjustment of the PCR amplification temperature zone, reduces the probability of false positive and false negative, and improves the specificity of the PCR amplification process. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is a structural schematic diagram of the centrifugal force driven disc PCR microfluidic chip in the application;

[0048] Figure 2 is a bottom view schematic diagram of the centrifugal force driven disc PCR microfluidic chip in the application;

[0049] Figure 3 is an exploded structural diagram of the centrifugal force driven disc PCR microfluidic chip in the application;

[0050] Figure 4 is a micro channel layer bottom view of the centrifugal force driven disc PCR microfluidic chip in the application;

[0051] Figure 5 is a temperature control layer top view of the centrifugal force driven disc PCR microfluidic chip in the application;

[0052] Figure 6 is a connection section view of the polyimide disc surface electrode and the back electrode terminal of the centrifugal force driven disc PCR microfluidic chip in the application.

[0053] wherein:

[0054] 1, micro channel layer, 2, bonding layer, 3, temperature control layer, 4, serpentine micro channel, 5, spiral micro channel, 501, PCR amplification area micro channel, 502, detection area micro channel, 6, arc-shaped micro channel, 701, first temperature zone, 702, second temperature zone, 703, third temperature zone, 8, heating electrode, 801, first heating electrode, 802, second heating electrode, 803, third heating electrode, 9, sensor electrode, 901, first sensor electrode, 902, second sensor electrode, 903, third sensor electrode, 10, electrode sheet, 11, conductive metal, 12, electrode terminal, 13, connecting piece, 14, flat key, 15, conductive slip ring, 16, screw, 17, screw hole, 18, bevel, 19, central sample inlet area, 20, micro column array, 21, collection port, 22, micro channel outlet. DETAILED DESCRIPTION

[0055] The application will be further described below in conjunction with the drawings:

[0056] As shown in Figure 1 , Figure 2 and Figure 3 , a disc PCR microfluidic chip based on centrifugal force driving includes a chip disc. The chip disc includes a microfluidic channel layer 1, a bonding layer 2 and a temperature control layer 3 arranged in sequence, the materials of the microfluidic channel layer 1 and the bonding layer 3 are PDMS, and the temperature control layer 2 adopts a polyimide disc with a thickness of 5 mm as a substrate, and a micro electrode is processed on the surface thereof. The microfluidic channel layer 1 has a thickness of 5 mm, a circular cavity with a radius of 25 mm in the middle, the center of the circular cavity coincides with the center of the chip, and the cavity wall slope is a slope with an angle of 45° (i.e. a bevel 18), and a horn-shaped collection port 21 with a height of 100 um is arranged on the cavity wall slope, the central axis of the collection port 21 points to the center of the chip, the outer end thereof is located on the cavity wall slope, the width thereof is 5 mm, and the inner end thereof is connected with the microfluidic channel in the microfluidic channel layer 1, and the width thereof is 200 um.

[0057] The diameter of the chip disc is 6 inches, the microfluidic channel layer 1 made of PDMS material and the temperature control layer 3 made of polyimide material are strongly bonded through the bonding layer 2, the bonding layer 2 is an ultra-thin PDMS film, which is prepared by high-speed spin coating, and a circular through hole with a radius of 25 mm and the same size as the central sample inlet area 19 is dug in the center. The bonding layer 2 is an ultra-thin PDMS spin coating film with a thickness of 20 um, and a circular hole with a radius of 25 mm is punched in the center to cooperate with the central sample inlet area, and is covered on the electrode surface of the bottom temperature control layer during the bonding process. At the same time, four threaded counterbores are processed on the back of the temperature control layer 3, four M5 screws 16 pass through the screw holes 17 on the connecting piece 13 of the chip disc to realize the connection of the chip and the connecting piece 13, a key groove is processed on the bottom cylindrical shaft of the connecting piece 13, and is connected with the conductive slip ring 15 through the flat key 14 and realizes power transmission. The microfluidic channel layer and the temperature control layer of the chip are strongly bonded through the ultra-thin PDMS film, then four M5 threaded counterbores with a diameter of 40 mm are processed on the back of the temperature control layer, the hole depth is 3 mm, so that the chip is fixed with the connecting piece through four M5 screws, and the connecting piece is connected with the conductive slip ring through the key groove and a 5×4×16 mm round head flat key. The conductive slip ring not only transmits the rotating torque of the disc, but also transmits the electrical signal of the external circuit to the electrode on the upper surface of the temperature control layer 3.

[0058] As shown in Figure 4As shown, a circular cavity with a radius of 25 mm is formed in the center of the microchannel layer 1. The area within the circular cavity is the central sample injection zone 19, serving as the generation and screening area for sample microdroplets. The outer peripheral wall of the circular cavity is beveled at 45° 18, and a microdroplet collection port 21 is located on the side wall of the bevel. The microchannel outlet 22 is located on the outer peripheral wall of the microchannel layer 1. The collection port is funnel-shaped and constricted, with a height of 100 μm, tapering from a 5 mm beginning on the bevel to a 200 μm end. The end of the collection port connects to the microchannel, and the microchannel outlet is located on the edge wall of the microchannel layer. The microchannel has a rectangular cross-section with a width and height of 200 μm and 100 μm, respectively. The presence of the bevel 18 causes larger diameter microdroplets to be ejected from the disk due to the greater centrifugal force, while smaller diameter microdroplets enter the microdroplet collection port 21.

[0059] The microchannel inlet and outlet are connected by three microchannel segments: the first segment is a serpentine microchannel 4, the second segment is a spiral microchannel 5, and the third segment is a tangential arc-shaped microchannel 6 connected to the microchannel outlet 22. The microchannel has a rectangular cross-section with a width of 200µm and a height of 100µm. The microchannel inlet is connected to the inner end of the horn-shaped collection port. Initially, it is a serpentine microchannel with the center of the arc coinciding with the center of the chip. Subsequently, the end of the serpentine microchannel is connected to a multi-turn spiral microchannel. The centerline of the spiral microchannel is a spiral line with its center coinciding with the center of the chip. The inner and outer radii of the spiral line are 36mm and 72mm, respectively. Finally, the end of the spiral microchannel is guided to the edge outlet of the microchannel layer through a tangential arc-shaped microchannel. The three microchannels are divided into two parts by a circle with a radius of 60 mm centered at the chip center. The inner part is a 16-turn PCR amplification microchannel 501, used for PCR amplification of sample microdroplets. The outer part is a 9-turn detection microchannel 502, where amplified sample microdroplets are subjected to subsequent fluorescence analysis and other detection processes. The PCR amplification microchannel includes a serpentine microchannel and a 16-turn spiral microchannel. The detection microchannel includes a 9-turn spiral microchannel and an arc-shaped outlet microchannel. The number of spiral microchannel turns can be increased according to actual needs to achieve more PCR cycles. The PCR amplification microchannel 501 is further divided into three parts by three 14 mm gaps. The two lower quarter-circular regions, clockwise, are the first temperature zone 701 and the second temperature zone 702, respectively. The upper half-circular region is the third temperature zone 703, corresponding to the denaturation, annealing, and extension processes required for PCR amplification. The first and second temperature zones are located in the lower semicircular sector area, each occupying 1 / 4 of the circular area. The third temperature zone is located in the upper semicircular sector area, occupying 1 / 2 of the circular area. The interval between each temperature zone is 14mm.

[0060] like Figure 5As shown, the upper surface of the temperature control layer 3 in the circular area with a radius of 25-60 mm is magnetron sputtered with three groups of electrode patterns with a thickness of 100 nm by the Lift-off process, which are arranged directly below the three temperature zones of the micro-channel layer and separated from the micro-channel by the PDMS film of the bonding layer. Each group of electrode patterns is divided into heating electrodes and sensor electrodes, corresponding to the three temperature zones, which are the first heating electrode 801 and the first sensor electrode 901, the second heating electrode 802 and the second sensor electrode 902, and the third heating electrode 803 and the third sensor electrode 903. The electrodes are arranged in a snake shape, and each group of sensor electrodes is arranged equidistantly from the corresponding heating electrode. Each segment of the heating electrode is arranged in a snake shape with a width of 3 mm, and each segment of the sensor electrode is arranged equidistantly from the heating electrode with a width of 1 mm except the starting segment which is a straight line. The thickness of the electrodes is 100 nm. All the electrodes are covered by the bonding layer PDMS film after the chip bonding, thereby being isolated from the fluid in the micro-channel layer. The heating electrodes provide the required temperature environment for the temperature zones, and the sensor electrodes, together with the external temperature control circuit, realize real-time temperature measurement of the temperature zones according to the measurement principle that the resistance of the electrode changes with temperature.

[0061] The starting and ending points of each segment of the heating electrode and the sensor electrode are connected to a rounded square electrode sheet with a side length of 6 mm. The electrode sheet has a thickness of 100 nm, and the heating electrode electrode sheet and the sensor electrode electrode sheet are arranged on two concentric circles with the circle center coinciding with the chip center. A conductive metal is embedded in the bottom of each electrode sheet to pass through the polyimide substrate and is connected to an electrode terminal on the back of the substrate. The electrode terminal has a diameter of 5 mm and a height of 10 mm, and 12 electrode terminals are arranged vertically below the 12 electrode sheets of the temperature control layer. The two ends of each electrode are connected to a rounded square electrode sheet 10 with a side length of 6 mm. A conductive metal 11 is embedded in the bottom of each electrode sheet 10 to pass through the polyimide disc, as shown in Figure 6 The heating electrode 8 on the upper surface of the temperature control layer 3 provides a specific temperature environment for the three temperature zones. The first heating electrode 801 provides the denaturation temperature (95°C) of the first temperature zone 701, the second heating electrode 802 provides the annealing temperature (60°C) of the second temperature zone 702, and the third heating electrode 803 provides the extension temperature (72°C) of the third temperature zone 703. To achieve the above-mentioned required accurate temperature values, the sensor electrode 9 combines with the external amplification circuit to monitor the real-time temperature values in the three temperature zones using the Wheatstone bridge principle. All the electrodes on the disc surface of the chip are connected to the electrode terminal 12 on the back of the temperature control layer 3 through the electrode sheet 10 on the upper surface of the temperature control layer 3 and the conductive metal 11 embedded in the temperature control layer made of polyimide. Then, the multi-channel conductive wire on the conductive slip ring is used to conduct to the external circuit, thereby realizing the circuit connection during the rotation of the disc PCR micro-fluidic chip.

[0062] like Figure 5 As shown, within a circular region with a radius of 10 mm at the center of the upper surface of the temperature control layer 3, six concentric micropillar arrays 20 are arranged. The spacing between each concentric array is equal, and the micropillars have a diameter of 800 μm and a height of 300 μm. The number of micropillars in each concentric array from the inside out is 1, 4, 8, 12, 18, and 20, respectively. The micropillar array 20 comprises multiple uniformly distributed micropillars with a diameter of 800 μm and a height of 300 μm. Located at the center of the central sample injection area 19, the micropillar array 20 promotes the impact and breakup of sample droplets during droplet injection, contributing to the generation of more numerous and smaller secondary sample microdroplets.

[0063] The working process of the centrifugally driven disc PCR microfluidic chip described above is as follows:

[0064] S1. The motor shaft drives the chip disk through the conductive slip ring 15 and the connector 13, and continuously injects mineral oil into the central sample injection area 19 of the chip as a continuous phase to isolate sample microdroplets. At the same time, a continuously renewed rotating oil film is formed to carry away the sample microdroplet waste liquid generated during one impact on the disk surface, so as to carry out the next sample injection.

[0065] S2. Using a pipette, a pre-prepared sample droplet is drawn and placed into the central sample injection area 19 of the rotating disk. The sample droplet contains the gene fragment to be amplified and various reaction substrates required for PCR amplification. Subsequently, the sample droplet impacts the rotating microcolumn array 20 and breaks into numerous oil-coated microdroplets of varying sizes. Under centrifugal force, larger oil-coated microdroplets are ejected from the disk through the bevel 18, while smaller oil-coated microdroplets enter the collection port 21. The entire process is completed within 2 seconds. After a 2-second interval, the continuously injected oil film carries away the previous sample microdroplet adhering to the disk surface, and the next sample injection process is performed. This cycle is repeated. The presence of the continuous phase oil film ensures that the sample microdroplets are encapsulated by the oil film, preventing cross-contamination between different samples, thereby achieving the purpose of parallel detection of multiple samples.

[0066] S2, the oil microdroplets entering the microfluidic channel will flow along the microfluidic channel to the outside of the chip due to the centrifugal force acting radially outward, first flowing through a section of the serpentine microfluidic channel 4, the sample microdroplets are pre-denatured in the channel, then flowing through the multi-turn spiral PCR amplification zone microfluidic channel 501, each turn sequentially passes through the first temperature zone 701 (denaturation), the second temperature zone 702 (annealing), and the third temperature zone 703 (elongation) to perform one PCR amplification reaction, when all sample microdroplets are amplified and flow into the detection zone microfluidic channel 502, the disc stops rotating and oil injection, at this time all sample microdroplets have undergone 16 amplifications, and all samples after amplification are detected in the detection zone microfluidic channel 502, after detection, the disc is rotated again, and all sample microdroplets are discharged from the chip disc through the arc-shaped microfluidic channel 6 and the microfluidic channel outlet 22.

[0067] The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. A centrifugal force driven disc PCR microfluidic chip, characterized in that, The chip comprises a chip disc; The chip disc comprises a microfluidic channel layer, a bonding layer and a temperature control layer arranged in sequence; The microfluidic channel layer is centrally provided with a circular cavity as a central sample injection area, and is internally arranged with a microfluidic channel; the microfluidic channel is divided into a PCR amplification area microfluidic channel and a detection area microfluidic channel along the radial direction of the chip from inside to outside; the PCR amplification area microfluidic channel comprises a plurality of temperature reaction areas; The bonding layer is used for bonding connection of the microfluidic channel layer and the temperature control layer, and a through hole is formed in the middle of the bonding layer; The temperature control layer is provided with a plurality of heating electrodes corresponding to the temperature reaction areas and a plurality of sensor electrodes corresponding to the heating electrodes; the surface of the temperature control layer is further provided with a micro column array, the micro column array penetrates through the through hole and extends into the central sample injection area; The first section of the microfluidic channel is a serpentine microfluidic channel, the second section is a multi-turn spiral line microfluidic channel, and the third section is an arc-shaped microfluidic channel tangent to the outside of the spiral line; The outlet of the microfluidic channel is located on the edge wall of the microfluidic channel layer; The arc-shaped microfluidic channel leads to the outlet of the microfluidic channel.

2. The microfluidic chip according to claim 1, wherein The side surface of the circular cavity has a bevel; The bevel side wall has a collection port for collecting microdroplets; The collection port is in the shape of a horn; The end of the collection port is connected to the inlet of the microfluidic channel.

3. The microfluidic chip according to claim 1, wherein The PCR amplification area microfluidic channel comprises three temperature zones, namely a first temperature zone, a second temperature zone and a third temperature zone; The first temperature zone and the second temperature zone are located in the lower half circular sector area, each occupying 1 / 4 of the circular ring area, and the third temperature zone is located in the upper half circular sector area, occupying 1 / 2 of the circular ring area, and each temperature zone is spaced apart by 14 mm.

4. The microfluidic chip according to claim 3, wherein The upper surface of the temperature control layer is arranged with heating electrodes corresponding to the three temperature zones and sensor electrodes corresponding to the heating electrodes, respectively, first heating electrodes, second heating electrodes, third heating electrodes and first sensor electrodes, second sensor electrodes and third sensor electrodes; The heating electrodes and the sensor electrodes are arranged in positions corresponding to the first temperature zone, the second temperature zone and the third temperature zone in the vertical direction in space.

5. The microfluidic chip according to claim 1 or 4, wherein The end of the heating electrode and the sensor electrode is connected with an electrode sheet; The bottom of the electrode sheet is embedded with a conductive metal; The conductive metal is used to connect the electrode sheet to the electrode terminal arranged on the back of the temperature control layer.

6. The microfluidic chip according to claim 1, wherein The microfluidic channel layer and the bonding layer are made of PDMS material; The temperature control layer is made of polyimide material.

7. The microfluidic chip according to claim 1, wherein The temperature control layer is provided with a connecting piece; The connecting piece is detachably connected with a conductive slip ring.

8. The microfluidic chip according to claim 7, wherein The back surface of the temperature control layer is provided with a threaded counterbore; The connecting piece is connected to the threaded counterbore through a screw; The connecting piece is provided with a key groove; The connecting piece is connected with the conductive slip ring through a flat key matched with the key groove.

9. The method according to any one of claims 1 to 8, wherein The method comprises: S1, the chip disc rotates, and mineral oil is continuously injected into the central sample injection area as a continuous phase for isolating sample microdroplets, while continuously forming a rotating oil film to take away sample microdroplet waste generated in a previous impact process on the chip disc surface for sample injection of the next sample; S2, a pre-prepared sample droplet is dropped into the central sample injection area of the rotating chip disc, the sample droplet contains a gene fragment to be amplified and various reaction substrates required for PCR amplification, and then the sample droplet is broken to generate a plurality of oil-in-microdroplets after impacting the rotating microcolumn array, under the action of centrifugal force, the oil-in-microdroplets with a larger particle size are thrown out of the disc, and the oil-in-microdroplets with a smaller particle size enter the microchannel; S3, the oil-in-microdroplets entering the microchannel flow to the outside of the chip along the microchannel under the action of centrifugal force radially outward, in the process of flowing through the microchannel, the sample microdroplets will be pre-denatured, denatured, annealed and elongated to perform a PCR amplification reaction, when all the sample microdroplets are amplified and flow into the detection area microchannel, the chip disc stops rotating and oil injection, and all the samples after amplification are detected in the microchannel, and after detection, the disc is rotated again, and all the sample microdroplets are discharged from the chip disc.

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