An integrated microfluidic chip for quantitative detection of circulating tumor DNA in whole blood

Abstract

The present invention discloses an integrated microfluidic chip for quantitatively detecting circulating tumor DNA in whole blood, including an upper cover plate, a first flow channel layer, a second flow channel layer, a third flow channel layer, a PDMS film layer, a control layer and a lower cover plate. Stacking the layers of the chip in sequence can realize multiple module functions, including a plasma separation module, a DNA purification module, a mixing module, a droplet digital LAMP module, and a control module. After the whole blood is mixed with a non-chaotropic reagent, it is added to the plasma separation module, and the plasma filtered out by the filter membrane enters the DNA purification module for free DNA extraction. After the purified DNA and the LAMP reagent are fully mixed in the mixing channel, they enter the droplet digital LAMP module to complete the droplet preparation and digital LAMP reaction, and finally the circulating tumor DNA is quantitatively determined according to the number of positive and negative droplets. The microfluidic chip involved in the present invention is simple to operate, has little pollution, and has a low production cost, and can realize the full on-chip integrated operation of pre-treatment, purification and quantitative detection of circulating tumor DNA in whole blood.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic chips, and in particular relates to an integrated microfluidic chip for quantitatively detecting circulating tumor DNA in whole blood. Background Art

[0002] Circulating tumor DNA (ctDNA) is a free DNA released by tumor cells into the blood circulation system, which contains information on mutations and gene variations originating from the tumor. Detecting circulating tumor DNA can effectively monitor and evaluate the occurrence, development and recovery of related diseases.

[0003] At present, conventional circulating tumor DNA testing needs to be performed separately on multiple instruments such as centrifuges, nucleic acid extractors, and nucleic acid amplifiers. The sample transfer process is prone to nucleic acid contamination and other problems. These devices are generally expensive, large in size, and complex to operate, making them difficult to use in resource-poor areas. Summary of the invention

[0004] Technical problem to be solved by the present invention: The purpose of the present invention is to solve the deficiencies in the prior art and to provide an integrated microfluidic chip for quantitatively detecting circulating tumor DNA in whole blood, which integrates the currently cumbersome circulating tumor DNA detection steps into the same microfluidic chip and has the characteristics of simple operation, low pollution and low production cost.

[0005] The technical solution of the present invention: The integrated microfluidic chip for quantitative detection of circulating tumor DNA in whole blood of the present invention comprises a plasma separation module, a DNA purification module, a mixing module, a droplet digital LAMP module and a control module;

[0006] The plasma separation module is used to separate the mixed reagent of whole blood and non-chaotropic reagent, filter out the plasma, and the filtered plasma enters the DNA purification module for free DNA extraction and purification;

[0007] The DNA purification module, under the action of the control module, discharges impurities other than DNA and sends the purified DNA to the mixing module;

[0008] After the purified DNA and LAMP reagents are fully mixed in the mixing module, they enter the droplet digital LAMP module to complete the droplet preparation and digital LAMP reaction.

[0009] Furthermore, the plasma separation module includes a sample inlet, a pre-filtration diversion channel, a filtration inlet, a filtration membrane, a post-filtration confluence channel, a filtration outlet, and a plasma outlet;

[0010] The plasma stock solution passing through the sample inlet is divided by the pre-filtration diversion flow channel, and then flows into the filter membrane from the plurality of filter inlets arranged at the end of the pre-filtration diversion flow channel; after being filtered by the filter membrane, it is collected by the post-filtration confluence flow channel arranged under the filter membrane, and then discharged from the plasma outlet;

[0011] The DNA purification module uses a non-chaotropic reagent and includes a DNA purification flow channel; a DNA purification flow channel inlet is provided at the starting end of the DNA purification flow channel; the DNA purification flow channel inlet is connected to the plasma outlet; a DNA purification flow channel outlet 1 for discharging a DNA-free washing solution and a DNA purification flow channel outlet 2 for discharging an eluent containing DNA are provided at the end of the DNA purification flow channel;

[0012] The mixing module comprises a mixing channel, a DNA eluent inlet and a LAMP reagent inlet arranged at the starting end of the mixing channel, and a mixing channel outlet channel arranged at the end of the mixing channel; the plasma outlet is connected to the DNA eluent inlet;

[0013] The droplet digital LAMP module adopts step emulsification to prepare droplets, including a multi-channel step emulsification inlet flow channel connected to the mixing flow channel outlet flow channel, and a multi-channel step emulsification flow channel arranged at the end of the multi-channel step emulsification inlet flow channel; the multi-channel step emulsification flow channel outlet end is connected to the droplet storage tank, the droplet digital LAMP waste liquid flow channel and the second waste liquid outlet;

[0014] The control module includes a waste liquid stop valve and an eluent stop valve;

[0015] The waste liquid stop valve is opened, the eluent stop valve is closed, the DNA purification flow channel outlet 1 is opened, the DNA purification flow channel outlet 2 is closed, and the waste liquid generated by the DNA purification module is discharged from the DNA purification flow channel outlet 1;

[0016] The waste liquid stop valve is closed, the eluent stop valve is opened, the first DNA purification channel outlet is closed, the second DNA purification channel outlet is opened, and the eluent containing DNA generated by the DNA purification module is discharged from the second DNA purification channel outlet.

[0017] Further, it includes an upper cover plate, a first flow channel layer, a second flow channel layer, a third flow channel layer, a PDMS film layer, a control layer, and a lower cover plate stacked in parallel from top to bottom;

[0018] A gas inlet of a waste liquid stop valve and a gas inlet of an eluent stop valve extend from the upper cover plate to the control layer;

[0019] A waste liquid outlet 1, a LAMP reagent inlet and a waste liquid outlet 2 extend from the upper cover plate to the third flow channel layer;

[0020] The first flow channel layer is provided with a DNA purification flow channel, a first DNA purification flow channel outlet and a second DNA purification flow channel outlet;

[0021] The third flow channel layer is provided with a mixing flow channel, a DNA eluent inlet and a LAMP reagent inlet;

[0022] The control module comprises an upper hole of a waste liquid shut-off valve and an upper hole of an eluent shut-off valve arranged on the second flow channel layer; the upper hole of the waste liquid shut-off valve is arranged below the first outlet of the DNA purification flow channel; the upper hole of the eluent shut-off valve is arranged below the second outlet of the DNA purification flow channel;

[0023] The control module further comprises a stop valve flow channel and an eluent stop valve flow channel arranged on the third flow channel layer; the stop valve flow channel is connected to the waste liquid stop valve gas inlet and the waste liquid stop valve lower hole; the eluent stop valve flow channel is connected to the eluent stop valve lower hole and the DNA eluent inlet;

[0024] The control module also includes a PDMS film layer, and a waste liquid shutoff valve gas path and an eluent shutoff valve gas path arranged on the control layer;

[0025] One end of the waste liquid stop valve gas circuit is connected to the waste liquid stop valve gas inlet, and the other end is connected to the waste liquid stop valve gas circuit end arranged below the DNA purification flow channel outlet;

[0026] One end of the eluent stop valve gas path is connected to the eluent stop valve gas inlet, and the other end is connected to the eluent stop valve gas path end arranged below the second outlet of the DNA purification flow channel;

[0027] The waste liquid shut-off valve gas inlet inputs gas to the waste liquid shut-off valve gas path and the end of the waste liquid shut-off valve gas path, and the PDMS film layer is deformed at the waste liquid shut-off valve gas path, and the deformed PDMS film layer passes through the waste liquid shut-off valve lower hole and the waste liquid shut-off valve upper hole, blocking the DNA purification flow channel outlet 1;

[0028] The gas inlet of the eluent stop valve inputs gas to the eluent stop valve gas path and the end of the eluent stop valve gas path, and the PDMS film layer is deformed at the eluent stop valve gas path. The deformed PDMS film layer passes through the lower hole of the eluent stop valve and the upper hole of the eluent stop valve, blocking the second outlet of the DNA purification flow channel.

[0029] Furthermore, the plasma separation module is a three-layer structure, wherein the sample inlet, the pre-filtration diversion channel, and the filtration inlet are arranged in the first channel layer;

[0030] The filter membrane is arranged in the second flow channel layer; the filter membrane is arranged below the filter inlet;

[0031] The post-filtration confluence channel, the filtration outlet and the plasma outlet are arranged on the third channel layer;

[0032] The filtration membrane is an asymmetric polyethersulfone membrane, a polycarbonate membrane or a cellulose acetate membrane; the pre-filtration diversion channel is a one-to-N channel; and the post-filtration confluence channel is an N-in-one channel matching the pre-filtration diversion channel.

[0033] Furthermore, the mixing channel is composed of Y monomer diamond-shaped channels that are wide at the top and narrow at the bottom and narrow at the top and wide at the bottom, wherein the width of the wide channel is 2 to 3 times the width of the narrow channel; the arrangement rule of the monomer diamond-shaped channels is an alternating arrangement of monomer diamond-shaped channels that are wide at the top and narrow at the bottom and monomer diamond-shaped channels that are narrow at the top and wide at the bottom.

[0034] Furthermore, the flow channel of the DNA purification flow channel is provided with a plurality of elliptical convex expansions for increasing the contact area of ​​the flow channel with the fluid.

[0035] Furthermore, the droplet digital LAMP module includes a multi-channel step emulsification flow channel arranged in the first flow channel layer, a multi-channel step emulsification inlet flow channel, a droplet digital LAMP waste liquid flow channel arranged in the third flow channel layer, and a droplet storage pool extending from the first flow channel layer to the third flow channel layer;

[0036] The multi-channel stepped emulsification flow channel adopts a multi-channel parallel structure, the number of channels is 1 to 16, and a triangular opening stepped emulsification structure is adopted at the steps. The prepared droplets are stored in the droplet storage pool, and the excess droplets flow out through the droplet digital LAMP waste liquid flow channel and the second waste liquid outlet.

[0037] Furthermore, the upper cover plate, the first flow channel layer, the second flow channel layer, the third flow channel layer, the PDMS film layer, the control layer, and the lower cover plate all have positioning holes for positioning and clamping the chip.

[0038] Furthermore, N and Y are any natural numbers from 4 to 16.

[0039] Furthermore, the upper cover plate, the second flow channel layer and the lower cover plate are made of a PET film coated with acrylic acid on both sides;

[0040] The first flow channel layer, the third flow channel layer and the control layer are made of silicone film;

[0041] The PDMS film layer is made of PDMS material film.

[0042] The present invention has the following beneficial effects compared with the prior art:

[0043] 1. The present invention can integrate the pre-treatment, DNA purification and quantitative detection operations of circulating tumor DNA detection on a microfluidic chip. Compared with the traditional operations on instruments such as centrifuges, nucleic acid extractors, and nucleic acid amplification instruments, it avoids the contamination of sample transfer, high instrument costs and complex instrument operations.

[0044] 2. The stop valve is realized by gas pushing the PDMS film to deform. When the gas enters the stop valve gas path, the PDMS film deforms, blocking the liquid path to achieve the stop effect. When the gas is not passed, the PDMS film does not deform, the liquid path is conductive, and the conductive effect is achieved. In a microchip, it is easy to control.

[0045] 3. The DNA purification channel contains multiple elliptical convex expansions, which are used to increase the contact area between the channel and the fluid and increase the incubation time of the fluid in the channel, which is conducive to the binding of DTBP to DNA and APDMS modified on the DNA purification channel.

[0046] 4. The arrangement rule of the asymmetric diamond structure is the staggered arrangement of the monomer diamond flow channel with a wide top and a narrow bottom and the monomer diamond flow channel with a narrow top and a wide bottom. The asymmetric diamond structure allows the DNA-carrying eluate and the LAMP reagent mixture to be continuously and unequally divided and converged, achieving the purpose of rapid mixing.

[0047] 5. The present invention uses thin film materials as microfluidic chip materials, which are simple to process, low in cost, and have the potential for mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the overall structure of the present invention;

[0049] Figure 2 It is an exploded schematic diagram of the overall structure of the present invention;

[0050] Figure 3 It is a schematic diagram of the structure of the upper cover plate in the present invention;

[0051] Figure 4 It is a structural schematic diagram of the first flow channel layer and a partial enlarged diagram of the mixing module in the present invention;

[0052] Figure 5 is a schematic structural diagram of the second flow channel layer in the present invention;

[0053] Figure 6 is a schematic structural diagram of the third flow channel layer in the present invention;

[0054] Figure 7 It is a schematic diagram of the structure of the PDMS film layer in the present invention;

[0055] Figure 8 It is a schematic diagram of the structure of the control layer in the present invention;

[0056] Fig. 9 It is a structural schematic diagram of the lower cover plate in the present invention;

[0057] Fig.10 This is an experimental diagram of cell concentration in plasma after separation by the plasma separation module of the present invention;

[0058] Fig.11 This is a simulation result diagram of the fluid flowing through the mixing module in the present invention.

[0059] Among them, 1-upper cover plate, 2-first flow channel layer, 3-second flow channel layer, 4-third flow channel layer, 5-PDMS film layer, 6-control layer, 7-lower cover plate, 8-sample inlet, 9-positioning hole, 10-waste liquid outlet 1, 11-waste liquid stop valve gas inlet, 12-eluent stop valve gas inlet, 13-LAMP reagent inlet, 14-waste liquid outlet 2, 15-pre-filtration diversion channel, 17-filtration inlet, 16-DNA purification channel inlet, 18-DNA purification channel, 19-mixing channel, 20-multi-channel step emulsification channel, 21-DNA purification channel outlet 1, 22-DNA purification channel outlet 2, 23-D NA eluent inlet, 24-droplet storage tank, 25-filter membrane, 26-upper hole of waste liquid shut-off valve, 27-upper hole of eluent shut-off valve, 28-mixing channel after filtration, 29-plasma outlet, 30-filter outlet, 31-lower hole of waste liquid shut-off valve, 32-flow channel of waste liquid shut-off valve, 33-lower hole of eluent shut-off valve, 34-flow channel of eluent shut-off valve, 35-waste liquid flow channel of droplet digital LAMP, 36-end of gas path of waste liquid shut-off valve, 37-gas path of waste liquid shut-off valve, 38-gas path of eluent shut-off valve, 39-end of gas path of eluent shut-off valve, 40-mixing channel outlet flow channel, 41-multi-channel stepped emulsification inlet flow channel, 42-eluent outlet. DETAILED DESCRIPTION

[0060] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0061] like Figure 1 and Figure 2 As shown, the microfluidic chip includes from top to bottom: an upper cover plate 1, a first flow channel layer 2, a second flow channel layer 3, a third flow channel layer 4, a PDMS film layer 5, a control layer 6, and a lower cover plate 7. Each layer is stacked in sequence to realize multiple functional modules, including

[0062] The plasma separation module comprises a first flow channel layer 2, a second flow channel layer 3, a third flow channel layer 4,

[0063] The DNA purification module comprises a first flow channel layer 2 and a second flow channel layer 3,

[0064] Mixing module, droplet digital LAMP module, control module.

[0065] like Figure 3 , Figure 4 (a) Figure 5 , Figure 6 As shown, the plasma separation module includes a sample inlet 8, a pre-filtration diversion channel 15, a filtration inlet 17, a filtration membrane 25, a post-filtration confluence channel 28, a filtration outlet 30 and a plasma outlet 29.

[0066] like Figure 4 (a) Figure 5 As shown, the DNA purification module includes a DNA purification flow channel inlet 16, a DNA purification flow channel 18, a DNA purification flow channel outlet 1 21 and a DNA purification flow channel outlet 22.

[0067] like Figure 4 (a) Figure 4 As shown in (b), the mixing module includes a DNA eluent inlet 23, a LAMP reagent inlet 13, a mixing channel 19 and a mixing channel outlet channel 40.

[0068] like Figure 4 (a) Figure 5 , Figure 6 As shown, the droplet digital LAMP module includes a multi-channel step emulsification inlet flow channel 41, a multi-channel step emulsification flow channel 20, a droplet storage pool 24, a droplet digital LAMP waste liquid flow channel 35 and a waste liquid outlet 14.

[0069] like Figure 5 , Figure 6 , Figure 7 , Figure 8 As shown, the control module includes an upper hole 26 of a waste liquid shut-off valve, an upper hole 27 of an eluent shut-off valve, a lower hole 31 of a waste liquid shut-off valve, a lower hole 33 of an eluent shut-off valve, a flow channel 32 of a waste liquid shut-off valve, a flow channel 34 of an eluent shut-off valve, a PDMS thin film layer 5, a gas inlet 11 of a waste liquid shut-off valve, a gas inlet 12 of an eluent shut-off valve, a gas path end 36 of a waste liquid shut-off valve, a gas path end 39 of an eluent shut-off valve, a gas path 37 of a waste liquid shut-off valve, a gas path 38 of an eluent shut-off valve, a waste liquid outlet 10 and an eluent outlet 42.

[0070] like Figure 3 , Figure 4 (a) Figure 5 , Figure 6 As shown, the plasma separation module is a three-layer structure, and the sample inlet 8, the pre-filtration diversion channel 15, and the filtration inlet 17 are arranged in the first channel layer 2;

[0071] The filter membrane 25 is disposed in the second flow channel layer 3; the filter membrane 25 is an asymmetric polyethersulfone membrane, a polycarbonate membrane or a cellulose acetate membrane;

[0072] The post-filtration confluence channel 28, the filtration outlet 30 and the plasma outlet 29 are arranged in the third channel layer;

[0073] The pre-filtration diversion channel 15 is an n-division channel, wherein n is 4 to 16; the post-filtration confluence channel 28 is an n-in-one channel, wherein n is consistent with the pre-filtration diversion channel 15 and is 4 to 16; the plasma outlet 29 is connected to the DNA purification channel inlet 16.

[0074] like Figure 4 (a) Figure 5 As shown, the DNA purification module is located in the first flow channel layer 2 and the second flow channel layer 3.

[0075] Principle of DNA purification module: Non-chaotropic reagent dimethyl dithiopropionate DTBP can bind to DNA in plasma, and can also bind to 3-aminopropyldiethoxymethylsilane APDMS modified on the DNA purification channel 18. After DTBP binds to DNA and APDMS, washing solution is used to wash away other impurities in plasma, while DNA is fixed on the DNA purification channel 18, and finally an eluent is used to disconnect DTBP from DNA and APDMS, thereby obtaining an eluent carrying DNA.

[0076] When the non-chaotropic reagent DTBP combines with DNA and the APDMS modified on the DNA purification flow channel 18, the DNA is fixed on the DNA purification flow channel 18, and the washing liquid is used to remove impurities in the plasma, and the waste liquid stop valve and the eluent stop valve are controlled to open the DNA purification flow channel outlet 1 21, close the DNA purification flow channel outlet 2 22, and the washing liquid flows out from the DNA purification flow channel outlet 1 21;

[0077] After flushing with the washing solution, an eluent is used to disconnect the non-chaotropic reagent DTBP from the DNA and the APDMS modified on the DNA purification channel outlet 21, thereby obtaining an eluent carrying the DNA. The waste liquid shut-off valve and the eluent shut-off valve are controlled to close the DNA purification channel outlet 21 and open the DNA purification channel outlet 22. The eluent carrying the DNA flows out from the DNA purification channel outlet 22, passes through the DNA eluent inlet 23, and flows into the mixing channel 19.

[0078] like Figure 4 (a) Figure 4As shown in (b), the mixing module, the asymmetric diamond structure of the mixing channel 19 is composed of n upper wide and lower narrow monomer diamond channels and upper narrow and lower wide monomer diamond channels, wherein the width of the wide channel is 2 to 3 times the width of the narrow channel, and n is 4 to 16. The arrangement rule of the asymmetric diamond structure is the staggered arrangement of upper wide and lower narrow monomer diamond channels and upper narrow and lower wide monomer diamond channels. The asymmetric diamond structure allows the DNA-carrying eluent and the LAMP reagent mixture to be continuously and unequally diverted and converged to achieve the purpose of rapid mixing. After the DNA-carrying eluent and the LAMP reagent mixture are evenly mixed, the liquid enters the outlet channel 40 of the mixing channel.

[0079] like Figure 4 (a) Figure 5 , Figure 6 As shown, the multi-channel step emulsification inlet flow channel 41 of the droplet digital LAMP module is connected to the mixing channel outlet flow channel 40;

[0080] The multi-channel stepped emulsification flow channel 20 adopts a multi-channel parallel structure with 1 to 16 channels. A triangular opening stepped emulsification structure is adopted at the steps. The prepared droplets are stored in the droplet storage pool 24, and the excess waste liquid flows out through the droplet digital LAMP waste liquid flow channel 35 and the waste liquid outlet 14.

[0081] like Figure 5 , Figure 6 , Figure 7 , Figure 8 As shown, the control module has a waste liquid shutoff valve and an eluent shutoff valve. The gas controls the PDMS film layer to deform, thereby opening or blocking the hole 26 on the waste liquid shutoff valve, so that the fluid therein can flow or cannot flow.

[0082] When the waste liquid shut-off valve is closed, gas is input from the waste liquid shut-off valve gas inlet 11 to the waste liquid shut-off valve gas path 37 and the waste liquid shut-off valve gas path end 36, so that the PDMS film layer 5 is deformed at the waste liquid shut-off valve gas path 37 and the waste liquid shut-off valve gas path end 36, and the deformed PDMS film layer bulges in the waste liquid shut-off valve lower hole 31 to form a spherical surface, blocking the waste liquid shut-off valve upper hole 26, so that the shut-off valve liquid path is blocked;

[0083] On the contrary, when the waste liquid shut-off valve is opened, the gas input is canceled, the PDMS film layer 5 does not deform, and the waste liquid generated by DNA purification, that is, other impurities in the plasma except DNA, flows into the waste liquid shut-off valve flow channel 32 through the waste liquid shut-off valve upper hole 26 and the waste liquid shut-off valve lower hole 31, and flows out through the waste liquid outlet 10.

[0084] When the eluent stop valve is closed, gas is input from the eluent stop valve gas inlet 12 to the eluent stop valve gas path 38 and the eluent stop valve gas path end 39, so that the PDMS film layer 5 is deformed at the eluent stop valve gas path 38 and the eluent stop valve gas path end 39, and the deformed PDMS film layer blocks the eluent stop valve upper hole 27 by stretching in the eluent stop valve lower hole 33;

[0085] On the contrary, when the eluent stop valve is opened, there is no gas input, the PDMS film layer 5 does not deform, and the waste liquid flows into the eluent stop valve flow channel 34 and the eluent outlet 42 through the eluent stop valve upper hole 27 and the eluent stop valve lower hole 33 .

[0086] like Figure 2 As shown, the upper cover plate 1, the first flow channel layer 2, the second flow channel layer 3, the third flow channel layer 4, the PDMS film layer 5, the control layer 6, and the lower cover plate 7 all have four positioning holes 9 for positioning and clamping the chip.

[0087] like Figure 4 As shown in (a), the DNA purification channel 18 contains a plurality of elliptical convex expansions for increasing the contact area between the channel and the fluid and increasing the incubation time of the fluid in the channel, which is beneficial for DTBP to bind to DNA and APDMS modified on the DNA purification channel.

[0088] Fig.10 The experimental results of the cell concentration in plasma after separation by the plasma separation module of the chip were obtained using Thermo's CountessII automatic cell counter. Compared with 10 9 Order of magnitude blood cell count, blood cells dropped to 10 5 The removal rate is above 99.9%.

[0089] Fig.11 This is the simulation result diagram of the mixing module obtained using COMSOL Multiphysics 5.5, where Q1 and Q2 are the input flows of the upper left and lower left inlets respectively. Fig.11 (a) Q1 = Q2 = 100 μL / min; Fig.11 (b) Q1 = Q2 = 200 μL / min; Fig.11 (c) Q1 = Q2 = 300 μL / min; Fig.11 (d) Q1 = Q2 = 400 μL / min; Fig.11 (e) Q1 = Q2 = 500 μL / min; Fig.11 (f) Q1 = Q2 = 600 μL / min; Fig.11 (g) Q1 = Q2 = 700 μL / min; Fig.11 (h) Q1 = Q2 = 800 μL / min; Fig.11 In (i), Q1=Q2=900 μL / min; it can be seen that in a larger range, the mixing structure can effectively mix the two fluids.

[0090] In the above embodiment, the seven-layer chip stacked up and down is made of materials such as silicone film, wherein the upper cover plate 1, the second flow channel layer 3 and the lower cover plate 7 are made of PET film with double-sided acrylic coating; the first flow channel layer 2, the third flow channel layer 4 and the control layer 6 are made of silicone film; the PDMS film layer 5 is made of PDMS film.

[0091] The layers are bonded together using a plasma bonder to form a Figure 1 Tight connection.

[0092] In the above embodiments, the corresponding holes between the layers are used to achieve fluid circulation in the vertical direction.

[0093] In the above embodiment, the specific detection process is as follows:

[0094] Before adding the sample, Novec 7500 oil containing 2% surfactant is first injected from the second waste liquid outlet 14 to fill the droplet storage pool 24 through the droplet digital LAMP waste liquid flow channel 35 .

[0095] Use a syringe to add the mixed whole blood sample and the non-chaotropic reagent DTBP into the sample inlet 8 of the upper cover plate 1. The sample flows into the pre-filtration diversion channel 15 in the first channel layer 2 through the sample inlet 8. After diversion, the sample flows into the filter membrane 25 of the second channel layer 3 through the filter inlet 17 at the end of the pre-filtration diversion channel 15.

[0096] The plasma is produced by the filtration of the filter membrane 25 and flows downward into the filtration outlet 30 of the third flow channel layer 4, and after confluence in the post-filtration confluence channel 28, flows out through the plasma outlet 29;

[0097] The plasma passes through the through holes on the second flow channel layer 3 , flows upward into the DNA purification flow channel inlet 16 located on the first flow channel layer 2 , and flows into the DNA purification flow channel 18 .

[0098] Since DTBP can connect DNA and the DNA purification flow channel 18, the DNA in the plasma is fixed in the DNA purification flow channel 18;

[0099] Add washing liquid from the sample inlet 8, use the washing liquid to remove impurities in the plasma, control the waste liquid stop valve and the eluent stop valve, open the DNA purification flow channel outlet 1 21, close the DNA purification flow channel outlet 2 22, and the waste liquid generated after the washing liquid washes the DNA purification flow channel 18, and flows out through the DNA purification flow channel outlet 1 21, the waste liquid stop valve upper hole 26, the waste liquid stop valve lower hole 31, the waste liquid stop valve flow channel 32, and the waste liquid outlet 10 in sequence;

[0100] After washing, add eluent from the sample inlet 8, use the eluent to disconnect the non-chaotropic reagent DTBP from the DNA and the DNA purification flow channel outlet 1 21, thereby eluting the DNA, control the waste liquid shut-off valve and the eluent shut-off valve to close the DNA purification flow channel outlet 1 21 and open the DNA purification flow channel outlet 2 22, and the eluent carrying the DNA flows out from the DNA purification flow channel outlet 2 22, through the DNA eluent inlet 23, and flows into the mixing flow channel 19 on the same layer.

[0101] The corresponding LAMP reagent and fluorescent dye are added to the LAMP reagent inlet 13, and the DNA eluate and the LAMP reagent are fully mixed in the mixing channel 19 and enter the multi-channel step emulsification channel 20 to generate droplets of uniform size; at this time, the chip is placed in a 65°C oven for one hour, and then the number of positive and negative droplets is observed and counted under a fluorescence microscope, and the amount of circulating tumor DNA is quantified according to the Poisson distribution.

[0102] The prepared droplets are stored in the droplet storage pool 24, and the excess Novec7500 oil containing 2% surfactant is squeezed out through the droplet digital LAMP waste liquid flow channel 35 in the third flow channel layer 4 and the waste liquid outlet 2 14.

[0103] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above specific embodiments. The above specific embodiments and the description in the specification are only for further illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of the present invention to be protected is defined by the claims and their equivalents.

Claims

1. An integrated microfluidic chip for quantitative detection of circulating tumor DNA in whole blood, characterized in that: It includes a plasma separation module, a DNA purification module, a mixing module, a droplet digital LAMP module and a control module; The plasma separation module is used to separate the mixed reagent of whole blood and non-chaotropic reagent, filter out the plasma, and the filtered plasma enters the DNA purification module for free DNA extraction and purification; The DNA purification module, under the action of the control module, discharges impurities other than DNA and sends the purified DNA to the mixing module; After the purified DNA and LAMP reagents are fully mixed in the mixing module, they enter the droplet digital LAMP module to complete the droplet preparation and digital LAMP reaction; The plasma separation module comprises a sample inlet (8), a pre-filtration diversion channel (15), a filtration inlet (17), a filtration membrane (25), a post-filtration confluence channel (28), a filtration outlet (30) and a plasma outlet (29); The plasma raw liquid passing through the sample inlet (8) is diverted by the pre-filtration diversion channel (15), and then flows into the filter membrane (25) from the plurality of filter inlets (17) arranged at the end of the pre-filtration diversion channel (15); after being filtered by the filter membrane (25), it is collected by the post-filtration confluence channel (28) arranged under the filter membrane (25), and then discharged from the plasma outlet (29); The DNA purification module comprises a DNA purification flow channel (18); a DNA purification flow channel inlet (16) is provided at the starting end of the DNA purification flow channel (18); the DNA purification flow channel inlet (16) is connected to the plasma outlet (29); a DNA purification flow channel outlet 1 (21) for discharging a washing solution not containing DNA and a DNA purification flow channel outlet 2 (22) for discharging an eluent containing DNA are provided at the end of the DNA purification flow channel (18); The mixing module comprises a mixing channel (19), a DNA eluent inlet (23) and a LAMP reagent inlet (13) arranged at the starting end of the mixing channel (19), and a mixing channel outlet channel (40) arranged at the end of the mixing channel (19); the plasma outlet (29) is connected to the DNA eluent inlet (23); The droplet digital LAMP module comprises a multi-channel stepped emulsification inlet flow channel (41) connected to the mixing flow channel outlet flow channel (40), and a multi-channel stepped emulsification flow channel (20) arranged at the end of the multi-channel stepped emulsification inlet flow channel (41); the liquid outlet end of the multi-channel stepped emulsification flow channel (20) is connected to the droplet storage tank (24), the droplet digital LAMP waste liquid flow channel (35) and the second waste liquid outlet (14); The control module includes a waste liquid stop valve and an eluent stop valve; The waste liquid shut-off valve is opened, the eluent shut-off valve is closed, the DNA purification flow channel outlet 1 (21) is opened, the DNA purification flow channel outlet 2 (22) is closed, and the waste liquid generated by the DNA purification module is discharged from the DNA purification flow channel outlet 1 (21); The waste liquid shut-off valve is closed, the eluent shut-off valve is opened, the DNA purification channel outlet 1 (21) is closed, the DNA purification channel outlet 2 (22) is opened, and the eluent containing DNA generated by the DNA purification module is discharged from the DNA purification channel outlet 2 (22).

2. The microfluidic chip according to claim 1, characterized in that: It comprises an upper cover plate (1), a first flow channel layer (2), a second flow channel layer (3), a third flow channel layer (4), a PDMS film layer (5), a control layer (6), and a lower cover plate (7) which are stacked in parallel from top to bottom; A waste liquid stop valve gas inlet (11) and an eluent stop valve gas inlet (12) penetrate from the upper cover plate (1) to the control layer (6); A waste liquid outlet 1 (10), a LAMP reagent inlet (13) and a waste liquid outlet 2 extend from the upper cover plate (1) to the third flow channel layer (4); The first flow channel layer (2) is provided with a DNA purification flow channel (18), a first DNA purification flow channel outlet (21) and a second DNA purification flow channel outlet (22); The third flow channel layer (4) is provided with a mixing flow channel (19), a DNA eluent inlet (23) and a LAMP reagent inlet (13); The control module comprises a waste liquid shut-off valve upper hole (26) and an eluent shut-off valve upper hole (27) arranged on the second flow channel layer (3); the waste liquid shut-off valve upper hole (26) is arranged below the first outlet (21) of the DNA purification flow channel; the eluent shut-off valve upper hole (27) is arranged below the second outlet (22) of the DNA purification flow channel; The control module further comprises a stop valve flow channel (32) and an eluent stop valve flow channel (34) arranged on the third flow channel layer (4); the stop valve flow channel (32) is connected to the waste liquid stop valve gas inlet (11) and the waste liquid stop valve lower hole (31); the eluent stop valve flow channel (34) is connected to the eluent stop valve lower hole (33) and the DNA eluent inlet (23); The control module further comprises a PDMS film layer (5), and a waste liquid stop valve gas path (37) and an eluent stop valve gas path (38) arranged on the control layer (6); One end of the waste liquid shut-off valve gas path (37) is connected to the waste liquid shut-off valve gas inlet (11), and the other end is connected to the waste liquid shut-off valve gas path end (36) arranged below the first outlet (21) of the DNA purification flow channel; One end of the eluent stop valve gas path (38) is connected to the eluent stop valve gas inlet (12), and the other end is connected to the eluent stop valve gas path end (39) arranged below the second outlet (22) of the DNA purification flow channel; The waste liquid shut-off valve gas inlet (11) inputs gas into the waste liquid shut-off valve gas path (37) and the waste liquid shut-off valve gas path end (36), and the PDMS film layer (5) is deformed at the waste liquid shut-off valve gas path (37). The deformed PDMS film layer (5) passes through the waste liquid shut-off valve lower hole (31) and the waste liquid shut-off valve upper hole (26), thereby blocking the DNA purification flow channel outlet 1 (21); The eluent stop valve gas inlet (12) inputs gas into the eluent stop valve gas path (38) and the eluent stop valve gas path end (39), and the PDMS film layer (5) is deformed at the eluent stop valve gas path (38). The deformed PDMS film layer (5) passes through the eluent stop valve lower hole (33) and the eluent stop valve upper hole (27), thereby blocking the second DNA purification flow channel outlet (22).

3. The microfluidic chip according to claim 2, characterized in that: The plasma separation module has a three-layer structure, wherein the sample inlet (8), the pre-filtration diversion channel (15), and the filtration inlet (17) are arranged in the first channel layer (2); The filter membrane (25) is arranged on the second flow channel layer (3); the filter membrane (25) is arranged below the filter inlet (17); The post-filtration confluence flow channel (28), the filtration outlet (30) and the plasma outlet (29) are arranged in the third flow channel layer; The filter membrane (25) is an asymmetric polyethersulfone membrane, a polycarbonate membrane or a cellulose acetate membrane; the pre-filtration diversion channel (15) is an N-channel; and the post-filtration confluence channel (28) is an N-in-one channel that matches the pre-filtration diversion channel (15).

4. The microfluidic chip according to claim 2, characterized in that: The mixing channel (19) is composed of Y single diamond-shaped channels that are wide at the top and narrow at the bottom, and narrow at the top and wide at the bottom, wherein the width of the wide channel is 2 to 3 times the width of the narrow channel, and the arrangement rule of the single diamond-shaped channels is an alternating arrangement of single diamond-shaped channels that are wide at the top and narrow at the bottom and single diamond-shaped channels that are narrow at the top and wide at the bottom.

5. The microfluidic chip according to claim 1, characterized in that: The flow channel of the DNA purification flow channel (18) is provided with a plurality of elliptical convex expansions for increasing the contact area of ​​the flow channel with the fluid.

6. The microfluidic chip according to claim 1, characterized in that: The droplet digital LAMP module comprises a multi-channel step emulsification channel (20) arranged on a first channel layer (2), a multi-channel step emulsification inlet channel (41), a droplet digital LAMP waste liquid channel (35) arranged on a third channel layer (4), and a droplet storage pool (24) extending from the first channel layer (2) to the third channel layer (4); The multi-channel stepped emulsification flow channel (20) adopts a multi-channel parallel structure, the number of channels is 1 to 16, and a triangular opening stepped emulsification structure is adopted at the steps. The prepared droplets are stored in the droplet storage pool (24), and the excess droplets flow out through the droplet digital LAMP waste liquid flow channel (35) and the second waste liquid outlet (14).

7. The microfluidic chip according to claim 2, characterized in that: The upper cover plate (1), the first flow channel layer (2), the second flow channel layer (3), the third flow channel layer (4), the PDMS film layer (5), the control layer (6), and the lower cover plate (7) all contain positioning holes (9) for positioning and clamping the chip.

8. The microfluidic chip according to claim 3, characterized in that: The N and Y are any natural numbers between 4 and 16.

9. The microfluidic chip according to any one of claims 2 to 8, characterized in that: The upper cover plate (1), the second flow channel layer (3) and the lower cover plate (7) are made of a PET film coated with acrylic acid on both sides; The first flow channel layer (2), the third flow channel layer (4) and the control layer (6) are made of silicone film; The PDMS thin film layer (5) is made of PDMS material.

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