Microfluidic chip, microfluidic chip assembly and digital nucleic acid detection sampling device

Abstract

The present invention provides a microfluidic chip, a microfluidic chip assembly and a digital nucleic acid detection sampling device. The microfluidic chip comprises: a flow channel layer, the flow channel layer is provided with a detection flow channel for accommodating a detection sample; the flow channel layer is also provided with a liquid inlet, the liquid inlet is connected to the detection flow channel; a reagent well plate, the reagent well plate is suitable for pre-installing detection reagents; the reagent well plate is provided with a plurality of micropores penetrating the reagent well plate from the thickness direction; the flow channel layer is provided with a receiving groove, the reagent well plate is suitable for being clamped in the receiving groove; the detection flow channel is located at the bottom of the receiving groove, when the reagent well plate is clamped in the receiving groove, the micropore is directly connected to the detection flow channel, and the surface of the reagent well plate facing away from the detection flow channel is flush with the surface of the flow channel layer; an encapsulation film layer, the encapsulation film layer is suitable for encapsulating the reagent well plate in the receiving groove, the encapsulation film layer is attached to and covers the surface of the reagent well plate and the surface of the flow channel layer around the reagent well plate; the encapsulation film layer has air permeability. The scheme of the present invention can improve the detection efficiency of the microfluidic chip.

Description

Technical Field

[0001] The present invention relates to the technical field of PCR detection, and in particular to a microfluidic chip, a microfluidic chip component and a digital nucleic acid detection sampling device. Background Art

[0002] In recent years, in PCR (Polymerase Chain Reaction) detection, especially in digital nucleic acid detection, microfluidic chips have attracted widespread attention due to their high sensitivity and accuracy, and have important applications in tumor research, virus detection and other fields. In view of the urgent need for fast sample detection speed and high accuracy in current in vitro diagnosis and other fields, microfluidic chip technology will develop in the direction of shortening detection time and improving detection efficiency. In the current microfluidic chip research, since the detection sample needs to be loaded into the flow channel of the microfluidic chip in digital nucleic acid detection, it is mixed with the detection reagent added later or pre-loaded to show the detection result. For the microfluidic chip with the detection reagent added later, it is necessary to reserve thickness and openings as the passage for the detection reagent to be added to the flow channel, thereby limiting the thickness of the microfluidic chip and making it difficult to miniaturize the microfluidic chip; for the microfluidic chip with pre-loaded reagents, it is usually necessary to set up an additional accommodation space on the side of the detection flow channel, or increase the thickness of the microfluidic chip to accommodate the pre-loaded detection reagent. Both of the above limit the miniaturization of microfluidic chips, which limits the batch and convenience of digital nucleic acid detection. In addition, in order to meet the injection and sealing effects, the heat transfer efficiency is sacrificed to varying degrees, thereby reducing the detection time. For example, some commercial biological detection chips need to be immersed in oil for heat transfer after the sample is brushed into the micropores; or a glass plate is used to slide to achieve liquid segmentation, and glass is a poor conductor of heat when used as a heat transfer medium; there are also various laboratory-prepared chips that use a large amount of PDMS as a chip material, sacrificing heat transfer capacity, all of which will reduce detection efficiency. Summary of the invention

[0003] The present invention provides a microfluidic chip, a microfluidic chip assembly and a digital nucleic acid detection sampling device to solve the problem of limited detection efficiency of the microfluidic chip.

[0004] The present invention provides a microfluidic chip, comprising: a flow channel layer, wherein the flow channel layer is provided with a detection flow channel for accommodating a detection sample; the flow channel layer is also provided with a liquid inlet, wherein the liquid inlet is a blind hole, and is connected with the detection flow channel and the environmental space on the surface of the flow channel layer; a reagent well plate, wherein the reagent well plate is suitable for pre-installing detection reagents; the reagent well plate is provided with a plurality of micropores penetrating the reagent well plate from the thickness direction; the flow channel layer is provided with a receiving groove, wherein the reagent well plate is suitable for being clamped in the receiving groove; the detection flow channel is located at the groove bottom of the receiving groove, and when the reagent well plate is clamped in the receiving groove, the micropores are directly connected with the detection flow channel, and the surface of the reagent well plate facing away from the detection flow channel is flush with the surface of the flow channel layer; and a packaging film layer, wherein the packaging film layer is suitable for packaging the reagent well plate in the receiving groove, wherein the packaging film layer is attached to and covers the surface of the reagent well plate and the surface of the flow channel layer around the reagent well plate; and the packaging film layer has air permeability.

[0005] Optionally, the microwells are arranged in an array on the reagent well plate.

[0006] Optionally, the detection channel includes a plurality of inlet channels and a plurality of amplification channels, the amplification channels are arranged in parallel or in a serpentine manner, the inlet channel connects the liquid inlet and each of the amplification channels; the position of the amplification channel in the channel layer corresponds to the arrangement position of the micropores in the reagent well plate.

[0007] Optionally, the flow channel layer is further provided with a sealing channel, the sealing channel surrounds the detection flow channel and is spaced apart from the detection flow channel; the sealing channel is filled with a sealing filler.

[0008] Optionally, the sealing filler includes one or more of silicone oil, fluorinated liquid, mineral oil or epoxy resin.

[0009] Optionally, the microfluidic chip further comprises: a support layer, the support layer being arranged on a surface of the flow channel layer facing away from the packaging film layer; the support layer being provided with a connecting hole penetrating the support layer in a thickness direction, the connecting hole connecting the liquid inlet and an external space on the surface of the support layer;

[0010] Optionally, the support layer is a transparent layer, and / or the support layer is provided with a plurality of observation holes, and positions of the observation holes correspond to the detection flow channel.

[0011] Optionally, the material of the support layer includes one or more of PDMS, glass, PMMA, PC or COC.

[0012] Optionally, the width of the detection channel is 10μm-10mm, and the height of the detection channel is 5μm-1mm; the material of the channel layer is PDMS, Si, COP or glass; the height of the reagent well plate is 10μm-1mm; the pore size of the micropore is 10μm-1mm; the material of the reagent well plate includes Si, glass, COP or metal; the thickness of the encapsulation film layer is 1μm-100μm; the material of the encapsulation film layer includes a high molecular polymer with a network cross-linked structure.

[0013] Optionally, the high molecular polymer having a network cross-linking structure includes PDMS, TPU or PE.

[0014] The present invention also provides a microfluidic chip assembly, comprising the microfluidic chip provided by the present invention; and also comprising: a liquid storage plate, wherein the liquid storage plate is provided with a liquid storage hole penetrating the liquid storage plate in the thickness direction, and the liquid storage hole is suitable for pre-installing detection reagents; the liquid storage plate is suitable for being arranged on the surface of the support layer facing away from the flow channel layer, and when the liquid storage plate is arranged on the surface of the support layer, the liquid storage hole is connected to the liquid inlet.

[0015] Optionally, a sealing layer is suitable for being arranged on a surface of the packaging film layer that is away from the flow channel layer, and the sealing layer covers the entire surface of the packaging film layer.

[0016] Optionally, the liquid storage plate is a transparent plate, or the liquid storage plate is adapted to be peelably arranged on a surface of the support layer on a side facing away from the flow channel layer.

[0017] Optionally, the liquid storage hole is a tapered hole.

[0018] Optionally, the material of the liquid storage plate includes PDMS or rubber.

[0019] Optionally, the material of the sealing layer includes thermosetting oil, epoxy resin or UV glue.

[0020] The present invention also provides a digital nucleic acid detection sampling device, comprising the microfluidic chip assembly provided by the present invention, and also comprising: a fixed splint, wherein the fixed splint is suitable for clamping and fixing the microfluidic chip and the liquid storage plate; the fixed splint is provided with a connector corresponding to the position of the liquid inlet and the connecting hole, the connector has a hollow ventilation tube, which is suitable for connecting with the connecting hole, and the connector is suitable for connecting to an air source through a connecting air tube to ventilate and pressurize the connecting hole; the fixed splint is also provided with an observation window, and when the microfluidic chip and the liquid storage plate are clamped and fixed on the fixed splint, the position of the observation window corresponds to the position of the detection flow channel.

[0021] Optionally, the fixed splint includes an upper splint and a lower splint; when the microfluidic chip and the liquid storage plate are clamped and fixed on the fixed splint, the upper splint contacts the liquid storage layer, and the lower splint contacts the microfluidic chip; the connector and the observation window are arranged on the upper splint; the upper splint is provided with a plurality of upper connecting holes, and the lower splint is provided with a plurality of lower connecting holes, and the positions of the upper connecting holes correspond to those of the lower connecting holes one by one, and the upper splint and the lower splint are suitable for being connected and fixed by passing a locking piece through the upper connecting holes and the corresponding lower connecting holes.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The microfluidic chip provided by the present invention can be pre-installed with detection reagents using the micropores on the reagent well plate in the microfluidic chip by using a reagent well plate that can be stuck in the flow channel layer and a packaging film layer that is suitable for attaching and covering the surface of the reagent well plate and the surface of the flow channel layer around the reagent well plate and has air permeability. On the one hand, since the reagent well plate is stuck in the flow channel layer by setting a card slot in the flow channel layer, the thickness of the entire flow channel layer is not increased, and the horizontal area of ​​the flow channel layer is not increased. At the same time, the space for pre-installed reagents is increased, which is conducive to the miniaturization of the microfluidic chip, and then to the batch and convenience of PCR detection; and, it is also possible to use the two-layer structure of the flow channel-micropore and use two incompatible solvents to realize the simultaneous loading of detection reagents and test samples into the microfluidic chip 100, which can save detection time and improve detection efficiency. On the other hand, by encapsulating the reagent well plate with a permeable encapsulation film layer, the pre-installed detection reagent can enter the micropore from the detection flow channel in the form of ventilation and pressurization, and because the permeability of the film encapsulation is good, the liquid circulation in the flow channel is better, and the sample and the detection reagent are mixed faster during detection. In addition, in PCR detection, it is usually necessary to use an external heat source to heat the microfluidic chip from the bottom to speed up the mixing of the sample and the detection reagent; the microfluidic chip of the present invention, due to the use of a permeable film as the bottom layer of the encapsulation, compared with the filler encapsulation or solid encapsulation, the heat source can be directly transferred to the flow channel (micropore) through the film, and the reagent / sample in the flow channel (micropore) is spaced smaller from the heat source, the thermal response speed is faster, and the heat transfer performance is better, so that the detection time can be shortened, thereby improving the detection efficiency. In addition, the thinner the film, the better the heat transfer effect, combined with the use of flexible thermosetting materials, can be not limited to traditional processing methods, and the thickness can be reduced to less than 50μm, or even to 10μm. The film thickness can be ignored during heat transfer.

[0024] 2. The microfluidic chip assembly provided by the present invention can realize the simple and rapid loading of pre-installed reagents in the microfluidic chip from the liquid reservoir plate into the microfluidic chip by combining the liquid reservoir plate with the microfluidic chip provided by the present invention. Separating the pre-installed reagents from the detection step can shorten the detection time and improve the detection efficiency.

[0025] 3. The digital nucleic acid detection sampling device provided by the present invention includes the microfluidic chip assembly provided by the present invention. Through the cooperation of the fixed splint, the microfluidic chip can be quickly pre-loaded with reagents before sampling, and the fixed splint is reserved with an observation window, through which the reaction results in the detection channel of the microfluidic chip can be directly observed. There is no need to take out the microfluidic chip after pre-loading the reagent and then perform additional sampling. The observation results can be directly reflected and observed, which is convenient to operate, can save detection time, and improve detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is an exploded view of a microfluidic chip according to an embodiment of the present invention:

[0028] Figure 2 A schematic diagram of the structure of a flow channel layer of a microfluidic chip according to an embodiment of the present invention;

[0029] Figure 3 A schematic diagram of the structure of a reagent well plate of a microfluidic chip according to an embodiment of the present invention;

[0030] Figure 4 This is a schematic diagram of the structure of a microfluidic chip according to an embodiment of the present invention, in which a reagent well plate and a flow channel layer are packaged together through a packaging film layer;

[0031] Figure 5 This is a schematic structural diagram of a liquid storage plate according to an embodiment of the present invention;

[0032] Figure 6 It is a schematic diagram of a partial transparent structure of a sample injection device according to an embodiment of the present invention;

[0033] Figure 7 An exploded view of a sample injection device according to an embodiment of the present invention;

[0034] Figure 8 It is a schematic structural diagram of an upper clamping plate of a sample injection device according to an embodiment of the present invention;

[0035] Figure 9A-9F A schematic diagram of a process for pre-loading reagents in a microfluidic chip assembly according to an embodiment of the present invention;

[0036] Fig.10 This is a diagram of thermal response simulation results of a microfluidic chip according to an embodiment of the present invention;

[0037] Fig.11 The thermal response measurement result of the microfluidic chip according to one embodiment of the present invention is shown in FIG.

[0038] Fig.12 This is a diagram showing the effect of using a microfluidic chip according to an embodiment of the present invention;

[0039] Fig.13 FIG. 1 is an amplification curve diagram of a microfluidic chip amplification point according to an embodiment of the present invention. DETAILED DESCRIPTION

[0040] In order to solve the problem of limited detection efficiency of a microfluidic chip, the present invention provides a microfluidic chip, a microfluidic chip assembly and a digital nucleic acid detection sampling device.

[0041] The present invention provides a microfluidic chip, comprising: a flow channel layer, the flow channel layer is provided with a detection flow channel for accommodating a detection sample; the flow channel layer is also provided with a liquid inlet, the liquid inlet is connected to the detection flow channel; a reagent well plate, the reagent well plate is suitable for pre-installing a detection reagent; the reagent well plate is provided with a plurality of micropores penetrating the reagent well plate from a thickness direction; the flow channel layer is provided with a receiving groove, the reagent well plate is suitable for being clamped in the receiving groove; the detection flow channel is located at the groove bottom of the receiving groove, when the reagent well plate is clamped in the receiving groove, the micropores are directly connected with the detection flow channel, and the surface of the reagent well plate facing away from the detection flow channel is flush with the surface of the flow channel layer; a packaging film layer, the packaging film layer is suitable for packaging the reagent well plate in the receiving groove, the packaging film layer is attached to and covers the surface of the reagent well plate and the surface of the flow channel layer around the reagent well plate; the packaging film layer has air permeability.

[0042] The present invention also provides a microfluidic chip assembly, including the microfluidic chip provided by the present invention; and also including: a liquid storage plate, the liquid storage plate is provided with a liquid storage hole that penetrates the liquid storage plate in the thickness direction, and the liquid storage hole is suitable for pre-installing detection reagents; the liquid storage plate is suitable for being arranged on the surface of the support layer on the side facing away from the flow channel layer, and when the liquid storage plate is arranged on the surface of the support layer, the liquid storage hole is connected with the liquid inlet.

[0043] The present invention also provides a digital nucleic acid detection sampling device, including the microfluidic chip assembly provided by the present invention, and also including: a fixed splint, which is suitable for clamping and fixing the microfluidic chip and the liquid storage plate; the fixed splint is provided with a connector at the position corresponding to the liquid inlet and the connecting hole, and the connector has a hollow ventilation tube, which is suitable for connecting with the connecting hole, and the connector is suitable for connecting to the air source through the connecting air tube to ventilate and pressurize the connecting hole; the fixed splint is also provided with an observation window, and when the microfluidic chip and the liquid storage plate are clamped and fixed on the fixed splint, the position of the observation window corresponds to the position of the detection channel.

[0044] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0046] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0047] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0048] Example 1

[0049] refer to Figure 1-Figure 4 This embodiment provides a microfluidic chip 100, comprising:

[0050] The flow channel layer 110 is provided with a detection flow channel for accommodating the detection sample. The flow channel layer 110 is also provided with a liquid inlet 112 , which is a blind hole connecting the detection flow channel and the environmental space on the surface of the flow channel layer 110 .

[0051] The reagent well plate 120 is suitable for pre-installing the detection reagent. The reagent well plate 120 is provided with a plurality of micropores 121 that penetrate the reagent well plate from the thickness direction. The flow channel layer 110 is provided with a receiving groove 111, and the reagent well plate 120 is suitable for being clamped in the receiving groove 111. The detection flow channel is located at the bottom of the receiving groove 111. When the reagent well plate 120 is clamped in the receiving groove 111, the micropores 121 are directly connected to the detection flow channel, and the surface of the reagent well plate 120 facing away from the detection flow channel is flush with the surface of the flow channel layer 110.

[0052] The packaging film layer 130 is suitable for packaging the reagent well plate 120 in the receiving groove 111. The packaging film layer 130 is attached to and covers the surface of the reagent well plate 120 and the surface of the flow channel layer 110 around the reagent well plate 120. The packaging film layer 130 is air permeable.

[0053] The microfluidic chip 100 provided in this embodiment, through the reagent well plate that can be stuck in the flow channel layer 110 and the packaging film layer 130 that is suitable for attaching and covering the surface of the reagent well plate 120 and the surface of the flow channel layer 110 around the reagent well plate 120 and having air permeability, can be used in the microfluidic chip 100 to pre-install the detection reagent using the micropores 121 on the reagent well plate 120. On the one hand, since the reagent well plate 120 is stuck in the flow channel layer 110 by providing the receiving groove 111 in the flow channel layer 110, the thickness of the entire flow channel layer 110 is not increased, nor is the horizontal area of ​​the flow channel layer 110. At the same time, the space for pre-installed reagents is increased, which is conducive to the miniaturization of the microfluidic chip 100, and then to the batch and convenience of PCR detection; and, through the two-layer structure of the flow channel-micropore, two incompatible solvents can be used to realize the simultaneous loading of the detection reagent and the detection sample into the microfluidic chip 100, which can save detection time and improve detection efficiency. On the other hand, by encapsulating the reagent well plate 120 with a permeable encapsulation film layer 130, the pre-installed detection reagent can enter the micropore 121 from the detection flow channel in the form of ventilation and pressurization, and because the permeability of the film encapsulation is good, the liquid circulation in the flow channel is better, and the sample and the detection reagent are mixed faster during detection. In addition, in PCR detection, it is usually necessary to use an external heat source to heat the microfluidic chip from the bottom to speed up the mixing of the sample and the detection reagent. The microfluidic chip 100 of the present invention, due to the use of a permeable film as the bottom package, compared to the filler encapsulation or solid encapsulation, the heat source can be directly transferred to the flow channel (micropore) through the film, and the reagent / sample in the flow channel (micropore) is spaced smaller from the heat source, the thermal response speed is faster, and the heat transfer performance is better, thereby shortening the detection time and improving the detection efficiency. In addition, the thinner the packaging film layer 130 is, the better the heat transfer effect is. By using a flexible thermosetting material, the thickness of the packaging film layer 130 can be reduced to less than 50 μm, or even to 10 μm, without being limited by traditional processing methods. This allows the thickness of the packaging film layer 130 to be ignored during heat transfer.

[0054] Specifically, the width of the detection channel is 10μm-10mm, for example, it can be 10μm, 20μm, 50μm, 100μm, 200μm, 500μm, 1mm, 2mm, 5mm, 10mm. The height of the detection channel is 5μm-1mm, for example, it can be 5μm, 10μm, 20μm, 50μm, 100μm, 200μm, 500μm, 1mm. If the height of the detection channel is too low or the width is too narrow, the flow of the liquid will be restricted, the upper limit of the liquid flow rate will be reduced, and the detection efficiency will be reduced; if the height of the detection channel is too high or the width is too wide, it will be difficult to fill the liquid in the channel, and after filling, only part of it will be in contact with the detection reagent, resulting in deviations in the detection results and reduced detection efficiency.

[0055] The material of the flow channel layer 110 is PDMS (Poly Dimethylsiloxane), Si, COP (Cyclo Olefin Polymer) or glass. The flow channel layer 110 made of these materials is easy to be thermally bonded with other layer structures.

[0056] The height of the reagent well plate 120 is 10 μm-1 mm, for example, it can be 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1 mm. The aperture of the micropore 121 is 10 μm-1 mm, for example, it can be 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1 mm; if the height of the reagent well plate 120 (i.e., the depth of the micropore 121) is too low, or the aperture of the micropore 121 is too small, the amount of pre-installed reagent filled in the micropore 121 is too small, and the detection result is inaccurate; if the height is too high, or the aperture of the micropore 121 is too large, the detection reagent at the bottom of the micropore 121 is actually difficult to mix with the sample in the flow channel, resulting in a waste of the detection reagent. The material of the reagent well plate 120 includes Si, glass, COP or metal. The reagent well plate 120 made of these materials has good heat transfer capacity.

[0057] The thickness of the encapsulation film layer 130 is 1 μm-100 μm, for example, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm. If the thickness of the encapsulation film layer 130 is too thin, it is easy to be damaged, it is difficult to achieve the encapsulation effect, and leakage occurs; if the thickness is too thick, the heat transfer effect is affected. The material of the encapsulation film layer 130 includes a polymer having a network cross-linked structure. Specifically, the polymer having a network cross-linked structure may be, for example, PDMS, TPU (thermoplastic polyurethane elastomer) or PE (polyethylene). The polymer having a network cross-linked structure is easier to form a film and can have good heat transfer performance and air permeability.

[0058] For further reference, Figure 3 In this embodiment, the micropores 121 are arranged in an array on the reagent well plate 120. The arrayed micropores 121 are evenly arranged on the reagent well plate 120, so that the amount of reagent contacted by each detection flow channel connected thereto is substantially the same, thereby avoiding deviation in the detection result due to different amounts of reagent contacted by samples in different flow channels.

[0059] For further reference, Figure 2In this embodiment, the detection flow channel includes a plurality of entry flow channels 113 and a plurality of amplification flow channels 114, and the amplification flow channels 114 are arranged in parallel or serpentine, and the entry flow channel 113 connects the liquid inlet 112 and each amplification flow channel 114. For example, in this embodiment, the entry flow channel 113 is an incremental thin flow channel in the form of two or four divisions, which diverts the liquid inlet of the liquid inlet to more amplification flow channels 114, and can well guide the liquid inlet to fully flow into each amplification flow channel 114. The flow channel width of the amplification flow channel 114 is significantly higher than that of the entry flow channel 113, so that the sample can be fully spread in the amplification flow channel 114, contact with the detection reagent, and then show the detection result. The amplification flow channel 114 can be divided into 4, 8, and 16. The wider the flow channel, the more micropores 121 are covered, and the less difficult it is to make. In addition, the position of the amplification flow channel 114 in the flow channel layer 110 corresponds to the arrangement position of the micropores 121 in the reagent well plate 120. The amplification flow channel 114 can be directly connected to the micropore 121 , and the sample entering the amplification flow channel 114 can be in rapid and sufficient contact with the detection reagent pre-installed in the micropore 121 .

[0060] Furthermore, in this embodiment, the flow channel layer 110 is further provided with a sealing channel 115, which surrounds the detection flow channel and is spaced apart from the detection flow channel. The sealing channel 115 is filled with a sealing filler. The sealing channel 115 is used to improve the sealing of the chip, which is beneficial to prevent the evaporation of the liquid in the chip.

[0061] Specifically, the sealing filler includes one or more of silicone oil, fluorinated liquid, mineral oil or epoxy resin.

[0062] Furthermore, in this embodiment, the microfluidic chip further includes: a support layer 140, which is disposed on the surface of the flow channel layer 110 on the side facing away from the encapsulation film layer 130. The support layer 140 is provided with a connecting hole that penetrates the support layer in the thickness direction, and the connecting hole connects the liquid inlet 112 and the external space on the surface of the support layer 140. The support layer 140 serves to enhance the strength of the microfluidic chip 100 and prevent the evaporation of the liquid in the flow channel. The connecting hole provided in the support layer 140 allows the reagent or sample to enter the detection flow channel of the flow channel layer 110 through the connecting hole.

[0063] Specifically, the support layer 140 is a transparent layer, and / or the support layer 140 is provided with a plurality of observation holes, and the positions of the observation holes correspond to the detection flow channel. In this way, when the support layer 140 is arranged above the flow channel layer 110, the sample loading progress or the detection result in the flow channel layer 110 is not affected.

[0064] The material of the support layer 140 includes one or more of PDMS, glass, PMMA (polymethyl methacrylate, acrylic), PC (Polycarbonate) or COC (Copolymers Of Cycloolefin), which are easy to be thermoformed and easily bonded to the flow channel layer 110 by hot pressing.

[0065] When assembling the microfluidic chip 100 of this embodiment, all structures (including the support layer 140, the flow channel layer 110, the reagent well plate 120 and the packaging film layer 130) are bombarded with oxygen plasma using an oxygen plasma cleaning machine, and then the reagent well plate 120 is placed in the receiving groove 111 of the flow channel layer 110, and then the packaging film layer 130 is covered on the reagent well plate 120 and the flow channel layer 110, and the bonding of the various layers of the microfluidic chip 100 is completed by hot pressing at 100°C for 1 hour.

[0066] Example 2

[0067] refer to Figure 1-Figure 5 This embodiment provides a microfluidic chip assembly, including the microfluidic chip 100 provided in the above-mentioned embodiment 1.

[0068] The liquid storage plate 200 is also included. The liquid storage plate 200 is provided with a liquid storage hole 210 that penetrates the liquid storage plate 200 in the thickness direction. The liquid storage hole 210 is suitable for pre-installing the detection reagent. The liquid storage plate 200 is suitable for being arranged on the surface of the support layer 140 on the side facing away from the flow channel layer 110. When the liquid storage plate 200 is arranged on the surface of the support layer 140, the liquid storage hole 210 is connected to the liquid inlet 112.

[0069] The microfluidic chip assembly provided in this embodiment is combined with the microfluidic chip 100 provided in the above embodiment 1 through the liquid reservoir plate 200, so that the pre-installed reagents in the microfluidic chip 100 can be simply and quickly loaded into the microfluidic chip 100 from the liquid reservoir plate 200. Separating the pre-installed reagents from the detection steps can shorten the detection time and improve the detection efficiency.

[0070] Specifically, the liquid storage plate 200 is a transparent plate, or the liquid storage plate 200 is suitable for being peelably arranged on the surface of the support layer on the side facing away from the flow channel layer. In this way, through the transparent or peelable liquid storage plate 200, the sample loading progress or the detection result in the flow channel layer 110 can be intuitively observed from above.

[0071] In this embodiment, the liquid storage hole 210 is a tapered hole. The liquid storage hole 210 is a tapered hole, which can make the liquid storage hole 210 have a larger volume to store enough pre-installed reagents, and can be easily connected to the liquid inlet 112. The tapered structure can make the injection of the stored liquid smoother than the straight structure.

[0072] In some embodiments, the material of the liquid reservoir plate 200 includes PDMS or rubber. The liquid reservoir plate 200 made of PDMS or rubber is easy to prepare and does not react with the reagent.

[0073] In some embodiments, the microfluidic chip assembly further includes a sealing layer, which is suitable for being arranged on the surface of the packaging film layer facing away from the flow channel layer, and the sealing layer covers the entire surface of the packaging film layer. The sealing layer is used to seal the microfluidic chip 100 on one side of the packaging film layer 130 after the microfluidic chip 100 is sampled to prevent liquid evaporation and to minimize the influence on the film layer thickness.

[0074] Specifically, the material of the sealing layer includes one or more of thermosetting oil, epoxy resin or UV glue.

[0075] Example 3

[0076] refer to Figure 6-Figure 8 , this embodiment provides a digital nucleic acid detection sampling device, including the microfluidic chip assembly provided in the above-mentioned embodiment 2, and also includes: a fixed splint, which is suitable for clamping and fixing the microfluidic chip 100 and the liquid storage plate 200. The fixed splint is provided with a connector at the position corresponding to the liquid inlet and the connecting hole. The connector has a hollow ventilation tube, which is suitable for connecting with the connecting hole. The connector is suitable for connecting to the air source through the connecting air tube to ventilate and pressurize the connecting hole. The fixed splint is also provided with an observation window. When the microfluidic chip and the liquid storage plate are clamped and fixed on the fixed splint, the position of the observation window corresponds to the position of the detection channel.

[0077] Specifically, in the present embodiment, the fixing clamp includes an upper clamp 410 and a lower clamp 420. When the microfluidic chip 100 and the liquid storage plate 200 are clamped and fixed on the fixing clamp, the upper clamp 410 contacts the liquid storage plate 200, and the lower clamp 420 contacts the microfluidic chip 100. The connector 413 and the observation window 412 are arranged on the upper clamp 410. The upper clamp 410 is provided with a plurality of upper connection holes 411, and the lower clamp 420 is provided with a plurality of lower connection holes 421. The positions of the upper connection holes 411 and the lower connection holes 421 correspond one to one. The upper clamp 410 and the lower clamp 420 are suitable for being connected and fixed by passing the locking member 500 through the upper connection hole 411 and the corresponding lower connection hole 421. The locking member 500 can be, for example, a bolt.

[0078] refer to Figure 9A-9D Taking the dual detection reagents for nucleic acid detection as an example, a process of injecting a sample into the microfluidic chip 100 using the digital nucleic acid detection injection device of this embodiment is as follows:

[0079] First, refer to Fig. 9A, provide a liquid storage plate 200, which is arranged on the surface of the support layer 140 of the microfluidic chip 100, and align the liquid storage hole 210 with the liquid inlet 112 (the liquid storage hole 210 is a straight hole for simplicity in the figure). Add the first reagent X to the bottom of the liquid inlet 112, and then inject the second reagent Y through the hole wall of the liquid storage hole 210. The first reagent X is usually an aqueous phase (it can also be an oil phase), and the second reagent Y is usually an oil phase (it can also be an oil phase). Since the first reagent X and the second reagent Y are immiscible with each other, the second reagent Y is suspended above the first reagent X. Then, the microfluidic chip 100 loaded with the two reagents is fixed in the injection device, and the connector in the injection device is connected to the gas source through a connection (the fixing clamp of the injection device is not shown in the figure). Reference Fig. 9B and Fig. 9C , driven by the positive pressure P, the liquid squeezes the air in the micropore 121 and the detection channel out of the thin film layer from the bottom of the micropore 121, thereby filling the first reagent X into the micropore 121, and the second reagent Y is continuously spread on the top of the micropore 121. In actual detection, the sample is mixed with the second reagent Y through the liquid inlet 112, and then contacts and reacts with the first reagent in the micropore 121 to show the detection result. Alternatively, one of the first reagent X or the second reagent Y has been mixed with the sample to be tested in advance, and the above steps are the steps of synchronous injection of the sample to be tested and the detection reagent. Reference Fig.9E and Fig.9F After the injection is completed, the bottom of the microfluidic chip 100 is finally sealed with a sealing layer. The sealed microfluidic chip 100 is then subjected to subsequent testing and result observation. When heating is required, the microfluidic chip 100 is heated from one side of the sealing layer.

[0080] For PCR testing with a single detection reagent, the first reagent is the detection reagent, and the second reagent can be a liquid such as water, ethanol, propanol, etc. that does not react with the detection reagent and the sample, which serves as a carrier solvent for the detection sample. Through steps similar to the above, the detection reagent and the sample to be tested are loaded simultaneously. Fig. 9C , the injection of the microfluidic chip 100 is completed, and for some embodiments, reference is made to Fig.9D After removing the liquid storage plate 200, the microfluidic chip 100 is taken out independently for subsequent sealing and observation of test results. Those skilled in the art can choose according to needs, such as if the liquid storage plate 200 is a transparent material, and the liquid storage plate 200 can be not removed and sealed directly.

[0081] refer to Figure 10-13 In order to demonstrate the practical application effect of the microfluidic chip provided by the present invention, the present invention has carried out simulation and actual testing, and the results are as follows Figure 10-13 shown.

[0082] refer to Fig.10The thermal response simulation results of the microfluidic chip 100 show that: the temperature control table is set at the bottom of the microfluidic chip through digital simulation to control the temperature rise and fall, and the thermal response curve of the microfluidic chip is measured through simulation. Fig.10 Curve A in the temperature control of the microfluidic chip, curve B is the simulated thermal response curve of the microfluidic chip 100 provided in Example 1 of the present invention, and curve C is the simulated thermal response curve of the comparison chip, wherein the comparison chip is a microfluidic chip with a micropore array as a storage space for reagents or patterns, but no flow channel connected thereto (i.e., no secondary layered setting of flow channel-micropore) and no thin film encapsulation. The simulation detection position selects the center position of any micropore in the microfluidic chip for simulation mapping. When heating up, when curve A (temperature control table) reaches 96°C at 7.2s, curve B (chip of the present application) reaches 95°C at 13.1s, and curve C (comparison chip) reaches 95°C at 86.4s; when cooling down, when curve A (temperature control table) drops to 60°C at 114.2s, curve B (chip of the present application) and curve C (comparison chip) drop to 60°C at 127.9s and 161.6s, respectively. It can be seen that the microfluidic chip 100 provided in Example 1 of the present invention has a faster thermal response speed and better thermal response performance than the comparison chip.

[0083] refer to Fig.11 , the actual measured results of the thermal response of the microfluidic chip 100: using 100nm CY5 dye to simulate the first reagent, the chip was heated from 60℃ to 96℃ and then dropped to 60℃ in the digital nucleic acid detection at 5℃ / s, and the fluorescence intensity reflected the temperature change in the well. D is the temperature change of the hot plate, E is the change of the relative intensity of fluorescence in the well of the device, and F is the change of the relative intensity of fluorescence in the microwell of the comparison chip (the same comparison chip as the above curve C) under the same conditions. When the temperature of the hot plate (curve D) begins to change (57s and 190s), the relative fluorescence intensity in the microwell of the microfluidic chip (curve E) provided in Example 1 of the present invention is compared with the comparison chip (curve F). It can be seen from the change trend that the response is faster. For example, when the temperature of the hot stage drops to 60℃, the speed at which the temperature of curve E reaches a stable state is about 50s faster than that of curve F. This further proves the advantage of the chip provided in Example 1 of the present invention in terms of thermal response speed. The experimental results show that the device has significant advantages over traditional commercial chips in heating and cooling.

[0084] The digital PCR experiment was performed using the PCR system as the first reagent and the solidified silicone oil as the second reagent. The experimental temperature conditions were as follows: 95°C (20s) for the pre-denaturation stage; 96°C (2s)-60°C (10s) for the cycle amplification stage, for a total of 40 cycles; and finally, 4°C for insulation. The obtained fluorescence image is shown in the figure below. Fig.12 As shown, Fig.12This is a fluorescence image of the digital PCR detection amplification endpoint of the chip provided in Example 1 of the present invention. The white dots are amplification positive points, corresponding to Fig.13 Black line, darker background points are negative points after amplification, corresponding to Fig.13 The gray lines on the chip are shown in Figure 1. The thick black straight lines are the areas between the flow channels, so there are no dots. The amplification curves of all reaction units on the chip are shown in Figure 1. Fig.13 Each line in the figure represents the change of fluorescence in each microwell. The dark line represents the well with amplification. Because of amplification, the fluorescence intensity in the well increases, corresponding to Fig.12 The bright dots; the light-colored lines represent the wells without amplification, and the fluorescence intensity changes are relatively stable, corresponding to Fig.12 Dark spots. Fig.12 and Fig.13 It is proved that the detection chip provided in the above embodiment can quickly realize the detection of samples and has good sealing and stability.

[0085] The present invention has been described above by way of embodiments. It is believed that those skilled in the art can understand the present invention through the above embodiments. Obviously, the above embodiments are merely examples for the purpose of clear description, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A microfluidic chip, characterized in that: include: A flow channel layer, wherein the flow channel layer is provided with a detection flow channel for accommodating a detection sample; the flow channel layer is also provided with a liquid inlet, which is a blind hole connecting the detection flow channel and an environmental space on the surface of the flow channel layer; A reagent well plate, wherein the reagent well plate is suitable for pre-loading detection reagents; the reagent well plate is provided with a plurality of micro-holes penetrating the reagent well plate in the thickness direction; The flow channel layer is provided with a receiving groove, and the reagent well plate is suitable for being clamped in the receiving groove; the detection flow channel is located at the bottom of the receiving groove, and when the reagent well plate is clamped in the receiving groove, the micropores are directly connected to the detection flow channel, and the surface of the reagent well plate facing away from the detection flow channel is flush with the surface of the flow channel layer; The packaging film layer is suitable for packaging the reagent well plate in the containing groove, and the packaging film layer is attached to and covers the surface of the reagent well plate and the surface of the flow channel layer around the reagent well plate; the packaging film layer is air permeable.

2. The microfluidic chip according to claim 1, characterized in that: The microwells are arranged in an array on the reagent well plate.

3. The microfluidic chip according to claim 1, characterized in that: The detection flow channel includes a plurality of inlet flow channels and a plurality of amplification flow channels, the amplification flow channels are arranged in parallel or in a serpentine manner, the inlet flow channel connects the liquid inlet and each of the amplification flow channels; the position of the amplification flow channel in the flow channel layer corresponds to the arrangement position of the micropores in the reagent well plate.

4. The microfluidic chip according to claim 1, characterized in that: The flow channel layer is further provided with a sealing channel, the sealing channel surrounds the detection flow channel and is spaced apart from the detection flow channel; the sealing channel is filled with a sealing filler.

5. The microfluidic chip according to claim 4, characterized in that: The sealing filler includes one or more of silicone oil, fluorinated liquid, mineral oil or epoxy resin.

6. The microfluidic chip according to claim 1, characterized in that: Also includes: A support layer is provided on a surface of the flow channel layer facing away from the packaging film layer; the support layer is provided with a connecting hole penetrating the support layer in a thickness direction, and the connecting hole connects the liquid inlet and an external space on the surface of the support layer.

7. The microfluidic chip according to claim 6, characterized in that: The support layer is a transparent layer, and / or the support layer is provided with a plurality of observation holes, and the positions of the observation holes correspond to the detection flow channel.

8. The microfluidic chip according to claim 7, characterized in that: The material of the support layer includes one or more of PDMS, glass, PMMA, PC or COC.

9. The microfluidic chip according to any one of claims 1 to 8, characterized in that: The width of the detection channel is 10 μm-10 mm, and the height of the detection channel is 5 μm-1 mm; The material of the flow channel layer is PDMS, Si, COP or glass; The height of the reagent well plate is 10 μm-1 mm; the pore size of the micropore is 10 μm-1 mm; The material of the reagent well plate includes Si, glass, COP or metal; The thickness of the packaging film layer is 1 μm-100 μm; The material of the encapsulation film layer includes a high molecular polymer with a network cross-linking structure.

10. The microfluidic chip according to claim 9, characterized in that: The high molecular polymer with a network cross-linking structure includes PDMS, TPU or PE.

11. A microfluidic chip assembly, characterized in that: The microfluidic chip according to any one of claims 1 to 9; and further comprising: A liquid storage plate, wherein the liquid storage plate is provided with a liquid storage hole penetrating the liquid storage plate in the thickness direction, and the liquid storage hole is suitable for pre-installing detection reagents; the liquid storage plate is suitable for being arranged on the surface of the support layer on the side facing away from the flow channel layer, and when the liquid storage plate is arranged on the surface of the support layer, the liquid storage hole is connected with the liquid inlet.

12. The microfluidic chip according to claim 11, characterized in that: The microfluidic chip further comprises a sealing layer, which is suitable for being arranged on a surface of the packaging film layer on a side facing away from the flow channel layer, and the sealing layer covers the entire surface of the packaging film layer.

13. The microfluidic chip according to claim 12, characterized in that: The liquid storage plate is a transparent plate, or the liquid storage plate is adapted to be peelably arranged on a surface of the support layer on a side facing away from the flow channel layer.

14. The microfluidic chip according to claim 13, characterized in that: The liquid storage hole is a tapered hole.

15. The microfluidic chip according to claim 13, characterized in that: The material of the liquid storage plate includes PDMS or rubber.

16. The microfluidic chip according to claim 13, characterized in that: The material of the sealing layer includes thermosetting oil, epoxy resin or UV glue.

17. A digital nucleic acid detection sampling device, characterized in that: The microfluidic chip assembly according to any one of claims 11 to 16 further comprises: A fixing clamp, the fixing clamp is suitable for clamping and fixing the microfluidic chip and the liquid storage plate; the fixing clamp is provided with a connector at a position corresponding to the liquid inlet and the communicating hole, the connector has a hollow vent pipe, suitable for communicating with the communicating hole, and the connector is suitable for connecting to an air source through a connecting air pipe to ventilate and pressurize the communicating hole; The fixing clamp is further provided with an observation window. When the microfluidic chip and the liquid storage plate are clamped and fixed on the fixing clamp, the position of the observation window corresponds to the position of the detection flow channel.

18. The digital nucleic acid detection sampling device according to claim 17, characterized in that: The fixed clamping plate comprises an upper clamping plate and a lower clamping plate; When the microfluidic chip and the liquid storage plate are clamped and fixed on the fixed clamping plate, the upper clamping plate contacts the liquid storage layer, and the lower clamping plate contacts the microfluidic chip; the connector and the observation window are arranged on the upper clamping plate; The upper clamping plate is provided with a plurality of upper connecting holes, and the lower clamping plate is provided with a plurality of lower connecting holes, and the positions of the upper connecting holes correspond to the positions of the lower connecting holes one by one. The upper clamping plate and the lower clamping plate are suitable for being connected and fixed by means of locking members passing through the upper connecting holes and the corresponding lower connecting holes.

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