A portable microfluidic analysis chip

Abstract

The invention discloses a kind of portable microfluidic analysis chip, belong to the field of microfluidic technology, including: sequential release functional area, micro-reaction functional area and fluid diversion functional area;Sequential release functional area includes M-level liquid storage chambers;The bottom of each liquid storage chamber is provided with micropore, and each micropore is connected with the input end of micro-reaction functional area;Only the top of the first-level liquid storage chamber is communicated with the atmosphere, and a float microvalve is provided in the front M-1 level liquid storage chambers;The bottom of the front-level liquid storage chamber is connected with the top of the rear-level liquid storage chamber;Fluid diversion functional area includes drainage flow channel, the recovery branch flow channel for recovering reaction solution, the drainage branch flow channel for discharging waste liquid, the first outlet and the second outlet, recovery branch flow channel and drainage branch flow channel are connected with the first outlet and the second outlet respectively, and are all connected with micro-reaction functional area by drainage flow channel.The present invention can automatically complete the sequential release of multiple reagents on chip, and flexibly control the flow rate and direction of microfluid.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and more specifically, relates to a portable microfluidics analysis chip. Background Art

[0002] As an emerging science and technology, microfluidics has been widely applied in fields such as chemistry, biology, engineering, and physics. With its strong interdisciplinary nature, it has achieved breakthroughs in the precise manipulation of time, space, and the objects being analyzed, and can address many key issues in life analysis. Microfluidics can integrate laboratory-based testing experiments onto a small chip, saving both consumables and time. More importantly, it can integrate multiple testing technologies, improving detection efficiency.

[0003] In order to realize automated analysis on millimeter-scale chips, it is first necessary to realize the automated sequential release of various reagents required for the analytical reaction, and then complete the removal of waste liquid and the recovery of special solutions. There are many ways of sequential release. For example, sequential release based on centrifugal force, sequential release based on capillary force, and gravity-driven solution release mode. Although these methods can realize the sequential release of solutions, the control of the flow rate and direction of the microfluid after release will be subject to certain limitations. For example, in most cases, the disc-type chip can only realize the flow of fluid from the center of the disc to the outside. At the same time, the flow rate is determined by the centrifugal speed. If the speed is too high, it is easy to break through the downstream valve, and if the speed is too low, the fluid will also be difficult to flow. Based on the capillary force and gravity-driven method, the direction of the fluid is also single, and is greatly affected by the size of the microchannel and the contact angle of the channel surface, and has higher requirements for the processing technology.

[0004] In the patent document with application publication number CN107541452A, a microfluidic chip and chip assembly are disclosed, which includes a liquid storage unit, an extraction and amplification unit, and a waste liquid recovery unit; wherein, the liquid storage unit includes a plurality of liquid reservoirs, and a first circulation pipe connected to the liquid outlet of each liquid reservoir, and a first control member is provided between each liquid outlet and the first circulation pipe, and the first control member is used to control the conduction state of the liquid outlet. When any liquid reservoir is in a pressurized state, the corresponding first control member is in an open state, and the remaining first control members are in a closed state. By controlling the pressurized state of multiple liquid reservoirs in sequence, it is possible to control the injection of multiple liquid reagents in sequence. However, in order to achieve the control of the injection of liquid reagents in sequence, each liquid storage chamber needs to be connected to a pressurizing device. Furthermore, to prevent gas from entering the fluid channel and contaminating the test sample, the reservoir in this solution is a conical funnel-shaped structure with a suspended ball inside. When liquid reagent is present in the reservoir, the suspended ball floats on the surface of the water; when the liquid reagent is exhausted, the suspended ball blocks the outlet of the conical reservoir. However, in practice, the seal formed by the suspended ball and the conical structure is not stable under this structure. Summary of the Invention

[0005] In response to the defects of the existing technology and the need for improvement, the present invention provides a microfluidic analysis chip based on a float microvalve. Its purpose is to improve the structure of the microfluidic chip so that the sequential release of multiple reagents can be automatically completed on the chip, while the flow rate and direction of the released microfluids can be flexibly controlled.

[0006] To achieve the above object, according to one aspect of the present invention, there is provided a portable microfluidic analysis chip, comprising: a sequential release functional area, a micro-reaction functional area, and a fluid diversion functional area;

[0007] The micro-reaction functional area is provided with reactants;

[0008] The sequential release functional area includes M levels of liquid storage chambers; each liquid storage chamber is provided with a micropore at the bottom, and each micropore is connected to the input end of the micro-reaction functional area through a fluid microchannel; the top of the first-level liquid storage chamber is connected to the atmosphere, while the tops of the remaining liquid storage chambers are not connected to the atmosphere; each of the first to M-1-level liquid storage chambers is provided with a float microvalves having a diameter larger than the diameter of the bottom micropores and a density less than the density of the reagent in the liquid storage chamber; in two adjacent liquid storage chambers, the bottom of the previous liquid storage chamber is connected to the top of the next liquid storage chamber through an air path microchannel; M is an integer greater than or equal to 2;

[0009] The fluid diversion functional area includes a drainage channel, a recovery branch channel, a drainage branch channel, a first outlet and a second outlet; one end of the drainage channel, the recovery branch channel and the drainage branch channel are connected together and the three channels are interconnected, the other end of the drainage channel is connected to the output end of the micro-reaction functional area, the other end of the recovery branch channel is connected to the first outlet, and the other end of the drainage branch channel is connected to the second outlet.

[0010] Furthermore, the fluid diversion functional area also includes a waste liquid chamber, in which a water-absorbing material is arranged, and a drainage microchannel is arranged in the water-absorbing material, one end of the drainage microchannel is connected to the second outlet, and the other end of the drainage branch flow channel is connected to the other end of the drainage microchannel.

[0011] Furthermore, one end of the drainage channel, the recovery branch channel and the drainage branch channel are connected through a diversion microstructure; the diversion microstructure is in a "T" shape, one end of the drainage channel is connected to the vertical port of the diversion microstructure, and one end of the recovery branch channel and the drainage branch channel are respectively connected to the two horizontal ports of the diversion microstructure; a buffer microcavity is formed at the horizontal and vertical intersection of the diversion microstructure, and the buffer microcavity bulges outward along the vertical direction to form a buffer steep slope.

[0012] Furthermore, the middle of the recovery branch flow channel is cut off to form a quantitative outlet closer to the drainage flow channel and a collection inlet closer to the first outlet;

[0013] In addition, the fluid diversion functional area also includes a quantitative distribution unit; the quantitative distribution unit includes: N levels of quantitative chambers; the bottom of each quantitative chamber is connected to the collection inlet through an air path microchannel; the top of the first-level quantitative chamber is connected to the quantitative outlet through a fluid microchannel, the tops of two adjacent quantitative chambers are connected through a fluid microchannel, and the top of the last-level quantitative chamber is connected to the collection inlet through a fluid microchannel;

[0014] Wherein, N is an integer greater than or equal to 1.

[0015] Furthermore, the air path microchannels in the quantitative distribution unit are subjected to hydrophobic treatment, or are made of a waterproof and breathable membrane.

[0016] Furthermore, the air path microchannels in the sequential release functional area are subjected to hydrophobic treatment, or are made of a waterproof and breathable membrane.

[0017] Furthermore, the structure for loading reactants in the micro-reaction functional area is a micro-array micro-column or a micro-channel filled with glass micro-beads.

[0018] Furthermore, in the sequential release functional area, the length of the fluid microchannel used to connect the micropores at the bottom of the liquid storage chamber and the input end of the microreaction functional area is set to meet the following requirements: there is a certain time interval between the time when the reagent in the previous liquid storage chamber completely flows into the microreaction functional area and the time when the reagent in the next liquid storage chamber begins to flow into the microreaction functional area.

[0019] Furthermore, the bottom of each liquid storage chamber is made of elastic material.

[0020] Furthermore, the bottom of each liquid storage chamber is made of silicone material.

[0021] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects:

[0022] (1) The microfluidic analysis chip based on the float microvalve provided by the present invention, when working, adds reaction reagents to each liquid storage chamber according to the reaction sequence, and after applying negative pressure through the first outlet or the second outlet, it will trigger the sequential release of the reagents in the liquid storage chambers of each level. Specifically, since the density of the float microvalve is less than the density of the reagent, it will float above the reagent. The first-level liquid storage chamber is connected to the atmosphere, and the reagent therein will flow from the bottom micropore through the fluid microchannel into the micro-reaction functional area under the action of the atmosphere, thereby realizing the release of the reagent in the first-level liquid storage chamber. The remaining chambers cannot be released due to the liquid sealing effect. As the reagent in the first-level liquid storage chamber is gradually released, the float microvalve will gradually drop with the liquid level until the reagent in the first-level liquid storage chamber is completely released, and the float microvalve will fall into the bottom micropore to form a seal. At the same time, the second-level liquid storage chamber is connected to the atmosphere through the air path microchannel and the first-level liquid storage chamber. Under the action of the atmosphere, the reagent therein will be released to the micro-reaction functional area, thereby realizing the release of the second-level reagent. By analogy, the reagents in the first to M-th level liquid storage chambers are finally released in sequence. During this process, the flow rate and direction of the fluid in the chip can be flexibly controlled by controlling the size and direction of the pressure at the first outlet or the second outlet. At the same time, by controlling the size and direction of the pressure at the two outlets, the waste liquid can be controlled to flow into the drainage branch channel to achieve the discharge of the waste liquid, and the post-reaction solution can flow into the recovery branch channel to achieve the recovery of the reaction solution.

[0023] (2) The present invention integrates a waste liquid chamber in the fluid diversion functional area, which can realize the recovery of waste liquid in the chip, reducing the pollution to the environment and the infection to the testing personnel during the detection process; at the same time, a water-absorbing material is provided in the waste liquid chamber, and a drainage microchannel is provided in the water-absorbing material, and the drainage branch flow channel and the waste liquid chamber, as well as the drainage branch flow channel and the second outlet are connected through the drainage microchannel, which can ensure that the pressure applied through the second outlet is not affected while the waste liquid is discharged, thereby better realizing fluid control.

[0024] (3) The present invention sets a "T"-shaped diversion microstructure at the connection between the drainage channel, the recovery branch channel and the discharge branch channel, and sets a buffer steep slope therein. Based on this structural design, the fluid entering the corresponding branch channel can be accurately controlled, which not only avoids part of the waste liquid from entering the recovery channel and affecting the recovery purity, but also avoids part of the sample from entering the waste liquid discharge channel and causing waste of rare samples.

[0025] (4) The present invention sets a multi-stage quantitative chamber in the middle of the recovery branch, the top of the first-stage quantitative chamber is connected to the quantitative outlet through a fluid microchannel, the tops of the two adjacent quantitative chambers are connected through a fluid microchannel, and the top of the last-stage quantitative chamber is connected to the collection inlet through a fluid microchannel. At the same time, the bottom of the quantitative chamber is connected to the collection inlet through an air path microchannel, resulting in the bottom air path connection path being shorter than the top fluid microchannel. Therefore, after the reaction solution flows out through the quantitative inlet, it will first flow into the first-stage quantitative chamber and gradually fill the first-stage quantitative chamber, and then flow into the second-stage quantitative chamber and gradually fill the second-stage quantitative chamber, and so on, and finally fill the quantitative chambers at each stage in sequence. Different analytical reactants are pre-set in the quantitative chambers at each stage, and the reaction solution can be subsequently subjected to high-throughput integrated analysis on the chip, thereby further improving the integration of the chip.

[0026] (5) In the present invention, the gas microchannels are all hydrophobic treated or made of a waterproof and breathable membrane, which can ensure that the gas is discharged normally through the gas microchannels and the liquid does not enter the gas microchannels.

[0027] (6) The present invention specially sets the length of the fluid microchannel connecting the micropores at the bottom of each liquid storage chamber and the microreaction functional area in the sequential release functional area, so that there is a certain time interval between the time when the reagent in the previous liquid storage chamber completely flows into the microreaction functional area and the time when the reagent in the next liquid storage chamber begins to flow into the microreaction functional area, thereby avoiding mutual interference between the reagents and ensuring the accuracy of analysis and detection.

[0028] (7) The present invention uses elastic material (preferably silicone material) to make the bottom of the liquid storage chamber. When the reagent in the liquid storage chamber is completely released and the float microvalve falls into the micropore, the microsphere and the micropore will have better sealing performance because the diameter of the microsphere is slightly larger than the diameter of the micropore. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of the planar structure of a microfluidic analysis chip based on a float microvalve provided in Example 1 of the present invention;

[0030] Figure 2 A schematic diagram of the three-dimensional structure of a microfluidic analysis chip based on a float microvalve provided in Example 1 of the present invention;

[0031] Figure 3This is a schematic structural diagram of the micro-reaction functional area provided in Example 1 of the present invention;

[0032] Figure 4 This is a schematic diagram of the principle of sequential release of reagents in each level of liquid storage chambers in the sequential release functional zone provided in Example 1 of the present invention;

[0033] Figure 5 A schematic diagram of the connection between the drainage branch channel and the waste liquid chamber in the fluid diversion functional area provided in Example 1 of the present invention;

[0034] Figure 6 Schematic diagram of the diversion microstructure provided in Example 1 of the present invention; wherein (a) is a schematic diagram of the three-dimensional structure, and (b) is a schematic diagram of the planar structure;

[0035] Figure 7 A schematic diagram of a layered structure of a microfluidic analysis chip based on a float microvalve provided in Example 1 of the present invention;

[0036] Figure 8 Schematic diagram of the microfluidic analysis chip based on the float microvalve provided in Example 1 of the present invention for use in nucleic acid purification and enrichment; wherein (A) to (L) respectively represent different states during the reaction process;

[0037] Figure 9 Schematic diagram of the test results of the microfluidic analysis chip based on the float microvalve provided in Example 1 of the present invention applied to nucleic acid purification and enrichment; wherein (a) shows the effect of different injection speeds on the nucleic acid enrichment effect, (b) shows the effect of different recovery speeds on the nucleic acid enrichment effect, and (c) shows the effect of different mixing reaction times on the nucleic acid enrichment effect;

[0038] Figure 10 The nucleic acid enrichment test results corresponding to different sample volumes when the microfluidic analysis chip based on the float microvalve provided in Example 1 of the present invention is used for nucleic acid purification and enrichment; wherein (a) shows the effect of different sample volumes on the nucleic acid enrichment effect, and (b) shows the effect of different sample volumes on the nucleic acid enrichment efficiency;

[0039] Figure 11 A schematic plan view of the sequential release of nine reagents provided in Example 2 of the present invention;

[0040] Figure 12 A schematic diagram of the three-dimensional structure of the sequential release of nine reagents provided in Example 2 of the present invention;

[0041] Figure 13 A schematic plan view of a microfluidic analysis chip based on a float microvalve provided in Example 3 of the present invention;

[0042] Figure 14A schematic diagram of the three-dimensional structure of a microfluidic analysis chip based on a float microvalve provided in Example 3 of the present invention;

[0043] Figure 15 This is an enlarged schematic diagram of the three-dimensional structure of the quantitative dispensing unit provided in Example 3 of the present invention;

[0044] Figure 16 Schematic diagram of the visualized quantitative dispensing principle of the quantitative dispensing unit provided in Example 3 of the present invention; wherein (a) is a top view and (b) is a side view;

[0045] Throughout the drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0046] 1-sequential release functional area; 11-liquid storage chamber, 12-micropore, 13-fluid microchannel, 14-gas microchannel;

[0047] 2-micro-reaction functional area;

[0048] 3-fluid diversion functional area; 31-drainage channel, 32-recovery branch channel, 33-drainage branch channel, 34-first outlet, 35-second outlet, 36-diversion microstructure, 37-waste liquid chamber, 38-drainage microchannel; 361-buffer microcavity, 362-buffer steep slope;

[0049] 4- quantitative distribution unit; 41- quantitative chamber, 42- fluid microchannel, 43- gas microchannel. DETAILED DESCRIPTION

[0050] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0051] Those skilled in the art should understand that these embodiments are merely used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. It should be noted that, in the description of the present invention, the terms "upper", "lower", "left", "right", "inner", "outer", etc., which indicate directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. This is merely for ease of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it should not be understood as limiting the present invention. In addition, it should be noted that, in the description of the present invention, unless otherwise expressly 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, an indirect connection through an intermediate medium, or it can be internal communication between two elements. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances. In addition, in the present invention, the terms "first", "second", etc. (if any) in the present invention and the accompanying drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0052] The present invention provides a portable microfluidic analysis chip that can be used to automatically complete nucleic acid extraction and amplification, enzyme-linked immunosorbent assay, immunochromatographic reaction, drug screening, cell stress response, etc.

[0053] Example 1:

[0054] A portable microfluidic analysis chip, such as Figure 1 and Figure 2 As shown, it includes: a sequential release functional area 1, a micro-reaction functional area 2 and a fluid diversion functional area 3.

[0055] The micro-reaction functional area 2 is provided with reactants;

[0056] The sequential release functional area 1 includes four levels of liquid storage chambers 11; a micropore 12 is provided at the bottom of each liquid storage chamber, and each micropore 12 is connected to the input end of the micro-reaction functional area 2 through a fluid microchannel 13; the top of the first-level liquid storage chamber is connected to the atmosphere, while the tops of the remaining liquid storage chambers are not connected to the atmosphere; a float microvalves having a diameter larger than the diameter of the bottom micropore and a density less than the density of the reagent in the liquid storage chamber is provided in the first to third-level liquid storage chambers; in two adjacent liquid storage chambers, the bottom of the previous-level liquid storage chamber is connected to the top of the next-level liquid storage chamber through an air path microchannel 14. Optionally, in this embodiment, the float microvalve is specifically a hollow ball structure; it should be noted that the float microvalve may not use a hollow structure while ensuring that the float can rise or fall with the liquid level and ensures sealing.

[0057] The fluid diversion functional area 3 includes a drainage channel 31, a recovery branch channel 32, a drainage branch channel 33, a first outlet 34, a second outlet 35 and a waste liquid chamber 37; a water-absorbing material is provided in the waste liquid chamber 37, and a drainage microchannel 38 is provided in the water-absorbing material, and one end of the drainage microchannel 38 is connected to the second outlet 35; the drainage channel 31, the recovery branch channel 32 and one end of the drainage branch channel 33 are connected together and the three channels are interconnected, the other end of the drainage channel 31 is connected to the output end of the micro-reaction functional area 2, the other end of the recovery branch channel 32 is connected to the first outlet 34, and the other end of the drainage branch channel 33 is connected to the other end of the drainage microchannel 38.

[0058] In actual applications, the reactants in the micro-reaction functional area 2 can be determined according to the actual reaction type, and the structure for loading the reactants can be determined according to the reactants, including but not limited to microarray microcolumns or microchannels filled with glass microbeads, etc. Optionally, the portable microfluidic analysis chip provided in this embodiment is used to achieve automated enrichment of low-abundance nucleic acid samples. Accordingly, the reactants in the micro-reaction functional area 2 are glass microbeads for capturing nucleic acids, and the glass microbeads are filled in the microchannel. One end of the microchannel constitutes the input end of the micro-reaction functional area, and the other end constitutes the output end of the micro-reaction functional area. In order to ensure that the nucleic acid sample is fully in contact with the glass microbeads within the limited chip space and to ensure that the nucleic acid is fully captured, in this embodiment, the channel filled with glass microbeads is specifically a serpentine winding channel, such as Figure 3 As shown, optionally, the channel has a length of 32 mm, a height of 300 μm, a width of 800 μm, and a diameter of the glass microbeads of 150 μm to 200 μm.

[0059] In practical applications, the number and volume of the liquid storage chambers 11 in the sequential release functional area 1 need to be set according to the reaction type. Figure 1 and Figure 2 As shown, in this embodiment, the sequential release functional area 1 includes four levels of liquid storage chambers 11, which are used to store nucleic acid samples in the nucleic acid enrichment process, cleaning liquid for the first cleaning, cleaning liquid for the second cleaning, and nucleic acid elution liquid.

[0060] Based on the above structural design, the sequential release functional area 1 of this embodiment can realize the sequential release of reagents in each level of liquid storage chamber. Figure 4 It should be noted that for the convenience of description, Figure 4Only three stages of liquid storage chambers are shown. Reagents are added to each liquid storage chamber according to the reaction sequence, and negative pressure is applied to the first or second outlet, triggering the sequential release of reagents from each stage. Specifically, because the density of the float microvalve is less than that of the reagent, it floats above the reagent. Since the first-stage liquid storage chamber is connected to the atmosphere, the reagent therein flows from the bottom micropores through the fluid microchannels into the microreaction functional area under the influence of the atmosphere, achieving the release of the first-stage reagent. The remaining chambers are sealed by the liquid and cannot release the reagent. As the reagent in the first-stage liquid storage chamber gradually releases, the float microvalve gradually descends with the liquid level until the reagent in the first-stage liquid storage chamber is completely released, at which point the float microvalve falls into the bottom micropores, forming a seal. Simultaneously, the second-stage liquid storage chamber is connected to the atmosphere through the air path microchannel and the first-stage liquid storage chamber. Under the influence of the atmosphere, the reagent therein is released into the microreaction functional area, achieving the release of the second-stage reagent. As the reagent in the second-stage liquid storage chamber gradually releases, the float microvalve gradually descends with the liquid level until the reagent in the second-stage liquid storage chamber is completely released, at which point the float microvalve falls into the bottom micropore to form a seal. Simultaneously, the third-stage liquid storage chamber is connected to the atmosphere through the air path microchannel and the first and second-stage liquid storage chambers. Under the influence of the atmosphere, the reagent within will be released into the micro-reaction functional area, completing the release of the third-stage reagent. Because the third-stage liquid storage chamber is the last liquid storage chamber, as the reagent in the third-stage liquid storage chamber gradually releases, the float microvalve gradually descends with the liquid level until the reagent in the third-stage liquid storage chamber is completely released, and the reaction is complete.

[0061] It is easy to understand that when the number of liquid storage chambers changes, the sequential release of reagents can still be achieved based on the above principles.

[0062] In order to prevent the solution from entering the gas microchannel 14, preferably, in this embodiment, the gas microchannel 14 used to connect the two adjacent liquid storage chambers 11 has been hydrophobically modified, and the gas microchannel 14 is 100 μm wide, 300 μm high, and 2 mm long. In some other embodiments of the present invention, a waterproof and breathable membrane can also be used to make the gas microchannel, or other methods that can increase liquid resistance can be used to achieve it. In order to prevent different reagents from interfering with each other, in the sequential release functional area 1 of this embodiment, the length of the fluid microchannel used to connect the micropores at the bottom of the liquid storage chamber and the input end of the microreaction functional area is set to meet the following requirements: there is a certain time interval between the time when the reagent in the previous liquid storage chamber completely flows into the microreaction functional area and the time when the reagent in the next liquid storage chamber begins to flow into the microreaction functional area.

[0063] In practical applications, pressure is applied by pumping or inflating the outlet using an air pump. By controlling the airflow direction and gas flow rate, the magnitude and direction of the pressure applied at the outlet can be flexibly controlled. Furthermore, by controlling the magnitude and direction of the pressure at the two outlets, the direction of the fluid can be reversed, allowing waste liquid to flow into the drainage branch channel for discharge, while the post-reaction solution flows into the recovery branch channel for recovery. Furthermore, by applying pressure in different directions at the same outlet, reagents can be caused to flow back and forth within the micro-reaction zone, allowing for multiple mixing.

[0064] like Figure 5 As shown, in this embodiment, a waste liquid chamber 37 is integrated in the fluid diversion functional area 3, which can realize the recovery of waste liquid in the chip, reduce the pollution to the environment and the infection to the test personnel during the detection process; at the same time, a water-absorbing material is set in the waste liquid chamber 37, and a drainage microchannel 38 is set in the water-absorbing material. When the waste liquid flows into the drainage microchannel 38 through the drainage branch flow channel 33, it will be absorbed by the water-absorbing material. At the same time, the drainage branch flow channel 33 and the waste liquid chamber 37 are connected through the drainage microchannel 38, which can ensure that the pressure applied through the second outlet 35 is not affected while the waste liquid is discharged, so as to better realize the fluid control. It should be noted that in some other embodiments of the present invention, when the chip is used for other reactions that do not involve harmful substances, the waste liquid chamber may not be integrated in the fluid diversion functional area, but the waste liquid is discharged into the waste liquid chamber through the second outlet.

[0065] Optionally, in this embodiment, a drainage microchannel 38 is specifically engraved on the absorbent paper board, and the drainage microchannel 38 is slightly larger than the drainage channel 31, so that the drainage channel 31 is not in direct contact with the absorbent paper. This can avoid the negative pressure suction generated by the absorbent paper absorbing water and affecting the control of the microfluid in the chip.

[0066] In this embodiment, the drainage channel 31, the recovery branch channel 32 and one end of the drainage branch channel 33 are connected by a diversion microstructure 36; Figure 1 、 Figure 2 and Figure 6 As shown, the diversion microstructure 36 is in a T-shape, and its three-dimensional structure schematic diagram and plane structure schematic diagram are respectively as shown in FIG. Figure 6 As shown in (a) and (b), one end of the drainage channel 31 is connected to the vertical port of the diversion microstructure 36, and one end of the recovery branch channel 32 and the drainage branch channel 33 are respectively connected to the two horizontal ports of the diversion microstructure 36; a buffer microcavity 361 is formed at the horizontal and vertical intersection of the diversion microstructure 36, and the buffer microcavity 361 protrudes outward along the vertical direction to form a buffer steep slope 362.

[0067] Based on this structural design, the buffer microcavity has sufficient buffer space, so that after the liquid flows into the drainage channel, it will first be fully buffered and then enter the corresponding branch channel. In this way, the fluid entering the corresponding branch channel can be accurately controlled. That is to say, when discharging waste liquid, part of the waste liquid is prevented from entering the recovery channel and affecting the recovery purity. When recovering the reaction solution, part of the sample is prevented from entering the waste liquid discharge channel and causing waste of rare samples.

[0068] As an optional implementation method, this embodiment adopts a layered design approach to implement various structures within the chip, such as Figure 7 As shown, the chip consists of an 8-layer structure, from top to bottom: an upper gas path connecting channel layer (0.3mm), a liquid reservoir layer (5mm), a lower gas path connecting channel (0.3mm), a silicone membrane layer (1mm), a silicone elastic buffer layer (0.3mm), a spacer layer (0.1mm), a microfluidic channel layer (0.3mm) and a bottom cover (0.4mm). Preferably, in this embodiment, except that the silicone membrane layer is preferably made of elastic silicone material and the spacer layer is preferably made of PET film, the remaining materials are preferably PMMA sheets, and the layers are combined with double-sided tape. It should be noted that the layered design scheme and the thickness parameters of each layer here are only exemplary descriptions. In other embodiments of the present invention, other layered design schemes can also be adopted.

[0069] Based on the above structural design, the microfluidic separation chip provided in this embodiment can automatically realize the whole process of extraction, purification, enrichment and recovery of low-abundance nucleic acids; Figure 8 The specific analysis example shown is further explained. During the analysis process, a visual pigment solution is used to demonstrate the corresponding process. The specific process is as follows:

[0070] 1. Provide negative pressure at the second outlet to allow the microfluid to enter the waste liquid chamber. First, the sample in the first-stage liquid storage chamber will be released into the micro-reaction functional area first to achieve nucleic acid extraction, such as Figure 8 As shown in (A) to (B);

[0071] 2. After the sample flows through the micro-reaction functional area, the cleaning liquid in the second-stage liquid storage tank enters the micro-reaction functional area again to clean the excess impurities and purify the nucleic acid sample; similarly, after the cleaning liquid in the third-stage liquid storage chamber enters the waste liquid chamber after the first cleaning, it will automatically enter the micro-reaction functional area to further clean the extracted nucleic acid, such as Figure 8 As shown in (C) to (F);

[0072] 3. After the nucleic acid purification is completed, the nucleic acid eluate in the fourth stage liquid storage chamber will also automatically enter the micro-reaction functional area. At the same time, the pressure of the second outlet is suspended and the pressure of the first outlet is opened. Figure 8As shown in (G) in .

[0073] 4. After the nucleic acid elution solution enters the micro-reaction functional area, the first outlet provides pressure in different directions to achieve mixing of the nucleic acid elution solution in the micro-reaction area, such as Figure 8 As shown in (H) to (J) in FIG, the extracted and purified nucleic acid then moves toward the outlet and is finally recovered at the first outlet. Figure 8 As shown in (K) to (L).

[0074] Based on the aforementioned fact that the chip system is compatible with different flow rates and fluid directions, and the shear stress generated by the fluid velocity is different, the effects of different injection speeds, recovery speeds, and mixing reaction times on the nucleic acid enrichment effect were explored for standard nucleic acid samples. The sample volume was fixed at 200 μL, and the test results are shown in the figure. Figure 9 As shown in (a), (b) and (c) in Figure 9 As shown in (a), when the injection speed exceeds 70μL / min and is lower than 10μL / min, the CT value of the enriched nucleic acid is significantly higher, indicating that the enrichment concentration is low. The injection speed should be controlled between 10μL / min and 70μL / min. Figure 9 As shown in (b), the injection speed was fixed at 30 μL / min to verify different recovery speeds. The results showed that as the recovery fluid speed increased, the obtained nucleic acid amplification CT value also increased. Only when the flow rate was 5 μL / min, the obtained CT value was lower than the CT value of the original standard nucleic acid concentration. Since 5 μL / min is too slow, in this embodiment, a flow rate of 10 μL / min was preferably selected to perform the mixing reaction. The effect of the number of times on the nucleic acid enrichment effect is shown in Figure 2. Figure 9 As shown in (c), by comparing the difference with the CT value of the standard nucleic acid concentration, at a flow rate of 10μL / min, mixing back and forth 3 times can surpass the nucleic acid enrichment effect at a low flow rate (5μL / min). When it reaches 7 times, the nucleic acid enrichment effect is significantly improved.

[0075] In this embodiment, the injection port is in an unsealed state and the microbeads in the micro-reaction functional area are in a fixed state. Therefore, in order to achieve the enrichment of low-abundance nucleic acid concentrations, the enrichment effect can be improved by increasing the sample volume and fixing the volume of the recovered nucleic acid sample, thereby achieving the enrichment of lower-abundance nucleic acid samples. Figure 10 As shown in (a), the nucleic acid enrichment effect increases linearly with the increase of sample volume, indicating that when the sample volume increases to 1 mL, the nucleic acid concentration in the micro-reaction functional area still does not reach saturation. This also shows that the chip provided in this embodiment has good stability in nucleic acid extraction. By calculating the specific concentration, as shown in Figure 10As shown in (b), when the sample volume increases from 0.1 mL to 1 mL, the nucleic acid enrichment efficiency can be increased by 2536.83%.

[0076] In general, the portable microfluidic analysis chip provided in this embodiment, through the ingenious combination of the above-mentioned three functional areas, can automatically complete the sequential release of reagents, accurately control and change the microfluidic flow rate and fluid direction on demand, and accurately remove waste liquid and recover rare samples.

[0077] Example 2:

[0078] A portable microfluidic analysis chip, such as Figure 11 and Figure 12 As shown, this embodiment is similar to the above-mentioned embodiment 1, except that the chip provided in this embodiment can realize the sequential release of reagents in 9 liquid storage chambers, and can be applied to biochemical reactions that require more reaction reagents, such as enzyme-linked immunosorbent assay.

[0079] In this embodiment, the specific implementation of the remaining structures can refer to the description in the above embodiment 1.

[0080] Example 3,

[0081] A portable microfluidic analysis chip, such as Figure 13 、 Figure 14 and Figure 15 As shown, this embodiment is similar to the above-mentioned embodiment 1, except that, in this embodiment, the middle of the recovery branch flow channel 32 is cut off to form a quantitative outlet closer to the drainage flow channel and a collection inlet closer to the first outlet;

[0082] In addition, the fluid diversion functional area 3 also includes a quantitative distribution unit 4; the quantitative distribution unit 4 includes: 3-level quantitative chambers 41; the bottom of each quantitative chamber 41 is connected to the collection inlet through an air path microchannel 43; the top of the first-level quantitative chamber is connected to the quantitative outlet through a fluid microchannel 42, the tops of the two adjacent quantitative chambers are connected through the fluid microchannel 42, and the top of the last-level quantitative chamber is connected to the collection inlet through the fluid microchannel 42.

[0083] Optionally, the gas path microchannel 43 is hydrophobically modified to achieve a larger resistance of the microchannel to the liquid microfluid, while maintaining substantially zero resistance to the gas. Therefore, when the liquid microfluid enters the quantitative chamber, the excess gas at the bottom of the reaction chamber will flow out through the outlet. Since the lower gas path microchannel is closer to the outlet than the upper fluid microchannel, the microfluid will first fill the quantitative chamber. Subsequently, due to the increased resistance of the gas path fluid channel to the liquid microfluid, the fluid will not flow out through the gas path fluid microchannel, but will enter the next quantitative chamber through the upper fluid microchannel. By analogy, multiple quantitative chambers can be filled, such as Figure 16 (a) and (b) in the figure. Optionally, the lower gas path fluid channel may also utilize a waterproof breathable membrane or other technology to achieve selective flow of liquid and gas. It should also be noted that in other embodiments of the present invention, a waterproof breathable membrane may also be used to implement the gas path microchannel 43, or other methods that increase liquid resistance may be used to implement the gas path microchannel 43.

[0084] In this embodiment, different nucleic acid primers or different amplification reagents are placed in the three-level quantitative chambers respectively, thereby cleverly realizing a portable fully integrated high-throughput multi-target nucleic acid analysis chip through the principle of microfluidic resistance balance.

[0085] It is easy to understand that in actual applications, the number of quantitative chambers in the quantitative distribution unit can also be set to other values ​​according to actual analysis requirements.

[0086] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A portable microfluidic analysis chip, characterized in that: include: Sequential release functional area, micro-reaction functional area and fluid diversion functional area; The micro-reaction functional area is provided with reactants; The sequential release functional area includes M levels of liquid storage chambers; each liquid storage chamber is provided with a micropore at the bottom, and each micropore is connected to the input end of the micro-reaction functional area through a fluid microchannel; the top of the first-level liquid storage chamber is connected to the atmosphere, while the tops of the remaining liquid storage chambers are not connected to the atmosphere; each of the first to M-1-level liquid storage chambers is provided with a float microvalves having a diameter larger than the diameter of the bottom micropores and a density smaller than the density of the reagent in the liquid storage chamber; in two adjacent levels of liquid storage chambers, the bottom of the previous level liquid storage chamber is connected to the top of the next level liquid storage chamber through an air path microchannel; M is an integer greater than or equal to 2; The fluid diversion functional area includes a drainage channel, a recovery branch channel, a drainage branch channel, a first outlet and a second outlet; one end of the drainage channel, the recovery branch channel and the drainage branch channel are connected together and the three channels are interconnected, the other end of the drainage channel is connected to the output end of the micro-reaction functional area, the other end of the recovery branch channel is connected to the first outlet, and the other end of the drainage branch channel is connected to the second outlet.

2. The portable microfluidic analysis chip according to claim 1, characterized in that: The fluid diversion functional area also includes a waste liquid chamber, in which a water-absorbing material is arranged, and a drainage microchannel is arranged in the water-absorbing material; one end of the drainage microchannel is connected to the second outlet, and the other end of the drainage branch flow channel is connected to the other end of the drainage microchannel.

3. The portable microfluidic analysis chip according to claim 2, characterized in that: The drainage channel, the recovery branch channel and one end of the drainage branch channel are connected through a diversion microstructure; the diversion microstructure is in a "T" shape, one end of the drainage channel is connected to the vertical port of the diversion microstructure, and one end of the recovery branch channel and the drainage branch channel are respectively connected to the two horizontal ports of the diversion microstructure; a buffer microcavity is formed at the horizontal and vertical intersection of the diversion microstructure, and the buffer microcavity bulges outward along the vertical direction to form a buffer steep slope.

4. The portable microfluidic analysis chip according to any one of claims 1 to 3, characterized in that: The middle of the recovery branch flow channel is cut off to form a quantitative outlet closer to the drainage flow channel and a collection inlet closer to the first outlet; Furthermore, the fluid diversion functional area further includes a quantitative distribution unit; the quantitative distribution unit includes: N levels of quantitative chambers; the bottom of each quantitative chamber is connected to the collection inlet via an air path microchannel; the top of the first-level quantitative chamber is connected to the quantitative outlet via a fluid microchannel, the tops of two adjacent quantitative chambers are connected via a fluid microchannel, and the top of the last-level quantitative chamber is connected to the collection inlet via a fluid microchannel; Wherein, N is an integer greater than or equal to 1.

5. The portable microfluidic analysis chip according to claim 4, characterized in that: The air path microchannels in the quantitative distribution unit are subjected to hydrophobic treatment, or are made of a waterproof and breathable membrane.

6. The portable microfluidic analysis chip according to any one of claims 1 to 3, characterized in that: The air path microchannels in the sequential release functional area are subjected to hydrophobic treatment, or are made of a waterproof and breathable membrane.

7. The portable microfluidic analysis chip according to any one of claims 1 to 3, characterized in that: The structure for loading reactants in the micro-reaction functional area is a micro-array micro-column or a micro-channel filled with glass micro-beads.

8. The portable microfluidic analysis chip according to any one of claims 1 to 3, characterized in that: In the sequential release functional area, the length of the fluid microchannel connecting the micropores at the bottom of the liquid storage chamber and the input end of the microreaction functional area is set to meet the requirement that there is a certain time interval between the time when the reagent in the previous liquid storage chamber completely flows into the microreaction functional area and the time when the reagent in the next liquid storage chamber starts to flow into the microreaction functional area.

9. The portable microfluidic analysis chip according to any one of claims 1 to 3, characterized in that: The bottom of each liquid storage chamber is made of elastic material.

10. The portable microfluidic analysis chip according to claim 9, characterized in that: The bottom of each liquid storage chamber is made of silicone material.

Images