Micro-fluidic chip, micro-fluidic system, and manufacturing method

Abstract

A micro-fluidic chip, a micro-fluidic system, and a manufacturing method. The chip comprises a chip housing (100) and a micro-porous layer (200), the micro-porous layer (200) is mounted inside the chip housing (100), a plurality of micro-through holes (210) are formed on the micro-porous layer (200), and a sample injection hole (111) is formed on the chip housing (100). In a sample loading process, a sample is subjected to capillary force effects from the micro-through holes (210) having a high aspect ratio, so that the sample is drawn into the micro-through holes (210); as the sample is pushed forward inside a micro-fluidic channel (300), the sample can finally fill all the micro-through holes (210); and therefore, the sample is finally highly uniformly segmented and distributed in the plurality of micro-through holes (210). In the flowing process of sealing oil, a residual sample attached to the surface of the micro-porous layer (200) can be dragged while the sample is pushed forward.

Description

Microfluidic chip, microfluidic system and manufacturing method Technical Field

[0001] The present invention belongs to the technical field of biochemistry and molecular biology, and in particular relates to a microfluidic chip, a microfluidic system and a manufacturing method. Background Art

[0002] To increase the speed, reduce costs, and simplify diagnostic assays while maintaining experimental accuracy, researchers are focusing on miniaturization and integration of experimental devices. This includes increasing the number of parallel assays on a single carrier device. However, the miniaturization of microfluidic devices introduces a series of challenges, such as liquid loss and issues related to the consistency of microunit volumes. In particular, microfluidic devices face challenges in sample partitioning and distribution uniformity when filling microfluidic structures, primarily due to the unique flow behavior of the sample liquid within the device. Microfluidic chips offer a solution by providing microchannels to process microliter or nanoliter volumes of sample liquid, allowing multiple assays to be performed in a miniaturized environment. These chips typically pre-load microliter-scale reagents into small cells that come into contact with the sample liquid flowing through the reaction flow channels. The sample volume or concentration of each cell must be precisely controlled. These assays are widely used with biological materials (such as peptides, nucleic acids, cells, or tissues). In particular, digital PCR, as an innovation, achieves more reliable and sensitive nucleic acid measurements by partitioning the sample into numerous small areas for independent reactions, marking a major advancement over traditional PCR methods.

[0003] To this end, the present invention provides a microfluidic chip, a microfluidic system and a manufacturing method.

[0004] Summary of the Invention

[0005] In order to overcome the defects in the prior art, the purpose is to provide a microfluidic chip that can achieve highly uniform segmentation and distribution of samples, with lower cost and higher loading efficiency.

[0006] The present invention solves the technical problems and achieves the purpose of overcoming the above technical problems. The technical solution adopted by the present invention is as follows: a microfluidic chip comprises: a chip housing; a microporous layer, wherein the microporous layer is installed inside the chip housing, the microporous layer has a plurality of micro-through holes, a microfluidic channel is provided in the chip housing, and an injection hole is provided on the chip housing; the chip is suitable for sequentially introducing a sample and a sealing oil into the interior of the microfluidic channel through the injection hole and applying pressure, so that the sample and the sealing oil sequentially pass through the micro-through holes, the sample is sucked into the micro-through holes, and the sealing oil fully seals the microporous layer.

[0007] In some embodiments, the microfluidic channel is provided at the bottom of the micro-through hole in the chip housing, and is suitable for the sample and the sealing oil to pass through the bottom of the micro-through hole in sequence.

[0008] In some embodiments, a first sealed flow channel is provided on the top of the microporous layer in the chip housing, and a first guide hole is provided inside the chip housing, wherein the first guide hole connects the microfluidic channel and the first sealed flow channel.

[0009] In some embodiments, the chip housing is provided with an exhaust hole at the position of the first sealed flow channel; the dynamic viscosity of the sealing oil is greater than the dynamic viscosity of water, which is suitable for dragging away the sample remaining on the inner wall of the microfluidic channel during the flow of the sealing oil.

[0010] In some embodiments, the chip shell includes a first chip cover and a first chip base plate, the first chip cover and the first chip base plate are adapted and connected to provide a first mounting cavity between the first chip cover and the first chip base plate, the microporous layer is arranged in the first mounting cavity, the microfluidic channel is provided between the microporous layer and the first chip base plate, and the first sealed flow channel is provided between the microporous layer and the first chip cover; the injection hole is provided on one side of the first chip cover close to the microporous layer, the first guide hole is provided on the other side of the microporous layer close to the microporous layer, and the microthrough hole is located between the injection hole and the first guide hole.

[0011] In some embodiments, the first chip base plate has a first microfluidic groove to form the microfluidic channel; the first microfluidic groove has a first connecting area on a side away from the injection hole, suitable for the first connecting area and the first sealed flow channel to be connected to each other without being blocked by the microporous layer.

[0012] In some embodiments, a first pad is provided on the first chip base plate near the bottom of the first mounting cavity, a microfluidic hole is opened on the first pad at the bottom of the micro-through hole to form the microfluidic channel, a plurality of limit blocks are provided inside the first mounting cavity near the edge of the first pad, the microporous layer is arranged on the top of the first pad and the microporous layer is positioned and restricted by the plurality of limit blocks.

[0013] In some embodiments, the microfluidic hole includes a drainage area, an expansion area and a flow forming area, the expansion area is located between the drainage area and the flow forming area and is interconnected; the width of the drainage area is smaller than the width of the flow forming area, the expansion area is trumpet-shaped, suitable for the narrow end of the expansion area to be connected to the drainage area, and the wide end of the expansion area to be connected to the flow forming area, suitable for the sample and the sealing oil to pass through the injection hole in turn and be introduced from the drainage area, the expansion area and the flow forming area to the first guide hole in turn; a plurality of the microthrough holes are distributed on the microporous layer to form a microporous area, the microporous area is located at the top of the expansion area and the flow forming area, and all the microthrough holes on the microporous area are connected to the microfluidic channel.

[0014] In some embodiments, a mounting groove is provided on the top surface of the first chip base plate, the first pad is provided at the bottom of the mounting groove, a plurality of limit blocks are provided on the top of the first pad close to the side of the mounting groove, and the first chip cover is installed at the notch position of the mounting groove and is limited and supported by the limit blocks; the top and bottom surfaces of the microporous layer and the first pad are both planes, the bottom of the mounting groove is plane, the first pad and the bottom of the mounting groove are plane-fitted, and the contact position between the first pad and the microporous layer is plane-fitted.

[0015] In some embodiments, a second guide hole is provided on the microporous layer at the bottom of the injection hole, and the second guide hole connects the injection hole and the microfluidic channel; an observation spacer is provided on the first chip cover at the top of the micro-through hole, and the observation spacer is made of a light-transmitting material.

[0016] In some embodiments, the micro-through hole is a through hole with a high aspect ratio; a first hydrophobic area is provided on the surface of the microporous layer, a hydrophilic area is provided on the inner wall of the micro-through hole, a second hydrophobic area is provided on the bottom plate of the first chip at the position of the microfluidic channel, and a third hydrophobic area is provided on the cover of the first chip at the position of the injection hole.

[0017] In some embodiments, the microfluidic channel is arranged at the top position of the microthrough hole in the chip shell, which is suitable for the sample and the sealing oil to pass through the top of the microthrough hole in sequence; the chip shell may also include a second chip cover and a second chip base plate, the second chip cover and the second chip base plate are adapted and connected to provide a second mounting cavity between the second chip cover and the second chip base plate, the microporous layer is arranged in the second mounting cavity, the microfluidic channel is provided between the microporous layer and the second chip cover, and a second sealing flow channel is provided between the microporous layer and the second chip base plate; it is suitable for the sample to be introduced into the microfluidic channel through the injection hole and to be separated and loaded at the top position of the microporous layer, and the sealing oil flows from the microfluidic channel into the second sealing flow channel and fully seals the microporous layer.

[0018] In some embodiments, a second pad is provided on the second chip cover near the top of the second mounting cavity, and a microfluidic hole is opened on the second pad at the top of the micro-through hole to form the microfluidic channel; or, the second pad is provided on the second chip cover near the top and bottom of the second mounting cavity, and a microfluidic hole is opened on the second pad at the micro-through hole to form the microfluidic channel or the second sealed flow channel.

[0019] In some embodiments, when a second pad is provided on the second chip cover only near the top of the second mounting cavity, a second microfluidic groove adapted to the microfluidic hole is provided on the second chip bottom plate to form the second sealed flow channel; the second microfluidic groove and the side of the microfluidic hole away from the injection hole both have a second connecting area, which is suitable for the two second connecting areas to be connected to each other without being blocked by the microporous layer.

[0020] A microfluidic system adopts the above-mentioned microfluidic chip, comprising: a plurality of the above-mentioned microfluidic chips; the plurality of the above-mentioned microfluidic chips are connected to each other.

[0021] A method for manufacturing a microfluidic chip, for preparing the above-mentioned microfluidic chip, comprises the following steps:

[0022] First, the sample is added into the injection hole, and then the sealing oil is added into the injection hole;

[0023] Applying positive pressure to the sample injection hole, so that the sealing oil pushes the sample into the microfluidic channel under the positive pressure;

[0024] The sample flows through the microporous layer along the microfluidic channel, and under the action of the capillary force of the micro-through-holes on the microporous layer, the sample is divided and sucked into the micro-through-holes;

[0025] The sealing oil continuously pushes the sample to flow along the microfluidic channel, and the sample is further sucked into the micro-through hole in the front of the flow direction, and the sealing oil removes the sample remaining on the inner wall of the microfluidic channel;

[0026] The sealing oil continues to move forward and enters the top of the microporous layer, filling the interior of the chip housing and fully sealing the microporous layer, thereby completing the preparation of the microfluidic chip.

[0027] Compared with the prior art, the microfluidic chip, microfluidic system and manufacturing method provided by the present invention have the following beneficial effects:

[0028] 1. The present invention provides a microfluidic chip, a microfluidic system and a manufacturing method. By setting a chip shell and a microporous layer, the microporous layer has multiple microthrough-holes, and the bottom of the microporous layer has a microfluidic channel for sample and sealing oil to flow and load. The sample will be affected by the capillary force of the high-aspect-ratio microthrough-holes, so that the sample is sucked into the microthrough-holes. As the sample moves forward inside the microfluidic channel, the sample will eventually fill all the microthrough-holes. Therefore, the final form of the sample is highly uniformly divided and distributed in a large number of microthrough-holes. The sealing oil effectively pushes the sample forward and drags the residual sample attached to the bottom surface of the microporous layer, thereby improving the chip preparation efficiency and quality. In addition, the microfluidic channel processing and preparation process are simpler and less expensive, and no complex external loading equipment is required for cooperation.

[0029] 2. The present invention provides a microfluidic chip, a microfluidic system and a manufacturing method. Through the drainage area, the expansion area and the flow forming area, when the sample and the sealing oil are introduced into the microfluidic channel formed by the microfluidic hole, they first enter the drainage area, then the expansion area, and then the flow forming area. Through the effective guidance of the expansion area, the sample is kept moving forward at approximately the same speed, reducing the problem of trapped air during the loading process, which is more conducive to the uniform separation of the samples and avoiding the situation where some samples have a faster flow rate and flow out of the microfluidic channel in advance, resulting in more samples being unable to be effectively utilized.

[0030] 3. The present invention provides a microfluidic chip, a microfluidic system and a manufacturing method. The high-viscosity sealing oil is used to better drag the water molecules attached to the bottom surface of the microporous layer during the process of promoting the flow of samples, so that the residual samples on the inner wall of the microfluidic channel can be effectively dragged away, thereby clearing the residual samples on the inner wall of the microfluidic channel, especially the bottom surface of the microporous layer, reducing the impact on the accuracy of subsequent detection and avoiding affecting the subsequent PCR reaction. Of course, the sealing oil can also drag the residual samples attached to the top surface of the microporous layer during the flow process. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below in conjunction with the accompanying drawings:

[0032] FIG1 is an exploded view of a microfluidic chip in an embodiment of bottom loading of the present invention;

[0033] FIG2 is an exploded view of a microfluidic chip in another embodiment of the present invention in which samples are loaded from the bottom;

[0034] FIG3 is an exploded view of a microfluidic chip in another embodiment of the present invention in which samples are loaded from the bottom;

[0035] FIG4 is an exploded view of a microfluidic chip in an embodiment of the present invention in which samples are loaded from the top;

[0036] FIG5 is an exploded view of a microfluidic chip in another embodiment of the present invention in which samples are loaded from the top;

[0037] FIG6 is a perspective view of a microfluidic system according to an embodiment of the present invention;

[0038] FIG7 is a flow chart of a method for manufacturing a microfluidic chip according to an embodiment of the present invention.

[0039] In the figure: chip housing 100, first chip cover 110, injection hole 111, exhaust hole 112, observation spacer 113, first chip base plate 120, mounting groove 121, first microfluidic groove 122, first connecting area 123, second chip cover 130, second chip base plate 140, second microfluidic groove 141, second connecting area 142,

[0040] Microporous layer 200, micro-through hole 210, first flow guide hole 220, microporous area 230, second flow guide hole 240, hydrophilic area 250,

[0041] Microfluidic channel 300,

[0042] The first sealed flow channel 400, the second sealed flow channel 410,

[0043] The first pad 500, the drainage area 510, the expansion area 520, the flow forming area 530, the second pad 540, the micro-flow hole 550,

[0044] Limit block 600,

[0045] The first hydrophobic area 700,

[0046] The second hydrophobic area 800,

[0047] The third hydrophobic region 900 . DETAILED DESCRIPTION

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the specific embodiments of the present invention will be described below with reference to the accompanying drawings. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings and other embodiments can be obtained based on these drawings without inventive work.

[0049] To simplify the drawings, only portions relevant to the invention are schematically depicted in each figure; they do not represent the actual structure of the product. Furthermore, to simplify the drawings and facilitate understanding, in some figures, only one component with the same structure or function is schematically depicted or labeled. In this document, "one" not only means "only one" but also "more than one."

[0050] It should be further understood that the term "and / or" used in the present description and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0051] It should be noted that, unless otherwise specified or limited, the terms "mounted," "connected," and "connected" should be understood broadly. For example, they can refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediary; and internal communication between two components. Those skilled in the art will understand the specific meanings of these terms in the present invention based on the specific circumstances.

[0052] In addition, in the description of the present invention, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0053] As shown in Figures 1 to 5, this embodiment provides a microfluidic chip, including a chip housing 100 and a microporous layer 200. A sample injection hole 111 is provided on the chip housing 100. When producing the microfluidic chip, a sample is first injected into the sample injection hole 111 and then a sealing oil is injected. The mouth of the sample injection hole 111 is pressurized so that the sealing oil is pushed by the pressure, and the sealing oil then pushes the sample to flow, so that the sample and the sealing oil enter the microfluidic channel 300 in sequence, and then the sample is separated and enters the microcavities on the surface of the microporous layer 200 respectively, and then the microporous layer 200 is sealed by the sealing oil.

[0054] Digital PCR (dPCR) is a molecular technology used to accurately quantify DNA or RNA. This technology divides the sample into thousands of independent small reactions, so that each reaction contains only zero or a small number of DNA molecules. It then achieves high-precision quantification through Poisson distribution calculation.

[0055] Digital PCR microfluidic chips are a type of dPCR technology platform. These chips utilize microfluidics to partition a DNA sample into a large number of tiny oil droplets or micro-reaction chambers. Each droplet or reaction chamber acts as an independent PCR reaction environment, containing few or no target DNA molecules. By statistically counting the amplification signals within these droplets or micro-reaction chambers, the number of target DNA molecules in the original sample can be very accurately estimated. The advantage of this method lies in its extremely high precision and sensitivity, making it very useful in areas such as low-abundance sample detection, single-cell analysis, gene expression level analysis, mutation detection, and pathogen detection.

[0056] However, this technology also faces a series of challenges, such as the loss of sample liquid, the consistency of the volume of the micro-reaction chamber, the uniformity of sample segmentation and distribution, the difficulty and cost of manufacturing microfluidics and micro-reaction chambers, and the complexity of the sample segmentation and distribution process.

[0057] In this embodiment, a microporous layer 200 is provided inside the chip housing 100, and a plurality of microthrough holes 210 are provided on the surface of the microporous layer 200. The microthrough holes 210 run through the entire microporous layer 200 from top to bottom. A microfluidic channel 300 is provided at the bottom of the microporous layer 200. The microfluidic channel 300 is a flow channel for the flow of samples and sealing oil formed between the inner wall of the chip housing 100 and the microporous layer 200. The microporous layer 200 as a whole separates the interior of the chip housing 100 to form two upper and lower chambers. The microfluidic channel 300 is formed in the bottom chamber of the microporous layer 200, and the sealing oil can also flow and fill in the top chamber of the microporous layer 200, thereby achieving sealing coverage of the top of the microporous layer 200. The upper and lower chambers of the microporous layer 200 can be connected. The connecting position is set at a position away from the injection hole 111; of course, in another embodiment, it is not ruled out that the microfluidic channel 300 is set at the top position of the microporous layer 200. During the loading process, the sample and the sealing oil are directly pressurized and introduced into the microfluidic channel 300 at the top position of the microporous layer 200 through the injection hole 111 in sequence, so that when the sample flows in the microfluidic channel 300, the sample is loaded at the top position of the microporous layer 200, uniformly divided and introduced, and sucked into the microthrough hole 210; in other embodiments, the microfluidic channel 300 can also be set at the bottom position of the microporous layer 200. When loading, the microfluidic chip can be flipped over as a whole so that the microfluidic channel 200 can also be flipped to the top position of the microfluidic channel 300 to meet the top loading requirements.

[0058] Furthermore, the injection hole 111 and the upper and lower chambers of the microporous layer 200 are connected at the two ends of the microporous layer 200. When preparing the microfluidic chip, the sample and the sealing oil are injected successively through the injection hole 111, wherein the sample is a biological material, preferably a polypeptide, nucleic acid, cell or tissue, and the sealing oil is preferably a high-viscosity silicone oil, and can also be a mineral oil, a fluorinated oil, etc., but is not limited thereto. The viscosity of the sealing oil is higher than the viscosity of the sample, such as: at 25 degrees Celsius, the dynamic viscosity of the sealing oil is greater than the dynamic viscosity of water (0.89cp). At this time, the sealing oil can have a better drag effect on the residual sample, etc., thereby improving the quality of the microfluidic chip after loading. ; Positive pressure is applied to the opening of the sample injection port, so that the sample and the sealing oil enter the microchannel 300 in turn, and the sealing oil is pushed by the positive pressure, and the sealing oil then pushes the sample, so that the sample flows from one end position of the bottom of the microporous layer 200 to the other end position, so the sample will also pass through the microthrough hole 210. Since the microthrough hole 210 is designed with a high aspect ratio and the microthrough holes 210 are concentrated in large quantities on the microporous layer 200, the microthrough holes 210 are all distributed at the top of the microchannel 300 and are adapted to the overall horizontal shape of the microchannel 300. The sample will be affected by the capillary force of the high aspect ratio microthrough hole 210, so that the sample is sucked into the microthrough hole 2 10. As the sample moves forward inside the microfluidic channel 300, the sample will eventually fill all the micro-through holes 210. Therefore, the final form of the sample is highly uniformly divided and distributed in a large number of micro-through holes 210. At the same time, under the action of positive pressure, the sealing oil will continue to move forward and fill the microfluidic channel 300. The sealing oil will also continue to flow upward through the upper and lower connecting positions of the microporous layer 200, that is, it will flow from the bottom position of the microporous layer 200 to the top position of the microporous layer 200, and finally fill the top chamber of the microporous layer 200. The filling here can be further understood as: its core purpose is to seal the microporous layer 200 on the upper and lower sides of a large number of micro-through holes 210. Therefore, in order to facilitate operation, meet the needs of later observation and meet the requirements of sample quality, it is preferred to fill the entire internal cavity of the chip shell 100 to achieve full sealing of the overall packaging of the microporous layer 200. Full sealing does not necessarily mean that all surfaces of the microporous layer 200 are wrapped, because some contact surfaces of the microporous layer 200 are not in an open state. Therefore, the core of full sealing is to seal the microporous layer 200 after the samples have been evenly separated and distributed with sealing oil to avoid continuous contact between the samples and the external environment, thereby ensuring the requirements of sample quality. The final sealing form is not specifically limited, and it is preferred that the chamber inside the chip shell 100 is completely filled.

[0059] Furthermore, there is no specific limitation on the method of applying positive pressure to the injection hole 111, as long as the positive pressure drives the sealing oil and sample to flow to prepare the microfluidic chip. For example, a syringe can be used to manually inject sealing oil into the injection hole 111 and apply positive pressure to push the sealing oil, etc., but it is not limited to this. Positive pressure refers to applying positive pressure toward the inside of the injection hole 111.

[0060] Furthermore, in this embodiment, when the sealing oil pushes the sample to flow inside the microfluidic channel 300, part of the liquid sample will remain on the bottom surface of the microporous layer 200. Since the density of the sealing oil given in this embodiment is lower than the sample density and the viscosity of the sealing oil is higher than the sample viscosity, the sealing oil can effectively maintain a certain pressure contact with the bottom surface of the microporous layer 200 and flow in the preset flow direction. Due to its relatively high viscosity, the sealing oil can remove the sample remaining on the bottom surface of the microporous layer 200. The specific removal principle is: the high-viscosity sealing oil has a slower fluidity and greater viscosity than the liquid sample. The sealing oil can more effectively drag the sample remaining on the bottom surface of the microporous layer 200, so that the sample on the bottom surface of the microporous layer 200 can be removed. Finally, the samples under observation are basically evenly concentrated in each micro-through hole 210, which effectively improves the detection accuracy and avoids affecting the subsequent PCR reaction. Of course, the sealing oil can also drag the sample remaining on the top surface of the microporous layer 200 during the flow process.

[0061] Furthermore, in order to ensure that the sample and sealing oil fill the internal cavity of the chip shell 100 and the internal air pressure of the chip shell 100 is balanced in this embodiment, a conventional exhaust hole 112 can be set on the chip shell 100. Of course, it is preferred to set the exhaust hole 112 at the top position of the chip shell 100 so that after the sealing oil fills the entire chip shell 100, any excess sealing oil can enter the exhaust hole 112, thereby reducing the risk of air trapped inside the chip shell 100 and ensuring the quality of the chip. In other embodiments, when the sealing oil is injected into the sample injection hole 111 and positive pressure is applied, a small gap can be appropriately left as a vent, etc., and the method is not limited to this; in another embodiment, the sealing oil can also be injected into the interior of the chip shell from the exhaust hole 112, while leaving enough gaps for exhaust.

[0062] Furthermore, the number and distribution of the injection holes 111 can be adaptively adjusted as needed. For example, multiple injection holes 111 can be used, or the same injection hole 111 can be divided into multiple channels inside the chip shell 100, so that the sample and sealing oil can flow into different positions of the microfluidic channel 300 respectively, and the sample flow separation and distribution can be achieved simultaneously through multiple positions. Finally, the sealing oil in multiple directions is collected and the microporous layer 200 is sealed to improve the chip preparation efficiency, etc., and the present invention is not limited to this.

[0063] In one embodiment, a first sealing channel 400 is provided at the top of the microporous layer 200 in the chip housing 100. On the one hand, the first sealing channel 400 can provide a pressure regulating effect for the micro-through-holes 210. When the sample is sucked into the micro-through-holes 210, the gas inside the micro-through-holes 210 can be discharged into the first sealing channel 400, ensuring that the sample is smoothly and evenly separated and introduced into a large number of micro-through-holes 210. On the other hand, when the sealing oil enters the top of the microporous layer 200, the sealing oil will flow in the first sealing channel 400 to achieve a covering and sealing of the top surface of the microporous layer 200.

[0064] Furthermore, a first guide hole 220 can be provided in the chip housing 100, and the first guide hole 220 connects the microfluidic channel 300 and the first sealing channel 400. After the sealing oil is introduced into the microfluidic channel 300 from the injection hole 111, it flows inside the microfluidic channel 300. The sealing oil enters the first guide hole 220 after passing through the micro-through hole 210 on the microporous layer 200, and is introduced into the first sealing channel 400 through the first guide hole 220, and then the sealing oil seals the top surface of the microporous layer 200 through the first sealing channel 400.

[0065] In one embodiment, the chip housing 100 is provided with an exhaust hole 112, which is arranged at a position corresponding to the first sealed flow channel 400, so that the chip housing 100 has the ability to exhaust upward. After the sealing oil is introduced into the interior of the microfluidic channel 300, as the sealing oil pushes the sample to flow, the gas inside the microfluidic channel 300 will enter the first sealed flow channel 400 through the first guide hole 220. Since the exhaust hole 112 is provided at the position of the first sealed flow channel 400 and the exhaust hole 112 connects the first sealed flow channel 400 with the outside world, the gas inside the first sealed flow channel 400 is discharged, thereby achieving the regulation of the air pressure inside and outside the chip housing 100;

[0066] In one embodiment, the sealing oil is high-viscosity silicone oil. Compared with mineral oil, high-viscosity silicone oil has better viscosity properties. In the process of promoting the flow of samples, it can better drag the water molecules attached to the bottom surface of the microporous layer 200, so that the residual samples on the inner wall of the microfluidic channel 300 can be effectively dragged away, thereby clearing the residual samples on the inner wall of the microfluidic channel 300, especially the bottom surface of the microporous layer 200, and reducing the impact on subsequent detection.

[0067] In one embodiment, the chip housing 100 includes a first chip cover 110 and a first chip base plate 120, so that the microporous layer 200 can be assembled in the first chip cover 110 and the first chip base plate 120. The first chip cover 110 and the first chip base plate 120 are sealed and connected to form the chip housing 100, and a first mounting cavity is formed between the first chip cover 110 and the first chip base plate 120. The microporous layer 200 can separate the first mounting cavity into a microfluidic channel 300 and a first sealed flow channel 400, wherein the microfluidic channel 300 is located at the bottom of the microporous layer 200, and the first sealed flow channel 400 is located at the top of the microporous layer 200.

[0068] Furthermore, the injection hole 111 is located near one end of the microporous layer 200, and the first guide hole 220 is located near the other end of the microporous layer 200. Therefore, a large number of micro-through holes 210 on the microporous layer 200 can be located between the injection hole 111 and the first guide hole 220. When the sample and the sealing oil are introduced into the microfluidic channel 300 from the injection hole 111, the sample and the sealing oil can effectively pass through the micro-through holes 210, thereby achieving uniform separation of the sample and effective introduction into the micro-through holes 210, and then the sealing oil enters the first guide hole 220, thereby achieving sealing of the upper and lower parts of the microporous layer 200 by the sealing oil.

[0069] In one embodiment, a first microfluidic groove 122 can be directly provided on the first chip base plate 120, and the first microfluidic groove 122 cooperates with the microporous layer 200 to form a microfluidic channel 300. Furthermore, by providing a first connecting area 123 at a position of the first microfluidic groove 122 away from the injection hole 111, the first connecting area 123 is wider than other areas of the first microfluidic groove 122, and the first connecting area 123 is not blocked by the microporous layer 200, and can effectively connect the microfluidic channel 300 and the first sealed flow channel 400.

[0070] In one embodiment, a first pad 500 is provided on the first chip base plate 120 at the bottom position of the first mounting cavity, and micropores 550 are provided on the first pad 500, and the microporous layer 200 is placed at the top position of the first pad 500, so that a microfluidic channel 300 is formed at the position of the micropores 550, and a limiting block 600 is provided on the first pad 500, and the limiting block 600 is used to limit the positioning of the microporous layer 200, and when the first chip base plate 120 and the first chip cover 110 are installed between them, a preset gap is set between the first chip cover 110 and the microporous layer 200 to form a first sealed flow channel 400, and the method of presetting the gap is not limited, such as installing the first chip cover 110 on the pad, and the height dimension of the pad is greater than the thickness dimension of the microporous layer 200, so that when the first chip cover 110 is placed on the pad, there will be a gap between the bottom surface of the first chip cover 110 and the microporous layer 200;

[0071] In other embodiments, a step surface may be provided on the opposite side of the first chip cover 110 or the first chip base plate 120 so that there is a gap between the bottom surface of the first chip cover 110 and the top surface of the microporous layer 200 after sealing and positioning, etc., but the present invention is not limited thereto.

[0072] In one embodiment, the microfluidic hole 550 includes a drainage area 510, a flow expansion area 520 and a flow forming area 530, and the drainage area 510, the flow expansion area 520 and the flow forming area 530 are sequentially connected to each other to form an integral microfluidic hole 550, and the microfluidic hole 550 passes through the first pad 500 from top to bottom. The purpose is to allow the sample and sealing oil entering the microfluidic hole 550 to flow on the internal top surface of the first chip base plate 120, so as to facilitate rapid assembly and form a preset microfluidic channel 300 shape to adapt to the distribution shape of the microthrough holes 210. For example, the overall distribution of the microthrough holes 210 can be a rectangular array structure or a hexagonal close-packed structure, etc., but is not limited to this. Of course, the shape of the micropores can also be selected according to actual conditions, such as using a circular or polygonal shape, etc., but is not limited to this. At the same time, the sample or sealing oil will not have steps during the flow inside the microfluidic channel 300, so that the sealing oil is always located on the top surface of the first chip base plate 120 in the microfluidic channel 300 and the top surface is flat;

[0073] Furthermore, the width of the drainage area 510 is smaller than that of the flow area 530. The width direction here refers to the direction along the horizontal plane and perpendicular to the overall flow direction of the sealing oil. The expansion area 520 can effectively and smoothly connect the drainage area 510 and the flow area 530. For example, a trumpet-shaped cross-section or an eight-shaped cross-section can be used. The micro-through holes 210 are distributed to form a microporous area 230. The microporous area 230 is located at the top of the expansion area 520 and the flow area 530. When the sample and After the sealing oil is introduced into the microfluidic channel 300 formed by the microfluidic hole 550, it first enters the drainage area 510, then enters the expansion area 520, and then enters the flow forming area 530. It enters the first flow guide hole 220 through the flow forming area 530 and flows toward the top of the microporous layer 200. The sample passing through the expansion area 520 and the flow forming area 530 will be sucked into the large number of micro-through holes 210 in the microporous area 230 to achieve uniform separation of the sample. In order to push the sample and the sealing oil forward at a relatively constant speed, that is, when the sample or the sealing oil flows in a preset direction, a flow separation line will be formed near the corresponding front end position of the sample or the sealing oil. For example, a separation line will be formed between the sample and the sample-free zone, and a separation line will be formed between the sample and the sealing oil. Through the effective guidance of the expansion zone 520, the separation line can be kept relatively straight and pushed forward at the same speed, so that the internal gas of the microfluidic channel 300 can be effectively introduced into the first guide hole 220 and finally discharged. Compared with if the drainage zone 510 has a larger width, the sample or sealing oil introduced into the drainage zone 510 through the injection hole 111 will first flow in an radiating shape toward the circumferential direction, which will easily cause local air entrapment problems, or will also have a certain impact on the uniform separation of the sample. In this embodiment, the expansion zone 520 is used to make the sample and the sealing oil flow in a fan shape, so that the forward flow of the sample and the sealing oil is more uniform.

[0074] In other embodiments, the microporous area 230 may also be located alone at the top of the flow forming area 530 , but is not limited thereto.

[0075] In one embodiment, a mounting groove 121 is defined on the top surface of the first chip base plate 120, a first pad 500 is disposed at the bottom of the mounting groove 121, a limiting block 600 is disposed near the top of the first pad 500 and near its periphery, the first chip cover 110 is disposed on top of the limiting block 600 and supported by the limiting block 600, and the first chip cover 110 is adapted to the notch of the mounting groove 121, so that the installed first chip cover 110 seals the notch of the mounting groove 121;

[0076] Furthermore, by setting the top and bottom surfaces of the microporous layer 200, the first pad 500 and the bottom of the mounting groove 121 to be flat, a better connection effect is achieved between the mounting groove 121, the first pad 500 and the microporous layer 200, and better sealing is achieved at the connection contact surface position, reducing the problem of sample and sealing penetration between the connection contact surface. Of course, it can also reduce deformation or internal stress problems during the installation process, and improve the accuracy and stability of the microfluidic chip after assembly.

[0077] In one embodiment, a second guide hole 240 is provided on the microporous layer 200 at the bottom of the injection hole 111, and the second guide hole 240 connects the injection hole 111 and the microfluidic channel 300. In this way, the microporous layer 200 can have a larger area except for the micro-through hole 210 area, thereby improving the connection stability and limiting effect after installation. On this basis, through the design of the second guide hole 240, the injection hole 111 can be designed to be close to the micro-through hole 210, and a higher loading efficiency can be achieved during the sample separation and sealing process of the sealing oil.

[0078] In one embodiment, an observation spacer 113 is provided on the first chip cover 110. The observation spacer 113 is made of a light-transmitting material, which facilitates observation of the micro-through hole 210 area before, during, and after sample loading, thereby facilitating sample loading operations and confirming sample quality.

[0079] Furthermore, the observation spacer 113 can be closer to the microporous layer 200 relative to the bottom surface of the first chip cover 110 , so as to more conveniently and clearly observe the sample loading situation.

[0080] In one embodiment, the micro-through hole 210 is a through hole with a high aspect ratio, so that the micro-through hole 210 has better capillary force, so that when the sample flows through the bottom of the micro-through hole 210, it can be more effectively sucked into the interior of the micro-through hole 210, thereby achieving uniform segmentation and distribution of the sample, and the area where the micro-through holes 210 in the microporous layer 200 are located contains tens of thousands of micro-through holes 210 with a high aspect ratio, such as: the diameter of the micro-through hole 210 is 50 microns, the height is 500 microns, etc., to achieve better capillary force and effectively meet the uniform segmentation and distribution of the sample, but is not limited to this.

[0081] Furthermore, the flow channel height of the first chip base plate 120 is 100 microns, the surface of the microporous layer 200 is provided with a first hydrophobic area 700, the inner wall of the microthrough hole 210 is provided with a hydrophilic area 250, the first chip base plate 120 is provided with a second hydrophobic area 800 at the position of the microfluidic channel 300, and the first chip cover 110 is provided with a third hydrophobic area 900 at the position of the injection hole 111, etc. Water transfer treatment can be performed at other positions that are in contact with or possible positions of the sample and sealing oil, so that the microthrough hole 210 with the hydrophilic area 250 can better absorb the liquid sample with a large amount of water, reducing the risk of sealing oil entering the microthrough hole 210, and can achieve the effect of better absorbing and locking the sample, and a hydrophobic layer is made on the contact wall of other areas through which the sample or sealing oil passes, which can further reduce the sample residue in other non-preset areas, such as the surface of the microporous layer 200, the inner wall of the injection hole 111, etc., but is not limited to this.

[0082] Furthermore, the material of the microporous layer 200 can be one or more of silicon, silicon oxide, glass, plastic, etc., but is not limited thereto. The material of the first chip base plate 120 can be one or more of silicon, silicon oxide, glass, plastic, metal, etc., but is not limited thereto. The material of the first chip cover 110 can be one or more of optically transparent injection moldable materials such as PP, COC, COP, etc., but is not limited thereto.

[0083] In one embodiment, when the microfluidic channel 300 in the chip housing 100 is located at the top of the microporous layer 200, when the sample passes through the microfluidic channel 300, the sample is evenly divided and loaded from the top of the micro-through hole 210;

[0084] There are two specific implementation methods that can be recommended, but are not limited to these;

[0085] First, the sample can be loaded on the top of the micro-through hole 210 by flipping the microfluidic chip. This has been explained in detail above and will not be repeated here.

[0086] Second, the microfluidic channel 300 is directly disposed on the top of the microporous layer 200. Specifically, a gap can be directly reserved between the microporous layer 200 and the inner top surface of the chip housing 100 to form the microfluidic channel 300. To ensure the position stability of the microporous layer 200, one or more combinations of methods such as snap-fitting, plug-in connection, welding, clamping structure, and bonding can be used, and no specific limitation is imposed here.

[0087] Furthermore, the chip housing 100 may further include a second chip cover 130 and a second chip base plate 140, with a second mounting cavity defined between the second chip cover 130 and the second chip base plate 140. A microporous layer 200 is disposed within the second mounting cavity. A microfluidic channel 300 is disposed between the microporous layer 200 and the second chip cover 130. An injection hole 111 is connected to the microfluidic channel 300, and a sample and sealing oil are sequentially injected into the microfluidic channel 300 through the injection hole 111, thereby achieving sample loading from the top of the microporous layer 200.

[0088] Furthermore, by setting a second pad 540 at the top position of the microporous layer 200, or setting a second pad 540 at both the top and bottom positions of the microporous layer 200, and having micropores 550 on the second pad 540, the second pad 540 at the top position is surrounded by the second chip cover 130 and the microporous layer 200, so that the micropores 550 at the top position form a microfluidic channel 300. Similarly, the second pad 540 at the bottom position can be surrounded by the second chip bottom plate 140 and the microporous layer 200, so that the micropores 550 at the bottom position form a second sealed flow channel 410. The second sealed flow channel 410 and the microfluidic channel 300 are connected, and the connection position is located away from the injection hole 111, so the injection hole 111 can be preferably It is located on one side of the microporous layer 200, and the connecting position of the microfluidic channel 300 and the second sealed flow channel 410 can be preferably located on the other side of the microporous layer 200. It should be emphasized that: the microthrough hole 210 can also connect the microfluidic channel 300 and the second sealed flow channel 410. The above-mentioned connecting position refers to other connecting positions except the microthrough hole 210; when the sample and sealing oil are introduced through the injection hole 111, the sample can be fully and uniformly separated and introduced and sucked into the microthrough hole 210, ensuring that all microthrough holes 210 are effectively loaded with samples, and then the sealing oil can enter the interior of the second sealed flow channel 410 through the connecting position, so as to realize the filling of the microfluidic channel 300 and the second sealed flow channel 410 with the sealing oil, thereby realizing the full sealing of the microporous layer.

[0089] When the second pad 540 is only provided at the top of the microporous layer 200, there is also a gap between the bottom of the microporous layer 200 and the second chip base plate 140, so that the second sealed flow channel 410 can be directly formed at the bottom of the microporous layer 200. For example, the position of the microporous layer can be restricted by one or more combinations of methods such as snap-fitting, plug-in connection, welding, clamping structure, and bonding, which are not specifically limited here.

[0090] In one embodiment, a second microfluidic groove 141 is provided on the second chip base plate 140, and a second sealed flow channel 410 is formed between the second microfluidic groove 141 and the microporous layer 200. Furthermore, a second connecting area 142 is provided between the microfluidic groove and the microfluidic hole 550 away from the injection hole 111. The width of the second connecting area 142 is greater than that of other areas on the second sealed flow channel 410, and the upper and lower second connecting areas 142 are not blocked by the microporous layer 200. Therefore, the two second connecting areas 142 can maintain mutual communication. In other embodiments, the microporous layer 200 can also partially block the second connecting area 142, but is not limited to this.

[0091] As shown in Figure 6, a microfluidic system is formed by combining and splicing multiple microfluidic chips. For example, multiple microfluidic chips can be arranged side by side and extended in a linear direction. Of course, a chessboard-like arrangement or other optional methods are not excluded. The main purpose is to facilitate the loading of multiple microfluidic chips. In other embodiments, in order to ensure the relative stability of the microfluidic chips, rapid assembly can be achieved. Alternatively, a mounting plate can be set up and an installation area can be arranged on the mounting plate as required. If the installation area is a mounting groove, the microfluidic chip can be directly installed in the mounting area to achieve rapid assembly between multiple microfluidic chips to form a stable overall system. Furthermore, in order to ensure that multiple microfluidic chips can be quickly loaded uniformly, a unified flow channel can be further arranged on the mounting plate. No specific restrictions are made here.

[0092] As shown in FIG7 , a method for manufacturing a microfluidic chip is used to prepare the above-mentioned microfluidic chip, comprising the following steps:

[0093] S100: firstly add the sample into the injection hole 111, and then add the sealing oil into the injection hole 111;

[0094] S200: applying positive pressure to the sample injection hole 111, and the sealing oil pushes the sample into the microfluidic channel 300 under the positive pressure;

[0095] S300: The sample flows along the microfluidic channel 300 under the microporous layer 200 , and under the capillary force of the micro-through holes 210 on the microporous layer 200 , the sample is divided and sucked into the micro-through holes 210 ;

[0096] S400: The sealing oil continuously pushes the sample to flow along the microfluidic channel 300. The sample is further sucked into the micro-through hole 210 in the front of the flow direction, and the sealing oil removes the sample remaining on the inner wall of the microfluidic channel 300.

[0097] S500: The sealing oil continues to move forward and enters the top of the microporous layer 200, filling the interior of the chip housing 100 and fully sealing the microporous layer 200, thereby completing the preparation of the microfluidic chip.

[0098] The above-mentioned manufacturing method can achieve highly uniform segmentation and distribution of samples without the need for complex microfluidic channel 300 processing, and the process is simple. At the same time, no complex external loading equipment is required for cooperation, and the loading time is short; the high aspect ratio micro-through hole 210 structure uses capillary force to achieve more uniform sample segmentation and distribution; the sample utilization rate is high, and there is almost no waste; the loading speed is faster, and the loading can generally be completed within 10 seconds; the chip structure is simple, the processing difficulty is low, and the cost is low; the loading equipment is simple, the implementation difficulty is low, and the cost is low.

[0099] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip, characterized in that: include: Chip housing; A microporous layer is installed inside the chip housing, the microporous layer has a plurality of micro-through holes, the chip housing is provided with a microfluidic channel, and the chip housing is provided with a sample injection hole; It is suitable for sequentially introducing samples and sealing oil into the interior of the microfluidic channel through the sample injection hole and applying pressure so that the samples and the sealing oil pass through the micro-through holes in sequence, the samples are sucked into the micro-through holes, and the sealing oil fully seals the microporous layer.

2. A microfluidic chip according to claim 1, characterized in that: The microfluidic channel is provided at the bottom of the micro-through hole in the chip housing, and is suitable for the sample and the sealing oil to pass through the bottom of the micro-through hole in sequence.

3. A microfluidic chip according to claim 2, characterized in that: A first sealed flow channel is provided on the top of the microporous layer in the chip housing, and a first guide hole is provided inside the chip housing. The first guide hole communicates with the microfluidic channel and the first sealed flow channel.

4. A microfluidic chip according to claim 3, characterized in that: The chip housing is provided with an exhaust hole at the position of the first sealed flow channel; The dynamic viscosity of the sealing oil is greater than the dynamic viscosity of water, and is suitable for dragging away the sample remaining on the inner wall of the microfluidic channel during the flow of the sealing oil.

5. The microfluidic chip according to claim 4, characterized in that: The chip housing includes a first chip cover and a first chip base plate. The first chip base plate is adapted and connected so that a first mounting cavity is defined between the first chip cover and the first chip base plate, the microporous layer is disposed in the first mounting cavity, the microfluidic channel is defined between the microporous layer and the first chip base plate, and the first sealed flow channel is defined between the microporous layer and the first chip cover; The injection hole is provided on one side of the first chip cover close to the microporous layer, the first guide hole is provided on the microporous layer close to the other side of the microporous layer, and the microthrough hole is located between the injection hole and the first guide hole.

6. The microfluidic chip according to claim 5, characterized in that: The first chip bottom plate has a first microfluidic groove to form the microfluidic channel; The first microfluidic channel has a first communication area on a side away from the injection hole, suitable for the first communication area and the first sealed flow channel to be connected to each other without being blocked by the microporous layer.

7. The microfluidic chip according to claim 5, characterized in that: A first pad is provided on the first chip base plate near the bottom of the first mounting cavity, and a microfluidic hole is opened on the first pad at the bottom of the micro-through hole to form the microfluidic channel. A plurality of limit blocks are provided inside the first mounting cavity near the edge of the first pad, and the microporous layer is arranged on the top of the first pad and is positioned and restricted by the plurality of limit blocks.

8. The microfluidic chip according to claim 7, characterized in that: The microfluidic hole comprises a drainage area, a flow expansion area and a flow forming area, wherein the flow expansion area is located between the drainage area and the flow forming area and is interconnected; The width of the drainage area is smaller than the width of the flow forming area. The expansion area is trumpet-shaped, suitable for the narrow end of the expansion area to be connected to the drainage area, and the wide end of the expansion area to be connected to the The flow forming area is connected, and is suitable for the sample and the sealing oil to pass through the injection hole in sequence and be introduced into the first guide hole from the drainage area, the expansion area and the flow forming area in sequence; A plurality of the micro-through holes are distributed on the micro-porous layer to form a micro-porous area, the micro-porous area is located at the top of the flow expansion area and the flow formation area, and all the micro-through holes on the micro-porous area are connected to the micro-flow channel.

9. The microfluidic chip according to claim 7, characterized in that: The top surface of the first chip base plate is provided with a mounting groove, the bottom of the mounting groove is provided with the first pad, the top of the first pad is provided with a plurality of the limiting blocks near the side of the mounting groove, and the first chip cover is installed at the notch of the mounting groove and is limited and supported by the limiting blocks; The top and bottom surfaces of the microporous layer and the first pad are both flat, the bottom of the mounting groove is flat, the first pad and the bottom of the mounting groove are flatly fitted, and the contact position between the first pad and the microporous layer is flatly fitted.

10. The microfluidic chip according to claim 7, characterized in that: A second guide hole is provided on the microporous layer at the bottom of the injection hole, and the second guide hole connects the injection hole and the microfluidic channel; An observation spacer is provided on the first chip at the top of the micro-through hole, and the observation spacer is made of a light-transmitting material.

11. The microfluidic chip according to claim 7, characterized in that: The micro through hole is a through hole with a high aspect ratio; The surface of the microporous layer is provided with a first hydrophobic area, the inner wall of the micro-through hole is provided with a hydrophilic area, the first chip bottom plate is provided with a second hydrophobic area at the position of the microfluidic channel, and the first chip is covered with A third hydrophobic area is provided at the position of the sample injection hole.

12. The microfluidic chip according to claim 1, characterized in that: The microfluidic channel is provided at the top of the micro-through hole in the chip housing, and is suitable for the sample and the sealing oil to pass through the top of the micro-through hole in sequence; The chip housing may further include a second chip cover and a second chip base plate, wherein the second chip cover and the second chip base plate are adapted and connected to form a second mounting cavity between the second chip cover and the second chip base plate, the microporous layer is disposed in the second mounting cavity, the microfluidic channel is disposed between the microporous layer and the second chip cover, and a second sealed flow channel is disposed between the microporous layer and the second chip base plate; The sample is introduced into the microfluidic channel through the injection hole and is separated and loaded at the top position of the microporous layer, and the sealing oil flows from the microfluidic channel into the second sealing flow channel and fully seals the microporous layer.

13. The microfluidic chip according to claim 12, characterized in that: A second pad is provided on the second chip cover near the top of the second mounting cavity, and a micro hole is opened on the second pad at the top of the micro through hole to form the micro flow channel; Alternatively, the second chip cover is provided with the second pads at the top and bottom positions close to the second mounting cavity, and the second pads are provided with microfluidic holes at the positions of the microthrough holes to form the microfluidic channel or the second sealed flow channel.

14. The microfluidic chip according to claim 13, characterized in that: When the second chip cover is provided with a second pad only near the top of the second mounting cavity, the second chip bottom plate is provided with a second micro flow groove adapted to the micro flow hole to form the second sealed flow channel; The second microfluidic channel and the microfluidic hole each have a second communication area on a side away from the injection hole, which is suitable for the two second communication areas to be connected to each other without being blocked by the microporous layer.

15. A microfluidic system, characterized in that: The microfluidic chip according to any one of claims 1 to 14 is used, comprising: a plurality of the microfluidic chips; A plurality of the microfluidic chips are connected to each other.

16. A method for manufacturing a microfluidic chip, characterized in that: The method for preparing the microfluidic chip according to any one of claims 1 to 14 comprises the following steps: Firstly, the sample is added into the injection hole, and then the sealing oil is added into the injection hole; Applying positive pressure to the sample injection hole, so that the sealing oil pushes the sample into the microfluidic channel under the positive pressure; The sample flows through the microporous layer along the microfluidic channel, and under the action of the capillary force of the micro-through-holes on the microporous layer, the sample is divided and sucked into the micro-through-holes; The sealing oil continuously pushes the sample to flow along the microfluidic channel, and the sample is further sucked into the micro-through hole in the front of the flow direction, and the sealing oil removes the sample remaining on the inner wall of the microfluidic channel; The sealing oil continues to move forward and enters the top of the microporous layer, filling the interior of the chip housing and fully sealing the microporous layer, thereby completing the preparation of the microfluidic chip.

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