Micro-fluidic chip for detecting potato-derived components

Abstract

The invention discloses a micro-fluidic chip for potato-derived component detection, and relates to the technical field of micro-fluidic chips, the micro-fluidic chip is in a circular plate shape and is divided into two layers from top to bottom, the two layers are a first layer of PVC film and a second layer of PMMA plate in sequence, a plurality of detection units are arranged in the micro-fluidic chip, and the first layer of PVC film and the second layer of PMMA plate are connected with the first layer of PMMA plate. Each detection unit comprises a sample introduction channel, a sample introduction port, a vent hole, a waste liquid cavity, a plurality of liquid storage cavities, a plurality of amplification cavities and a plurality of capillary channels which are arranged on the top surface of the second layer of PMMA plate, and a liquid injection hole corresponding to the sample introduction port and an exhaust hole corresponding to the vent hole are formed in the first layer of PVC film. According to the micro-fluidic chip capable of being used for detecting the potato-derived components, multiple steps such as reagent sample adding and mixing, accurate split charging, nucleic acid amplification and fluorescence detection are integrated through the size of a flow channel and design of a micro valve and a cavity, so that miniaturization and automation of the detection process are achieved, and the process is rapid, convenient and capable of being regulated and controlled in real time.

Description

Technical Field

[0001] The patent of this invention relates to the field of microfluidic chip technology, and in particular to a microfluidic chip for detecting potato-derived components. Background Art

[0002] As an important food crop worldwide, the difference in the cost of raw starch and processing technology of potatoes leads to a wide price gap, which has triggered the deliberate adulteration of unscrupulous merchants, which not only harms the interests of consumers, but also may cause food safety problems. At present, the research on the identification and detection methods of adulteration of potato-derived ingredients at home and abroad mainly focuses on near-infrared spectroscopy, scanning electron microscopy, hyperspectral imaging and other technologies, but these methods are easily affected by factors such as potato varieties, origins, and processing methods. At the same time, there are also problems such as complex instrument and equipment operation and high technical requirements for personnel.

[0003] Microfluidics is a technology that precisely controls and manipulates microscale fluids, especially submicron structures, also known as chip labs or microfluidic chip technology. It integrates the basic operating units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, into a micron-scale chip to automatically complete the entire analysis process. Due to its huge potential in the fields of biology, chemistry, and medicine, it has developed into a new research field that intersects biology, chemistry, medicine, fluids, electronics, materials, and mechanics. Due to the micron-scale structure, fluids in microfluidic chips show and produce special properties that are different from those at the macroscale, thus developing unique analytical performance. At the same time, it also has the advantages of light size, small amount of samples and reagents, low energy consumption, fast reaction speed, large-scale parallel processing, and disposable. Microfluidic technology has a wide range of applications in food testing, including microbial detection, trace residue detection, food component analysis, food authenticity verification and traceability, and food processing detection. These applications make the food testing process more efficient, accurate, and reliable, helping to ensure food quality and food safety. With the continuous development and innovation of technology, the application prospects of microfluidic technology in the field of food testing will be broader.

[0004] At present, the detection of potato-derived products on the market mainly includes physical detection, chemical detection and biotechnology-based detection methods. There is no microfluidic device specifically for the detection of potato-derived ingredients, and there are few controllable variables in the detection, which makes it difficult to meet experimental needs. Summary of the invention

[0005] In view of the shortcomings of existing products and technologies, the present invention proposes a microfluidic chip that can be used for the detection of potato-derived ingredients. Multiple steps such as reagent sampling and mixing, precise packaging, nucleic acid amplification and fluorescence detection are integrated through the size of the flow channel, microvalves and cavity design, thereby realizing the miniaturization and automation of the detection process. The process is fast, convenient and can be controlled in real time.

[0006] In order to solve the above technical problems, the present invention is implemented by the following technical solutions: A microfluidic chip for detecting potato-derived components, the microfluidic chip is in the shape of a circular plate, and is divided into two layers from top to bottom, which are a first layer of PVC film and a second layer of PMMA board, respectively. The two adjacent layers are bonded up and down and overlapped at the edges to form a sealed state. A plurality of detection units are arranged in the microfluidic chip, and each detection unit includes an injection channel, an injection port, a vent, a waste liquid chamber, a plurality of liquid storage chambers, a plurality of amplification chambers and a plurality of capillary channels arranged on the top surface of the second layer of PMMA board. The injection channel is in the shape of an arc and is arranged around the center of the microfluidic chip. The microfluidic chip is arranged in a manner that one end of the injection channel is provided with an injection port, and the other end is provided with an air vent and a waste liquid chamber, wherein the waste liquid chamber is located on a side of the injection channel away from the center of the microfluidic chip; the plurality of liquid storage chambers are distributed in an arc array on a side of the injection channel away from the center of the microfluidic chip and are all connected to the injection channel; the amplification chamber corresponds to the liquid storage chamber one by one, the amplification chamber is located on a side of the corresponding liquid storage chamber away from the injection channel, and the amplification chamber and the corresponding liquid storage chamber are connected through a capillary channel; the first layer of PVC film is provided with a liquid injection hole corresponding to the injection port and an exhaust hole corresponding to the air vent.

[0007] Preferably, the first layer of PVC film is a polyvinyl chloride film, and the second layer of PMMA board is a polymethyl methacrylate PMMA board.

[0008] Preferably, the injection channel, an injection port, an air vent, a waste liquid chamber, a plurality of liquid storage chambers, a plurality of amplification chambers and a plurality of capillary channels on the second layer of PMMA board are all holes carved on the surface of the PMMA board using CNC technology.

[0009] Preferably, the injection channel is a microfluidic channel with a length of 20-200 mm and a width of 2-20 mm, which leads from the position below the injection port to the waste liquid chamber.

[0010] Preferably, the liquid storage chamber is a microfluidic channel with a length of 1-10 mm and a width of 2-20 mm, which leads from the injection channel to the position above the capillary channel.

[0011] Preferably, the capillary channel is a groove with a length of 4-40 mm and a width of 0.3-3 mm, which extends from the liquid storage chamber to the position above the amplification chamber and is sunk into the second layer of PMMA board.

[0012] Preferably, the amplification chamber is a microchamber with a diameter of 1.5-15 mm and connected to one end of the capillary channel, and the inner diameter of the amplification chamber is larger than the inner diameter of the capillary channel.

[0013] Preferably, the number of the detection units is 6.

[0014] The microfluidic chip of the present invention can overcome the difficulties of traditional potato-derived component detection experiments such as complex operation, easy contamination, long detection time, and low throughput, and can simplify the operation process of potato-derived component detection experiments, reduce the use of samples and reagents, and reduce the error and contamination risks during the operation. The operation is convenient and fast, the efficiency is high, the throughput is large, and the reagent consumption is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A schematic structural diagram of the microfluidic chip of the present invention in a disassembled state.

[0016] Figure 2 A schematic diagram of the structure of a single detection unit in the present invention.

[0017] 1-the first layer of PVC film, 2-the second layer of PMMA board, 3-the injection port, 4-the injection channel, 5-the liquid storage chamber, 6-the amplification chamber, 7-the capillary channel, 8-the waste liquid chamber, 9-the vent hole. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0019] A microfluidic chip for detecting potato-derived ingredients, see Figure 1 , Figure 2 The microfluidic chip is in the shape of a circular plate, and is divided into two layers from top to bottom, which are the first layer of PVC film 1 and the second layer of PMMA board 2. The first layer of PVC film is a polyvinyl chloride film, and the second layer of PMMA board is a polymethyl methacrylate PMMA board. The two adjacent layers are bonded up and down and the edges overlap to form a sealed state. There are 6 detection units in the microfluidic chip, and each detection unit includes an injection channel 4, an injection port 3, a vent 9, a waste liquid chamber 8, eight storage chambers 5, eight amplification chambers 6 and eight capillary channels 7 arranged on the top surface of the second layer of PMMA board 2.

[0020] The injection channel 4 is arranged in an arc shape around the center of the microfluidic chip, one end of the injection channel 4 is provided with an injection port 3, and the other end is provided with a vent 9 and a waste liquid chamber 8, the waste liquid chamber 8 is located on the side of the injection channel 4 away from the center of the microfluidic chip; the plurality of liquid storage chambers 5 are distributed in an arc array on the side of the injection channel 4 away from the center of the microfluidic chip and are all connected to the injection channel 4, the amplification chamber 6 corresponds to the liquid storage chamber 5 one by one, the amplification chamber 6 is located on the side of the corresponding liquid storage chamber 5 away from the injection channel 4, and the amplification chamber 6 is connected to the corresponding liquid storage chamber 5 through a capillary channel 7. The inner diameter of the capillary channel 7 is smaller than the inner diameter of the liquid storage chamber 5, one end of which is connected to the liquid storage chamber 5, and the other end of which is connected to the amplification chamber 6. The capillary channel 7 can open and close all liquid flows in the corresponding liquid storage chamber 5 and the amplification chamber 6; the amplification chamber 6 is a circular chamber, one end of which is connected to the capillary channel 7; the injection channel 4 on the second layer of PMMA board 2 is a channel engraved on the surface of the PMMA board using CNC technology, and the channel is a microfluidic channel with a length of 20-200 mm and a width of 2-20 mm, which leads from the position below the injection port 3 to the waste liquid chamber 8. The number of microfluidic channels set in the present invention is 6, and the number can be increased or decreased according to actual needs.

[0021] Each injection channel 4 is provided with 8 parallel liquid storage chambers 5, and the number of the liquid storage chambers 5 can be increased or decreased according to actual needs. Each liquid storage chamber 5 is connected to the amplification chamber 6 through a capillary channel 7; the liquid storage chamber 5 is a microfluidic channel with a length of 1-10 mm and a width of 2-20 mm, which leads from the injection channel 4 to the position above the capillary channel 7; the liquid storage chamber 5 is a long strip chamber, one end of which is connected to the liquid inlet channel 4, and the other end is connected to the capillary channel 7; the capillary channel 7 is a groove sunk into the second layer of PMMA board with a length of 4-40 mm and a width of 0.3-3 mm, which leads from the liquid storage chamber 5 to the position above the amplification chamber 6; the inner diameter of the capillary channel 7 is smaller than the inner diameter of the liquid storage chamber 5, one end is connected to the liquid storage chamber 5, and the other end is connected to the amplification chamber 6; the amplification chamber 6 is a micro chamber with a diameter of 1.5-15 mm, which is connected to one end of the capillary channel 7; the amplification chamber 6 is a circular chamber, one end of which is connected to the capillary channel 7, and the inner diameter of the amplification chamber 6 is larger than the inner diameter of the capillary channel 7.

[0022] The first PVC film 1 is provided with a liquid injection hole corresponding to the sample inlet 3 and an exhaust hole corresponding to the vent hole 9 .

[0023] In the present invention, 6 groups of capillary channel groups each consisting of 8 capillary channels 7 are provided on the second layer of PMMA plate 2, which are used to open and close the flow of liquid between the liquid storage chamber 5 and the amplification chamber 6. The capillary channels 7 correspond to the amplification chamber 6 one by one, and the capillary channels 7 can open and close all liquid flows in the corresponding liquid storage chamber 5 and the amplification chamber 6. The microfluidic chip is placed on a centrifuge, and the liquid is added to the injection port 3 with a pipette. When the centrifuge is in a low-speed operation state, the liquid will be divided into 8 equal parts and transferred to the liquid storage chamber 5, but it cannot break through the capillary channel 7 between the liquid storage chamber 5 and the amplification chamber 6; when the centrifuge is in a high-speed operation state, the liquid in the liquid storage chamber 5 enters the amplification chamber 6, thereby completing the fluid driving process on the chip.

[0024] When in use, a self-test variable temperature amplification heating module is provided at the bottom of the microfluidic chip of the present invention, the temperature control accuracy is within 1 degree, the temperature range is adjustable from 32 to 98 degrees, the time control range is 1 to 180 hours, and it has the functions of start reminder and end reminder.

[0025] The first stage is high-throughput precise liquid separation and sampling: the sample to be tested, primer set, fluorescent probe, buffer, DNA polymerase and enzyme-free sterile water and other reagents are mixed, the mixed solution is sucked up with a pipette, and inserted into the microfluidic chip sampling port 3, the centrifuge is started, and the first low-speed centrifugation is started, so that the sample reaches the sampling channel 4 from the sampling port 3, and then reaches the liquid storage chamber 5 from the sampling channel 4; under normal circumstances, the flow resistance of the sampling channel 4 and the liquid storage chamber 5 is not much different, and the mixed solution will be divided into 8 equal parts and transferred to the liquid storage chamber 5, but it cannot break through the capillary channel 7 between the liquid storage chamber 5 and the amplification chamber 6, and the excess mixed liquid finally enters the waste liquid chamber 8.

[0026] The second stage of fluorescent PCR amplification reaction: stop injecting samples, start the centrifuge, start the second high-speed centrifugation, use centrifugal force to throw the liquid in the liquid storage chamber 5 into the amplification chamber 6, start the self-test variable temperature amplification heating module, and perform nucleic acid amplification and fluorescent PCR reaction; in the fluorescent PCR reaction, the primer set and the fluorescent probe specifically bind to the target potato-derived DNA component and amplify under the action of DNA polymerase; as the reaction proceeds, the fluorescent signal will gradually increase, and the target potato-derived DNA component can be quantitatively analyzed by real-time monitoring of the intensity change of the fluorescent signal, and compared with the negative control sample according to the presence and change of the real-time fluorescent quantitative PCR amplification specificity curve, so as to judge the authenticity of the test sample.

[0027] The present invention provides a microfluidic chip for detecting potato-derived components and a matching fluorescent PCR primer system, which integrates multiple steps such as reagent sample addition and mixing, precise packaging, nucleic acid amplification and fluorescent detection through the size of the flow channel, microvalve and cavity design, thereby realizing miniaturization and automation of the detection process, and the process is fast and convenient, and can be controlled in real time. The chip mainly designs the flow channel size, capillary channel and cavity size based on various parameters of the reagent system, flow resistance and centrifugal force, performs high-throughput rapid fluid control and packaging in a short time, and performs fluorescent PCR reaction and fluorescent signal detection in a stable and suitable environment, so that some rapid on-site detection requirements that were originally technically unsatisfactory are solved, and 48 repeated microchambers on each chip can react and detect samples at the same time, and the experimental environment formed in each individual chamber is highly similar. In a single experiment, one chip can complete the detection of up to 6 samples of different concentrations, and the detection time is about 25 minutes, which significantly improves the experimental efficiency compared with traditional methods.

[0028] The microfluidic chip greatly reduces the requirements for precision instruments and laboratory environment for nucleic acid amplification. The chip itself is small in size, easy to operate and carry, and the entire detection process is simple and quick.

[0029] The microfluidic chip has low requirements for the professional skills required of the experimental operators. Traditional laboratory experiments usually require operators to have rich experimental skills and experience, such as mastering the use of pipettes, accurately controlling the volume of liquids, and accurately adjusting experimental conditions. However, the microfluidic chip adopts an integrated and automated design, which simplifies the experimental operation into a few simple steps. The operator only needs to inject samples and reagents into the inlet of the chip to achieve the processes of sample processing, reaction, and detection. The entire experimental process can be automatically controlled by a computer program, reducing the manual intervention of the operator, and reducing the difficulty and error of the operation. The present invention does not rely on other instruments and equipment, has a fast operating speed, high efficiency, strong specificity, and is easy to operate.

[0030] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A microfluidic chip for detecting potato-derived ingredients, the microfluidic chip being in the shape of a circular plate and being divided into two layers from top to bottom, namely, a first layer of PVC film (1) and a second layer of PMMA plate (2), the two adjacent layers being bonded to each other and overlapping at the edges to form a sealed state, characterized in that: A plurality of detection units are arranged in the microfluidic chip, each detection unit comprising an injection channel (4), an injection port (3), a vent (9), a waste liquid chamber (8), a plurality of liquid storage chambers (5), a plurality of amplification chambers (6) and a plurality of capillary channels (7) arranged on the top surface of the second PMMA plate (2). The injection channel (4) is arranged in an arc shape around the center of the microfluidic chip, one end of the injection channel (4) is provided with an injection port (3), and the other end is provided with a vent (9) and a waste liquid chamber (8), wherein the waste liquid chamber (8) is located on a side of the injection channel (4) away from the center of the microfluidic chip; the plurality of liquid storage chambers (5) are distributed in an arc-shaped array on a side of the injection channel (4) away from the center of the microfluidic chip and are all connected to the injection channel (4); the amplification chamber (6) corresponds to the liquid storage chamber (5) one by one, the amplification chamber (6) is located on a side of the corresponding liquid storage chamber (5) away from the injection channel (4), and the amplification chamber (6) is connected to the corresponding liquid storage chamber (5) through a capillary channel (7); The first layer of PVC film (1) is provided with a liquid injection hole corresponding to the sample inlet (3) and an exhaust hole corresponding to the vent hole (9).

2. A microfluidic chip for detecting potato-derived components as claimed in claim 1, characterized in that: The first layer of PVC film (1) is a polyvinyl chloride film, and the second layer of PMMA board (2) is a polymethyl methacrylate (PMMA) board.

3. A microfluidic chip for detecting potato-derived components as claimed in claim 1, characterized in that: The injection channel (4), an injection port (3), an air vent (9), a waste liquid chamber (8), a plurality of liquid storage chambers (5), a plurality of amplification chambers (6) and a plurality of capillary channels (7) on the second layer of PMMA board (2) are all holes carved on the surface of the PMMA board using CNC technology.

4. A microfluidic chip for detecting potato-derived components as claimed in claim 1, characterized in that: The injection channel (4) is a microfluidic channel with a length of 20-200 mm and a width of 2-20 mm, extending from the lower position of the injection port (3) to the waste liquid chamber (8).

5. A microfluidic chip for detecting potato-derived components as claimed in claim 1, characterized in that: The liquid storage chamber (5) is a microfluidic channel with a length of 1-10 mm and a width of 2-20 mm, which leads from the injection channel (4) to the position above the capillary channel (7).

6. A microfluidic chip for detecting potato-derived components as claimed in claim 1, characterized in that: The capillary channel (7) is a groove sunk into the second layer of PMMA board (2) and is 4-40 mm long and 0.3-3 mm wide, leading from the liquid storage chamber (5) to the upper position of the amplification chamber (6).

7. A microfluidic chip for detecting potato-derived components as claimed in claim 1, characterized in that: The amplification chamber (6) is a micro-chamber with a diameter of 1.5-15 mm and connected to one end of the capillary channel (7). The inner diameter of the amplification chamber (6) is larger than the inner diameter of the capillary channel (7).

8. A microfluidic chip for detecting potato-derived components according to any one of claims 1 to 7, characterized in that: The number of the detection units is 6.

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