CN119614357A - A microfluidic chip for detecting potato-derived ingredients - Google Patents

Abstract

The present invention discloses a microfluidic chip for detecting potato-derived components, and relates to the technical field of microfluidic chips. The microfluidic chip is in the shape of a circular plate, and is divided into a two-layer structure from top to bottom, which is a first layer of PVC film and a second layer of PMMA board. A number 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 number of liquid storage chambers, a number of amplification chambers and a number of capillary channels arranged on the top surface of the second layer of PMMA board. 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 vent. The present invention proposes a microfluidic chip that can be used for detecting potato-derived components, and integrates multiple steps such as reagent sample addition and mixing, precise packaging, nucleic acid amplification and fluorescence detection through the size of the flow channel, microvalve and cavity design, so as to realize the miniaturization and automation of the detection process, and the process is fast and convenient, and can be controlled in real time.

Description

Microfluidic chip for detecting potato source components

Technical Field

The invention relates to the technical field of microfluidic chips, in particular to a microfluidic chip for detecting potato-derived components.

Background

The potato is used as an important food crop in the global scope, the cost of starch raw materials and the difference of processing technology cause great price difference, and the phenomenon of deliberate adulteration of illegal merchants is caused, so that the benefits of consumers are damaged, and the food safety problem is possibly caused. At present, research on potato source component adulteration identification detection methods at home and abroad is mainly focused on near infrared spectrum, scanning electron microscope, hyperspectral imaging and other technologies, but the methods are easily affected by potato varieties, production places, processing modes and other factors, and meanwhile, the problems of complex instrument and equipment operation, high technical requirements on personnel and the like exist.

Microfluidic is a technology for precisely controlling and manipulating micro-scale fluid, especially submicron structures, and is also called as lab-on-a-chip or microfluidic chip technology. The basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes are integrated on a micron-scale chip, and the whole analysis process is automatically completed. Because of its great potential in biological, chemical, medical and other fields, it has been developed into a new research field where the disciplines of biology, chemistry, medicine, fluids, electronics, materials, machinery and the like are crossed. Due to the micro-scale structure, the fluid shows and produces special properties different from macro-scale in the microfluidic chip, thus developing unique analytical properties. Meanwhile, the method has the advantages of light volume, small amount of used samples and reagents, low energy consumption, high reaction speed, capability of carrying out a large amount of parallel treatment, disposability and the like. Microfluidic technology has wide application in food detection, including microbiological detection, trace residue detection, food component analysis, food authenticity verification and traceability, and detection of food processing. These applications make the food detection process more efficient, accurate and reliable, helping to ensure food quality and food safety. With the continuous development and innovation of the technology, the application prospect of the microfluidic technology in the food detection field is wider.

At present, detection for potato-derived products in the market mainly comprises physical detection, chemical detection and a detection method based on biotechnology, a microfluidic device special for potato-derived component detection is not available, and controllable variables in detection are few, so that the requirements of experiments are difficult to meet.

Disclosure of Invention

Aiming at the defects of the existing products and technologies, the invention provides the microfluidic chip for detecting the potato source components, and the steps of reagent sample adding, sample mixing, accurate split charging, nucleic acid amplification, fluorescence detection and the like are integrated through the size of a flow channel, the design of a micro valve and a cavity, so that the miniaturization and automation of the detection process are realized, the process is rapid and convenient, and the real-time regulation and control can be realized.

In order to solve the technical problems, the invention is realized by the following technical scheme:

The microfluidic chip for detecting potato source components is in a circular plate shape, is divided into two layers from top to bottom, sequentially comprises a first layer of PVC film and a second layer of PMMA plate, wherein the adjacent two layers of structures are adhered up and down and the edges of the two layers of structures are overlapped to form a sealing state, a plurality of detection units are arranged in the microfluidic chip, each detection unit comprises a sample injection channel, a sample injection 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, the sample injection channels are arranged in an arc shape around the center of the microfluidic chip, one end of each sample injection channel is provided with the sample injection port, the other end of each sample injection channel is provided with the vent hole and the waste liquid cavity, the waste liquid cavities are located on one side of each sample injection channel away from the center of the microfluidic chip in an arc array, the amplification cavities and the liquid storage cavities are in one-to-one correspondence, the amplification cavities are located on one side of the corresponding sample injection channels, the amplification cavities and the corresponding liquid storage cavities are communicated through one channel, one capillary channel is arranged on the first layer of the capillary film, and the vent holes are corresponding to the vent holes are formed in the first layer of the sample injection film.

Preferably, the first layer of PVC film is a polyvinyl chloride film, and the second layer of PMMA plate is a polymethyl methacrylate PMMA plate.

Preferably, the sample injection channel, the sample injection port, the vent hole, the waste liquid cavity, the liquid storage cavities, the amplification cavities and the capillary channels on the second layer PMMA plate are all pore channels which are characterized on the surface of the PMMA plate by adopting CNC technology.

Preferably, the sample injection channel is a micro-flow channel which is 20-200mm long and 2-20mm wide and is communicated with the waste liquid cavity from the lower position of the sample injection port.

Preferably, the liquid storage cavity is a microfluidic channel with the length of 1-10mm and the width of 2-20mm, and the microfluidic channel is led to a position above the capillary channel from the sample introduction channel.

Preferably, the capillary channel is a groove which is 4-40mm long and 0.3-3mm wide and is sunk into the PMMA plate of the second layer and is communicated with a position above the amplification cavity from the liquid storage cavity.

Preferably, the amplification cavity is a micro-cavity with the diameter of 1.5-15mm and connected with one end of the capillary channel, and the inner diameter of the amplification cavity is larger than that of the capillary channel.

Preferably, the number of the detection units is 6.

The microfluidic chip can overcome the difficulties of complex operation, easy pollution, long detection time, low flux and the like of the traditional potato source component detection experiment, realize simplification of the operation flow of the potato source component detection experiment, reduce the use amount of samples and reagents, reduce the error and pollution risk in the operation process, and has the advantages of convenient and quick operation, high efficiency, large flux and less reagent consumption.

Drawings

Fig. 1 is a schematic structural view of an exploded state of a microfluidic chip of the present invention.

FIG. 2 is a schematic diagram of the structure of a single detection unit in the present invention.

1-First layer PVC membrane, 2-second layer PMMA board, 3-inlet, 4-inlet channel, 5-stock solution chamber, 6-amplification chamber, 7-capillary channel, 8-waste liquid chamber, 9-air vent.

Detailed Description

The invention is further described below with reference to the drawings and the detailed description.

Referring to fig. 1 and 2, the microfluidic chip for detecting potato source components is in a disc shape and is divided into two layers from top to bottom, namely a first layer of PVC film 1 and a second layer of PMMA plate 2 in sequence, wherein the first layer of PVC film is a polyvinyl chloride film, the second layer of PMMA plate is a polymethyl methacrylate PMMA plate, and the upper and lower adhesion and edge overlapping between the two adjacent layers of structures form a sealing state. The microfluidic chip is internally provided with 6 detection units, and each detection unit comprises a sample injection channel 4, a sample injection port 3, a vent hole 9, a waste liquid cavity 8, eight liquid storage cavities 5, eight amplification cavities 6 and eight capillary channels 7 which are arranged on the top surface of the second layer PMMA plate 2.

The sample injection channel 4 is arranged in an arc shape around the center of the microfluidic chip, a sample injection port 3 is arranged at one end of the sample injection channel 4, a vent hole 9 and a waste liquid cavity 8 are arranged at the other end of the sample injection channel 4, the waste liquid cavity 8 is located at one side of the sample injection channel 4 away from the center of the microfluidic chip, the plurality of liquid cavities 5 are distributed in an arc array on one side of the sample injection channel 4 away from the center of the microfluidic chip and are communicated with the sample injection channel 4, the amplification cavities 6 are in one-to-one correspondence with the liquid cavities 5, the amplification cavities 6 are located at one side of the corresponding liquid cavities 5 away from the sample injection channel 4, and the amplification cavities 6 are communicated with the corresponding liquid cavities 5 through a capillary channel 7. The inner diameter of the capillary channel 7 is smaller than that of the liquid storage cavity 5, one end of the capillary channel 7 is connected with the liquid storage cavity 5, the other end of the capillary channel 7 is connected with the amplification cavity 6, the capillary channel 7 can be connected with all liquid circulation conditions on the liquid storage cavity 5 and the amplification cavity 6 correspondingly, the amplification cavity 6 is a round cavity, one end of the capillary channel 7 is connected with the capillary channel 7, the sample injection channel 4 on the second layer PMMA plate 2 is a channel which is characterized on the surface of the PMMA plate by adopting CNC technology, the channel is a micro-flow channel which is 20-200mm long and 2-20mm wide, and is led to the waste liquid cavity 8 from the position below the sample injection port 3, the quantity of the micro-flow channel is 6, and the quantity of the micro-flow channel can be increased or decreased according to actual needs.

Each sample injection channel 4 is provided with 8 liquid storage cavities 5 which are connected in parallel, and the number of the liquid storage cavities can be increased or decreased according to actual needs. Each liquid storage cavity 5 is connected with an amplification cavity 6 through a capillary channel 7, the liquid storage cavity 5 is a micro-flow channel which is 1-10mm long and 2-20mm wide and is led to a position above the capillary channel 7 by a sample introduction channel 4, the liquid storage cavity 5 is a strip-shaped cavity, one end of the liquid storage cavity is connected with the liquid inlet channel 4, the other end of the liquid storage cavity is connected with the capillary channel 7, the capillary channel 7 is a groove which is 4-40mm long and 0.3-3mm wide and is sunk into the interior of a second layer PMMA plate and is led to a position above the amplification cavity 6 by the liquid storage cavity 5, the inner diameter of the capillary channel 7 is smaller than the inner diameter of the liquid storage cavity 5, one end of the capillary channel 7 is connected with the liquid storage cavity 5, the other end of the capillary channel 6 is connected with the amplification cavity 6, the amplification cavity 6 is a micro-cavity which is 1.5-15mm in diameter and is connected with one end of the capillary channel 7, the amplification cavity 6 is a round cavity, one end of the capillary channel 7 is connected with the inner diameter of the amplification cavity 6, and the inner diameter of the amplification cavity 6 is larger than the inner diameter of the capillary channel 7.

The first layer 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.

In the invention, 6 groups of capillary channel groups which are respectively formed by combining 8 capillary channels 7 are arranged on the second PMMA plate 2 and used for switching on and off the liquid circulation between the liquid storage cavity 5 and the amplifying cavity 6, the capillary channels 7 are in one-to-one correspondence with the amplifying cavity 6, the capillary channels 7 can switch on and off all the liquid circulation conditions on the liquid storage cavity 5 and the amplifying cavity 6, a microfluidic chip is arranged on a centrifugal machine, liquid is added into the sample inlet 3 by a liquid-transferring gun, when the centrifugal machine is in a low-speed running state, the liquid is equally divided into 8 parts and transferred to the liquid storage cavity 5, but the capillary channels 7 between the liquid storage cavity 5 and the amplifying cavity 6 cannot be broken, and when the centrifugal machine is in a high-speed running state, the liquid in the liquid storage cavity 5 enters the amplifying cavity 6, so that the fluid driving process on the chip is completed.

When the micro-fluidic chip is used, the bottom of the micro-fluidic chip is provided with a set of self-testing variable-temperature amplification heating module, the temperature control precision is within 1 ℃, the temperature range is adjustable and controllable within 32-98 ℃, the time control range is 1-180 hours, and the micro-fluidic chip has the functions of reminding and reminding to end.

The first stage of high flux accurate liquid-separating and sample-adding comprises mixing the sample to be detected, primer group, fluorescent probe, buffer solution, DNA polymerase and non-enzyme sterile water, sucking the mixed solution by using a liquid-transferring gun, inserting the mixed solution into a sample inlet 3 of a microfluidic chip, starting a centrifugal machine, starting the first low speed centrifugation, enabling the sample to reach a sample-feeding channel 4 from the sample-feeding inlet 3 and then reach a liquid-storing cavity 5 from the sample-feeding channel 4, and under normal condition, the flow resistance of the sample-feeding channel 4 and the liquid-storing cavity 5 is not greatly different, the mixed solution is equally divided into 8 parts and transferred to the liquid-storing cavity 5, but the mixed solution cannot break through a capillary channel 7 between the liquid-storing cavity 5 and an amplifying cavity 6, and the redundant mixed solution finally enters a liquid-storing cavity 8.

The second stage fluorescent PCR amplification reaction comprises stopping sample injection, starting a centrifugal machine, starting a second high-speed centrifugation, throwing the liquid in the liquid storage cavity 5 into the amplification cavity 6 by utilizing centrifugal force, starting a self-testing variable-temperature amplification heating module, and performing nucleic acid amplification and fluorescent PCR reaction, wherein in the fluorescent PCR reaction, a primer group and a fluorescent probe are specifically combined with target potato source DNA components and are amplified under the action of DNA polymerase, along with the progress of the reaction, fluorescent signals are gradually enhanced, the quantitative analysis of the target potato source DNA components can be performed by monitoring the intensity change of the fluorescent signals in real time, and the presence or absence and change of a real-time fluorescent quantitative PCR amplification specificity curve are compared with a negative control sample, so that the authenticity of a detection sample is judged.

The invention provides a microfluidic chip for detecting potato source components and a matched fluorescent PCR primer system, wherein a plurality of steps such as reagent sample adding and mixing, accurate split charging, nucleic acid amplification, fluorescent detection and the like are integrated through the size of a flow channel, a micro valve and cavity design, so that the miniaturization and automation of a detection process are realized, the process is rapid and convenient, and the real-time regulation and control can be realized. The chip mainly designs the size of a flow channel, the size of a capillary channel and the size of a cavity according to various parameters of a reagent system, flow resistance and centrifugal force, performs high-flux rapid fluid control and split charging in a short time, performs fluorescent PCR reaction and fluorescent signal detection in a stable and proper environment, solves the problem of rapid field detection requirements which cannot be met in technology originally, enables 48 repeated micro-chambers of each chip to simultaneously perform reaction and detection of samples, enables experimental environments formed in each independent chamber to be highly similar, enables one chip to perform detection of 6 samples with different concentrations at most in a single experiment, has detection time of about 25 minutes, and obviously improves experimental efficiency compared with the traditional method.

The microfluidic chip greatly reduces the requirements on nucleic acid amplification precise instruments and laboratory environments, and the chip has the characteristics of small volume, easiness in operation, convenience in carrying and the like, and the whole detection process is simple and rapid.

The microfluidic chip has low requirements on the expertise required by experimental operators. Conventional laboratory experiments typically require extensive skill and experience from the operator, such as a skilled handling of the pipette, accurate control of the liquid volume, accurate adjustment of the experimental conditions, etc. The microfluidic chip adopts an integrated and automatic design, so that experimental operation is simplified into a few simple steps. An operator can realize the processes of sample treatment, reaction, detection and the like by only injecting a sample and a reagent into an inlet of the chip. The whole experimental process can be automatically controlled through a computer program, so that manual intervention of operators is reduced, and operation difficulty and error are reduced. The invention does not depend on other instruments and equipment, has high running speed, high efficiency, strong specificity and simple operation.

It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (8)

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 up and down 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) arranged on the top surface of the second layer of PMMA plate (2), 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), 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 ingredients 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), 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) on the second layer of PMMA board (2) are all channels engraved 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, which leads from the position below the injection port (3) to the waste liquid chamber (8). 5. A microfluidic chip for detecting potato-derived ingredients 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 a 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 with a length of 4-40 mm and a width of 0.3-3 mm, which is sunken into the second layer of PMMA board (2) and leads from the liquid storage chamber (5) to the position above 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 microchamber with a diameter of 1.5-15 mm, connected to one end of the capillary channel (7), and 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.